Background
==========

The defects in neonatal adaptive immunity are relatively easy to understand *a priori*. Although there are complexities to be considered \[[@B1],[@B2]\], experimental evidence demonstrates that newborns, lacking prior antigen exposure, must develop immunologic memory based on postnatal experience with phogens and environmental immunogens \[[@B3]-[@B5]\].

It is less clear why there should be defects in newborns\' innate immunity, although these defects are well documented. For example, newborns have long been known to exhibit defects in phagocytosis \[[@B6]\], chemotaxis \[[@B7],[@B8]\], and adherence \[[@B9]\], the latter possibly due to aberrant regulation of critical cell-surface proteins that mediate leukocyte-endothelial interactions \[[@B10]\]. Newborn monocytes also exhibit diminished secretion of numerous cytokines under both stimulated and basal conditions \[[@B11]-[@B13]\].

Elucidating the causes of these defects is a crucial question in neonatal medicine, since infection remains a major cause of morbidity and mortality in the newborn period. However, unravelling the complex events in monocyte and/or neutrophil activation, from ligand binding to activation of effector responses, is clearly a daunting challenge. Any one of numerous pathways from the earliest cell signalling events to protein synthesis or secretion could be relevant, and focusing on any one may overlook critical aspects of cellular regulation. In this context, genomic and/or proteomic approaches may offer some important advantages, at least in the initial phases of investigation, by allowing investigators to survey the panoply of biological processes that may be relevant to identifying critical biological distinctions.

Recently published work has documented differences in gene expression between adult and cord blood monocytes \[[@B14]\], although these studies did not elucidate the fundamental, functional differences between cord blood and adult cells. The studies we report here demonstrate how computational analyses, applied to microarray data, can elucidate critical biological functions when analysis extends beyond the identification of differentially-expressed genes.

Methods
=======

Cells and cellular stimulation
------------------------------

Monocytes were purified from cord blood of healthy, term infants and from the peripheral blood of healthy adults by positive selection using anti-CD-14 mAb-coated magnetic beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer\'s instructions. Informed consent was obtained from adult volunteers; collection of cord blood was ruled exempt from consent after review by the Oklahoma Health Sciences Center IRB. In brief, blood was collected into sterile tubes containing sodium citrate as an anticoagulant (Becton Dickinson, Franklin Lakes, NJ). Peripheral blood mononuclear cells (PBMC) were prepared from the anti-coagulated blood using gradient separation on Histopaque-1077 performed directly in the blood collection tubes. Cells were washed three times in Ca^2+^and Mg^2+^-free Hanks\'s balanced salt solution. PBMC were incubated for 20 min at 4°C with CD14 microbeads at 20 *μ*l/1 × 10^7^cells. The cells were washed once, re-suspended in 500 *μ*l Ca^2+^and Mg^2+^-free PBS containing 5% FBS/1 × 10^8^cells. The suspension was then applied to a MACs column. After unlabeled cells passed through, the column was washed with 3 × 500 μl Ca^2+^and Mg^2+^-free PBS. The column was removed from the separator and was put on a new collection tube. One ml of Ca^2+^and Mg^2+^-free PBS was then added onto the column, which was immediately flushed by firmly applying the plunger supplied with the column.

Purified monocytes were incubated with LPS from *Escherichia coli*0111:4B (Sigma, St. Louis, MO) at 10 ng/ml for 45 min and 2-hours in RPMI 1640 with 10% fetal bovine serum or studied in the absence of stimulation (\"zero time\"). It should be noted that this product is not \"pure,\" and stimulates both TLR-4 and TRL-2 signaling pathways \[[@B15]\]. A smaller number of replicates (n = 5) was analyzed after 24 hr incubation. After the relevant time points, monocytes were lysed with TriZol (Invitrogen, Carlsbad, CA, USA) and RNA was isolated as recommended by the manufacturer. Cells from eight different term neonates and eight different healthy adults were used for these studies.

Gene microarrays
----------------

The microarrays used in these experiments were developed at the Oklahoma Medical Research Foundation Microarray Research Facility and contained probes for 21,329 human genes. Slides were produced using commercially available libraries of 70 nucleotide long DNA molecules whose length and sequence specificity were optimized to reduce the cross-hybridization problems encountered with cDNA-based microarrays (Qiagen-Operon). The oligonucleotides were derived from the UniGene and RefSeq databases. The RefSeq database is an effort by the NCBI to create a true reference database of genomic information for all genes of known function. All 11,000 human genes of known or suspected function were represented on these arrays. In addition, most undefined open reading frames were represented (approximately 10,000 additional genes).

Oligonucleotides were spotted onto Corning^®^UltraGAPS™ amino-silane coated slides, rehydrated with water vapor, snap dried at 90°C, and then covalently fixed to the surface of the glass using 300 mJ, 254 nm wavelength ultraviolet radiation. Unbound free amines on the glass surface were blocked for 15 min with moderate agitation in a 143 mM solution of succinic anhydride dissolved in 1-methyl-2-pyrolidinone, 20 mM sodium borate, pH 8.0. Slides were rinsed for 2 min in distilled water, immersed for 1 min in 95% ethanol, and dried with a stream of nitrogen gas.

Labeling, hybridization, and scanning
-------------------------------------

Fluorescently labeled cDNA was separately synthesized from 2.0 μg of total RNA using an oligo dT~12--18~primer, PowerScript reverse transcriptase (Clontech, Palo Alto, CA), and Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) for 1 hour at 42°C in a volume of 40 μl. Reactions were quenched with 0.5 M EDTA and the RNA was hydrolyzed by addition of 1 M NaOH for 1 hr at 65°C. The reaction was neutralized with 1 M Tris, pH 8.0, and cDNA was then purified with the Montage PCR~96~Cleanup Kit (Millipore, Billerica, MA). cDNA was added to ChipHybe™ hybridization buffer (Ventana Medical Systems, Tucson, AZ) containing Cot-1 DNA (0.5 mg/ml final concentration), yeast tRNA (0.2 mg/ml), and poly(dA)~40--60~(0.4 mg/ml). Hybridization was performed on a Ventana Discovery system for 6 hr at 42°C. Microarrays were washed to a final stringency of 0.1× SSC, and then scanned using a dual-color laser (Agilent Biotechnologies, Palo Alto, CA). Fluorescent intensity was measured by Imagene™ software (BioDiscovery, El Segundo, CA).

PCR validation of array data
----------------------------

### Reverse transcription

Three cord blood samples (C1, C2, and C5) and three adult samples (A1, A5, and A6) from the 45 minute time point were used for PCR validation. First strand cDNA was generated from 3.6 μg of total RNA per sample using the OmniScript Reverse Transcriptase and buffer (Qiagen, Valencia, CA), 1 μl of 100 μM oligo dT primer (dT~15~) in a 40 μl volume. Reactions were incubated 60 min at 37° and inactivated at 93° for 5 min. cDNA was diluted 1:100 in water and stored at -20°C.

Quantitative PCR
----------------

Gene-specific primers for 10 genes (*Erbb3, Tmod, Dscr1l1, Sp1, Scya4, Gro2, Cri1, Scya3, Scya3l1*, and *Il-1a*) were designed with a 60°C melting temperature and a length of 19--25 bp for PCR products with a length of 90--140 bp, using Applied Biosystems Inc (ABI, Foster City, CA) Primer Express 1.5 software. PCR was run with 2 μl cDNA template in 15 μl reactions in triplicate on an ABI SDS 7700 using the ABI SYBR Green I Master Mix and gene specific primers at a concentration of 1 μM each. The temperature profile consisted of an initial 95°C step for 10 minutes (for Taq activation), followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min, and then a final melting curve analysis with a ramp from 60°C to 95°C over 20 min. Gene-specific amplification was confirmed by a single peak in the ABI Dissociation Curve software. No template controls were run for each primer pair. Since equal amounts of total RNA were used for cDNA synthesis, Ct values should reflect relative abundance \[[@B16]\]. These values were used to calculate the average group Ct (Cord vs. Adult) and the relative ΔCt was used to calculate fold change between the two groups \[[@B17]\].

Apoptosis assays
----------------

Exposed membrane phospholipids (a marker for early apoptosis) were detected in adult and neonatal monocytes after LPS stimulation using a commercially available annexin V binding assay. Monocytes from cord blood and adult peripheral blood were obtained as outlined above. Isolated monocytes were either labeled immediately with annexin V-FITC or were stimulated for 14 hours with LPS 10 ng/ml prior to labeling (this time point was derived empirically to maximize apoptosis). Annexin V-FITC staining was completed via the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) using 5 μl of propidium iodine and 5 μl annexin V-FITC as recommended by the manufacturer. Analysis by flow cytometry was accomplished on a FACS Calibur automated benchtop flow cytometer. Data obtained by flow cytometry was analyzed by non-parametric t-test (Mann-Whitney test). An alpha level of 0.05 was considered statistically significant.

Statistical analysis
--------------------

Microarrays were normalized and tested for differential expression using methods described previously \[[@B18]\]. Differential expression was concluded if the genes met the following criteria: a minimum expression level at least 10 times above background at one or more time points, a minimum 1.5-fold difference in the mean expression values between groups at one or more time points, and a minimum of 80% reproducibility using the jack-knife method. A jack-knife is the most common type of Leave-one-out-cross-validation (LOOCV); it is used here to cross-validate genes selected by differential analysis \[[@B19]\]. Time series analysis was performed using the hypervariable (HV) gene method previously described by our group \[[@B20]\].

After selection, HV genes are clustered and interrogated for gene-gene interactions. K-means clustering, an unsupervised technique, was performed on the HV genes to create unbiased clusters. Discriminate function analysis (DFA), a supervised technique, was used to determine and spatially map gene-to-gene interactions \[[@B21]\].

All statistical analysis was performed in Matlab R14 (Natick, MA) and Statistica v7 (Tulsa, OK, USA). An alpha level of 0.05 was considered statistically significant for all analyses.

Analysis of the apoptosis assays was undertaken using both parametric and non-parametric analysis methods. Parametric analysis was undertaken using the student\'s t-test; non-parametic analysis used the Mann-Whitney U-test. A p-value of \> 0.05 was the threshold for rejecting the null hypothesis.

### Discriminant function analysis

DFA is a method that identifies a subset of genes whose expression values can be linearly combined in an equation, denoted a root, whose overall value is distinct for a given characterized group. DFA therefore, allows the genes that maximally discriminate among the distinct groups analyzed to be identified. In the present work, a variant of the classical DFA, named the Forward Stepwise Analysis, was used to select the set of genes whose expression maximally discriminated among experimentally distinct groups. The Forward Stepwise Analysis was built systematically in an iterative manner. Specifically, at each step all variables were reviewed to identify the one that most contributes to the discrimination between groups. This variable was included in the model, and the process proceeded to the next iteration. The statistical significance of discriminative power of each gene was also characterized by partial Wilk\'s Lambda coefficients, which are equivalent to the partial correlation coefficient generated by multiple regression analyses. The Wilk\'s Lambda coefficient used a ratio of within-group differences and the sum of within-plus between-group differences. Its value ranged from 1.0 (no discriminatory power) to 0.0 (perfect discriminatory power).

### Computer analysis of functional associations between differentially expressed genes

In addition to the above analyses, genes showing the most significant differences between neonatal and adult cells were characterized functionally using pre-existing databases such as PubMed, BIND, KEGG, and Ontoexpress. Biological associations of the differentially expressed genes were modelled using Ingenuity Pathways Analysis (Redwood City, CA). Data analyzed through this technique can then be resolved into cogent models of the specific biological pathways activated under the experimental conditions used in the microarray analyses.

Results
=======

Differential gene expression analysis
-------------------------------------

Table [1](#T1){ref-type="table"} lists genes determined to be differentially expressed between cord and adult peripheral blood monocytes, as described above. No genes were found to be statistically significantly differentially expressed between adult and cord monocytes in the absence of LPS exposure. 168 genes were differentially expressed between adult and cord monocytes after 45 min incubation with LPS. 95% of these genes (159 of 168) were over-expressed in adult relative to cord monocytes. After 120 minutes of LPS exposure, 24 genes were differentially expressed between adult and cord monocytes. Of the latter genes, 23 were more highly expressed in cord than adult monocytes. This pattern of differentially expressed genes suggested an initial delayed response to LPS followed by an enhanced transcription of genes in cord relative to adult monocytes. To test this hypothesis, k-means clustering was used to categorize differentially expressed genes based on their temporal profiles. Relative decreases in gene transcription by cord monocytes at 45 min were seen in 6 of the 9 clusters (Figure [1](#F1){ref-type="fig"}). Each of these clusters contained between 15 and 46 genes. Examination of the clusters showed that differences between groups after 45 minutes of LPS exposure were attributable to a) genes in certain clusters that were up-regulated in adult monocytes only, b) genes in other clusters that were down-regulated in cord monocytes only, or c) genes in yet other clusters that were up-regulated in adult and down-regulated in cord monocytes. These results, summarized in a heat map in Figure [2](#F2){ref-type="fig"}, indicated a high complexity of gene expression differences between adult monocytes and cord blood monocytes in response to LPS.

###### 

Differentially expressed genes between adult and cord monocytes at specific time points. T = time (min) at which the sample was taken. Numbers indicate corrected expression values.

                                                                                                                                                                                                   **Adult**   **Adult**    **Adult**    **Cord**   **Cord**     
  ------------------------------------------------ ------------------------ --------------- ------------------------------------------------------------------------------------------------------ ----------- ------------ ------------ ---------- ------------ -------------
  **Apoptosis**                                                                                                                                                                                                                                                  
                                                   [NM_033423](NM_033423)   CTLA1           Similar to granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1)                317         **419**      299          199        **193**      264
                                                   [AB037796](AB037796)     PDCD6IP         Programmed cell death 6 interacting protein                                                            75          **155**      68           79         **70**       81
                                                   [NM_024969](NM_024969)   TAIP-2          TGFb-induced apoptosis protein 2                                                                       63          **113**      107          53         **68**       116
                                                   [NM_003127](NM_003127)   SPTAN1          Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin)                                                     713         842          ***1171***   724        824          ***2093***
  **Protein synthesis, processing, degradation**                                                                                                                                                                                                                 
                                                   [AK001313](AK001313)     RPLP0           Ribosomal protein, large, P0                                                                           704         **1465**     947          703        **756**      669
                                                   [NM_006799](NM_006799)   PRSS21          Protease, serine, 21 (testisin)                                                                        204         **789**      457          169        **360**      400
                                                   [NM_003774](NM_003774)   GALNT4          UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 4 (GalNAc-T4)         576         **651**      648          528        **378**      578
                                                   [AK057790](AK057790)                     cDNA FLJ25061 fis, clone CBL04730                                                                      245         **373**      302          244        **215**      200
                                                   [NM_004223](NM_004223)   UBE2L6          Ubiquitin-conjugating enzyme E2L 6                                                                     128         **191**      146          108        **99**       109
                                                   [NM_014710](NM_014710)   GPRASP1         KIAA0443 gene product                                                                                  122         **182**      106          113        **119**      95
                                                   [NM_021090](NM_021090)   MTMR3           Myotubularin related protein 3                                                                         109         **171**      137          108        **87**       138
                                                   [AF339824](AF339824)     HS6ST3          Heparan sulfate 6-O-sulfotransferase 3                                                                 89          **112**      91           94         **46**       76
                                                   [NM_012180](NM_012180)   FBXO8           F-box only protein 8                                                                                   40          **67**       42           45         **33**       43
                                                   [U66589](U66589)         RPL5            Ribosomal protein L5                                                                                   34          **48**       37           30         **26**       36
                                                   [NM_001870](NM_001870)   CPA3            Carboxypeptidase A3 (mast cell)                                                                        183         ***495***    610          146        ***949***    756
                                                   [NM_006145](NM_006145)   DNAJB1          DnaJ (Hsp40) homolog, subfmaily B, member 1                                                            179         277          ***408***    168        299          ***745***
                                                   [AK025547](AK025547)     MRPL30          Mitochondrial ribosomal protein L30                                                                    83          118          ***126***    81         101          ***211***
                                                   [NM_000439](NM_000439)   PCSK1           Proprotein convertase subtilisin/kexin type 1                                                          39          55           ***53***     40         78           ***88***
  **Cell/Organism Movement**                                                                                                                                                                                                                                     
                                                   [NM_002067](NM_002067)   GNA11           Guanine nucleotide binding protein (G protein), alpha 11 (Gq class)                                    555         **870**      607          540        **468**      664
                                                   [NM_002465](NM_002465)   MYBPC1          Myosin binding protein C, slow type                                                                    81          **140**      154          88         **80**       161
                                                   [NM_003275](NM_003275)   TMOD            Tropomodulin                                                                                           276         ***151***    481          257        ***344***    503
                                                   [AK026164](AK026164)     MYL6            Myosin, light polypeptide 6, alkali, smooth muscle and non-muscle                                      7           ***6***      48           5          ***16***     11
  **Small Molecule Interactions**                                                                                                                                                                                                                                
                                                   [NM_006030](NM_006030)   CACNA2D2        Calcium channel, voltage-dependent, alpha 2/delta subunit 2                                            670         **1390**     1021         641        **639**      946
                                                   [AK025170](AK025170)     SFXN5           FLJ21517 fis, clone COL05829                                                                           431         **537**      437          405        **295**      374
                                                   [NM_021097](NM_021097)   SLC8A1          Solute carrier family 8 (sodium/calcium exchanger), member 1                                           396         **456**      458          412        **276**      369
  **Signal Transduction**                                                                                                                                                                                                                                        
                                                   [NM_032144](NM_032144)   RAB6C           RAB6C                                                                                                  827         **1658**     1307         626        **773**      1251
                                                   [NM_001982](NM_001982)   ERBB3           V-erb-b2 erythroblastic leukemia viral oncogene homolog 3                                              603         **1375**     671          555        **584**      643
                                                   [AK026479](AK026479)     SNX14           Sorting nexin 14                                                                                       682         **1207**     879          624        **567**      883
                                                   [NM_018979](NM_018979)   PRKWNK1         Protein kinase, lysine deficient 1                                                                     451         **813**      782          516        **480**      792
                                                   [NM_004811](NM_004811)   LPXN            Leupaxin                                                                                               329         **539**      445          323        **298**      503
                                                   [BC005365](BC005365)                     clone IMAGE:3829438, mRNA, partial cds                                                                 257         **418**      275          275        **275**      206
                                                   [NM_004723](NM_004723)   ARHGEF2         Rho/rac guanine nucleotide exchange factor (GEF) 2                                                     215         **300**      228          197        **176**      186
                                                   [AF130093](AF130093)     MAP3K4          Mitogen-activated protein kinase kinase kinase 4                                                       237         **285**      275          221        **171**      223
                                                   [AK000383](AK000383)     MKPX            Mitogen-activated protein kinase phosphatase x                                                         218         **221**      244          233        **126**      197
                                                   [NM_022304](NM_022304)   HRH2            Histamine receptor H2                                                                                  45          **121**      86           42         **74**       79
                                                   [NM_030753](NM_030753)   WNT3            Wingless-type MMTV integration site family member 3                                                    105         **117**      92           109        **63**       81
                                                   [AB024574](AB024574)     GTPBP2          GTP binding protein 2                                                                                  89          **90**       99           74         **57**       92
                                                   [NM_002836](NM_002836)   PTPRA           Protein tyrosine phosphatase, receptor type, A                                                         8           ***6***      80           6          ***16***     28
                                                   [NM_003656](NM_003656)   CAMK1           Calcium/calmodulin-dependent protein kinase I                                                          4940        10131        ***4446***   4785       4907         ***7190***
  **Cellular Metabolism & Cell Division**                                                                                                                                                                                                                        
                                                   [NM_006170](NM_006170)   NOL1            Nucleolar protein 1 (120 kD)                                                                           575         **1815**     1021         499        **896**      1093
                                                   [AL133115](AL133115)     COVA1           Cytosolic ovarian carcinoma antigen 1                                                                  1381        **1294**     848          1309       **658**      808
                                                   [D86962](D86962)         GRB10           Growth factor receptor-bound protein 10                                                                619         **906**      200          609        **512**      179
                                                   [NM_005628](NM_005628)   SLC1A5          Solute carrier family 1 (neutral amino acid transporter), member 5                                     338         **801**      600          311        **397**      524
                                                   [D17525](D17525)         MASP1           Mannan-binding lectin serine protease 1 (C4/C2 activating component of Ra-reactive factor)             372         **654**      43           361        **325**      55
                                                   [NM_016518](NM_016518)   PIPOX           Pipecolic acid oxidase                                                                                 240         **545**      330          221        **293**      286
                                                   [NM_012157](NM_012157)   FBXL2           F-box and leucine-rich repeat protein 2                                                                274         **501**      374          249        **277**      298
                                                   [NM_018446](NM_018446)   AD-017          Glycosyltransferase AD-017                                                                             301         **369**      337          288        **223**      327
                                                   [NM_001609](NM_001609)   ACADSB          Acyl-Coenzyme A dehydrogenase, short/branched chain                                                    354         **368**      325          273        **211**      276
                                                   [NM_001647](NM_001647)   APOD            Apolipoprotein D                                                                                       259         **358**      289          261        **202**      205
                                                   [NM_012113](NM_012113)   CA14            Carbonic anhydrase XIV                                                                                 218         **356**      279          251        **194**      270
                                                   [AB067472](AB067472)     DKFZP434L1435   KIAA1885 protein                                                                                       150         **213**      186          166        **119**      163
                                                   [NM_002916](NM_002916)   RFC4            Replication factor C (activator 1) 4 (37 kD)                                                           102         **177**      119          105        **86**       132
                                                   [NM_004889](NM_004889)   ATP5J2          ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2                          106         **147**      76           102        **76**       62
                                                   [AK057066](AK057066)                     cDNA FLJ32504 fis, clone SMINT1000016, weakly similar to 2-hydroxyacylsphingosine 1b                   69          **121**      126          64         **75**       84
                                                   [AK021722](AK021722)     AGPAT5          Lysophosphatidic acid acyltransferase, epsilon                                                         37          **71**       48           42         **39**       46
                                                   [NM_003664](NM_003664)   AP3B1           Adaptor-related protein complex 3, beta 1 subunit                                                      34          **52**       29           37         **24**       30
                                                   [AF146760](AF146760)     Sept10          Septin 10                                                                                              22          **36**       23           26         **16**       28
                                                   [NM_004910](NM_004910)   PITPNM          Phosphatidylinositol transfer protein, membrane-associated                                             2611        ***2809***   2410         2974       ***4590***   2675
                                                   [NM_018216](NM_018216)   FLJ10782        Pantothenic acid kinase                                                                                10          ***9***      10           9          ***18***     15
                                                   [NM_001714](NM_001714)   BICD1           Bicaudal D homolog 1 (Drosophila)                                                                      230         562          ***407***    197        447          ***691***
                                                   [AK054944](AK054944)     LENG5           Leukocyte receptor cluster (LRC) member 5                                                              67          100          ***91***     78         74           ***158***
  **Gene Expression**                                                                                                                                                                                                                                            
                                                   [NM_005088](NM_005088)   DXYS155E        DNA segment on chromosome X and Y (unique) 155 expressed sequence                                      4857        **3489**     3214         5177       **2241**     2725
                                                   [NM_006298](NM_006298)   ZNF192          Zinc finger protein 192                                                                                552         **988**      761          537        **578**      820
                                                   [NM_004991](NM_004991)   MDS1            Myelodysplasia syndrome 1                                                                              401         **691**      480          390        **361**      420
                                                   [NM_021784](NM_021784)   HNF3B           Hepatocyte nuclear factor 3, beta                                                                      320         **632**      367          347        **361**      391
                                                   [AF153201](AF153201)     LOC58502        C2H2 (Kruppel-type) zinc finger protein                                                                288         **532**      335          244        **297**      324
                                                   [NM_025212](NM_025212)   IDAX            Dvl-binding protein IDAX (inhibition of the Dvl and Axin complex)                                      297         **490**      311          303        **254**      241
                                                   [AK022962](AK022962)     PBX1            Pre-B-cell leukemia transcription factor 1                                                             237         **456**      326          245        **261**      345
                                                   [NM_017617](NM_017617)   NOTCH1          Notch-1 homolog                                                                                        309         **358**      353          324        **208**      370
                                                   [NM_001451](NM_001451)   FOXF1           Forkhead box F1                                                                                        165         **347**      306          177        **208**      328
                                                   [NM_007136](NM_007136)   ZNF80           Zinc finger protein 80 (pT17)                                                                          199         **269**      203          205        **143**      177
                                                   [NM_021975](NM_021975)   RELA            V-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene   184         **221**      139          150        **124**      122
                                                   [NM_031214](NM_031214)   TARDBP          TAR DNA binding protein                                                                                76          **154**      109          74         **91**       90
                                                   [NM_014007](NM_014007)   ZNF297B         Zinc finger protein 297B                                                                               109         **137**      122          109        **77**       111
                                                   [NM_014938](NM_014938)   MONDOA          Mlx interactor                                                                                         74          **90**       92           69         **53**       86
                                                   [NM_005822](NM_005822)   DSCR1L1         Down syndrome critical region gene 1-like 1                                                            45          **80**       30           40         **27**       26
                                                   [NM_004289](NM_004289)   NFE2L3          Nuclear factor (erythroid-derived 2)-like 3                                                            73          **63**       41           64         **39**       38
                                                   [NM_054023](NM_054023)   SCGB3A2         Secretoglobin family 3a, member 2                                                                      37          **59**       45           43         **34**       49
                                                   [NM_012107](NM_012107)   BP75            Bromodomain containing protein 75 kDa human homolog                                                    44          **51**       34           37         **22**       30
                                                   [NM_007212](NM_007212)   RNF2            Ring finger protein 2                                                                                  48          **40**       30           45         **18**       26
                                                   [D89859](D89859)         ZFP161          Zinc finger protein 161 homolog (mouse)                                                                500         596          ***4280***   458        481          ***6699***
                                                   [NM_014335](NM_014335)   CRI1            CREBBP/EP300 inhibitory protein 1                                                                      52          84           ***86***     57         72           ***196***
  **Immune Function**                                                                                                                                                                                                                                            
                                                   [NM_014889](NM_014889)   MP1             Metalloprotease 1 (pitrilysin family)                                                                  352         **401**      398          379        **260**      351
                                                   [NM_014312](NM_014312)   CTXL            Cortical thymocyte receptor (X. laevis CTX) like                                                       386         **370**      375          392        **224**      299
                                                   [NM_002053](NM_002053)   GBP1            Guanylate binding protein 1, interferon-inducible, 67 kD                                               259         **369**      334          245        **214**      251
                                                   [NM_005356](NM_005356)   LCK             Lymphocyte-specific protein tyrosine kinase                                                            186         **206**      187          235        **124**      181
                                                   [NM_000564](NM_000564)   IL5RA           Interleukin 5 receptor, alpha                                                                          112         **106**      124          121        **63**       150
                                                   [NM_001311](NM_001311)   CRIP1           Cysteine-rich protein 1 (intestinal)                                                                   45          ***31***     39           49         ***60***     43
                                                   [NM_002984](NM_002984)   SCYA4           Small inducible cytokine A4 MIP1B                                                                      492         2001         ***2483***   517        1523         ***3897***
                                                   [NM_002983](NM_002983)   SCYA3           Small inducible cytokine A3 MIP1A                                                                      248         1798         ***2207***   185        1364         ***3673***
                                                   [NM_014443](NM_014443)   IL17B           Interleukin 17B                                                                                        663         696          ***681***    706        703          ***1155***
                                                   [NM_006018](NM_006018)   HM74            Putative chemokine receptor-GTP-binding protein                                                        13          25           ***19***     15         26           ***34***
  **Miscellaneous Functions**                                                                                                                                                                                                                                    
                                                   [AB033041](AB033041)     VANGL2          Vang, van gogh-like 2 (Drosophila)                                                                     983         **1246**     1351         981        **796**      1304
                                                   [AK021444](AK021444)     POSTN           Periostin, osteoblast specific factor                                                                  569         **917**      789          522        **479**      629
                                                   [NM_003691](NM_003691)   STK16           Serine/threonine kinase 16                                                                             403         **777**      458          395        **348**      393
                                                   [NM_006438](NM_006438)   COLEC10         Collectin sub-family member 10 (C-type lectin)                                                         284         **762**      500          260        **351**      528
                                                   [AK057699](AK057699)                     FLJ33137 fis, clone UTERU1000077                                                                       375         **637**      613          369        **392**      616
                                                   [NM_017671](NM_017671)   C20orf42        Chromosome 20 open reading frame 42                                                                    362         **557**      551          280        **323**      478
                                                   [AK054683](AK054683)     DCLRE1C         DNA cross-link repair 1C                                                                               486         **555**      574          476        **293**      515
                                                   [NM_033060](NM_033060)   KAP4.10         Keratin associated protein 4.10                                                                        210         **245**      197          154        **123**      172
                                                   [AF319045](AF319045)     CNTNAP2         Contactin associated protein-like 2                                                                    112         **215**      173          120        **113**      176
                                                   [NM_001046](NM_001046)   SLC12A2         Solute carrier family 12 (sodium/potassium/chloride transporters), member 2                            158         **148**      184          146        **86**       161
                                                   [NM_016279](NM_016279)   CDH9            Cadherin 9, type 2 (T1-cadherin)                                                                       77          **112**      69           65         **51**       64
                                                   [NM_014208](NM_014208)   DSPP            Dentin sialophosphoprotein                                                                             60          **90**       64           57         **53**       59
                                                   [NM_015669](NM_015669)   PCDHB5          Protocadherin beta 5                                                                                   92          **83**       62           98         **42**       47
                                                   [AK023198](AK023198)     OPRK1           Opioid receptor, kappa 1                                                                               58          **76**       41           48         **46**       38
                                                   [NM_018240](NM_018240)   KIRREL          Kin of IRRE like (Drosophila)                                                                          60          **75**       47           66         **43**       46
                                                   [AK056781](AK056781)     ROCK1           Rho-associated, coiled-coil containing protein kinase 1                                                54          **62**       42           47         **41**       42
                                                   [NM_022123](NM_022123)   NPAS3           Basic-helix-loop-helix-PAS protein                                                                     17          **22**       9            16         **12**       13
                                                   [NM_001246](NM_001246)   ENTPD2          Ectonucleoside triphosphate diphosphohydrolase 2                                                       3438        3272         ***3731***   3767       3590         ***6309***
  **Unknown Function**                                                                                                                                                                                                                                           
                                                   [AK056884](AK056884)                     FLJ32322 fis, clone PROST2003577                                                                       2007        **2878**     2008         1825       **1548**     1958
                                                   [NM_017812](NM_017812)   FLJ20420        Coiled-coil-helix-coiled-coil-helix domain containing 3                                                1105        **1915**     1370         1125       **940**      1358
                                                   [AJ420459](AJ420459)     LOC51184        Protein x 0004                                                                                         661         **1579**     881          603        **771**      768
                                                   [BC011575](BC011575)                     Similar to RIKEN cDNA 0610031J06 gene, clone IMAGE:4639306                                             974         **1556**     1412         1020       **844**      1261
                                                   [AK057357](AK057357)     FLJ32926        DKFZp434D2472                                                                                          1188        **1378**     1159         1043       **515**      1136
                                                   [NM_025019](NM_025019)   TUBA4           tubulin, alpha 4                                                                                       1446        **1173**     1330         1477       **782**      1366
                                                   [AK023150](AK023150)                     FLJ13088 fis, clone NT2RP3002102                                                                       798         **1087**     905          845        **564**      785
                                                   [NM_017833](NM_017833)   C21orf55        Chromosome 21 open reading frame 55                                                                    741         **1079**     799          687        **508**      665
                                                   [BC001407](BC001407)                     Similar to cytochrome c-like antigen                                                                   524         **1004**     629          506        **502**      577
                                                   [AK023104](AK023104)                     FLJ22648 fis, clone HSI07329                                                                           441         **984**      621          488        **471**      495
                                                   [AK024617](AK024617)                     FLJ20964 fis, clone ADSH00902                                                                          824         **955**      745          788        **535**      824
                                                   [BC009536](BC009536)                     IMAGE:3892368                                                                                          553         **924**      775          597        **498**      671
                                                   [AK056287](AK056287)                     FLJ31725 fis, clone NT2RI2006716                                                                       435         **862**      907          405        **459**      893
                                                   [AK021611](AK021611)                     FLJ11549 fis, clone HEMBA1002968                                                                       535         **812**      675          545        **392**      630
                                                   [BC015119](BC015119)                     IMAGE:3951139                                                                                          445         **784**      487          455        **435**      439
                                                   [AK056492](AK056492)                     FLJ31930 fis, clone NT2RP7006162                                                                       252         **651**      525          266        **367**      457
                                                   [AB058711](AB058711)     KIAA1808        KIAA1808 protein                                                                                       208         **637**      357          199        **339**      366
                                                   [BC011266](BC011266)                     IMAGE:4156795                                                                                          354         **632**      432          356        **328**      460
                                                   [AK023316](AK023316)                     FLJ13254 fis, clone OVARC1000787                                                                       416         **596**      357          400        **290**      352
                                                   [NM_024696](NM_024696)   FLJ23058        Hypothetical protein FLJ23058                                                                          456         **541**      346          436        **313**      359
                                                   [AF253316](AF253316)                     Pheromone receptor (PHRET) pseudogene                                                                  136         **520**      425          128        **301**      347
                                                   [AK056007](AK056007)     BICD1           Bicaudal D homolog 1 (Drosophila)                                                                      704         **505**      439          624        **243**      305
                                                   [AB020632](AB020632)     KIAA0825        KIAA0825 protein                                                                                       249         **498**      353          246        **272**      339
                                                   [NM_017609](NM_017609)   DKFZp434A1721   Hypothetical protein DKFZp434A1721                                                                     182         **485**      319          190        **298**      304
                                                   [NM_018190](NM_018190)   FLJ10715        Hypothetical protein FLJ10715                                                                          202         **483**      310          174        **206**      266
                                                   [AK057046](AK057046)                     FLJ32484 fis, clone SKNMC2001555                                                                       229         **473**      294          261        **302**      228
                                                   [NM_013395](NM_013395)   AD013           Proteinx0008                                                                                           448         **461**      496          403        **304**      378
                                                   [BC008501](BC008501)     MGC14839        Similar to RIKEN cDNA 2310030G06                                                                       379         **414**      329          443        **264**      290
                                                   [AK021988](AK021988)                     FLJ11926 fis, clone HEMBB1000374                                                                       321         **411**      399          280        **218**      288
                                                   [AF119872](AF119872)                     PRO2272                                                                                                257         **405**      327          257        **205**      250
                                                   [NM_022744](NM_022744)   FLJ13868        Hypothetical protein FLJ13868                                                                          267         **376**      239          270        **212**      172
                                                   [AK022364](AK022364)                     FLJ12302 fis, clone MAMMA1001864                                                                       172         **355**      316          164        **184**      332
                                                   [BC002644](BC002644)     MGC4859         Hypothetical protein MGC4859 similar to HSPA8                                                          282         **335**      382          257        **223**      331
                                                   [AK022201](AK022201)                     FLJ12139 fis, clone MAMMA1000339                                                                       267         **302**      152          235        **123**      131
                                                   [NM_017953](NM_017953)   FLJ20729        Hypothetical protein FLJ20729                                                                          170         **290**      258          138        **170**      218
                                                   [AK057473](AK057473)                     FLJ32911 fis, clone TESTI2006210                                                                       160         **268**      265          163        **123**      247
                                                   [U50383](U50383)         RAI15           Retinoic acid induced 15                                                                               206         **265**      236          198        **159**      186
                                                   [AK027027](AK027027)                     FLJ23374 fis, clone HEP16126                                                                           134         **261**      170          134        **152**      141
                                                   [AK057288](AK057288)                     FLJ32726 fis, clone TESTI2000981                                                                       206         **249**      312          216        **152**      244
                                                   [U79280](U79280)         PIPPIN          Ortholog of rat pippin                                                                                 274         **229**      189          238        **117**      134
                                                   [AK023628](AK023628)                     FLJ13566 fis, clone PLACE1008330                                                                       140         **195**      230          133        **128**      193
                                                   [NM_025263](NM_025263)   CAT56           CAT56 protein                                                                                          126         **194**      147          127        **101**      130
                                                   [AF311324](AF311324)                     Ubiquitin-like fusion protein                                                                          191         **189**      179          190        **106**      138
                                                   [NM_005708](NM_005708)   GPC6            Glypican 6                                                                                             107         **185**      144          109        **88**       146
                                                   [AB037778](AB037778)     KIAA1357        KIAA1357 protein                                                                                       153         **180**      156          149        **118**      146
                                                   [AK055939](AK055939)                     FLJ31377 fis, clone NESOP1000087                                                                       152         **167**      179          136        **105**      173
                                                   [NM_018316](NM_018316)   FLJ11078        Hypothetical protein FLJ11078                                                                          89          **145**      118          73         **94**       103
                                                   [AF402776](AF402776)     BIC             BIC noncoding mRNA                                                                                     82          **136**      171          96         **88**       153
                                                   [BC003416](BC003416)                     IMAGE:3450973                                                                                          64          **133**      93           83         **73**       111
                                                   [AL137491](AL137491)                     DKFZp434P1530                                                                                          62          **130**      88           57         **72**       74
                                                   [AK057770](AK057770)                     FLJ25041 fis, clone CBL03194                                                                           110         **130**      114          108        **83**       84
                                                   [AB058769](AB058769)     KIAA1866        KIAA1866 protein                                                                                       89          **126**      122          102        **83**       91
                                                   [AB058747](AB058747)     WAC             WW domain-containing adapter with a coiled-coil region                                                 60          **124**      103          57         **76**       77
                                                   [AK054885](AK054885)     C6orf31         Chromosome 6 open reading frame 31                                                                     51          **119**      108          41         **68**       119
                                                   [AK022235](AK022235)                     FLJ12173 fis, clone MAMMA1000696                                                                       109         **103**      94           90         **62**       77
                                                   [AK026853](AK026853)     AOAH            Acyloxyacyl hydrolase (neutrophil)                                                                     59          **98**       64           59         **61**       56
                                                   [AK024877](AK024877)                     FLJ21224 fis, clone COL00694                                                                           53          **96**       110          55         **54**       103
                                                   [NM_003171](NM_003171)   SUPV3L1         Suppressor of var1, 3-like 1 (S. cerevisiae)                                                           65          **93**       60           60         **55**       58
                                                   [NM_052933](NM_052933)   TSGA13          Testis specific, 13                                                                                    66          **80**       70           68         **44**       71
                                                   [AK057907](AK057907)                     FLJ25178 fis, clone CBR09176                                                                           42          **77**       31           47         **43**       41
                                                   [AK055748](AK055748)                     FLJ31186 fis, clone KIDNE2000335                                                                       88          **67**       68           79         **44**       71
                                                   [BC013757](BC013757)                     IMAGE:4525041                                                                                          40          **54**       39           43         **33**       32
                                                   [AL365511](AL365511)                     Novel human gene mapping to chomosome 22                                                               19          **48**       29           20         **27**       37
                                                   [AK026889](AK026889)     APRIN           Androgen-induced proliferation inhibitor                                                               31          **35**       42           34         **21**       34
                                                   [AK057423](AK057423)                     FLJ32861 fis, clone TESTI2003589                                                                       36          **32**       34           30         **18**       31
                                                   [AK055543](AK055543)     MLSTD1          Male sterility domain containing 1                                                                     31          **31**       32           27         **18**       30
                                                   [AK056513](AK056513)                     FLJ31951 fis, clone NT2RP7007177                                                                       33          **29**       20           22         **13**       20
                                                   [NM_013319](NM_013319)   TERE1           Transitional epithelia response protein                                                                22          **28**       19           24         **17**       22
                                                   [AK026456](AK026456)                     FLJ22803 fis, clone KAIA2685                                                                           15          **26**       14           16         **13**       17
                                                   [AK021610](AK021610)                     cDNA FLJ11548 fis, clone HEMBA1002944                                                                  34          **26**       29           31         **15**       28
                                                   [AK026823](AK026823)                     FLJ23170 fis, clone LNG09984                                                                           15          **22**       14           19         **8**        18
                                                   [AK056805](AK056805)                     FLJ32243 fis, clone PROST1000039                                                                       400         ***177***    186          343        ***314***    160
                                                   [NM_012238](NM_012238)   SIRT1           Sirtuin silent mating type information regulation 2 homolog 1 (S. cerevisiae)                          149         156          **170**      178        134          **109**
                                                   [NM_016099](NM_016099)   GOLGA7          golgi autoantigen, golgin subfamily a, 7                                                               10493       15165        ***9882***   11947      11564        ***15698***
                                                   [AK022482](AK022482)                     FLJ12420 fis, clone MAMMA1003049                                                                       6052        9099         ***5803***   6362       7620         ***9309***
                                                   [AK026490](AK026490)     RAB32           RAB32, member RAS oncogene family                                                                      3677        7044         ***4641***   3671       5553         ***7561***
                                                   [NM_020684](NM_020684)   NPD007          NPD007 protein                                                                                         674         794          ***764***    630        720          ***1215***
                                                   [AL390158](AL390158)     ATXN7L3         Ataxin 7-like 3                                                                                        319         460          ***378***    339        403          ***598***
                                                   [NM_017752](NM_017752)   FLJ20298        Hypothetical protein FLJ20298                                                                          146         237          ***282***    133        233          ***493***
                                                   [AB037743](AB037743)     KIAA1322        KIAA1322 protein                                                                                       236         202          ***199***    239        246          ***319***
                                                   [AF339819](AF339819)                     clone IMAGE:38177                                                                                      77          111          ***110***    96         125          ***174***
                                                   [AK055215](AK055215)                     FLJ30653 fis, clone DFNES2000143                                                                       47          48           ***58***     43         80           ***92***

![LPS-stimulated genes in cord blood and adult monocytes can be differentiated on the basis of kinetics of expression. Expression level (in relative intensity units) is shown of the y-axis and time on the x-axis. At the 45 min time point, significant differences in expression level were seen between adult and neonatal monocytes for each of the gene groups A-H.](1476-9255-4-4-1){#F1}

![Heat map representation of differences in gene expression of adult and cord blood monocytes in response to LPS. Z-transformed scores of the mean expression values for adult monocytes prior to (A0), after 45 min (A45), and after 120 min (A120) of LPS exposure are graphically shown to the left. Similar scores from cord blood monocytes prior to (C0), after 45 min (C45), and after 120 min C120) of LPS exposure, respectively. The heat map was produced using software from Spotfire Decision Site (Somerville, MA).](1476-9255-4-4-2){#F2}

In addition to the above genes which differed in expression between groups following LPS exposure, 516 genes were also identified that were differentially expressed over time within a group. A supplementary table containing these data is available upon request. For these genes, a similar pattern of dynamic expression was seen as was observed in the other group. Therefore, these genes reflect common responses to LPS in monocytes from both sources.

A subset of genes that were differentially expressed either between adult and cord blood monocytes were selected for validation using the quantitative real-time polymerase chain reaction method (QRT-PCR). These included four genes that differed between groups after 45 min of LPS exposure (*Erbb3, Tmod, Dscr1l1*, and *Sp1*), and six genes that differed in expression after 2 hours of LPS exposure (*Scya4, Gro2, Cri1, Scya3, Scya3l1*, and *Il-1a*). Nine of the ten genes tested for QRT-PCR validation demonstrated similar levels of relative expression in QRT-PCR experiments as in the microarrays. Only CRI1 failed to corroborate the microarray data.

Hypervariable gene analysis
---------------------------

One hundred eighty-eight hypervariable (HV) genes were selected from expressed genes in adult and cord blood monocytes based on their changes across three time points. These genes exhibited significantly higher expression variation over time than the majority of genes. Differences in variation between two experimental sample sets, in this case adult and neonatal samples, can represent differences in homeostatic control mechanisms between these two sets \[[@B20]\]. The selected genes were hypervariable in both sample groups. HV genes with highly correlated expression levels in a given population are likely to share function \[[@B20]\]. A correlation based clustering procedure was carried out for these HV genes as described in the methods section. Genes belonging to the 5 largest clusters were used for creation of a graphical output, denoted a correlation mosaic. A correlation mosaic allows identification of the genes within clusters by visual inspection and subsequent functional analysis of genes within clusters (Figures [3A](#F3){ref-type="fig"} &[3B](#F3){ref-type="fig"}). Figure [3A](#F3){ref-type="fig"} represents 110 genes of the same cluster allocation between adult and cord blood monocyte samples, demonstrating a very high similarity between cells from these two groups, as measured by the correlation coefficients between genes from adult and cord monocytes with value \> 0.90 (figure [3A](#F3){ref-type="fig"}, black and white graph to the right). Three genes on this list (\#101--103) were the exception: transcriptional regulator interacting with the PHS-bromodomain 2 (*Trip-Br2*), interleukin 1 beta (*Il1b*), and the GRO2 oncogene(*Gro2*). These genes may play a critical role in differentiation between adult and cord monocyte behaviour \[[@B22],[@B23]\]. The high similarity of these mosaics presents evidence for the presence of fundamental processes in monocyte development that appear to be quite similar in both groups of samples. The details of the genes used in Figure [3A](#F3){ref-type="fig"} are presented as Table [2](#T2){ref-type="table"}. Another group of 78 genes were found that have different cluster designations between adult and cord blood monocytes (Figure [3B](#F3){ref-type="fig"}). Details of these genes are listed in Table [3](#T3){ref-type="table"}.

![Correlative mosaic for genes selected as HV-genes in cord blood and adult monocytes, belonging to five clusters of highest content. A. Genes of the same cluster in cord and adult; B. Genes of different cluster in cord and adult. Correlation coefficients are color-coded according to the key in the upper right. The correlation between the adult and cord blood monocyte profiles for each gene are shown in black and white, lower right.](1476-9255-4-4-3){#F3}

###### 

Genes from which correlation mosaics in Figure 3A were derived. Genes in this table show the highest level of correlation by DFA analysis comparing adult and cord blood monocytes.

  **Order in mosaic**   **Accession No.**         **Gene symbol**   **Description**
  --------------------- ------------------------- ----------------- ------------------------------------------------------------------------------------------------------
  1                     [NM_017614](NM_017614)    BHMT2             Betaine-homocysteine methyltransferase 2
  2                     [NM_001651](NM_001651)    AQP5              Aquaporin 5
  3                     [NM_020163](NM_020163)    LOC56920          Semaphorin sem2
  4                     [NM_012343](NM_012343)    NNT               Nicotinamide nucleotide transhydrogenase
  5                     [NM_000096](NM_000096)    CP                Ceruloplasmin (ferroxidase)
  6                     [NM_005819](NM_005819)    STX6              Syntaxin 6
  7                     [NM_052951](NM_052951)    C20orf167         Chromosome 20 open reading frame 167
  8                     [NM_001348](NM_001348)    DAPK3             Death-associated protein kinase 3
  9                     [X73502](X73502)          KRT20             Cytokeratin 20
  10                    [NM_052887](NM_052887)    TIRAP             Toll-interleukin 1 receptor (TIR) domain-containing adapter protein
  11                    [NM_019555](NM_019555)    ARHGEF3           Rho guanine nucleotide exchange factor (GEF) 3
  12                    [NM_014380](NM_014380)    NGFRAP1           Nerve growth factor receptor (TNFRSF16) associated protein 1
  13                    [NM_001272](NM_001272)    CHD3              Chromodomain helicase DNA binding protein 3
  14                    [NM_005842](NM_005842)    SPRY2             Sprouty homolog 2 (Drosophila)
  15                    [NM_012332](NM_012332)    MT-ACT48          Mitochondrial Acyl-CoA Thioesterase
  16                    [BC015041](BC015041)      VATI              Vesicle amine transport protein 1
  17                    [NM_003872](NM_003872)    NRP2              Neuropilin 2
  18                    [NM_005849](NM_005849)    IGSF6             Immunoglobulin superfamily, member 6
  19                    [NM_014323](NM_014323)    ZNF278            Zinc finger protein 278
  20                    [NM_030674](NM_030674)    SLC38A1           Solute carrier family 38, member 1
  21                    [NM_004153](NM_004153)    ORC1L             Origin recognition complex, subunit 1-like (yeast)
  22                    [NM_005249](NM_005249)    FOXG1B            Forkhead box G1B
  23                    [NM_021048](NM_021048)    MAGEA10           Melanoma antigen, family A, 10
  24                    [M60502](M60502)          FLG               Filaggrin
  25                    [NM_004997](NM_004997)    MYBPH             Myosin binding protein H
  26                    [J05046](J05046)          INSRR             Insulin receptor-related receptor
  27                    [M33987](M33987)          CA1               Carbonic anhydrase I
  28                    [D31886](D31886)          RAB3GAP           RAB3 GTPase-ACTIVATING PROTEIN
  29                    [L24498](L24498)          GADD45A           Growth arrest and DNA-damage-inducible, alpha
  30                    [L07590](L07590)          PPP2R3            Protein phosphatase 2 (formerly 2A), regulatory subunit B\" (PR 72), alpha isoform and (PR 130), bet
  31                    [D87024](D87024)          IGLV4-3           Immunoglobulin lambda variable 4-3
  32                    [L35848](L35848)          MS4A3             Membrane-spanning 4-domains, subfamily A, member 3 (hematopoietic cell-specific)
  33                    [M18216](M18216)          CEACAM6           Carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen)
  34                    [M11952](M11952)          TRBV7--8          T cell receptor beta variable 7--8
  35                    [D89094](D89094)          PDE5A             Phosphodiesterase 5A, cGMP-specific
  36                    [M77140](M77140)          GAL               Galanin
  37                    [D13628](D13628)          ANGPT1            Angiopoietin 1
  38                    [M81635](M81635)          EPB72             Erythrocyte membrane protein band 7.2 (stomatin)
  39                    [D89859](D89859)          ZFP161            Zinc finger protein 161 homolog (mouse)
  40                    [D26069](D26069)          CENTB2            Centaurin, beta 2
  41                    [L10717](L10717)          ITK               IL2-inducible T-cell kinase
  42                    [L04282](L04282)          ZNF148            Zinc finger protein 148 (pHZ-52)
  43                    [L41944](L41944)          IFNAR2            Interferon (alpha, beta and omega) receptor 2
  44                    [M82882](M82882)          ELF1              E74-like factor 1 (ets domain transcription factor)
  45                    [L26339](L26339)          RCD-8             Autoantigen
  46                    [D87328](D87328)          HLCS              Holocarboxylase synthetase (biotin-\[proprionyl-Coenzyme A-carboxylase (ATP-hydrolysing)\] ligase)
  47                    [D00943](D00943)          MYH6              Myosin, heavy polypeptide 6, cardiac muscle, alpha (cardiomyopathy, hypertrophic 1)
  48                    [D00099](D00099)          ATP1A1            ATPase, Na+/K+ transporting, alpha 1 polypeptide
  49                    [L36531](L36531)          ITGA8             Integrin, alpha 8
  50                    [D42084](D42084)          METAP1            Methionyl aminopeptidase 1
  51                    [M76766](M76766)          GTF2B             General transcription factor IIB
  52                    [J04621](J04621)          SDC2              Syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan)
  53                    [D31888](D31888)          RCOR              REST corepressor
  54                    [L32832](L32832)          ATBF1             AT-binding transcription factor 1
  55                    [D86981](D86981)          APPBP2            Amyloid beta precursor protein (cytoplasmic tail) binding protein 2
  56                    [M94362](M94362)          LMNB2             Lamin B2
  57                    [M54968](M54968)          KRAS2             V-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog
  58                    [D42046](D42046)          DNA2L             DNA2 DNA replication helicase 2-like (yeast)
  59                    [D86964](D86964)          DOCK2             Dedicator of cyto-kinesis 2
  60                    [D50683](D50683)          TGFBR2            Transforming growth factor, beta receptor II (70--80 kD)
  61                    [M96843](M96843)          ID2B              Striated muscle contraction regulatory protein
  62                    [M61906](M61906)          PIK3R1            Phosphoinositide-3-kinase, regulatory subunit, polypeptide 1 (p85 alpha)
  63                    [M12679](M12679)          HUMMHCW1A         Cw1 antigen
  64                    [M63623](M63623)          OMG               Oligodendrocyte myelin glycoprotein
  65                    [J04162](J04162)          FCGR3B            Fc fragment of IgG, low affinity IIIb, receptor for (CD16)
  66                    [L48516](L48516)          PON3              Paraoxonase 3
  67                    [M54927](M54927)          PLP1              Proteolipid protein1 (Pelizaeus-Merzbacher disease, spastic paraplegia 2, uncomplicated)
  68                    [D86973](D86973)          GCN1L1            GCN1 general control of amino-acid synthesis 1-like 1 (yeast)
  69                    [D43968](D43968)          RUNX1             Runt-related transcription factor 1 (acute myeloid leukemia 1-aml1 oncogene)
  70                    [L05500](L05500)          ADCY1             Adenylate cyclase 1 (brain)
  71                    [D80010](D80010)          LPIN1             Lipin 1
  72                    [D50918](D50918)          SEPT6             Septin 6
  73                    [D86988](D86988)          RENT1             Regulator of nonsense transcripts 1
  74                    [M90391](M90391)          IL16              Interleukin 16 (lymphocyte chemoattractant factor)
  75                    [M62324](M62324)          MRF-1             Modulator recognition factor I
  76                    [L77565](L77565)          DGS-H             DiGeorge syndrome gene H
  77                    [D86970](D86970)          TIAF1             TGFB1-induced anti-apoptotic factor 1
  78                    [D38169](D38169)          ITPKC             Inositol 1,4,5-trisphosphate 3-kinase C
  79                    [D87684](D87684)          UBXD2             UBX domain-containing 2
  80                    [D84454](D84454)          SLC35A2           Solute carrier family 35 (UDP-galactose transporter), member 2
  81                    [M97496](M97496)          GUCA2A            Guanylate cyclase activator 2A (guanylin)
  82                    [M95585](M95585)          HLF               Hepatic leukemia factor
  83                    [L38517](L38517)          IHH               Indian hedgehog homolog (Drosophila)
  84                    [L20860](L20860)          GP1BB             Glycoprotein Ib (platelet), beta polypeptide
  85                    [M26880](M26880)          UBC               Ubiquitin C
  86                    [D86962](D86962)          GRB10             Growth factor receptor-bound protein 10
  87                    [D63481](D63481)          SCRIB             Scribble
  88                    [D17525](D17525)          MASP1             Mannan-binding lectin serine protease 1 (C4/C2 activating component of Ra-reactive factor)
  89                    [L26584](L26584)          RASGRF1           Ras protein-specific guanine nucleotide-releasing factor 1
  90                    [M65066](M65066)          PRKAR1B           Protein kinase, cAMP-dependent, regulatory, type I, beta
  91                    [J05158](J05158)          CPN2              Carboxypeptidase N, polypeptide 2, 83 kD
  92                    [L36861](L36861)          GUCA1A            Guanylate cyclase activator 1A (retina)
  93                    [L11239](L11239)          GBX1              Gastrulation brain homeo box 1
  94                    [D90145](D90145)          SCYA3L1           Small inducible cytokine A3-like 1
  95                    [M96739](M96739)          NHLH1             Nescient helix loop helix 1
  96                    [M12959](M12959)          TRA@              T cell receptor alpha locus
  97                    [D80005](D80005)          C9orf10           C9orf10 protein
  98                    [M13231](M13231)          TRGC2             T cell receptor gamma constant 2
  99                    [D28588](D28588)          SP2               Sp2 transcription factor
  100                   [M57732](M57732)          TCF1              Transcription factor 1, hepatic-LF-B1, hepatic nuclear factor (HNF1), albumin proximal factor
  101                   [NM_014755](NM_014755)    TRIP-Br2          Transcriptional regulator interacting with the PHS-bromodomain 2
  102                   [NM_000576](NM_000576)    IL1B              Interleukin 1, beta
  103                   [NM_002089](NM_002089)    GRO2              GRO2 oncogene
  104                   [NM_002089](NM_002089)x   GPRC5D            G protein-coupled receptor, family C, group 5, member D
  105                   [NM_002713](NM_002713)    PPP1R8            Protein phosphatase 1, regulatory (inhibitor) subunit 8
  106                   [NM_014383](NM_014383)    TZFP              Testis zinc finger protein
  107                   [NM_012248](NM_012248)    SPS2              Selenophosphate synthetase 2
  108                   [AL137438](AL137438)      SEC15L            SEC15 (S. cerevisiae)-like
  109                   [NM_005387](NM_005387)    NUP98             Nucleoporin 98 kD
  110                   [NM_003476](NM_003476)    CSRP3             Cysteine and glycine-rich protein 3 (cardiac LIM protein)

###### 

Genes from which the mosaic in Figure 3B were derived. Genes from which correlation mosaics in Figure 3B were derived. Genes in this table show the greatest differences by DFA analysis comparing adult and cord blood monocytes.

  **Order in Mosaic**   **Accession No.**        **Gene Symbol**   **Description**
  --------------------- ------------------------ ----------------- ----------------------------------------------------------------------------------------------------
  1                     [AK055855](AK055855)     CLDN10            Claudin 10
  2                     [NM_000565](NM_000565)   IL6R              Interleukin 6 receptor
  3                     [NM_006150](NM_006150)   LMO6              LIM domain only 6
  4                     [NM_022787](NM_022787)   NMNAT             NMN adenylyltransferase-nicotinamide mononucleotide adenylyl transferase
  5                     [NM_002743](NM_002743)   PRKCSH            Protein kinase C substrate 80K-H
  6                     [NM_004847](NM_004847)   AIF1              Allograft inflammatory factor 1
  7                     [NM_021073](NM_021073)   BMP5              Bone morphogenetic protein 5
  \* 8                  [AK025306](AK025306)     CLK1              CDC-like kinase 1
  9                     [NM_004280](NM_004280)   EEF1E1            Eukaryotic translation elongation factor 1 epsilon 1
  \* 10                 [NM_004432](NM_004432)   ELAVL2            ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B)
  11                    [NM_012181](NM_012181)   FKBP8             FK506 binding protein 8 (38 kD)
  12                    [NM_002091](NM_002091)   GRP               Gastrin-releasing peptide
  13                    [NM_016355](NM_016355)   LOC51202          Hqp0256 protein
  14                    [NM_021204](NM_021204)   MASA              E-1 enzyme
  15                    [NM_004204](NM_004204)   PIGQ              Phosphatidylinositol glycan, class Q
  16                    [NM_002928](NM_002928)   RGS16             Regulator of G-protein signalling 16
  17                    [NM_005839](NM_005839)   SRRM1             Serine/arginine repetitive matrix 1
  18                    [NM_003166](NM_003166)   SULT1A3           Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 3
  19                    [NM_000356](NM_000356)   TCOF1             Treacher Collins-Franceschetti syndrome 1
  20                    [NM_016437](NM_016437)   TUBG2             Tubulin, gamma 2
  \* 21                 [NM_022568](NM_022568)   ALDH8A1           Aldehyde dehyrdogenase 8 family, member A1
  22                    [AF209930](AF209930)     CHRD              Chordin
  23                    [NM_005274](NM_005274)   GNG5              Guanine nucleotide binding protein (G protein), gamma 5
  24                    [NM_018384](NM_018384)   IAN4L1            Immune associated nucleotide 4 like 1 (mouse)
  25                    [NM_000640](NM_000640)   IL13RA2           Interleukin 13 receptor, alpha 2
  26                    [AK021692](AK021692)     LOC51141          Insulin induced protein 2
  27                    [NM_012443](NM_012443)   SPAG6             Sperm associated antigen 6
  28                    [NM_003155](NM_003155)   STC1              Stanniocalcin 1
  29                    [NM_022003](NM_022003)   FXYD6             FXYD domain-containing ion transport regulator 6
  30                    [NM_002763](NM_002763)   PROX1             Prospero-related homeobox 1
  31                    [NM_002836](NM_002836)   PTPRA             Protein tyrosine phosphatase, receptor type, A
  32                    [AL136835](AL136835)     TOLLIP            Toll-interacting protein
  33                    [AB058691](AB058691)     ALX4              Aristaless-like homeobox 4
  34                    [AF112345](AF112345)     ITGA10            Integrin, alpha 10
  35                    [NM_022788](NM_022788)   P2RY12            Purinergic receptor P2Y, G protein-coupled, 12
  36                    [NM_001213](NM_001213)   C1orf1            Chromosome 1 open reading frame 1
  37                    [NM_005860](NM_005860)   FSTL3             Follistatin-like 3 (secreted glycoprotein)
  38                    [NM_013320](NM_013320)   HCF-2             Host cell factor 2
  39                    [NM_058246](NM_058246)   LOC136442         Similar to MRJ gene for a member of the DNAJ protein family
  40                    [NM_020169](NM_020169)   LXN               Latexin protein
  41                    [BC008993](BC008993)     MGC17337          Similar to RIKEN cDNA 5730528L13 gene
  42                    [BC002712](BC002712)     MYCN              V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)
  43                    [AK026164](AK026164)     MYL6              Myosin, light polypeptide 6, alkali, smooth muscle and non-muscle
  44                    [NM_006215](NM_006215)   SERPINA4          Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 4
  45                    [NM_004790](NM_004790)   SLC22A6           Solute carrier family 22 (organic anion transporter), member 6
  46                    [NM_022911](NM_022911)   SLC26A6           Solute carrier family 26, member 6
  47                    [NM_003374](NM_003374)   VDAC1             Voltage-dependent anion channel 1
  48                    [NM_017818](NM_017818)   WDR8              WD repeat domain 8
  49                    [NM_003416](NM_003416)   ZNF7              Zinc finger protein 7 (KOX 4, clone HF.16)
  50                    [NM_002313](NM_002313)   ABLIM             Actin binding LIM protein
  51                    [NM_012074](NM_012074)   CERD4             Cer-d4 (mouse) homolog
  52                    [NM_000787](NM_000787)   DBH               Dopamine beta-hydroxylase (dopamine beta-monooxygenase)
  \* 53                 [NM_000561](NM_000561)   GSTM1             Glutathione S-transferase M1
  54                    [BC014075](BC014075)     GTPBP1            GTP binding protein 1
  55                    [NM_033260](NM_033260)   HFH1              Winged helix/forkhead transcription factor
  56                    [NM_033033](NM_033033)   KRTHB2            Keratin, hair, basic, 2
  57                    [NM_004789](NM_004789)   LHX2              LIM homeobox protein 2
  58                    [NM_014106](NM_014106)   PRO1914           PRO1914 protein
  \* 59                 [NM_006799](NM_006799)   PRSS21            Protease, serine, 21 (testisin)
  \* 60                 [NM_002900](NM_002900)   RBP3              Retinol binding protein 3, interstitial
  61                    [NM_033022](NM_033022)   RPS24             Ribosomal protein S24
  \* 62                 [AB029021](AB029021)     TRIM35            Tripartite motif-containing 35
  \* 63                 [NM_020989](NM_020989)   CRYGC             Crystallin, gamma C
  \* 64                 [BI198124](BI198124)     HMG1L10           High-mobility group (nonhistone chromosomal) protein 1-like 10
  65                    [NM_014163](NM_014163)   HSPC073           HSPC073 protein
  66                    [AF181985](AF181985)     JIK               STE20-like kinase
  67                    [NM_017607](NM_017607)   PPP1R12C          Protein phosphatase 1, regulatory (inhibitor) subunit 12C
  \* 68                 [NM_002873](NM_002873)   RAD17             RAD17 homolog (S. pombe)
  69                    [NM_022095](NM_022095)   ZNF335            Zinc finger protein 335
  \* 70                 [M90355](M90355)         BTF3L2            Basic transcription factor 3, like 2
  71                    [NM_002079](NM_002079)   GOT1              Glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1)
  72                    [NM_004146](NM_004146)   NDUFB7            NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7 (18 kD, B18)
  73                    [L38486](L38486)         MFAP4             Microfibrillar-associated protein 4
  \* 74                 [AF111848](AF111848)     ACTB              Actin, beta
  75                    [NM_001916](NM_001916)   CYC1              Cytochrome c-1

We analyzed these genes using DFA in order to find those genes most likely to highlight the differences between cord and adult monocytes. DFA identified genes having high discriminatory capabilities. The DFA software selected genes from Table [3](#T3){ref-type="table"} with highest discriminatory capabilities for this case. A total of 12 genes (marked with asterisk in Table [3](#T3){ref-type="table"}) were used by the DFA program to differentiate dynamical changes in both cord and adult monocytes after LPS stimulation. Values of the roots obtained by DFA analysis were used to graphically depict the differences of the gene expression values obtained in cord and adult samples in different stages after stimulation (Fig. [4](#F4){ref-type="fig"}). The spatial organization of the elements in this representation provides a measure of the overall similarity of the dynamic behaviour of these samples. The greatest temporal changes in gene expression for cord and adult monocytes noted above after 45 min of LPS exposure were also observed in the analysis using these 12 genes. However, almost no differences occurred at the 2 hr time point between cord and adult cells suggesting that the global behavior of the cells is similar, but the kinetics of change differ i.e. many of the changes are the same in both groups, but they occur at different rates.

![DFA analysis of phases of monocyte activation comparing cord and adult cells. DFA identified a subset of genes (see Table 3) whose expression values can be linearly combined in an equation, denoted a root, whose overall value is distinct for a given characterized group. These roots used as coordinate for presentation of these groups of samples in scatterplot. Results from individual samples for adult monocyte (circles) and cord monocytes (triangles) are discussed in the text. Results from individual samples for adult monocyte (circles) and cord monocytes (triangles) are shown.](1476-9255-4-4-4){#F4}

Apoptosis assays
----------------

The products of a subset of genes that were differentially expressed between groups after 45 min exposure to LPS are involved in apoptosis. We therefore performed a series of functional experiments comparing apoptosis in adult (n = 10) and neonatal (n = 10) cord bloods. Results of these assays are shown in Table [4](#T4){ref-type="table"}. Annexin assays demonstrated that adult monocytes display different kinetics for both apoptosis and necrosis as compared with neonatal monocytes. Flow cytometry revealed that 43 ± 5% (mean + SD) of adult and 53 + 8% of neonatal monocytes are undergoing apoptosis after stimulation with LPS for 14 hours (p \< 0.002), while 38 + 8% of adult and 25 + 9% of neonatal monocytes are necrotic after 14 hours of LPS stimulation (p \< 0.003). The number of live monocytes after 14 hours of LPS stimulation was not statistically different between the two groups. There was also no statistically significant difference in the number of live, apoptotic, or necrotic monocytes between adult and neonatal samples prior to LPS stimulation (data not shown).

###### 

Results of Annexin Binding Assays

  **Cell Type**          **Apoptotic Cells**   **Necrotic Cells**   **Significance**
  ---------------------- --------------------- -------------------- ------------------
  Adult monocytes        43 ± 5%               38 % ± 8%            P \< 0.002
  Cord blood monocytes   53 ± 8%               25% ± 9%             P \< 0.003

Discussion
==========

Following a given physiologic stimulus, signalling kinase activation, transcription factor translocation, and gene transcription all occur in rapid order. However, like all biological processes, mRNA accumulation (or decreases) does not occur uniformly, and we hypothesized that examining the kinetics of mRNA accumulation or disappearance might provide clues into relevant cellular dynamics. We used a well-developed and validated gene expression microarray to examine the dynamics of mRNA accumulation and differences between adult and neonatal monocytes in that process.

Genes were found to be differentially expressed between adult and cord monocytes after either 45 or 120 minutes of LPS exposure, with little difference at 24 hr (see Figure [4](#F4){ref-type="fig"}). Interestingly, no statistically significant differences in gene expression were observed between these groups in untreated cells. Previous reports by others indicated altered functions of cord blood monocytes in cytokine secretion and cellular adhesion. Results from this study cast new light on these findings and add complexity to understanding such differences. In some cases, our data support previous speculations about neonatal immune function. For example, the increased expression of IL-17B in neonatal monocytes is consistent with the observations of Vanden Eijnden and colleagues that newborns compensate for their relative immune deficiency by over-expression of the IL23-IL-17 signalling pathway in dendritic cells \[[@B24]\]. Similarly, we found significant elevations in cord monocyte transcripts of the chemokines MIP1B and MIP1A after 2 hrs of LPS exposure, consistent with Sullivan and colleagues\' report of higher amounts of MIPα in cord blood samples compared with adults \[[@B25]\]. On the other hand, transcripts for cadherin 9, *Rock1*, periostin, heparin sulfate 6-O-sulfotransferase 3, and C20orf42, whose products participate in various mechanisms that are associated with adhesion \[[@B26]-[@B28]\] were statistically significantly increased in adult monocytes after 45 min of LPS exposure, although no differences in expression for these genes between groups were detected at the later time point. These data suggest complex, dynamic relations for genes whose products are associated with cellular adhesion, and collectively highlight the importance of examining gene expression profiles (or related protein expression levels) over time.

The limits of gene expression profiling as a technique, albeit a very useful technique, must be acknowledged. The technique examines only RNA transcripts, not protein synthesis. Thus, alterations in other critical inflammatory mediators, such as eicosanoids, remain unobserved with this method. Furthermore, it is well known that there are many proteins, including critical inflammatory mediators, whose synthesis and secretion is not directly related in mRNA accumulation \[[@B29]\]. Thus, gene expression profiling should be complemented with other methods in order to maximize there potential.

In the final analysis, the utility of gene expression profiling will be demonstrated only if they provide insights into relevant physiologic or pathophysiologic function. For that reason, we elected to test the validity of the array data by examining a physiologic mechanism implicated by computer modelling of the array data. As noted in Table [1](#T1){ref-type="table"}, adult monocytes over-expressed a small number of genes associated with the regulation of apoptosis. Since monocyte activation is a \"balancing act\" between signals inducing apoptosis and those inducing activation and differentiation \[[@B30],[@B31]\], differences in the kinetics of expression or activation of enzymes or transcription factors that regulate apoptosis could have a crucial outcome on whether monocyte responses are pro- or anti-inflammatory. Annexin assays confirmed that there are significant differences in the appearance of apoptotic cells between adults and newborn monocytes (Table [4](#T4){ref-type="table"}). Since apoptotic cells dampen the inflammatory response, it is interesting to speculate that the related blunted neonatal response to inflammatory stimuli (including infection) may result, at least in part, from the excessive production of apoptotic cells during monocyte activation.

There has been, to our knowledge, one previously published paper using gene expression arrays to study neonatal monocyte function \[[@B14]\]. Our findings differ somewhat from those described by these authors. The most obvious difference was our finding of no statistically significant differences between adult and cord blood samples in the resting state. We should note, however, that it is otherwise difficult to compare the two studies. Jiang and colleagues used a 1000-fold greater dose of LPS to stimulate the monocytes, and RNA was prepared after 18 hr of stimulation. Thus, it is difficult to determine which of the effects observed by these authors were the direct result of LPS activation or were mediated through autocrine activation by proteins secreted in response to LPS. Furthermore, the non-physiologic dose of LPS used by those authors makes the biological/pathological relevance of that study difficult to interpret. Finally, we should note that the study by Jiang and colleagues used different methodologies for purifying monocytes. While our method, positive selection using CD14-coated microbeads, carries the theoretical risk of activating the cells through TLR-4/CD14 signaling pathways, adherence procedures carry the greater risk of activating the cells, as β2 integrins are activated during the adherence process.

From the bioinformatics standpoint, our data demonstrate how gene microarray experiments can quickly move from the generation of gene lists to the development of plausible and testable models of relevant biology and physiology. Specifically, they demonstrate that computer-assisted, physiologic modelling is another means of corroborating array findings and provides the advantage of providing an approach for immediately testing the biological relevance of microarray data before embarking on the sometimes laborious task of confirming differential expression of dozens or even hundreds of genes identified in a microarray experiment. As described in the results section, the differences between groups in gene expression at 45 min were attributable to a unique up-regulation of specific genes in adult monocytes, a unique down-regulation of other genes in cord monocytes, or a combination of both processes for other genes. We have searched for mechanisms that account for these patterns. Specifically, we have analyzed the genes within derived k-means clusters to determine if a large number of genes within a cluster are related to overlapping functions using Ingenuity Pathway Assist software, or alternatively to shared transcriptional response elements upstream of these genes. However, these strategies have failed to elucidate reasons to explain these findings.

Our studies also suggest that, while expensive and time-consuming to undertake, studying the kinetics of gene expression using microarrays can be highly informative. The previously reported study \[[@B14]\] examining gene expression differences between adult and cord blood monocytes was performed at only a single time point (18 hr after activation with a non-physiologic dose of LPS). Our studies suggest that the relevant biology may lie not in the specific genes that are differentially expressed at one particular time point, but, as one would predict with a dynamic system, which genes are expressed when. Timing of mRNA accumulation could determine, among other things, whether pro-apoptotic signals are processed in monocytes before cellular necrosis ensues.

The validity of the dynamic/kinetic approach is further supported by the correlation analyses (Figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}). These analyses demonstrate clearly that the accumulation of a specific mRNA is not an independent event. Gene transcription and mRNA degradation are dynamic processes closely tied to the accumulation or degradation of other mRNAs and the transcription of their cognate proteins. We contend that, without this dynamic view of cellular activity, investigators attempting to use microarray data to elucidate relevant biological or pathological processes will encounter unnecessary obstacles in attempts to move from the generation of gene lists to testing specific hypotheses.

Abbreviations
=============

LPS -- Lipopolysaccharide

DFA -- Discriminant function analysis

HV -- Hypervariable

Acknowledgements
================

Supported in part by the National Institutes of Health (NIH), National Center for Research Resources, a component of the NIH, General Clinical Research Center Grant MO1 RR-14467, NIH grants P20 RR020143-01, P20 RR15577, P20 RR17703, and P20 R016478-04 and by the Oklahoma Center for Science and Technology (OCAST).

The authors also wish to extend their thanks to Julie McGhee, M.D., for her review and thoughtful comments on this manuscript.
