Immuno-related Genes Research Articles

Peptide Degrader-Based Targeting of METTL3/14 Improves Immunotherapy Response in Cutaneous Melanoma.

METTL3 has emerged as a promising therapeutic target in cancer treatment, although its oncogenic functions in melanoma development and potential for therapeutic targeting drug have not been fully explored. In this study, we define the oncogenic role of METTL3 in melanoma development and progression. Building on this insight, we examine our recently designed peptide inhibitor RM3, which targets the binding interface of METTL3/14 complex for disruption and subsequent ubiquitin-mediated proteasomal degradation via the E3 ligase STUB1. RM3 treatment reduces proliferation, migration, and invasion, and induces apoptosis in melanoma cells in vitro and in vivo. Subsequent transcriptomic analysis identified changes in immuno-related genes following RM3-mediated suppression of METTL3/14 N6-methyladenosine (m6A) methyltransferase activity, suggesting a potential for interaction with immunotherapy. A combination treatment of RM3 with anti-PD-1 antibody results in significantly higher beneficial tumor response in vivo, with a good safety profile. Collectively, these findings not only delineate the oncogenic role of METTL3 in melanoma but also showcase RM3, acting as a peptide degrader, as a novel and promising strategy for melanoma treatment.

Effects of immunorelated gene polymorphisms on trastuzumab targeting breast cancer cell in vitro.

Aim: To investigate the associations between genetic polymorphisms in immunorelated genes and PBMC-induced cytotoxicity to breast cancer cell with the treatment of trastuzumab in vitro.Methods: Trastuzumab-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) from 148 healthy donors and 13 BC patients was analyzed by flow cytometry. 16 SNPs in 7 immunorelated genes were genotyped by Sequenom Mass Array Genotype Platform.Results: Cytotoxicity in the trastuzumab treated PBMCs were significantly higher than those of the basal group. A wide variability in trastuzumab-mediated cytotoxicity was observed, and PBMC from individuals with the CD247 rs16859030 T genotype generated increased cytotoxicity than those with the CC genotype.Conclusion: The CD247 rs16859030 polymorphism affects trastuzumab-mediated cytotoxicity in vitro.

Identifying immuno-related diagnostic genes and immune infiltration signatures for periodontitis and alopecia areata

BackgroundAlthough there have been indications that periodontitis (PD) may be susceptible to alopecia areata (AA), the underlying mechanism of its pathogenesis remains poorly understood. The objective of our study is to conduct further research into the occurrence of this complication. MethodsThe gene expression omnibus (GEO) database was the source of acquisition for both PD and AA datasets. Various methods, including the differentially expressed genes (DEGs) analysis, functional enrichment analysis, protein–protein interaction (PPI) network construction, Cytohubba algorithms, and RandomForest algorithms, were utilized to identify candidate hub immuno-related genes (IRGs) for diagnosing AA with PD. The diagnostic efficacy was assessed by constructing receiver operating characteristic (ROC) curves. To further deepen our understanding, immune cell infiltration, flow cytometry assay, and immunofluorescence techniques were employed to uncover immune cell dysregulation in PD and AA. Results899 and 803 DEGs were detected in AA and PD, respectively, with an intersection of 150 common DEGs enriched in immune regulation. Further analysis of the junction of shared DEGs and IRGs was analyzed using the PPI network, Mcode, and Cytohubba algorithms. Three hub genes (CTSS, IL2RG, and ITGAL) were subsequently selected by Cytohubba and RandomForest algorithms and were found to be promising candidate hub genes with high diagnostic values (AUC ranging from 0.776 to 0.909) for diagnosing AA with PD. Additionally, various dysregulated immune cells were observed, with mast cells potentially serving as markers for AA and plasma for PD. ConclusionThree candidate hub IRGs (CTSS, IL2RG, and ITGAL) were identified with considerable diagnostic values. Besides, mast cells could serve as markers for AA, while plasma may indicate PD. Our research has the potential to identify shared diagnostic candidate genes and immune cells for AA and PD patients.

Bioinformatics analysis of immune-related prognostic genes and immunotherapy in renal clear cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is an immunogenic tumor, and investigating the immunorelated genes is essential. To investigate the immunoprognostic genes of ccRCC, we analyzed the data assimilated from a public database (The Cancer Genome Atlas (TCGA) database and the gene expression omnibus (GEO) database) using bioinformatics. Then, an immunoprognosis model was constructed to identify four hub genes with moderate predictive values for the prognosis of ccRCC patients. These four genes were associated with the prognosis of ccRCC patients based on Oncomine and Gena Expression Profiling Interactive Analysis (GEPIA) databases. The correlation analysis between the immune infiltrate, immune checkpoints, and immunotherapy and this immunoprognosis model showed that immune infiltration could predict the immunotherapy effects. We also conducted a quantitative real-time polymerase chain reaction analysis and found that the expressions of three hub genes were associated with tumor progression (P<0.1). In conclusion, four genes that may serve as potential biomarkers in ccRCC were identified with respect to prognosis.

Involvement of galectin-9 from koi carp (Cyprinus carpio) in the immune response against Aeromonas veronii infection

Galectins are β-galactoside sugar binding proteins which function as important pattern recognition receptors (PRRs) in innate immunity. Here, we identified a galectin-9 gene from koi carp (Cyprinus carpio), named kGal-9. The ORF of kGal-9 is 963 bp in length, which encodes a polypeptide of 320 amino acids without either signal peptide. The predicted molecular weight is 36.25 kDa, and the isoelectric point is 8.3. Multiple sequence alignment showed that the putative kGal-9 contains two carbohydrate recognition domains (CRD), which are conserved in Galectin-9s. The phylogenetic tree showed that kGal-9 clustered to Galectin-9s from other teleosts, and shared the highest identity of 87.5% with Qihe crucian (Carassius auratus). kGal-9 mRNA was abundant in head kidney, gills, and gut, but low in liver and muscle. Further, the expression level of kGal-9 in the head kidney and liver increased significantly after Aeromonas veronii (abbreviated A.v) infection. Unexpectedly, kGal-9 showed a remarkable downregulation in the spleen at various time points post A.v infection. Intramuscular injection of pckGal-9 not merely reduced the bacterial load of spleen tissue, but also improved the survival rate of koi carp post A.v challenge. Besides, administration of pckGal-9 stimulated the expression of several immuno-related genes including proinflammatory cytokines (IL-1β, IL-6), anti-inflammatory cytokine (IL-10), complement components (C4, C9), with fluctuation in spleen and head kidney. Taken together, the obtained results suggest that kGal-9 occupies an important role in innate immunity and defense against bacterial infection in koi carp.

Metastatic Immune-Related Genes for Affecting Prognosis and Immune Response in Renal Clear Cell Carcinoma.

Background: In renal clear cell carcinoma, a common cancer of the urinary system, 25–30% patients are metastatic at initial diagnosis and 20–30% patients have a tendency of recurrence and metastasis after local surgery. With the rapid development of tumor immunology, immune agents have brought new directions to tumor therapy. However, no relevant studies have explored the role of immune-related genes in kidney cancer metastasis. Methods: Co-expressed metastatic immune-related differentially expressed genes (mIR-DEGs) were screened by GSE12606, GSE47352, and immunorelated genes. Then, differential expression analysis, prognostic analysis, and univariate and multivariate Cox regression analysis in KIRC were performed to determine independent prognostic factors associated, and the risk prognostic model was established. The correlation of hub mIR-DEGs with clinicopathological factors, immune invasion, and immune checkpoints was analyzed, and the expression of hub mIR-DEGs and their effect on tumor were re-evaluated by OCLR scores in KIRC. Results: By comprehensive bioassay, we found that FGF17, PRKCG, SSTR1, and SCTR were mIR-DEGs with independent prognostic values, which were significantly associated with clinicopathological factors and immune checkpoint–related genes. The risk prognostics model built on this basis had good predictive potential. In addition, targeted small molecule drugs, including calmidazolium and sulfasalazine, were predicted for mIR-DEGs. Further experimental results were consistent with the bioinformatics analysis. Conclusion: This study preliminarily confirmed that FGF17, PRKCG, SSTR1, and SCTR were targeted genes affecting renal cancer metastasis and related immune responses and can be used as potential therapeutic targets and prognostic biomarkers for renal cancer. Preliminary validation found that PRKCG and SSTR1 were consistent with predictions.

Immunorelated gene polymorphisms associated with acute myeloid leukemia.

Although the pathogenesis of acute myeloid leukemia (AML) is still unknown, accumulating evidence has revealed that immune response plays a vital part in the pathogenesis. Here, we investigated the involvement of 21single nucleotide polymorphisms (SNPs) of immunorelated genes, including cytokines [interleukin (IL)-2, IL-4,IL-9, IL-12A, IL-22, interferon (IFN-α) and transforming growth factor (TGF)-β1], transcriptional regulatory genes (TBX21, STAT1, STAT3, STAT5B, STAT6, GATA3, FOXP3 and IRF4)and others (IL2RA,IL6R, NFKBIA) in 269 AML in-patients and 200 healthy controls. Furthermore, we analyzed the relationship between the SNPs and clinical characteristics. Immunorelated SNP genotyping was performed on the Sequenom MassARRAY iPLEX platform. All the SNPs in healthy controls were consistent with Hardy-Weinberg equilibrium. All final P-values were adjusted by Bonferroni multiple testing. Our results showed that IL-22 (rs2227491) was significantly associated with the white blood cell (WBC) counts. Signal transducer and activator of transcription 5B (STAT-5B) (rs6503691) showed a close relationship with the recurrent genetic abnormalities in patients with AML. We verified the negatively independent effect of age and risk of cytogenetics on overall survival (OS). More importantly, the GG genotype of IL-12A (rs6887695) showed a negative impact on AML prognosis independently. Furthermore, the relative expression of IL-12 was decreased in GG genotype, no matter under a co-dominant or recessive model. However, no correlation was observed between the SNPs mentioned above and disease susceptibility, risk stratification and survival. Our findings suggest that immunorelated gene polymorphisms are associated with prognosis in AML, which may perform as novel inspection targets for AML patients.

Interleukin-6 in Siberian sturgeon (Acipenser baeri): Molecular characterization and immune functional activity

Interleukin-6 (IL-6) is a pleiotropic cytokine with crucial immunoregulatory functions in both innate and adaptive immune responses. However, the IL-6 sequence and function remain unknown in sturgeon, one chondrostean fish. In the present study, we identified an interleukin-6 homolog from Siberian sturgeon (Acipenser baeri), named AbIL-6. Its open reading frame (ORF) was 657 nucleotides in length, encoding a polypeptide of 218 amino acids, which contains a signal peptide and the IL-6 family domain. Phylogenetic analysis showed that sturgeon IL-6 had close relationship with both teleost and chondrichthyes IL-6s. Abil-6 mRNA was highly expressed in spleen, brain and liver tissues of healthy sturgeon, and significantly up-regulated in the spleen, head kidney and liver by A.hydrophila (A.h) challenge. Heat-killed A.h and LPS effectively stimulated Abil-6 transcripts in primary spleen cells in vitro. In order to understand the bioactivity and influence of AbIL-6 on immune responses, recombinant AbIL-6 (rAbIL-6) was synthesized by prokaryotes and demonstrated to promote the proliferation of spleen cells and head kidney cells in vitro. Additionally, intraperitoneal injection of rAbIL-6 induced significantly higher expression of four immuno-related genes including il-1β, cxcl10, mhcIIβ and igm. rAbIL-6 improved the survival rate and reduced the tissue bacterial load after A.h infection. Taken together, these results suggest that AbIL-6 plays an important role in inflammatory responses and immune defense against bacterial infection of sturgeon.

Molecular characterization and biological functioning of interleukin-8 in Siberian sturgeon (Acipenser baeri)

Interleukin-8, otherwise known as CXCL8, is a CXC chemokine that plays a pivotal regulatory role in immune and inflammation responses of animals. Here, we identified an interleukin-8 homologue from Siberian sturgeon (Acipenser baeri), named AbIL-8, which belongs to the lineage 1 group of teleost fish IL-8s. The cDNA of Abil-8 is 1130 bp in length, containing a 5′- untranslated region (UTR) of 50 bp, a 3′- UTR of 783 bp, and an open reading frame (ORF) of 297 bp that encodes a protein consisting of 98 amino acids. The deduced AbIL-8 contained five cysteines, four of which are highly conserved, and an ELR motif typical of known mammalian CXC chemokines was also found preceding the CXC motif. Our phylogenetic analysis showed that AbIL-8 clustered with the CXCL8_L1 sequences from other teleosts, being clearly distinct from those of either birds or mammals. Abil-8 mRNA was constitutively expressed in all tested tissues and significantly up-regulated in the liver and spleen tissues by the bacteria Aernomas hydrophila. The in vitro experiment using primary spleen cells stimulated with heat-killed Aernomas hydrophila or lipopolysaccharide (LPS) revealed a similar expression pattern to that found in vivo, whereas stimulation on spleen cells with β-glucan or polyI:C elicited negligible changes in levels of Abil-8 mRNA. Purified recombinant AbIL-8 not only exhibited chemotactic activity for lymphocytes and monocytes in peripheral blood leukocytes (PBLs) and, to a lesser extent, spleen cells, but also stimulated the proliferation of spleen cells at 10 ng/mLor above. Furthermore, intraperitoneal injection of rAbIL-8 also up-regulated the expression of immuno-related genes (IL-6, IgM and MHCIIβ) at 24 h. Collectively, these results enhance our understanding of how IL-8 functions in the regulation of the immune responses in sturgeon.

In the shadow: The emerging role of long non-coding RNAs in the immune response of Atlantic salmon

The genomic era has increased the research effort to uncover how the genome of an organism, and specifically the transcriptome, is modulated after interplaying with pathogenic microorganisms and ectoparasites. However, the ever-increasing accessibility of sequencing technology has also evidenced regulatory roles of long non-coding RNAs (lncRNAs) related to several biological processes including immune response. This study reports a high-confidence annotation and a comparative transcriptome analysis of lncRNAs from several tissues of Salmo salar infected with the most prevalent pathogens in the Chilean salmon aquaculture such as the infectious salmon anemia (ISA) virus, the intracellular bacterium Piscirickettsia salmonis and the ectoparasite copepod Caligus rogercresseyi. Our analyses showed that lncRNAs are widely modulated during infection. However, this modulation is pathogen-specific and highly correlated with immuno-related genes associated with innate immune response. These findings represent the first discovery for the widespread differential expression of lncRNAs in response to infections with different types of pathogens in Atlantic salmon, suggesting that lncRNAs are pivotal player during the fish immune response.

Immunogenic proteins and their vaccine development potential evaluation in outer membrane proteins (OMPs) of Flavobacterium columnare

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide. Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease. In this study, an immunoblotting approach was employed to identify immunogenic outer membrane proteins (OMPs) from F. columnare in two-dimensional electrophoresis (2-DE) map gels using antibacterial sera obtained from grass carp (Ctenopharyngodon idella), and anti-grass carp-recombinant Ig (rIg) monoclonal antibodies. Five unique immunogenic proteins, including the gliding motility lipoprotein GldJ (GldJ), hypothetical protein FCOL_13420 (Fco1), lipoprotein (Lip), F0F1 ATP synthase subunit beta (F0f1) and outer membrane efflux protein precursor (Omep), were characterized. Over-expression of these proteins in Escherichia coli DE3, and their immunogenicity and protective efficacy were evaluated in grass carp. The relative percent survival (RPS) of the groups immunized separately with recombinant GldJ, Lip and Omep was 72%, 64% and 68%, respectively when compared to control fish. Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F. columnare. The results suggest that GldJ, Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.

The expression and role of CD59 in human extravillous trophoblast

The objective of the study was to investigate the effects of an in ovo injection of CuNano and the timing of injection on metabolic rate (O2 consumption and heat production, HP) and development of layer hatchlings. On day 1 of incubation, 192 fertile eggs from 29-week-old Lohmann breeder strain chickens were distributed into four groups that were administered colloidal CuNano on: day 1 and/or 10. Gaseous exchange was measured in an open-air-circuit respiration unit, and HP was calculated for 16- and 19-day-old embryos. Yolk free body weight (YFBW) at 24 h after hatching and the relative organ weights were used as a measure of hatchling development. In ovo injection of CuNano on different days during incubation significantly decreased O2 consumption and HP compared with the control group. The residual yolk sac weight in the treated groups was significantly higher than in the control group (P<0.0001), indicating that CuNano injection reduced lipid oxidation, which could be associated with the lower O2 consumption (P=0.001). Accordingly, the organ weights (intestine, heart, liver) relative to YFBW were also lower in embryos injected with CuNano (all; P<0.05). Interestingly, the difference in metabolic rate and organ weights between treatments was not reflected in YFBW (P>0.05). Furthermore, the plasma concentrations of IgM and IgG and the mRNA expression of NF-kB and TNF-α were not affected (both; P>0.05), indicating the absence of inflammatory modulation by CuNano. These preliminary results demonstrated that CuNano, regardless of the day of injection, altered the metabolic rate of embryos and depressed the development of organs; however, it did not affect YFBW, immunoglobulin concentrations and the expression of immuno-related genes.

The Sex Bias in Systemic Sclerosis: on the Possible Mechanisms Underlying the Female Disease Preponderance

Systemic sclerosis is a multifactorial and heterogeneous disease. Genetic and environmental factors are known to interplay in the onset and progression of systemic sclerosis. Sex plays an important and determinant role in the development of such a disorder. Systemic sclerosis shows a significant female preponderance. However, the reason for this female preponderance is incompletely understood. Hormonal status, genetic and epigenetic differences, and lifestyle have been considered in order to explain female preponderance in systemic sclerosis. Sex chromosomes play a determinant role in contributing to systemic sclerosis onset and progression, as well as in its sex-biased prevalence. It is known, in fact, that X chromosome contains many sex- and immuno-related genes, thus contributing to immuno tolerance and sex hormone status. This review focuses mainly on the recent progress on epigenetic mechanisms--exclusively linked to the X chromosome--which would contribute to the development of systemic sclerosis. Furthermore, we report also some hypotheses (dealing with skewed X chromosome inactivation, X gene reactivation, acquired monosomy) that have been proposed in order to justify the female preponderance in autoimmune diseases. However, despite the intensive efforts in elucidating the mechanisms involved in the pathogenesis of systemic sclerosis, many questions remain still unanswered.

Effect of copper nanoparticles on metabolic rate and development of chicken embryos

The objective of the study was to investigate the effects of an in ovo injection of CuNano and the timing of injection on metabolic rate (O2 consumption and heat production, HP) and development of layer hatchlings. On day 1 of incubation, 192 fertile eggs from 29-week-old Lohmann breeder strain chickens were distributed into four groups that were administered colloidal CuNano on: day 1 and/or 10. Gaseous exchange was measured in an open-air-circuit respiration unit, and HP was calculated for 16- and 19-day-old embryos. Yolk free body weight (YFBW) at 24h after hatching and the relative organ weights were used as a measure of hatchling development. In ovo injection of CuNano on different days during incubation significantly decreased O2 consumption and HP compared with the control group. The residual yolk sac weight in the treated groups was significantly higher than in the control group (P<0.0001), indicating that CuNano injection reduced lipid oxidation, which could be associated with the lower O2 consumption (P=0.001). Accordingly, the organ weights (intestine, heart, liver) relative to YFBW were also lower in embryos injected with CuNano (all; P<0.05). Interestingly, the difference in metabolic rate and organ weights between treatments was not reflected in YFBW (P>0.05). Furthermore, the plasma concentrations of IgM and IgG and the mRNA expression of NF-kB and TNF-α were not affected (both; P>0.05), indicating the absence of inflammatory modulation by CuNano. These preliminary results demonstrated that CuNano, regardless of the day of injection, altered the metabolic rate of embryos and depressed the development of organs; however, it did not affect YFBW, immunoglobulin concentrations and the expression of immuno-related genes.

Human leukocyte antigen in primary biliary cirrhosis: An old story now reviving

Primary biliary cirrhosis (PBC) is an autoimmune biliary disease characterized by injury of small and medium size bile ducts, eventually leading to liver cirrhosis and death. Although the causes remain enigmatic, recent evidence has strengthened the importance of genetic factors in determining the susceptibility to the disease. Besides the strong heritability suggested by familial occurrence and monozygotic twins concordance, for decades there has not been a clear association with specific genes, with the only exception of a low risk conferred by a class II human leukocyte antigen (HLA) variant, the DRB1*08 allele, at least in some populations. The picture has become more complete when strong protective associations between PBC and the HLA DRB1*11 and DRB1*13 alleles were found in Italian and UK series. However, HLA genes have begun again to attract interest thanks to recent genome-wide association studies (GWAS), which clearly demonstrated that the major components of the genetic architecture of PBC are within the HLA region. As expected in a genetically complex disease, GWAS also identified several novel non-HLA variants, but it is worth noting that all of them are in immuno-related genes. In this review, the paradigmatic tale of what, and how, we learned about HLA genes in PBC will be retraced with particular focus on how GWAS are enabling a rewriting the story of PBC pathogenesis. These recent discoveries will not only drive functional studies but will also hold the promise of developing novel disease-specific treatments.

Searching for immunomodulatory sequences in sea bass ( Dicentrarchus labrax L.): Transcripts analysis from thymus

The thymus is a key organ of the immune system in most vertebrates and, for this reason, it has been used in this paper for the generation of a normalized cDNA library from sea bass ( Dicentrarchus labrax), one of the most extensively cultured species in South Mediterranean aquaculture. A total of 1632 ESTs from this library were initially analysed for sequence quality and vector sequences and, after this control, 1264 (77% of total clones sequenced) high-quality ESTs were further processed. The total collection of D. labrax thymus ESTs has been deposited in the EBI-GenBank-DBJ database (GenBank accession numbers from FN565576 to FN566839). The functional classification of ESTs was performed by Gene Ontology and KEGG annotation and, successively, the sequences were analysed using the ImmunomeBase software to identify potentially immuno-related genes. Using this approach, we found about 100 putative genes involved in immune system responses, most new in sea bass, that were analysed more in detail. Some of the potentially interesting genes identified by these in silico analyses were studied by real-time PCR to verify their expression both at basal level and after in vitro stimulation of sea bass head kidney leukocytes. The used strategy has been confirmed as a good approach to discover new immuno-related genes and to improve the knowledge of specific markers that could help the discrimination of T-cell subpopulations in sea bass and, in general, in Teleosts.

Identification of differentially expressed genes in haemocytes of the crayfish ( Procambarus clarkii) infected with white spot syndrome virus by suppression subtractive hybridization and cDNA microarrays

By using suppression subtractive hybridization (SSH) and cDNA microarrays, we studied the differentially expressed genes in haemocytes of the crayfish ( Procambarus clarkii) infected with white spot syndrome virus (WSSV). Thirty three differentially expressed genes were detected in which 31 were up-regulated and 2 were down-regulated. The up-regulated genes include serine protease inhibitors, chaperonin, synaptasome-associated protein of 25 kD(SNAP25), tubulin, zinc-finger protein, intracellular fatty acid binding protein, extracellular superoxide dismutase precursor, arginine kinase, 70 kD heat shock like protein and Bax inhibitor-1. A lot of genes including the 2 down-regulated genes are still unknown. All these immuno-related genes responding to the virus infection provide a new insight for further study in the shrimp innate immunity.

Impact of a polyomavirus (BKV) infection on mRNA expression in human endothelial cells

Polyomavirus BK-associated nephropathy (PVAN) is an emerging cause of early renal transplant failure. In order to learn more about the cellular response to BK virus, microarrays were used to study its effect on mRNA expression in human endothelial cells. The oligo-based, 35 k arrays used cover the predicted 25,000 human protein-expressing genes, and distinguish between a number of alternatively spliced mRNAs. Four parallel experiments were performed for each of two time-points (24 and 40 h) during the first round of the 48 h viral replicative cycle. Immunoperoxidase staining demonstrated that the pulse exposure to virus caused infection in at least 75% of the cells. At 24 h, 55 genes were more than doubly up-regulated and 249 genes were similarly down-regulated; at 40 h, the numbers were 242 and 104, respectively. Gene ontology analyses suggested that immune/defence response genes were selectively down-regulated. Genes involved in cell division and DNA replication tended to be up-regulated, which may reflect an attempt on behalf of the virus to promote viral replication. Genes associated with PVAN were not induced, suggesting that these genes are not required for viral replication, but rather reflect circumstances specific for the disease. Only a few immuno-related genes were turned on, including the interferon response genes G1P2 and IFIT3. However, some of the up-regulated genes of unknown function may be involved in viral defence.

Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp ( Penaeus japonicus) by suppression subtractive hybridization

In order to find the immune-relevant factors responsible for the virus resistance in the WSSV-resistant shrimp, a suppression subtractive hybridization method was employed to identify differentially expressed genes and their expression profile in the hepatopancreas of the virus-resistant penaeid shrimp. Thirty five genes were identified from more than 400 clones, of which eight are found for the first time in penaeid shrimp. βGBP is the most abundant gene in our subtractive library except hemocyanin. Lectin, ferritin, oxygenase and chitinase of the virus-resistant shrimp all showed up-regulation in expression compared with those of normal shrimp. In addition, Ranbp, Rho and Rab were found in the subtractive library. This is the first evidence indicating that small GTPases are involved in the signal transduction in shrimp defense response. Furthermore, a number of genes encoding apoptotic-related proteins and antioxidant enzymes were expressed at a higher level in the virus-resistant shrimp. In short, the high expression of immuno-related genes in response to the virus infection and the involvement of small GTPases in the antiviral response provide a new insight for further study in the crustacean innate immunity.

Normalization and analysis of cDNA microarrays using within-array replications applied to neuroblastoma cell response to a cytokine.

The quantitative comparison of two or more microarrays can reveal, for example, the distinct patterns of gene expression that define different cellular phenotypes or the genes that are induced in the cellular response to certain stimulations. Normalization of the measured intensities is a prerequisite of such comparisons. However, a fundamental problem in cDNA microarray analysis is the lack of a common standard to compare the expression levels of different samples. Several normalization protocols have been proposed to overcome the variabilities inherent in this technology. We have developed a normalization procedure based on within-array replications via a semilinear in-slide model, which adjusts objectively experimental variations without making critical biological assumptions. The significant analysis of gene expressions is based on a weighted t statistic, which accounts for the heteroscedasticity of the observed log ratios of expressions, and a balanced sign permutation test. We illustrated the use of the techniques in a comparison of the expression profiles of neuroblastoma cells that were stimulated with a growth factor, macrophage migration inhibitory factor (MIF). The analysis of expression changes at mRNA levels showed that approximately 99 genes were up-regulated and 24 were reduced significantly (P <0.001) in MIF-stimulated neuroblastoma cells. The regulated genes included several oncogenes, growth-related genes, tumor metastatic genes, and immuno-related genes. The findings provide clues as to the molecular mechanisms of MIF-mediated tumor progression and supply therapeutic targets for neuroblastoma treatment.