Chronic Refractory Immune Thrombocytopenia Is Associated With Variants in Immune Genes.

The pathogenesis of chronic refractory immune thrombocytopenia (C/RITP) is mechanistically complex and considerably varies across patients. Few studies have focused on the genetic characteristics of C/RITP in children. The aim of this study was to analyze and summarize the clinical manifestations and genetic characteristics of C/RITP children with mutations in immune-related genes. In the study, 51 children with variants in immune-related genes (mutation group) and 103 children with no abnormal mutations (control group) were enrolled. Children in the mutation group showed severity of hemorrhage, a higher incidence of abnormal immunological indices, and an increased expression of SLE biomarkers. The number of peripheral T and B lymphocytes in the mutation group significantly increased. Nine patients (17.6%) had probable pathogenic variant genes associated with primary immunodeficiencies (TNFRSF13B, CARD11, CBL, and RAG2), and 42 patients (82.4%) had variants of uncertain significance in 23 genes. C/RITP patients with variants in immune-related genes had more severe bleeding, abnormal immunological indices, and an increased expression of SLE biomarker. Next-generation sequenciong (NGS) might be a useful way to differentiate those patients from C/RITP.

Reduced expression of innate immunity-related genes in lymph node metastases of luminal breast cancer patients.

Immune system plays a dual role in cancer by either targeting or supporting neoplastic cells at various stages of disease, including metastasis. Yet, the exact immune-related transcriptome profiles of primary tumours (PT) and lymph node metastases (LNM) and their evolution during luminal breast cancer (BCa) dissemination remain undiscovered. In order to identify the immune-related transcriptome changes that accompany lymphatic spread, we analysed PT-LNM pairs of luminal BCa using NanoString technology. Decrease in complement C3-one of the top-downregulated genes, in LNM was validated at the protein level using immunohistochemistry. Thirty-three of 360 analysed genes were downregulated (9%), whereas only 3 (0.8%) upregulated in LNM when compared to the corresponding PT. In LNM, reduced expression was observed in genes related to innate immunity, particularly to the complement system (C1QB, C1S, C1R, C4B, CFB, C3, SERPING1 and C3AR1). In validation cohort, complement C3 protein was less frequently expressed in LNM than in PT and it was associated with worse prognosis. To conclude, local expression of the complement system components declines during lymphatic spread of non-metastatic luminal BCa, whilst further reduction of tumoral complement C3 in LNM is indicative for poor survival. This points to context-dependent role of complement C3 in BCa dissemination.

Construction of an immune-related gene signature for prediction of prognosis in patients with cervical cancer.

Cervical cancer (CeCa) is becoming an intractable public health issue worldwide. Emerging evidence uncovers that the tumor progression and prognosis of patients with CeCa are tightly associated with the abundance of tumor-infiltrating immune cells. In the current study, the abundance of tumor-infiltrating immune cells in CeCa samples was assessed by using the ssGSEA, thereby generating two immune-related groups according to the immune status. A 4-gene prognostic signature (RIPOR2, DAAM2, SORBS1, and CXCL8) was next established based on the grouping and its predictive capability was validated by multiple analyses. The TIMER database was used to evaluate the association between 4 hub gene expression and immune cell infiltration. Immunophenoscore (IPS) was used to assess response to immune checkpoint inhibitors in CeCa samples. As the results, a novel grouping strategy based on immune cell infiltration was developed and validated. Based on the grouping, a 4-gene signature was identified to be an independent prognostic indicator for overall survival (OS) in CeCa patients. Among the 4 hub genes, RIPOR2 and CXCL8 expression were significantly correlated with immune cell infiltration. Besides, higher immune checkpoints expression and IPS scores were found in the 4-gene signature low-risk group, suggesting a more immunoactive status that tended to respond to immune checkpoint inhibitors. To sum up, a novel immune-related signature is established to predict CeCa patients' prognosis and also associated with response to immune checkpoint inhibitors, which might be a promising prognostic stratification strategy and innovate therapeutic management.

A prognostic signature based on immune-related genes for cervical squamous cell carcinoma and endocervical adenocarcinoma.

Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) is the fourth commonest female malignancy worldwide. CESC progresses in immune-microenvironment mainly composed of infiltrating immune and stromal cells. Here, we performed an integrated analysis incorporating the expression profiles from the Cancer Genome Atlas (TCGA) database and scores of immune and stromal cells calculated by Estimation of Stromal and Immune cells in Malignant Tumours using Expression data (ESTIMATE) algorithm. A two-gene signature (CD1C and CD6 genes) was established to predict the prognosis of CESC. Based on this signature, patients were divided into the high- and low-risk groups, and this signature showed good prognostic performance according to the results of Kaplan-Meier analysis and receiver operating characteristic (ROC) analysis in train set and two validation sets. A nomogram was built for evaluating the clinical applicability of this signature. In addition, based on Tumor Immune Estimation Resource (TIMER) database, 2 hub genes showed negative correlations with tumor purity and positive correlations with infiltrating levels of immune filtrating cells. What's more, we propose new treatment strategies for the two prognostic subtypes. Low- risk patients were found presenting with a higher level of immune checkpoint molecules and showing higher immunogenicity in immunophenoscore (IPS) analysis, which indicated a better response for immunotherapy. Meanwhile, estimated by Genomics of Drug Sensitivity in Cancer (GDSC) database, the high-risk patients showed sensitive responses to five chemotherapy drugs. Finally, 10 candidate small-molecule drugs for CESC were defined. In summary, the CD1C-CD6 signature can accurately predict the prognosis of CESC.

Carbapenemases novas ou emergentes em Pseudomonas aeruginosa / New or emerging carbapenemases in Pseudomonas aeruginosa

Pseudomonas aeruginosa é um importante agente de infecções relacionadas à assistência à saúde em todo o mundo. O tratamento empírico de infecções graves causadas por esta espécie usualmente inclui os carbapenêmicos. Essa terapia, se inadequada, está relacionada a um aumento significativo da mortalidade. Os principais mecanismos de resistência aos carbapenêmicos em bacilos Gram-negativos são a redução da permeabilidade da membrana externa, hiperexpressão de bombas de efluxo e produção de betalactamases, sendo este último, o mais eficiente. O objetivo deste trabalho consistiu na caracterização de carbapenemases novas ou emergentes e seu contexto genético em P. aeruginosa. Foram utilizados 11 isolados de P. aeruginosa capazes de hidrolisar o imipenem em ensaio espectrofotométrico e negativos para genes de carbapenemases conhecidos e uma cepa produtora de KPC-2. Tais isolados foram submetidos à confirmação do perfil de resistência aos carbapenêmicos pelos métodos de disco-difusão e microdiluição em caldo (CIM), bloqueio enzimático com EDTA e Blue-Carba. O perfil plasmidial foi avaliado utilizando-se os métodos de lise alcalina e eletroforese em campos pulsados (PFGE). Os genomas foram sequenciados utilizando-se o sistema MiSeq. O perfil clonal dos isolados foi avaliado por PFGE e Multilocus Sequence Typing (MLST). Dez isolados apresentaram Blue-Carba negativo, compatível com a presença de carbapenemases fracas. Quatro isolados apresentaram plasmídeos, visualizados em gel de agarose após PFGE. Os plasmídeos do isolado D5303023 foram eletroporados em E. coli TOP 10 e selecionados em meio LB contendo 1µg/mL imipenem, contudo não foram obtidos transformantes. A análise por PFGE identificou sete grupos clonais distintos. Quanto à tipagem por MLST, foi detectado um novo tipo ST3187 e os ST277 e ST1560 foram os predominantes. O isolado D9203039 apresentou uma carbapenemase GES-20 associada a uma betalactamase de espectro estendido GES-19, sendo os genes que as codificam localizados no integron In724. Esse isolado pertence ao ST309, clone de potencial alto risco, associado ao fenótipo de resistência a múltiplos antimicrobianos no México. O genoma da cepa positiva para KPC-2 foi digerido com a S1 nuclease e submetido à PFGE, evidenciando um plasmídeo de aproximadamente 453 kb. Após análise do sequenciamento confirmou-se que a cepa pertence ao ST446 e foi observada a presença do gene blaKPC-2 em um contig de 128.487 bp. Este contig apresentou 99,9% de similaridade com o plasmídeo 1 da cepa P. aeruginosa RW109; no entanto, ensaios de conjugação bacteriana falharam em obter colônias de transconjugantes. O gene blaKPC-2 foi encontrado flanqueado à montante por uma estrutura truncada de um transpóson da família Tn3 e à jusante por uma ISKpn6 truncada. As análises das sequências de DNA das demais cepas evidenciaram a presença de sequências gênicas, que traduzidas, apresentavam similaridade para sete metalobetalactamases (MBL), indicando a potencial presença de novos genes de carbapenemases. Foi caracterizada pela primeira vez no Brasil cepa produtora da carbapenemase GES-20 e o novo ST3187. Foram detectados potenciais novos genes de carbapenemases. O gene blaKPC-2 no isolado J5083553 tem provável localização plasmidial

Pseudomonas aeruginosa is a major agent of healthcare-related infections worldwide. Empirical treatment of serious infections caused by this species usually includes carbapenems. This therapy, if inadequate, is related to a significant increase in mortality. The main mechanisms of carbapenem resistance in Gram-negative bacteria are the reduction of outer membrane permeability, overexpression of efflux pumps and beta-lactamase production, the latter being the most efficient. The aim of this work was the characterization of new or emerging carbapenemases and their genetic context in P. aeruginosa. Eleven P. aeruginosa isolates capable of hydrolyzing imipenem in spectrophotometric assay and negative for known carbapenemases genes were used, as well as a KPC-2 producing strain. These isolates were subjected to confirmation of carbapenem resistance profile by disk diffusion, broth microdilution (MIC) and enzyme inhibition by EDTA and Blue-Carba methods. Plasmid profile was evaluated using alkaline lysis and pulsed field electrophoresis (PFGE) methods. Genomes were sequenced using the MiSeq system. The clonal profile of the isolates was evaluated by PFGE and Multilocus Sequence Typing (MLST). Ten isolates presented negative Blue-Carba, compatible with the presence of weak carbapenemases. Four isolates presented plasmids visualized on agarose gel after PFGE. The plasmids of isolate D5303023 were electroporated into E. coli TOP 10 and selected in LB medium containing 1 µg/mL imipenem, however no transformants were obtained. The PFGE analysis identified 7 distinct clonal groups. As for MLST typing, a new type ST3187 was detected and the ST277 and ST1560 were the predominant types. The genome of the KPC-2 positive strain was digested with S1 nuclease and subjected to PFGE, evidencing a plasmid of approximately 453 kb. Isolate D9203039 presented a GES-20 carbapenemase associated with a GES-19 extended spectrum beta-lactamase. The genes encoding them were located in the In724 integron. This isolate belonged to ST309, a potential high risk clone, associated with the multidrug resistant strain in Mexico. After sequencing it was confirmed that the strain belongs to ST446 and it was observed the presence of the blaKPC-2 gene in a contig of 128,487 bp. This contig was 99.9% similar to plasmid 1 of the P. aeruginosa RW103 strain. However, bacterial conjugation assays failed to obtain transconjugant colonies. The blaKPC-2 gene was found flanked upstream by a truncated structure of a Tn3 family transposon and downstream by a truncated ISKpn6. DNA sequence analysis of the other strains showed the presence of gene sequences, which translated, showed similarity to seven metallo-beta-lactamases (MBL), indicating the potential presence of new carbapenemase genes. It was characterized for the first time in Brazil carbapenemase producing strain GES-20 and the new ST3187. Potential new carbapenemase genes were detected. The blaKPC-2 gene in isolate J5083553 has plasmid localization

Modelo predictivo multiparamétrico del estatus axilar en pacientes con cáncer de mama: carga tumoral total y perfil molecular. Estudio multicéntrico / A multiparametric predictive model of axillary status in patients with breast cancer: total tumoral load and molecular signature. A multicenter study

Objetivo. El objetivo de nuestro estudio es evaluar el impacto combinado de cada uno de los perfiles moleculares del cáncer de mama, subrogados inmunohistoquímicamente junto con la carga tumoral total del ganglio centinela como predictores de afectación metastásica en los ganglios axilares no centinela. Material y métodos. Se incluyeron 373 pacientes de carcinoma infiltrante de mama con ganglio centinela metastásico y linfadenectomía axilar, procedentes de seis hospitales españoles. Se aplicaron los criterios de ST Gallen para definir el perfil molecular. Se realizó un análisis multivariante para definir diferentes modelos predictivos y se estudiaron las distribuciones de densidad de probabilidad de la carga tumoral para cada perfil molecular en los casos con axila metastásica y no metastásica en los ganglios no centinela. Resultados. Hubo un 66% de linfadenectomías axilares metastásicas. Se obtuvieron 7 modelos predictivos cuyas áreas bajo la curva ROC oscilaron entre 0,65 y 0,77. El mejor modelo fue el basado en la carga tumoral total, tipo histológico, diámetro tumoral, grado, invasión linfovascular, perfil molecular y número total de ganglios centinela. Las mayores diferencias de densidad de probabilidad de la carga tumoral total se producen entre las distribuciones de casos positivos y negativos de los perfiles moleculares BH, TN y HER2. Conclusión. La inclusión del perfil molecular en el modelo mejora el área bajo la curva ROC, especialmente si se incluye también el número total de cganglios centinela. Se observan diferencias entre los distintos perfiles moleculares para el valor predictivo de la carga tumoral total (AU)

Objective. To evaluate the combined impact of each of the immunohistochemically surrogated molecular signatures (PM) of breast cancer subtype along with the total tumor load (CTT) of the sentinel node (SN) as a predictor of non-SN metastatic involvement. Methods. We included 373 patients diagnosed with infiltrating breast cancer with metastatic SN who underwent subsequent axillary lymph node dissection (ALND) from six hospitals. The surrogate MS for each case was defined as per ST Gallen definitions. A multivariate analysis was conducted to estimate the predictive model and normal kernel functions to fit the density distributions of the total tumoral load for each molecular signatures. Results. Metastatic involvement of the axillary lymph node was identified in 66% of the patients. We obtained seven different predictive models with an area under curve (AUC) ranging from 0.65 to 0.77. The best model was based on the CTT, histological type, tumor size, stage, lymphatic invasion, MS, and the total number of SN. The greatest differences in the density functions of the CTT were found in the PM for positive and negative cases of the BH, TN and HER2 subtypes. Conclusions. The inclusion of PM in the multivariate model improved the AUC, especially when the total number of sentinel nodes were included. Differences were observed in the impact of the CTT among the different smolecular profiles subtypes (AU)

Cirugía reductora de riesgo en el cáncer de mama / Risk-reducing surgery in breast cancer

Objetivo. Revisión de la bibliografía sobre la mastectomía profiláctica contralateral a nivel de riesgos y costes, así como su probable efecto en la supervivencia. Material y método. Se realizó una estrategia de búsqueda bibliográfica de mastectomía profiláctica con los siguientes descriptores: cirugía reductora de riesgo, cáncer de mama, incidencia, complicaciones, supervivencia libre de enfermedad, supervivencia global, mortalidad por cáncer de mama y costes. Resultados. Existe una tendencia general al aumento de la mastectomía profiláctica como cirugía reductora de riesgo contralateral en el cáncer de mama. Los principales factores que influyen en este proceso son: edad joven, raza blanca, estadio inicial de la enfermedad, expresión de receptores hormonales, uso de la RMN, nivel educacional alto, acceso a la reconstrucción, historia familiar de cáncer de mama y/o susceptibilidad genética para la mutación BRCA 1 y 2. Esta cirugía puede presentar comorbilidades que pueden dar lugar a reintervenciones y/o retraso del tratamiento adyuvante, además de la probabilidad de un impacto psicosocial al influir en variables como la imagen corporal y la sexualidad. La mastectomía profiláctica podría reducir el riesgo de cáncer de mama contralateral favoreciendo la simetría pero sin beneficios evidentes en la supervivencia. Las mejoras en el tratamiento adyuvante han favorecido la supervivencia de este tipo de pacientes con una tendencia a una menor proporción de cánceres de mama contralaterales. En pacientes portadoras de mutación BRCA 1/2 la mastectomía profiláctica contralateral ha resultado coste-efectiva. Conclusiones. La mastectomía profiláctica parece reducir el riesgo de aparición de cáncer de mama contralateral pero no está exenta de complicaciones. Es necesario un proceso de toma de decisiones donde se facilite una información realista de la cirugía, de la enfermedad individual de la paciente y de la posibilidad de un consejo genético (AU)

Objective. To conduct a literature review of contralateral prophylactic mastectomy in unilateral breast cancer as risk reducing surgery and its probable effect on survival. Material and method. We performed a systematic search of the literature on prophylactic mastectomy by using the following keywords risk reducing surgery, breast cancer, incidence, complications, disease-free survival, overall survival, mortality due to breast cancer, and costs. Results. There is a general trend to increased use of contralateral prophylactic mastectomy to reduce the risk of contralateral breast cancer. The main factors influencing this process are young age, Caucasian race, early-stage breast cancer, estrogen receptor positivity, the use of magnetic resonance imaging at diagnosis, high educational level, access to immediate breast reconstruction, and family history of breast cancer and/or genetic testing for the BRCA 1 and BRCA 2 mutations. This surgery may have complications, which can lead to reoperation and/or delay adjuvant therapy and may also have a psychosocial impact, influencing body image and sexuality. Mastectomy may reduce the risk of contralateral breast cancer, favoring symmetry, but with no apparent survival benefit. Improvements in adjuvant treatments have improved survival in these patients with a lower proportion of contralateral breast cancers. Contralateral prophylactic mastectomy is clearly cost-effective in patients carrying the BRCA 1/2 mutation. Conclusion. Prophylactic mastectomy appears to reduce the risk of contralateral breast cancer, although this procedure should be reconsidered because it is not free of complications. Realistic information on the intervention and the disease, as well as genetic counseling, are required for proper decision-making (AU)

The genes and the environment in nasal allergy.

PURPOSE OF REVIEW: In this review, we summarize the latest publications on the genetic and environmental determinants of allergic rhinitis. RECENT FINDINGS: Recent advances in genetic technology and bioinformatics have enabled simultaneous unbiased analysis of the entire genome regarding DNA sequence variants, epigenetic modifications and gene expression, providing functional correlates for DNA variants and phenotypes. As a result, new genes of mitochondrial and B-lymphocyte metabolism have been associated with allergic rhinitis phenotypes. Epidemiological studies recently showed an increased risk to develop allergic rhinitis in all age groups with reduction in farm exposure and in children with few older siblings. Climate changes seem to have also influenced pollen exposure and pollen-induced allergic disease. Lastly, occupational rhinitis has been increasingly recognized as a large burden to society. SUMMARY: In summary, new high throughput genetics research technologies have pointed to new previously unsuspected pathways that may modulate the risk of developing allergic rhinitis such as mitochondrial metabolism. In addition, recent environmental factors found to influence the risk of developing allergic rhinitis include exposure to farm, pollution, occupational agents, and changes in climate.

Síndrome Schinzel-Giedion: nueva mutación en SETBP1 / Schinzel-Giedion syndrome: A new mutation in SETBP1

El síndrome Schinzel-Giedion (SSG) (#MIM 269150) es una enfermedad genética infrecuente, caracterizada por dismorfia cráneo-facial específica, anomalías congénitas múltiples y discapacidad intelectual grave. La mayoría de los pacientes fallece en los primeros años de vida. Se debe a mutaciones en el gen SETBP1, habiéndose descrito a la fecha un reducido número de pacientes con confirmación molecular. Presentamos a un paciente de 4 años con SSG asociado a la mutación c.2608G>T (p.Gly870Cys) en el gen SETBP1, no descrita previamente. Se revisan las características clínicas de esta enfermedad y su diagnóstico diferencial. Los rasgos dismórficos son muy característicos en el SSG. Su reconocimiento clínico es fundamental para alcanzar un diagnóstico precoz, planificar un correcto seguimiento y ofrecer asesoramiento genético familiar adecuado. A la fecha, este es el decimoséptimo paciente ublicado con mutación en el gen SETBP1, primero en España, contribuyendo a ampliar el conocimiento clínico y molecular de esta entidad

Schinzel-Giedion syndrome (SGS) (#MIM 269150) is a rare genetic disorder characterized by very marked craniofacial dysmorphism, multiple congenital anomalies and severe intellectual disability. Most affected patients die in early childhood. SETBP1 was identified as the causative gene, but a limited number of patients with molecular confirmation have been reported to date. The case is reported of a 4 and a half year-old male patient, affected by SGS. SETBP1 sequencing analysis revealed the presence of a non-previously described mutation: c.2608G>T (p.Gly870Cys). The clinical features and differential diagnosis of this rare condition are reviewed. Dysmorphic features are strongly suggestive of SGS. Its clinical recognition is essential to enable an early diagnosis, a proper follow-up, and to provide the family with genetic counseling. To date, this is the seventeenth SGS patient published with SETBP1 mutation, and the first in Spain, helping to widen clinical and molecular knowledge of the disease

Immune-related functions of the Hivep gene family in East African cichlid fishes.

Immune-related genes are often characterized by adaptive protein evolution. Selection on immune genes can be particularly strong when hosts encounter novel parasites, for instance, after the colonization of a new habitat or upon the exploitation of vacant ecological niches in an adaptive radiation. We examined a set of new candidate immune genes in East African cichlid fishes. More specifically, we studied the signatures of selection in five paralogs of the human immunodeficiency virus type I enhancer-binding protein (Hivep) gene family, tested their involvement in the immune defense, and related our results to explosive speciation and adaptive radiation events in cichlids. We found signatures of long-term positive selection in four Hivep paralogs and lineage-specific positive selection in Hivep3b in two radiating cichlid lineages. Exposure of the cichlid Astatotilapia burtoni to a vaccination with Vibrio anguillarum bacteria resulted in a positive correlation between immune response parameters and expression levels of three Hivep loci. This work provides the first evidence for a role of Hivep paralogs in teleost immune defense and links the signatures of positive selection to host-pathogen interactions within an adaptive radiation.

Sequence polymorphism and expression variability of Crassostrea gigas immune related genes discriminate two oyster lines contrasted in term of resistance to summer mortalities.

Summer mortalities of Crassostreagigas are a major concern in oyster aquaculture. They are the result of a complex interaction between the host, pathogens and environmental factors. Oyster genetics have been identified as an essential determinant of oyster susceptibility to summer mortalities. As the capability of oysters to circumvent diseases depends in part on their immune defenses, we aimed to analyze the gene expression and sequence polymorphism of 42 immune related genes in two oyster lines selected for their "High" (H) and "Low" (L) survival to summer mortalities. Results showed that the variability of gene expression and the sequence polymorphism acting on particular genes could enable the discrimination between H and L oyster lines. Besides, a higher sequence polymorphism was observed on the L line affecting 11 of the 42 analyzed genes. By analyzing gene expression, sequence polymorphism and gene copy number of two antimicrobial peptide families (Cg-Defs and Cg-Prp), and an antimicrobial protein (Cg-BPI) on individual oysters, we showed that gene expression and/or sequence polymorphism could also discriminate H and L oyster lines. Finally, we observed a positive correlation between the gene expression and the gene copy number of antimicrobials and that sequence polymorphism could be encoded in the genome. Overall, this study gives new insights in the relationship between oyster immunity and divergent phenotypes, and discusses the potential implication of antimicrobial diversity in oyster survival to summer mortalities.

Polymorphic genetic variation in immune system genes: a study of two populations of Espirito Santo, Brazil.

Mapping single nucleotide polymorphisms (SNPs) in genes potentially involved in immune responses may help understand the pathophysiology of infectious diseases in specific geographical regions. In this context, we have aimed to analyze the frequency of immunogenetic markers, focusing on genes CD209 (SNP -336A/G), FCγRIIa (SNP -131H/R), TNF-α (SNP -308A/G) and VDR (SNP Taq I) in two populations of the Espirito Santo State (ES), Brazil: general and Pomeranian populations. Peripheral blood genomic DNA was extracted from one hundred healthy individuals of the general population and from 59 Pomeranians. Polymorphic variant identification was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). SNP genotype frequencies were in Hardy-Weinberg Equilibrium. There was no statistically significant difference in allelic and genotypic distributions between the two populations studied. Statistically significant differences were observed for SNP genotype distribution in genes CD209, TNF-α and VDR when comparing the ES populations with other Brazilian populations. This is the first report of CD209, FcγRIIa, TNF-α and VDR allelic frequencies for the general and Pomeranian populations of ES.

Immune markers and differential signaling networks in ulcerative colitis and Crohn's disease.

BACKGROUND: Cytokine signaling pathways play a central role in the pathogenesis of inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn's disease (CD) have unique as well as overlapping phenotypes, susceptibility genes, and gene expression profiles. This study aimed to delineate patterns within cytokine signaling pathways in colonic mucosa of UC and CD patients, explore molecular diagnostic markers, and identify novel immune mediators in IBD pathogenesis. METHODS: We quantified 70 selected immune genes that are important in IBD signaling from formalin-fixed, paraffin-embedded (FFPE) colon biopsy samples from normal control subjects and UC and CD patients having either severe colitis or quiescent disease (n = 98 subjects). We utilized and validated a new modified real-time reverse-transcription polymerase chain reaction (RT-PCR) technique for gene quantification. RESULTS: Expression levels of signaling molecules including IL-6/10/12/13/17/23/33, STAT1/3/6, T-bet, GATA3, Foxp3, SOCS1/3, and downstream inflammatory mediators such as chemokines CCL-2/11/17/20, oxidative stress inducers, proteases, and mucosal genes were differentially regulated between UC and CD and between active and quiescent disease. We also document the possible role of novel genes in IBD, including SHP-1, IRF-1,TARC, Eotaxin, NOX2, arginase I, and ADAM 8. CONCLUSIONS: This comprehensive approach to quantifying gene expression provides insights into the pathogenesis of IBD by elucidating distinct immune signaling networks in CD and UC. Furthermore, this is the first study demonstrating that gene expression profiling in FFPE colon biopsies might be a practical and effective tool in the diagnosis and prognosis of IBD and may help identify molecular markers that can predict and monitor response to individualized therapeutic treatments.

Identification of differentially expressed protective genes in liver of two rainbow trout strains.

Since 1975, the rainbow trout strain BORN (Germany) has been bred in brackish water from a coastal form imported from Denmark. Accompanying phenotypic monitoring of the adapted BORN trout until now revealed that this selection strain manifested a generally elevated resistance towards high stress and pathogenic challenge including lower susceptibility towards Aeromonas salmonicida infections in comparison to other trout strains in local aqua farms. We focus on the elucidation of both, genetic background and immunological basis for the increased survivorship to infections. A first comparison of gene expression profiles in liver tissue of healthy rainbow trout from the local selection strain BORN and imported trout using a GRASP 16K cDNA microarray revealed six differentially expressed genes evoking pathogen and wounding responses, LEAP2A (encoding for liver-expressed antimicrobial peptide), SERPINA1 (alpha-1 antitrypsin), FTH1 (middle subunit of ferritin), FGL2 (fibroleukin), CLEC4E (macrophage-inducible C-type lectin), and SERPINF2 (alpha-2 antiplasmin). Since the latter gene is not described in salmonid species so far, our first aim was to characterize the respective sequence in rainbow trout. Two trout SERPINF2 genes were identified, which share only 48% identical amino acid residues and a characteristic SERPIN domain. Second, we aimed to analyse the expression of those genes after temperature challenge (8 °C and 23 °C). Only FTH1 was upregulated in BORN and import trout after increase of temperature, while SERPINA1 and FGL2 were only elevated in import trout. Third, the expression of all named genes was analyzed after pathogen challenge with A. salmonicida subsp. salmonicida. As a main finding, we detected a comparably faster regeneration of LEAP2A mRNA abundance in BORN trout following bacterial infection. Ingenuity Pathways Analysis suggested a functional interplay among the mentioned factors and the pro-inflammatory cytokine TNF, whose stronger expression was validated in liver of BORN trout. This data indicate that the examined genes contribute to an improved first barrier against invading pathogens in BORN trout.

Global gene expression analysis of chicken caecal response to Campylobacter jejuni.

Campylobacter jejuni colonises the caecum of more than 90% of commercial chickens. Even though colonisation is asymptomatic, we hypothesised that it is mediated by activation of several biological pathways. We therefore used chicken-specific 20K oligonucleotide microarrays to examine global gene expression in C. jejuni-challenged birds. Microarray results demonstrate small but significant fold-changes in expression of 270 genes 20 h post-challenge, corresponding to a wide range of biological processes including cell growth, nutrient metabolism and immunological activity. Expression of NOX1 (2.3-fold) and VCAM1 (1.5-fold) were significantly increased in colonised birds (P<0.05), indicating oxidative burst and endothelial cell activation, respectively. Microarray results, supplemented by qRT-PCR analyses demonstrated increased TOPK (1.9-fold), IL17 (3.6-fold), IL21 (2.1-fold), IL7R (4-fold) and CTLA4 (2.5-fold) gene expression (P<0.05), which was suggestive of T cell mediated activity. Combined these results suggest that C. jejuni has nominal effects on global caecal gene expression in the chicken but significant changes detected are suggestive of a protective intestinal T cell response.

Molecular cloning, characterization and expression analysis of the complement component C6 gene in grass carp.

The complement system, as a representative of innate immunity, plays a key role in the host defense against infections. C6 is the member of complement components creating the membrane attack complex (MAC). In this study, we cloned and characterized the grass carp complement component C6 (gcC6) gene. Our data showed that gcC6 gene contained a 2724bp open reading frame (ORF), a 237bp 5'-untranslated region (UTR) and a 219bp 3'-UTR. The deduced amino acid sequence of gcC6 showed 77.6% and 58.9% identity to zebrafish C6 and rainbow trout C6, respectively. GcC6 gene was expressed in a wide range of grass carp tissues, and the highest expression level of gcC6 was detected in the spleen and liver. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in muscle, trunk kidney, liver, head kidney, spleen, heart and intestine, whereas it was down-regulated in the brain and skin. The expression level of gcC6 was high at the unfertilized egg stage. It was significantly increased at 1 day post-hatching, but it was decreased at 10 days post-hatching. This result suggested that the complement C6 transcripts in early embryos were of maternal origin.

Induction of interferon-stimulated genes by Chlamydia pneumoniae in fibroblasts is mediated by intracellular nucleotide-sensing receptors.

BACKGROUND: Recognition of microorganisms by the innate immune system is mediated by pattern recognition receptors, including Toll-like receptors and cytoplasmic RIG-I-like receptors. Chlamydia, which include several human pathogenic species, are obligate intracellular gram-negative bacteria that replicate in cytoplasmic vacuoles. The infection triggers a host response contributing to both bacterial clearance and tissue damage. For instance, type I interferons (IFN)s have been demonstrated to exacerbate the course of Chlamydial lung infections in mice. METHODS/PRINCIPAL FINDINGS: Here we show that Chlamydia pneumoniae induces expression of IFN-stimulated genes (ISG)s dependent on recognition by nucleotide-sensing Toll-like receptors and RIG-I-like receptors, localized in endosomes and the cytoplasm, respectively. The ISG response was induced with a delayed kinetics, compared to virus infections, and was dependent on bacterial replication and the bacterial type III secretion system (T3SS). CONCLUSIONS/SIGNIFICANCE: Activation of the IFN response during C. pneumoniae infection is mediated by intracellular nucleotide-sensing PRRs, which operate through a mechanism dependent on the bacterial T3SS. Strategies to inhibit the chlamydial T3SS may be used to limit the detrimental effects of the type I IFN system in the host response to Chlamydia infection.

Expression of immune-related genes in one phase of embryonic development of freshwater crayfish, Pacifastacus leniusculus.

Crayfish do not have larval stage as other crustacean such as penaeid shrimp they spawn their eggs until hatching and what hatches out from the eggs are miniature crayfish known as juveniles. In order to address the question whether immune genes are initially expressed during the embryo development in the egg stage, the expression of some immune-related genes: prophenoloxidase (proPO), peroxinectin, hemocyanin, anti-lipopolysaccharide factor (ALF), plcrustin, astakine-1, 2 and transglutaminase (TGase) were determined in the middle phase of crayfish embryo development. Furthermore, immune challenge was used to determine the immune response of eggs by immersing them in a solution of the highly pathogenic bacterium Aeromonas hydrophila. Semi-quantitative RT-PCR analysis showed that all tested genes are present except proPO in this phase of crayfish embryo development and none of the genes tested changed their expression following immersion in A. hydrophila. The proPO transcript has been reported from hemocytes in crustaceans and it plays crucial roles in crustacean immune response. This may indicate that the development of immune-competent hemocytes in this stage of crayfish embryo is not completed and the egg shell as such plays an important role as a shield in protecting the embryo from bacteria and maybe also other pathogens.

Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection.

Edwardsiella tarda is an important Gram-negative bacterium that causes systemic infections in a wide range of hosts including fish. The pathogenic mechanisms in this disease are still poorly understood in fish. Indian major carp, Labeo rohita were intraperitoneally challenged with a pathogenic isolate of E. tarda to measure sequential changes in immunity level. A significant decrease in the superoxide production, myeloperoxidase, alternative complement activity, total protein levels and antiprotease activity of serum was marked in the infected fish. However, the serum lysozyme activity and haemagglutination titre were raised in the infected fish. Similarly, a significant rise in specific antibody titre was noticed on and after 10 days post-challenge. This study also elucidates the changes in the relative expression of some immune-related genes viz., interleukin 1-beta (IL-1beta), inducible nitric oxide synthase (iNOS), complement component C3, beta(2)-microglobulin, CXCa, tumor necrosis factor-alpha (TNFalpha), and C-type and G-type lysozymes during the infection. Significant up-regulation of IL-1beta, iNOS, C3, CXCa and expression of both types of lysozyme genes was noticed at 6-12 h post-challenge (h.p.c.) whereas down-regulation of beta(2)-microglobulin and TNFalpha genes was observed after 48 h p.c. The results obtained here strengthen the understanding on molecular pathogenesis of edwardsiellosis in L. rohita.