ABSTRACT
STUDY QUESTION: Which transcriptomic alterations in mid-luteal endometrial scratch biopsies, taken prior to the assisted reproductive treatment (ART) treatment cycle are associated with unsuccessful pregnancy? SUMMARY ANSWER: Dysregulated interleukin-17 (IL-17) pathway components are demonstrated in women who fail to become pregnant after ART. WHAT IS KNOWN ALREADY: Implantation failure is now recognised as a critical factor in unexplained infertility and may be an important component of failed ART. STUDY DESIGN, SIZE, DURATION: Using a prospective longitudinal study design, 29 nulliparous women with unexplained infertility undergoing ART were recruited between October 2016 and February 2018. Mid-luteal stage endometrium and matched serum samples were collected, and patients underwent a single embryo transfer in the subsequent cycle. RNA-seq analysis of endometrial biopsies was performed on the discovery cohort (n = 20). PARTICIPANTS/MATERIALS, SETTING, METHODS: Gene set enrichment analysis of the differentially expressed genes (DEGs) was performed. Endometrium and serum were then prepared for IL-17A analysis by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: There were 204 differentially expressed protein-coding genes identified in tissue from women who became pregnant (n = 9) compared with tissue from women who failed to become pregnant (n = 11) (false discovery rate; P < 0.05). Of the 204 DEGs, 166 were decreased while 38 were increased in the pregnant compared to the non-pregnant groups. Gene set enrichment analysis of the DEGs identified an over-representation of IL-17 and Pl3K-Akt signalling pathways. All the DEGs within the IL-17 signalling pathway (MMP3, MMP1, IL1ß, LCN2, S100A9 and FOSL1) demonstrated decreased expression in the pregnant group. Serum IL-17 protein levels were increased in the non-pregnant discovery cohort (n = 11) and these findings were confirmed a validation cohort (n = 9). LIMITATIONS, REASONS FOR CAUTION: Limitations of our study include the cohort size and the lack of aneuploidy data for the embryos; however, all embryos transferred were single good or top-quality blastocysts. WIDER IMPLICATIONS OF THE FINDINGS: These findings demonstrate dysregulated IL-17 pathway components in women who fail to become pregnant after ART. Elevated serum levels of the pro-inflammatory cytokine IL-17 may predict failure of ART in women with unexplained infertility. Future trials of anti-IL-17 therapies in this cohort warrant further investigation. STUDY FUNDING/COMPETING INTEREST(S): Funding from the UCD Wellcome Institutional Strategic Support Fund, which was financed jointly by University College Dublin and the SFI-HRB-Wellcome Biomedical Research Partnership (ref 204844/Z/16/Z), is acknowledged. The authors have no competing interests. TRIAL REGISTRATION NUMBER: NA.
Subject(s)
Infertility , Interleukin-17 , Endometrium , Female , Humans , Interleukin-17/genetics , Longitudinal Studies , Pregnancy , Pregnancy Outcome , Prospective Studies , Reproductive Techniques, AssistedABSTRACT
BACKGROUND: ß-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. ß-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the ß-defensin gene repertoire in sheep using the bovine genome as the primary reference. RESULTS: All 57 currently predicted bovine ß-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (>70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the ß-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 ß-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 ß-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential ß-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species. CONCLUSIONS: This is the first comprehensive analysis of ß-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of ß-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.
Subject(s)
Sheep, Domestic/genetics , beta-Defensins/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , Gene Expression , Male , Multigene Family , Phylogeny , Sequence Analysis, DNA , Testis/metabolism , beta-Defensins/chemistry , beta-Defensins/metabolismABSTRACT
Natural killer (NK) cells are highly heterogeneous innate lymphocytes with a diverse repertoire of phenotypes and functions. Their role in organ transplantation has been poorly defined due to conflicting clinical and experimental data. There is evidence that NK cells can contribute to graft rejection and also to tolerance induction. In most solid organ transplantation settings, the role of NK cells is only considered from the perspective of the recipient immune system. In contrast to other organs, the liver contains major resident populations of immune cells, particularly enriched with innate lymphocytes such as NK cells, NKT cells, and gamma-delta T cells. Liver transplantation therefore results in a unique meeting of donor and recipient immune systems. The unusual immune repertoire and tolerogenic environment of the liver may explain why this potentially inflammatory "meeting" often results in attenuated immune responses and reduced requirement for immunosuppression. Recent trials of immunosuppression withdrawal in liver transplant patients have identified NK cell features as possible predictors of tolerance. Here we propose that hepatic NK cells play a key role in the induction of tolerance post-liver transplant and examine potential mechanisms by which these cells influence liver transplant outcome.
Subject(s)
Adaptive Immunity/immunology , Graft Rejection/immunology , Immune Tolerance/immunology , Killer Cells, Natural/immunology , Liver Transplantation , Animals , HumansABSTRACT
Recent analysis of the bovine genome revealed an expanded suite of ß-defensin genes that encode what are referred to as antimicrobial or host defense peptides (HDPs). Whereas primate genomes also encode α- and θ-defensins, the bovine genome contains only the ß-defensin subfamily of HDPs. ß-Defensins perform diverse functions that are critical to protection against pathogens but also in regulation of the immune response and reproduction. As the most comprehensively studied subclass of HDPs, ß-defensins possess the widest taxonomic distribution, found in invertebrates as well as plants, indicating an ancient point of origin. Cross-species comparison of the genomic arrangement of ß-defensin gene repertoire revealed them to vary in number among species presumably due to differences in pathogenic selective pressures but also genetic drift. ß-Defensin genes exist in a single cluster in birds, but four gene clusters exist in dog, rat, mouse, and cow. In humans and chimpanzees, one of these clusters is split in two as a result of a primate-specific pericentric inversion producing five gene clusters. A cluster of ß-defensin genes on bovine chromosome 13 has been recently characterized, and full genome sequencing has identified extensive gene copy number variation on chromosome 27. As a result, cattle have the most diverse repertoire of ß-defensin genes so far identified, where four clusters contain at least 57 genes. This expansion of ß-defensin HDPs may hold significant potential for combating infectious diseases and provides opportunities to harness their immunological and reproductive functions in commercial cattle populations.
Subject(s)
Cattle/genetics , Health , Multigene Family , beta-Defensins/genetics , Animals , Anti-Infective Agents/metabolism , Evolution, Molecular , Immunomodulation/genetics , beta-Defensins/metabolismABSTRACT
ß-defensins are effector molecules of the innate immune system, found in many diverse species. Their presence in invertebrates as well as vertebrates suggests highly conserved functional roles. Most ß-defensins are believed to act as antimicrobial agents at epithelial surfaces, although additional functions have also been described, including immune regulatory activity, wound repair and a role in coat-colour determination. High expression of ß-defensins have been found in testis and epididymidal epithelium as well as in the seminal fluid of humans, macaque, rat, mouse and cow. Human and macaque ß-defensins have recently been shown to affect sperm motility while a mutation in ß-defensin 126 is associated with reduced fertility in men. Genetic variation in bovine defensin genes may explain the increased incidence of low fertility in cattle. Here, we present a summary of the known functions of ß-defensins as well as their emerging role in reproduction and their potential to improve fertility in cattle.
Subject(s)
Immunity, Innate/genetics , Reproduction , beta-Defensins/physiology , Amino Acid Sequence , Animals , Cattle , Genitalia, Male/immunology , Genitalia, Male/metabolism , Humans , Macaca , Male , Mice , Molecular Sequence Data , Rats , Reproduction/genetics , Reproduction/immunology , Sequence Homology, Amino Acid , beta-Defensins/classificationABSTRACT
Epigenetic regulation of gene expression could help explain variation in responses to infection and differences in disease susceptibility in cattle. The aim of this study was to examine epigenetic mechanisms in the regulation of LPS-induced innate immune gene expression in peripheral blood mononuclear cells (PBMCs) from five healthy calves. Firstly, epigenetic enzyme gene expression (histone deacetylase (HDAC) and DNA methyltransferase (DNMT)) was measured after LPS stimulation. Secondly, the effect of the histone deacetylase inhibitor Trichostatin A (TSA) on histone H3 acetylation and on innate immune gene expression was also measured. Results showed differential expression of HDAC6, HDAC7 and DNMT3A genes in response to LPS in cells from all animals, while TSA significantly inhibited pro-inflammatory cytokine (TNF, IL2 and IFNG) expression (P<0.05), presumably by histone acetylation. These results suggest an important role for the HDAC family of enzymes in the regulation of bovine innate immune gene expression.
Subject(s)
Cattle , Epigenesis, Genetic/immunology , Hydroxamic Acids/pharmacology , Immunity, Innate , Leukocytes, Mononuclear/drug effects , Lipopolysaccharides/toxicity , Animals , Cell Survival , Computational Biology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Leukocytes, Mononuclear/metabolism , PhylogenyABSTRACT
BACKGROUND: The response to the treatment with pegylated interferon (PEG IFN)-α combined with ribavirin in chronic hepatitis C virus (HCV) infection varies with some patients having a rapid or early response which is not sustained. AIMS: To investigate the rates of rapid virological response (RVR), early virological response (EVR) and sustained virological response (SVR) in an Irish cohort of HCV infected patients receiving IFN-α/ribavirin. METHODS: Rates of RVR, EVR and SVR were examined in 123 patients undergoing standard treatment for chronic HCV infection between 2001 and 2007 at a Dublin Teaching Hospital. RESULTS: The rates of RVR, EVR and SVR in genotype 1 patients were 48, 68 and 50%, while in genotype 2/3 patients they were 87, 93 and 87%, respectively. The positive predictive values (PPV) of RVR for SVR in genotype 1 and genotype 2/3 patients were 90 and 92.4%, respectively. CONCLUSION: The rates of response to PEG IFN-α/ribavirin in Irish patients are consistent with other international reports. We support the regular monitoring of rapid and early virological response as a standard of care in treating chronic hepatitis C patients.
Subject(s)
Antiviral Agents/therapeutic use , Hepacivirus , Hepatitis C, Chronic/drug therapy , Interferon-alpha/therapeutic use , Polyethylene Glycols/therapeutic use , Ribavirin/therapeutic use , Adult , Drug Therapy, Combination , Female , Hepacivirus/genetics , Hepatitis C, Chronic/blood , Hepatitis C, Chronic/virology , Humans , Interferon alpha-2 , Ireland , Male , Middle Aged , Predictive Value of Tests , RNA, Viral/blood , Recombinant Proteins/therapeutic use , Viral LoadABSTRACT
Dendritic cells (DCs) are likely to play a key role in the compromised T-cell function associated with hepatitis C Virus (HCV) infection. However, studies of DC function in HCV-infected patients to date have yielded conflicting findings possibly because of patient and virus heterogeneity. Here, we report the characterization of monocyte-derived DCs obtained from a homogenous cohort of women who were infected with HCV genotype 1b following exposure to contaminated anti-D immunoglobulin from a single donor source. Patients included in the study had not received anti-viral therapy and all had mild liver disease. We show that phenotypically normal monocyte-derived dendritic cells (MDDCs) (CD11c(+) HLA(-) DR(+) CD1a(+) CD14(lo) ) can be obtained from these patients. These cells respond to both Poly(I:C) and LPS, by up-regulating expression of CD86. They secrete high levels of IL-8 and CCL5 in response to LPS, an indication that the MyD88-dependent and MyD88-independent signalling pathways downstream of TLR4 ligation are functioning normally. However, these cells are poor stimulators of T-cell proliferation in allogeneic mixed lymphocyte reactions. Furthermore, patient MDDCs fail to secrete IFN-α in response to poly(I:C) or IFN-ß stimulation. Altered DC function may contribute to impaired cellular immune responses and chronicity of disease following HCV infection in this cohort. An effective therapeutic vaccine for chronic HCV infection will most likely need to target DCs to elicit an appropriate cellular response; therefore, it is important to resolve how the DCs of different patient cohorts respond to stimulation via TLRs.
Subject(s)
Cell Proliferation , Dendritic Cells/immunology , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/pathology , Interferon-alpha/metabolism , T-Lymphocytes/immunology , Aged , Female , Humans , Immune Tolerance , Male , Middle AgedABSTRACT
Chronic hepatitis C virus (HCV) infection occurs in patients who fail to mount an effective T-cell response against the virus. One hypothesis for poor anti-viral immunity in these patients is that the virus impedes the immune response by disabling dendritic cells (DCs), cells that play a key role in pathogen recognition and initiation of adaptive immunity. Initial studies in the 1990s supported this hypothesis, as they clearly demonstrated that monocyte-derived DCs obtained from patients with chronic HCV infection displayed a reduced ability to stimulate lymphocyte proliferation. However, over the last 20 years, the situation has become more ambiguous. Many studies support the initial observation of a DC defect, while others using different patient cohorts or technologies have clearly demonstrated intact DC function in patients with chronic HCV. It is likely that the true situation lies somewhere in between. Just as there is a spectrum of disease in patients with chronic HCV, DCs obtained from different patients may display different properties. It is important to reconcile these divergent findings, as a clearer understanding of how the virus affects DC function will facilitate the development of immunotherapy and therapeutic vaccination strategies for patients with chronic HCV infection.
Subject(s)
Dendritic Cells/immunology , Hepacivirus/immunology , Hepatitis C, Chronic/immunology , Viral Proteins/immunology , Cell Line , Cell Proliferation , Cytokines/metabolism , Dendritic Cells/virology , Hepacivirus/pathogenicity , Hepatitis C, Chronic/virology , Humans , Immunity, Cellular , T-Lymphocytes/immunology , Toll-Like Receptors/immunologyABSTRACT
Several new automated methods have recently become available for high-throughput DNA extraction, including the Maxwell 16 System (Promega UK, Southampton, UK). The purpose of this report is to compare automated with manual DNA extraction methods, and invasive with noninvasive sample collection methods, in terms of DNA yield and quality. Milk, blood, and nasal swab samples were taken from 10 cows for DNA extraction. Nasal swabs were also taken from 10 calves and semen samples from 15 bulls for comparative purposes. The Performagene Livestock (DNA Genotek, Kanata, Ontario, Canada) method was compared with similar samples taken from the same animal using manual extraction methods. All samples were analyzed using both the Qubit Quantification Platform (Invitrogen Ltd., Paisley, UK) and NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) to accurately assess DNA quality and quantity. In general, the automated Maxwell 16 System performed best, consistently yielding high quantity and quality DNA across the sample range tested. Average yields of 28.7, 10.3, and 19.2 µg of DNA were obtained from 450 µL of blood, 400 µL of milk, and a single straw of semen, respectively. The quality of DNA obtained from buffy coat and from semen was significantly higher with the automated method than with the manual methods (260/280 ratio of 1.9 and 1.8, respectively). Centrifugation of whole blood facilitated the concentration of leukocytes in the buffy coat, which significantly increased DNA yield after manual extraction. The Performagene method also yielded 18.4 and 49.8 µg of high quality (260/280 ratio of 1.8) DNA from the cow and calf nasal samples, respectively. These results show the advantages of noninvasive sample collection and automated methods for high-throughput extraction and biobanking of high quality DNA.
Subject(s)
Cattle/genetics , DNA/isolation & purification , Genome , Animals , Automation/methods , Female , Male , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinaryABSTRACT
Psoriasis is one of the most common immune-mediated disorders. There is evidence that it is mediated by Th1 and, more recently, Th17 cells. The cytokine pattern, particularly the dominance of TNF-α, implicates the innate immune system in psoriasis pathogenesis. Of the many components of the innate immune system known to be involved in psoriatic lesions, natural killer and natural killer T cells appear to have a unique role. We review the evidence supporting a role for natural killer cells in psoriasis.
Subject(s)
Killer Cells, Natural/immunology , Psoriasis/immunology , Psoriasis/physiopathology , Humans , Immunity, Innate , Psoriasis/pathology , Skin/immunology , Skin/pathologyABSTRACT
BACKGROUND: Type 1 interferons upregulate oligoadenylate synthetase 1 (OAS1). A single nucleotide polymorphism (SNP) in exon 7 of OAS1 results in differential RNAseL enzyme activity, the A allele coding for a truncated form with low activity and the G conferring high activity. We hypothesized that OAS1 genotypes would influence both susceptibility to multiple sclerosis (MS) and disease activity with the AA genotype being overrepresented and the GG genotype underrepresented in relapsing-remitting MS (RRMS) with increased disease activity. METHODS: We examined OAS1 genotype distribution in 401 patients with MS, 394 healthy controls, and 178 patients with RRMS receiving interferon-beta (IFNbeta) assessed as 1) having no or minimal disease activity on IFNbeta, 2) having disease activity despite IFNbeta, and 3) 65 patients with RRMS with highly active disease. RESULTS: The OAS1 genotype distribution differed between patients with MS and controls (p = 0.000003), with lower frequency of GG homozygotes in patients with MS (6%) compared with controls (17%). In relation to disease severity, 34 (32%) patients with no or minimal disease activity on IFNbeta had the AA and 8 (8%) the GG genotype; of patients with disease activity despite IFNbeta, 27 (51%) were AA, while only 1 (2%) was GG (p = 0.03). Median time to first relapse on IFNbeta was 24 months in patients with RRMS with AA genotype and 33 months with AG or GG genotype (p = 0.04). The GG genotype was absent in 65 patients with highly active RRMS (p = 0.03). CONCLUSIONS: A functional OAS1 SNP, AA genotype, confers susceptibility to MS and the GG genotype may protect against increased disease activity.
Subject(s)
2',5'-Oligoadenylate Synthetase/genetics , Genetic Predisposition to Disease , Multiple Sclerosis, Relapsing-Remitting/genetics , Multiple Sclerosis/genetics , Polymorphism, Single Nucleotide , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cohort Studies , Female , Genotype , Humans , Immunologic Factors/therapeutic use , Interferon-beta/therapeutic use , Male , Middle Aged , Multiple Sclerosis/drug therapy , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Severity of Illness Index , Young AdultABSTRACT
Studies of postpartum endometrial physiologic and immune mechanisms in cows are compromised by the difficulty in acquiring tissue of suitable quality and in sufficient quantity (Bos taurus). Endometrial biopsy sampling has attracted concern regarding potential animal ill-health and perturbed subsequent fertility. Here, we describe a method of endometrial biopsy that obtains high-quality tissue samples and does not compromise fertility. Using a Hauptner instrument, endometrial biopsies were taken at 15, 30, and 60 d postpartum from 13 mixed-breed beef cows. The effects of repeat biopsy on health (heart rate, respiration rate, color of mucous membranes, rectal temperature), onset of estrous cyclicity, and first service conception rate were monitored. Extensive daily clinical examinations revealed no signs of ill-health. All cows had resumed estrous cyclicity at 60 d postpartum. A conception rate of 77% was achieved after estrus synchronization and artificial insemination. Each biopsy yielded intact endometrial tissue and nucleic acid suitable for extensive histologic and molecular analysis, respectively. We conclude that when carried out appropriately, bovine endometrial biopsy is a safe and reliable technique for assessing postpartum uterine function or health.
Subject(s)
Biopsy/veterinary , Cattle , Endometrium/pathology , Reproduction , Animals , Biopsy/adverse effects , Biopsy/methods , Endometrium/chemistry , Estrus Synchronization , Female , Insemination, Artificial/veterinary , Postpartum Period , RNA/analysis , Reverse Transcriptase Polymerase Chain Reaction/veterinaryABSTRACT
The chicken lysozyme gene encodes a hydrolase that has a key role in defence, especially in ovo. This gene was resequenced in global chicken populations [red, grey, Ceylon and green jungle fowl (JF)] and related bird species. Networks, summary statistics and tests of neutrality indicate that although there is extensive variation at the gene, little is present at coding sites, with the exception of one non-synonymous site. This segregating site and a further fixed non-synonymous change between red JF and domestic chicken populations are spatially close to the catalytic sites of the enzyme and so might affect its activity.
Subject(s)
Chickens/genetics , Muramidase/genetics , Animals , Chickens/metabolism , Genetics, Population , Models, Molecular , Muramidase/chemistry , Phylogeny , Polymorphism, Single NucleotideABSTRACT
Bovine tuberculosis (BTB), caused by Mycobacterium bovis, continues to pose a threat to livestock worldwide and, as a zoonotic infection, also has serious implications for human health. The implementation of comprehensive surveillance programmes to detect BTB has been successful in reducing the incidence of infection in many countries, yet BTB has remained recalcitrant to eradication in several EU states, particularly in Ireland and the UK. There are well-recognized limitations in the use of the current diagnostics to detect all infected animals and this has led to renewed efforts to uncover novel diagnostic biomarkers that may serve to enhance the performance of the tests. Studies of single immunological parameters have so far been unable to unlock the complexities of the immune response to mycobacterial infection. However, the development of high-throughput methods including pan-genomic gene expression technologies such as DNA microarrays has facilitated the simultaneous identification and analysis of thousands of genes and their interactions during the immune response. In addition, the application of these new genomic technologies to BTB has identified pathogen-associated immune response signatures of host infection. The objective of these investigations is to understand the changing profile of immune responses throughout the course of infection and to identify biomarkers for sensitive diagnosis, particularly during the early stages of infection. Transcriptional profiling via microarray and more recently via next-generation sequencing technologies may lead to the development of specific and sensitive diagnostics for M. bovis infection and will enhance the prospect of eradication of tuberculosis from cattle populations.
Subject(s)
Gene Expression Profiling , Mycobacterium bovis/genetics , Mycobacterium bovis/immunology , Tuberculosis, Bovine/genetics , Tuberculosis, Bovine/immunology , Animals , Antigens, Bacterial , Cattle , Gene Expression/immunology , Genetic Markers , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Leukocytes, Mononuclear/immunology , Tuberculosis, Bovine/diagnosis , Zoonoses/microbiologyABSTRACT
BACKGROUND: Natural killer (NK) cells may be impaired in patients with persistent hepatitis C virus (HCV) infection, but studies to date have yielded inconsistent findings due to patient and virus heterogeneity and difficulties obtaining appropriate controls. AIMS: To overcome these variables, we have examined numbers, phenotypes, cytotoxic activities and cytokine profiles of circulating NK cells from Irish women who acquired infection through administration of HCV genotype 1b-contaminated anti-D immunoglobulin from a single source and matched controls. RESULTS: Comparing 29 women who developed persistent infection with 21 who spontaneously resolved infection and 26 controls, we found that NK cell numbers were consistently lower in the persistently infected group (p = 0.02 and 0.002). This decrease was due to depletions of NK cells expressing low levels of CD56 (CD56(dim) NK cells; p = 0.004 and 0.0001), whilst CD56(bright) NK cells were expanded (p = 0.004 and 0.0001). Compared to HCV resolvers, CD56(dim) NK cells from persistently infected patients less frequently expressed CD16 and more frequently expressed NKG2A/C/E. These phenotypic changes did not significantly affect natural or interleukin-2-induced cytotoxicity by peripheral blood mononuclear cells against K562 and Daudi targets. Greater frequencies of CD56(bright) NK cells from chronic HCV patients produced interferon-gamma compared with HCV responders (p = 0.05) and controls (p = 0.0001) after phorbol ester stimulation in vitro. CONCLUSIONS: Alterations in NK subset distributions in chronic HCV infection may explain why previous reports of impaired NK cell functions were difficult to confirm. Altered NK cell functions may contribute to impaired cellular immune responses and chronicity of disease following HCV infection.
Subject(s)
Hepatitis C, Chronic/immunology , Killer Cells, Natural/immunology , Adult , Aged , CD56 Antigen/blood , Cytotoxicity, Immunologic , Female , Follow-Up Studies , Humans , Immunity, Cellular , Immunity, Innate , Immunophenotyping , Interferon-gamma/biosynthesis , Killer Cells, Natural/cytology , Middle Aged , T-Lymphocyte Subsets/immunologyABSTRACT
Intrahepatic thrombotic events have been postulated to play a key role in the pathogenesis of hepatic fibrosis. Genetic and acquired thrombotic risk factors may therefore contribute to the varying rates of fibrosis progression observed in patients with chronic hepatitis C virus (HCV) infection. The aim of this study was to assess the impact of inherited mutations in factor V and factor II (prothrombin) on hepatic fibrosis progression rates in individuals infected with HCV. Two hundred and ten Irish women infected with HCV genotype 1b, contracted from a single source (HCV-contaminated anti-D immunoglobulin) were genotyped for the factor V Leiden G1691A and prothrombin G20210A polymorphisms, and compared with Irish Caucasoid controls. Index and subsequent liver biopsies were scored (Ishak scoring system) by a single pathologist. Statistical analysis was performed using SPSS. Factor V Leiden and prothrombin G20210A heterozygosity were determined in 3.7% and 1.85%, respectively, of the study population. There was no association between these polymorphisms and fibrotic score on the index biopsy, or degree of change in fibrotic score on subsequent biopsies. The mean fibrotic score for factor V wild type was 1.06 vs 0.71 for the heterozygotes (P = 0.89). The mean change in fibrotic scores between subsequent biopsies was 0.72 for factor V wild type vs 0.50 for heterozygotes (P = 0.68). Similarly, there was no significant difference in fibrotic score for those with the prothrombin G20210A polymorphism (P = 0.936). Alanine aminotransferase levels for factor V wild type were significantly lower than those for the heterozygotes, 45.9 vs 57 (P = 0.032). Factor V Leiden and prothrombin G20210A heterozygosity rates were infrequently detected in this HCV cohort and were similar to rates seen in a Caucasian Irish control population. In this cohort, neither factor V Leiden nor prothrombin G20210A polymorphisms had a significant impact on fibrotic scores or degree of change between subsequent biopsies. These data do not support a key role for thrombotic risk factors in fibrogenesis in HCV-infected patients.
Subject(s)
Factor V/genetics , Hepacivirus , Hepatitis C, Chronic/blood , Hepatitis C, Chronic/genetics , Prothrombin/genetics , Thrombosis/genetics , Disease Progression , Female , Genetic Predisposition to Disease , Hepatitis C, Chronic/pathology , Humans , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Liver Cirrhosis/virologyABSTRACT
INTRODUCTION: Cigarette smoking is the strongest modifiable risk factor for developing thyroid-associated ophthalmopathy (TAO), and the severity of TAO is related to the current number of cigarettes smoked per day. We aimed to establish the effects of cigarette smoke extract (CSE) on an in vitro model of TAO. METHODS: Orbital tissue was taken during surgery from 10 patients with TAO and nine control subjects. Orbital fibroblasts were cultured and exposed to CSE, and intercellular adhesion molecule 1 (ICAM1) expression was measured by flow cytometry. Glycosaminoglycan production was measured by hyaluronic acid ELISA. Orbital fibroblasts were grown in adipogenic media with or without CSE and/or IL-1, and the degree of adipogenesis was quantified. RESULTS: Fibroblasts from patients with TAO and controls showed similar responses. ICAM1 expression was not affected by CSE. Hyaluronic acid production was stimulated by CSE in a dose-dependent manner (correlation coefficient, 0.978; P = 0.022), with 5% CSE causing an increase of 44% (P = 0.001). CSE increased adipogenesis in a dose-related manner, as did IL-1. The effects of CSE and IL-1 on adipogenesis were synergistic, with the degree of adipogenesis in the well containing both 5% CSE and 0.1 ng/ml IL-1 being double the magnitude of the sum of the values obtained from either stimulus alone (P < 0.001). Addition of an anti-IL-1 antibody to the well containing both 5% CSE and 0.1 ng/ml IL-1 reduced the degree of adipogenesis by 82% (P < 0.001). CONCLUSION: These findings may help explain how cigarette smoking has a detrimental effect in TAO and suggests that IL-1 may be an attractive therapeutic target in TAO.
Subject(s)
Graves Ophthalmopathy/etiology , Smoking/adverse effects , Adipogenesis , Glycosaminoglycans/biosynthesis , Humans , Intercellular Adhesion Molecule-1/analysis , Interleukin-1/pharmacology , Smoke/adverse effects , Nicotiana/adverse effects , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
OBJECTIVE: Cytokines are likely to play a key pathogenic role in thyroid-associated ophthalmopathy (TAO). Anti-cytokine therapy has been proposed to be a possible treatment for active TAO. We aimed to establish the effects of selected cytokines on intercellular adhesion molecule 1 (ICAM1) expression, glycosaminoglycan (GAG) production and adipogenesis in orbital fibroblasts (OFs) from patients with TAO. METHODS: Orbital tissue was taken during surgery from eight patients with TAO and five control subjects. OFs were cultured and ICAM1 expression measured by flow cytometry. GAG production was measured by hyaluronic acid ELISA. OFs were grown in adipogenic media and the degree of adipogenesis quantified. RESULTS: Responses were similar in OFs from patients with and without TAO. Tumour necrosis factor-alpha (TNFalpha) and interleukin1 (IL1) (0.1 ng/ml) stimulated ICAM1 expression by eight- to ten-fold. Anti-cytokine agents inhibited the cytokine-upregulated ICAM1 expression by 90-99% (P<0.01). TNFalpha and IL1 (0.1 ng/ml) increased hyaluronic acid production by 44 and 95% (P<0.01) respectively. Anti-cytokine agents inhibited these responses by 79-138% (P<0.04).0.013 AU and -1.0; P<0.03) whilst IL1 (0.1 ng/ml) stimulated adipogenesis (+0.05 AU and +5.7; P<0.02) measured by oil-red-O extraction and visual assessment respectively. The anti-IL1 agent inhibited IL1-mediated adipogenesis by 69-106% (P<0.04). CONCLUSION: TNFalpha and IL1 stimulate ICAM1 expression and GAG production, but have opposite effects on adipogenesis in OFs in vitro. IL1 promotes adipogenesis and its effects can be blocked by anti-IL1 agents in vitro. These agents may be the anti-cytokine treatment of choice for clinical trials in active TAO.
Subject(s)
Adipogenesis/drug effects , Graves Ophthalmopathy/metabolism , Interleukin-1/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Adult , Aged , Cells, Cultured , Cytokines/biosynthesis , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Products, gag/metabolism , Histocytochemistry , Humans , Hyaluronic Acid/metabolism , Male , Middle AgedABSTRACT
In addition to hematopoietic progenitors, human bone marrow contains mature T/NK lymphocytes. Valpha24Vbeta11 NKT-cells, a subset of NK receptor+ (NKR+) T-cells in humans, are rare in bone marrow, suggesting the presence of other NKR+ T-cells which may contribute to tumor surveillance. NKR+/- T-cells were examined in blood (PB), and bone marrow from donors (DM) and patients with active hematopoietic malignancy (PM), or in remission (PR). T-cells in PR & PM were enriched for CD56+ and CD57+ subsets, compared to DM. All marrow NKR+/- T-cell subsets were more activated than PB. PM and, surprisingly, PR marrow contained more activated cells than DM. CD8+ cells were significantly increased in all patient marrows and there was evidence of the formation of an effector/memory pool in malignant marrow. These data suggest that NKR+ T-cell enrichment in human bone marrow that has been exposed to neoplastic transformation is compatible with a role in localized tumor surveillance/eradication.