Prednisolone does not affect direct-acting antivirals against hepatitis C, but inhibits interferon-alpha production by plasmacytoid dendritic cells.

BACKGROUND: Chronic hepatitis C virus (HCV) infection compromises long-term outcomes of liver transplantation. Although glucocorticosteroid-based immunosuppression is commonly used, discussion is ongoing on the effect of prednisolone (Pred) on HCV recurrence and response to antiviral therapy post transplantation. Recently, new drugs (direct-acting antivirals) have been approved for the treatment of HCV, however, it remains unknown whether their antiviral activity is affected by Pred. The aim of this study was to investigate the effects of Pred on the antiviral activity of asunaprevir (Asu), daclatasvir (Dac), ribavirin (RBV), and interferon-alpha (IFN-α), and on plasmacytoid dendritic cells (PDCs), the main IFN-α-producing immune cells. METHODS: The effects of Pred and antiviral compounds were tested in both a subgenomic and infectious HCV replication model. Furthermore, effects were tested on human PDCs stimulated with a Toll-like receptor-7 ligand. RESULT: Pred did not directly affect HCV replication and did not inhibit the antiviral action of Asu, Dac, RBV, or IFN-α. Stimulated PDCs potently suppressed HCV replication. This suppression was reversed by treating PDCs with Pred. Pred significantly decreased IFN-α production by PDCs without affecting cell viability. When Asu and Dac were combined with PDCs, a significant cooperative antiviral effect was observed. CONCLUSION: This study shows that Pred acts on the antiviral function of PDCs. Pred does not affect the antiviral action of Asu, Dac, RBV, or IFN-α. This implies that there is no contraindication to combine antiviral therapies with Pred in the post-transplantation management of HCV recurrence.

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions.

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg ). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (-64%) but TLR-9-induced IFN-α secretion only slightly (-20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4(+) forkhead box protein 3 (FoxP3)(+) regulatory T cells, but did not affect the generation of suppressive CD8(+) CD38(+) lymphocyte activation gene (LAG)-3(+) Treg . In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4(+) T cell proliferation and induce CD4(+) FoxP3(+) regulatory T cell generation.

Allele-sharing of cytokine genes in familial inflammatory bowel disease.

BACKGROUND/AIMS: The pathogenesis of inflammatory bowel disease is complex, multifactorial, and involves genetic predisposition. This predisposition is likely to include various chromosomal loci, but simple Mendelian inheritance cannot be excluded in a subset of families with inflammatory bowel disease. METHODOLOGY: We evaluated allele-sharing in 17 sib-pairs with inflammatory bowel disease as an approach to select candidate genes for further studies in individual families. It was determined whether each sib-pair had inherited the same alleles for interleukin-2, interleukin-2 receptor beta, interleukin-4, interleukin-4 receptor, interleukin-10, interleukin-10 receptor, tumor necrosis factor alpha, tumor necrosis factor alpha receptor 1 and 2. RESULTS: The results were very different per individual family. The estimated probability of sharing both alleles identical-by-descent at interleukin-4 receptor, interleukin-10, interleukin-10 receptor, and tumor necrosis factor alpha were 50%, 39%, 40%, and 33% respectively. The LOD score was significant for interleukin-4 receptor (p = 0.04). CONCLUSIONS: In this small group of sib-pairs with inflammatory bowel disease a modestly increased allele-sharing was found for some inflammatory related genes. Different results per family may suggest genetic heterogeneity. This method can be useful as a first step to further evaluation of specific candidate genes which may play a pathogenetic role in individual families.

Prednisolone suppresses the function and promotes apoptosis of plasmacytoid dendritic cells.

Organ transplant recipients are highly susceptible to viral infections early after transplantation. Plasmacytoid dendritic cells (PDC) play a major role in antiviral immunity. Therefore, we determined the numbers of circulating PDC after liver transplantation (LTX) and established the effects of immunosuppressive drugs on PDC survival and function. PDC were determined longitudinally in 13 LTX recipients treated with prednisone and cyclosporin or tacrolimus. Purified PDC were cultured with or without clinically relevant concentrations of cyclosporin, tacrolimus or prednisolone. Apoptosis induction was monitored by determination of active caspase-3, nuclear condensation and annexin-V/7AAD staining. After LTX, a 4-fold reduction in the number of circulating PDC was observed (p < 0.01), which recovered partially after discontinuation of prednisone treatment. In vitro, prednisolone induced apoptosis in PDC, while cyclosporin and tacrolimus did not. Higher doses of prednisolone were needed to induce apoptosis in Toll-like receptor (TLR)-stimulated PDC. However, non-apoptosis inducing concentrations of prednisolone suppressed interferon-alpha production, upregulation of co-stimulatory molecules and allo-stimulatory capacity of TLR-stimulated PDC. In conclusion, prednisolone induces apoptosis in PDC, which explains the decline in circulating PDC numbers after transplantation. Moreover, prednisolone suppresses the functions of TLR-stimulated PDC. Therefore, corticosteroid-free immunosuppressive therapy may reduce the number and severity of viral infections after transplantation.

Hepatitis B immunoglobulins inhibit dendritic cells and T cells and protect against acute rejection after liver transplantation.

The efficacy of anti-viral intravenous immunogobulins (anti-HBs Ig and anti-CMV Ig) in preventing acute rejection after liver transplantation was assessed in a retrospective analysis, and correlated to their effects on immune cells in vitro. HBs Ag-positive liver graft recipients (n = 40) treated prophylactically with anti-HBs Ig had a significantly lower incidence of acute rejection compared with recipients without viral hepatitis (n = 147) (12% vs. 34%; p = 0.012), while the incidence of rejection in HCV-positive recipients (n = 29) was similar to that in the control group. Treatment with anti-CMV Ig (n = 18) did not protect against rejection. In vitro, anti-HBs Ig suppressed functional maturation of and cytokine production by human blood-derived dendritic cells (DC) at concentrations similar to the serum concentrations reached during anti-HBs Ig treatment of liver graft recipients. In addition, anti-HBs Ig inhibited allo-antigen- and lectin-stimulated proliferation of peripheral T cells. Anti-CMV Ig suppressed functional DC-maturation and alloantigen-stimulated T-cell proliferation, but not lectin-driven T-cell proliferation. In conclusion, anti-HBs Ig protects against acute rejection after liver transplantation, probably by functional inhibition of the two principal immune cells involved in allograft rejection, DC and T cells.

Human liver myeloid dendritic cells maturate in vivo into effector DC with a poor allogeneic T-cell stimulatory capacity.

We hypothesized that the relatively low immunogenicity of liver grafts might be related to a special maturation program of hepatic myeloid dendritic cells (MDC), yielding relatively immature effector MDC with weak allogeneic T-cell stimulatory capacity. To investigate whether maturation of human liver-derived MDC in vivo differs from maturation of MDC at another anatomical location, we compared the immunophenotypes and allogeneic T-cell stimulatory capacity of MDC from hepatic with those from inguinal lymph nodes (LN). MDC were purified by immunomagnetic selection from hepatic LN obtained from multi-organ donors (n = 8) and from inguinal LN of kidney transplant recipients (n = 7). MDC from hepatic LN had a significantly reduced capacity to stimulate allogeneic T-cell proliferation compared to MDC from inguinal LN. However, this was not due to an immaturity, since MDC from hepatic LN had significantly higher expressions of HLA-DR, CD80, and CD86 compared to MDC from inguinal LN. Hepatic MDC maturate in vivo to a mature type of effector MDC with relatively poor allogeneic T-cell stimulatory capacity.

A functional interleukin-10 mutation in Dutch patients with Crohn's disease.

BACKGROUND AND AIMS: Interleukin-10 is an anti-inflammatory and immunomodulatory cytokine. Interleukin-10 deficient mice are prone to develop chronic colitis. Administration of recombinant human interleukin-10 has been proposed to have a beneficial effect in a subgroup of patients with Crohn's disease. Recently, we found an interleukin-10 Gly15Arg mutation in a family with Crohn's disease which is associated with reduced interleukin-10 secretion by in vitro stimulated monocytes and lymphocytes. We hypothesised that this interleukin-10 mutation plays a role in maintaining the inflammatory process in Crohn's disease in some families. PATIENTS AND METHODS: We evaluated interleukin-10 Gly15Arg in 379 patients with Crohn's disease, and 75 unrelated healthy controls. Also, first degree family members of interleukin-10 Gly15Arg carriers were evaluated. Additionally, mutation carriers and their relatives were evaluated for CARD15 R702W, G908R, and 1007fs. RESULTS: Two patients with Crohn's disease were heterozygous for the interleukin-10 Gly15Arg mutation. No homozygotes were found. The Gly15Arg mutation was not observed in the controls. In first degree family members of the Crohn's disease-affected interleukin-10 Gly15Arg carriers, the mutation was found in Crohn's disease-affected as well as in their apparently healthy individuals. All family members carried one or two CARD15 mutation(s). CONCLUSION: The interleukin-10 Gly15Arg mutation is rare in patients with Crohn's disease, and is not associated with the disease in the Netherlands.

A Gly15Arg mutation in the interleukin-10 gene reduces secretion of interleukin-10 in Crohn disease.

BACKGROUND: Genetic susceptibility, probably involving cytokines and their receptors, plays an important role in inflammatory bowel disease (IBD). In this study we examine the potential role of the interleukin-10 (IL-10) gene as a susceptibility gene in IBD. METHODS: We studied 17 sib-pairs with either Crohn disease (CD) or ulcerative colitis. After microsatellite analysis for allele-sharing, the IL-10 gene of sib-pairs who shared alleles was screened for nucleotide alterations in and around exons and the promoter region. The IL-10 promoter polymorphism at position -1082 was also determined. Function was evaluated by measuring IL-10 secretion by peripheral blood mononuclear cells stimulated with lipopolysaccharide or phorbol ester. The activity of recombinant immature wild-type and mutated IL-10 was tested in a proliferation assay with a human monocytic leukaemia cell line (HL60 cells). RESULTS: DNA sequencing revealed a G --> A point mutation in exon 1 at base position 43 in one sib-pair, both affected with CD. It was also found in 2 of their healthy siblings, but not in 75 unrelated healthy controls. This mutation results in a glycine to arginine substitution at amino acid position 15 of the leader sequence (Gly15Arg). The in vitro IL-10 secretion by mononuclear cells of the IL-10 Gly15Arg carriers was about 50% of healthy controls, matched for the -1082 polymorphism in the IL-10 promoter region. Incubation of HL60 cells with recombinant mutated IL-10 showed a markedly reduced cell proliferation compared to wild-type IL-10. CONCLUSION: A Gly15Arg mutation in the leader sequence of IL-10 was found in a multiple CD-affected family. This altered leader sequence decreases IL-10 secretion, thereby reducing the anti-inflammatory effect.

Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations.

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts and flow cytometry with a Vbeta antibody panel covering more than 65% of Vbeta domains was validated using 28 SB-defined clonal T-cell receptor (TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V(beta) RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR products were detected in all 47 samples, whereas single Vbeta domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vbeta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vbeta gene segments not covered by the applied Vbeta monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta products and single Vbeta domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vbeta antibody studies can be used as a powerful screening method for the detection of single Vbeta domain expression, followed by molecular studies in patients with more than 20% single Vbeta domain expression or large suspect T-cell populations (more than 50%-60%) without Vbeta reactivity.

Flow cytometric analysis of the Vbeta repertoire in healthy controls.

BACKGROUND: Analysis of the T-cell receptor (TCR)-Vbeta repertoire has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens. For the interpretation of these studies, we need information about the Vbeta repertoire usage in healthy individuals. METHODS: We analyzed blood T-lymphocyte (sub)populations of 36 healthy controls (age range: from neonates to 86 years) with a carefully selected most complete panel of 22 Vbeta monoclonal antibodies, which together recognized 70-75% of all blood TCRalphabeta(+) T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vbeta antibody combinations for easy and rapid detection of single ("clonal") Vbeta domain usage in large T-cell expansions. RESULTS: The mean values of the Vbeta repertoire usage were stable during aging in blood TCRalphabeta(+) T lymphocytes as well as in the CD4(+) and CD8(+) T-cell subsets, although the standard deviations increased in the elderly. The increased standard deviations were caused by the occurrence of oligoclonal T-cell expansions in the elderly, mainly consisting of CD8(+) T lymphocytes. The 15 detected T-cell expansions did not reach 40% of total TCRalphabeta(+) T lymphocytes and represented less than 0.4 x 10(9) cells per liter in our study. Vbeta usage of the CD4(+) and CD8(+) subsets was comparable for most tested Vbeta domains, but significant differences (P < 0.01) between the two subsets were found for Vbeta2, Vbeta5.1, Vbeta6.7, Vbeta9.1, and Vbeta22 (higher in CD4(+)), as well as for Vbeta1, Vbeta7.1, Vbeta14, and Vbeta23 (higher in CD8(+)). Finally, single Vbeta domain expression in large T-cell expansions can indeed be detected by the six-tube test kit. CONCLUSIONS: The results of our study can now be used as reference values in studies on distortions of the Vbeta repertoire in disease states. The six-tube test kit can be used for detection of single Vbeta domain expression in large T-cell expansions (>2.0 x 10(9)/l), which are clinically suspicious of T-cell leukemia.

Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia.

Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality assessment. For polymerase chain reaction analysis, primers directed against framework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segments and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolution fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the assays generated polyclonal patterns in the reactive tissues. The sensitivity in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% (high-resolution fingerprinting analysis with FR2 primers). Comparison of the three FR3 assays revealed that gene scanning had the highest sensitivity (69%), probably because it could detect small aberrant monoclonal amplicons. False-negative results were especially frequent in follicular center lymphoma (n = 20), but also in diffuse large B-cell lymphoma (n = 18), both renowned for having mutated variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combining these techniques, 17 (85%) of 20 follicular center lymphoma samples showed monoclonality. Therefore, especially in follicular center lymphoma, diffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, multiple variable gene segment primer sets must be used for a reliable assessment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however, is a reliable alternative within a diagnostic setting, avoiding the use of radioactivity or expensive automated sequencing equipment and fluorochrome-labeled (oligo)nucleotides.

Novel mutations in the LKB1/STK11 gene in Dutch Peutz-Jeghers families.

The Peutz-Jeghers syndrome (PJS) is a rare hereditary disorder in which gastrointestinal hamartomatous polyposis, mucocutaneous pigmentation, and a predisposition for developing cancer are transmitted in an autosomal dominant fashion. The recently identified LKB1/STK11 gene located at chromosome 19p13.3 is mutated in a number of PJS pedigrees. We performed mutation analysis in 19, predominantly Dutch, PJS families. In 12 of these families, we identified LKB1/STK11 mutations, none of which has been described before. These 12 novel LKB1/STK11 mutations consist of one nonsense mutation, three frameshift deletions, three frameshift insertions, two acceptor splice site mutations, and three missense mutations. In addition, we detected four polymorphisms in LKB1/STK11. In the remaining seven PJS families, we found no apparent abnormalities of the LKB1/STK1I gene, which could reflect the existence of locus heterogeneity in PJS. None of the mutations occurred in more than one family, and a number were demonstrated to have arisen de novo. The diverse array of mutations found, the apparent high mutation rate, as well as the existence of a possible second PJS locus, renders diagnostic or predictive genetic testing in individual patients difficult, although future identification of additional mutations or even gene(s) will help in increasing the yield of direct mutation analysis.

Peutz-Jeghers syndrome: 78-year follow-up of the original family.

BACKGROUND: The association between heredity, gastrointestinal polyposis, and mucocutaneous pigmentation in Peutz-Jeghers syndrome (PJS) was first recognised in 1921 by Peutz in a Dutch family. This original family has now been followed-up for more than 78 years. We did mutation analysis in this family to test whether the recently identified LKB1 gene is indeed the PJS gene in this family. METHODS: The original family was retraced and the natural history of PJS was studied in six generations of this kindred by interview, physical examination, chart view, and histological review of tissue specimens. DNA-mutation analysis was done in all available descendants. FINDINGS: Clinical features in this family included gastrointestinal polyposis, mucocutaneous pigmentation, nasal polyposis, and rectal extrusion of polyps. Survival of affected family members was reduced by intestinal obstruction and by the development of malignant disease. A novel germline mutation in the LKB1 gene was found to cosegregate with the disease phenotype in the original family. The mutant LKB1 allele carried a T insertion at codon 66 in exon 1 resulting in frameshift and stop at codon 162 in exon 4. INTERPRETATION: The morbidity and mortality in this family suggest that PJS is not a benign disease. An inactivating germline mutation in the LKB1 gene is involved in the PJS phenotype in the original and oldest kindred known to be affected by PJS.

Correction of abnormal T-cell receptor repertoire during interferon-alpha therapy in patients with hairy cell leukemia.

Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-alpha (IFN-alpha) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-alpha in the past, at initiation, and at several intervals up to 6 years of IFN-alpha treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+ cells; 600 to 700/microL whole blood), which decreased during IFN-alpha therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex-unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-alpha can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.