Patrick P. C. Boor

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.

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/STK11 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. Hum Mutat 13:476–481, 1999.

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.

Superior immunomodulatory effects of intravenous immunoglobulins on human T-cells and dendritic cells: comparison to calcineurin inhibitors.

Background. Prophylactic administration of anti-HBs intravenous immunoglobulins (IVIg) in hepatitis B infected-liver transplant patients protects against acute rejection. To explore the suitability of intravenous immunoglobulins (IVIg) as prophylaxis of acute rejection and graft-versus-host disease (GVHD) after allograft transplantation, the effects of IVIg and calcineurin inhibitors (CNI) on human blood-derived T-cells and DC were compared. Methods. T-cells were stimulated with phytohemagglutinin (PHA) or allogeneic spleen antigen-presenting cells (APC) and T-cell proliferation and cytokine production were determined in presence or absence of IVIg or CNI. Immature blood dendritic cells (DC) were stimulated in presence or absence of IVIg or CNI, and allogeneic T-cell stimulatory capacity, cell death, and phenotypic maturation were established. Results. IVIg and CNI equally inhibited T-cell proliferation and IFN-&ggr; production after PHA stimulation or allogeneic stimulation. CD8+ T-cells were preferentially affected by both IVIg and CNI after allogeneic stimulation. Like CNI, addition of IVIg at later time points after T-cell activation suppressed mitotic progression of responding T-cells. IVIg-treated DC were suppressed in their capacity to stimulate allogeneic T-cell proliferation by 73±12%, whereas DC-function was not affected by CNI. The decreased allogeneic T-cell stimulatory capacity of IVIg-treated DC correlated to induction of cell death in DC and decreased up-regulation of CD40 and CD80. Conclusions. In vitro IVIg functionally inhibit the two principal immune cell-types involved in rejection and GVHD, i.e. T-cells and DC, whereas CNI only suppress T-cells. By targeting both T-cells and DC, IVIg may be a promising candidate for immunosuppressive treatment after allograft transplantation.

Recipient CTLA-4 +49 G/G Genotype Is Associated with Reduced Incidence of Acute Rejection After Liver Transplantation

The aim of this pilot study was to investigate whether acute rejection after liver transplantation is associated with known single‐nucleotide polymorphisms (SNPs) in the CD86‐ and CTLA‐4 genes of liver‐transplant donors and recipients. Single nucleotide polymorphisms were determined in 135 liver transplant recipients and in 73 donors. Acute rejection was not associated with CD86 + 1057 G/A genotype distributions in donors and in recipients. In univariate analysis recipient CTLA‐4 –318 G/T and + 49 A/G genotype distributions were both weakly associated with acute rejection. Multivariate analysis revealed that the CTLA‐4 + 49 SNP, but not the –318 SNP, was independently of other risk factors associated with acute rejection. Only one out of 13 CTLA‐4 + 49 G‐homozygotes (8%) experienced acute rejection(s) compared with 40% of A/A or A/G recipients. The CTLA‐4 + 49 A/G SNP, which results in an amino acid substitution in the signal peptide of the protein, did not, however, affect intracellular expression or trafficking of CTLA‐4 in T cells, nor soluble serum CTLA‐4 concentrations of the liver transplant recipients. In conclusion, this pilot study suggests that liver transplant recipients homozygous for CTLA‐4 + 49 G have a reduced risk of acute rejection.

Human plasmacytoid dendritic cells induce CD8+LAG-3+Foxp3+CTLA-4+ regulatory T cells that suppress allo-reactive memory T cells

Allo‐reactive memory T cells are a major barrier for induction of immunological tolerance to allografts in humans. Here, we report that stimulation of unfractionated human T cells with TLR‐stimulated allogeneic plasmacytoid dendritic cells (pDCs) induces CD8+ regulatory T cells (Tregs) that inhibit T‐cell allo‐responses, including those of memory T cells. CD3+ T cells were primed for 7 days with allogeneic pDCs that had been pre‐stimulated with TLR‐7 or TLR‐9 ligands. While the T cells proliferated and produced cytokines during the priming culture, they were profoundly hypo‐responsive to re‐stimulation with the same allo‐antigen in a second culture. Moreover, T cells primed by pDCs exerted donor‐specific suppression on allo‐responses of both unfractionated and memory CD3+ T cells. The regulatory capacity of pDC‐primed T cells was confined to CD8+LAG‐3+Foxp3+CTLA‐4+ T cells, which suppressed allogeneic T‐cell responses through a CTLA‐4‐dependent mechanism. Induction of CD8+ Tregs by pDCs could be partially prevented by 1‐methyl tryptophan, an inhibitor of indoleamine 2,3‐dioxygenase. In conclusion, stimulation of human T cells by TLR‐stimulated allogeneic pDCs induces CD8+ Tregs that inhibit allogeneic T‐cell responses, including memory T cells. Donor‐derived pDCs may be considered as an immunotherapeutic tool to prevent activation of the recipient allo‐reactive (memory) T‐cell repertoire after allogeneic transplantation.

CARD15 mutations in Dutch familial and sporadic inflammatory bowel disease and an overview of european studies

Objectives The single nucleotide variations R702W, G908R and L1007fs in the CARD15 gene have been found to be independently associated with Crohns disease. The aim of this study was to evaluate the prevalence of these gene variations in Dutch multiple inflammatory bowel disease-affected families, in sporadic inflammatory bowel disease patients and in healthy controls. Methods Dutch Caucasians from multiple inflammatory bowel disease-affected families were recruited, including 78 probands with Crohns disease, 34 probands with ulcerative colitis and 71 inflammatory bowel disease-affected and 100 non-affected family members. In addition, 45 sporadic inflammatory bowel disease patients (36 Crohns disease and nine ulcerative colitis), and 77 unrelated healthy controls were included. Genomic DNA was isolated to determine CARD15 R702W, G908R and L1007fs. For these mutations, we evaluated disease susceptibility and correlation with inflammatory bowel disease phenotypes. Results In all included unrelated inflammatory bowel disease-affected probands, the R702W, G908R and L1007fs allele frequencies were 8.8, 6.1 and 11.0%, respectively, for Crohns disease, and 4.7, 0 and 2.3% for ulcerative colitis. In controls, the allele frequencies were 5.9, 0.7 and 1.9%, respectively. G908R and L1007fs were associated with Crohns disease (P=0.006 and 0.001, respectively). Compound heterozygotes for any of the three mutations were 11 (9.2%) in Crohns disease patients, but none in ulcerative colitis patients nor controls. Carriage of CARD15 mutations was not associated with familial disease (P≥0.38). Inflammatory bowel disease-affected family members of Crohns disease probands carrying L1007fs, however, were carriers significantly more often than expected (P<0.001). In Crohns disease patients, a significant trend was found between carriage of at least one CARD15 mutation and between carriage of L1007fs and behaviour of disease, including more carriers with stricturing and even more with penetrating disease (P=0.006 and 0.017, respectively). Conclusion In the Dutch population, CARD15 G908R and L1007fs are associated with Crohns disease. Although no difference was found between sporadic and familial cases, in L1007fs-positive multiple affected families the inflammatory bowel disease-affected relatives are more likely than expected to carry this mutation. In Crohns disease, carriage of at least one CARD15 mutation is associated with a more complicated disease behaviour.

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.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 m 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 r → r 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 m 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.

Migration of allosensitizing donor myeloid dendritic cells into recipients after liver transplantation

It is thought, but there is no evidence, that myeloid dendritic cells (MDCs) of donor origin migrate into the recipient after clinical organ transplantation and sensitize the recipients immune system by the direct presentation of donor allo‐antigens. Here we show prominent MDC chimerism in the recipients circulation early after clinical liver transplantation (LTx) but not after renal transplantation (RTx). MDCs that detach from human liver grafts produce large amounts of pro‐inflammatory [tumor necrosis factor alpha and interleukin 6 (IL‐6)] and anti‐inflammatory (IL‐10) cytokines upon activation with various stimuli, express higher levels of toll‐like receptor 4 than blood or splenic MDCs, and are sensitive to stimulation with a physiological concentration of lipopolysaccharide (LPS). Upon stimulation with LPS, MDCs detaching from liver grafts prime allogeneic T cell proliferation and production of interferon gamma but not of IL‐10. Soluble factors secreted by liver graft MDCs amplify allogeneic T helper 1 responses. In conclusion, after clinical LTx, but not after RTx, prominent numbers of donor‐derived MDCs migrate into the recipients circulation. MDCs detaching from liver grafts produce pro‐inflammatory and anti‐inflammatory cytokines and are capable of stimulating allogeneic T helper 1 responses, and this suggests that MDC chimerism after clinical LTx may contribute to liver graft rejection rather than acceptance. Liver Transpl 16:12–22, 2010.

GITR engagement in combination with CTLA-4 blockade completely abrogates immunosuppression mediated by human liver tumor-derived regulatory T cells ex vivo

In liver cancer tumor-infiltrating regulatory T cells (Ti-Treg) are potent suppressors of tumor-specific T-cell responses and express high levels of the Treg-associated molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR). In this study, we have evaluated the capacity of GITR-ligation, CTLA-4-blockade and a combination of both treatments to alleviate immunosuppression mediated by Ti-Treg. Using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC) or liver metastases from colorectal cancer (LM-CRC) we show that treatment with a soluble form of the natural ligand of GITR (GITRL), or with blocking antibodies to CTLA-4, reduces the suppression mediated by human liver tumor-infiltrating CD4+Foxp3+ Treg, thereby restoring proliferation and cytokine production by effector T cells. Importantly, combined treatment with low doses of both molecules exhibited stronger recovery of T cell function compared with either treatment alone. Our data suggest that in patients with primary and secondary liver cancer both GITR-ligation and anti-CTLA-4 mAb can improve the antitumor immunity by abrogating Ti-Treg mediated suppression.