Immune responses against islet allografts during tapering of immunosuppression--a pilot study in 5 subjects.

Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity.

Four-year metabolic outcome of a randomised controlled CD3-antibody trial in recent-onset type 1 diabetic patients depends on their age and baseline residual beta cell mass.

AIMS/HYPOTHESIS: The aim of the study was to examine the 48 month outcome of treating recent-onset type 1 diabetic patients for 6 days with humanised CD3-antibody, ChAglyCD3. METHODS: Eighty patients, aged 12-39 years, were recruited for a phase 2 multicentre trial and randomised to placebo (n=40) or ChAglyCD3 (n=40) treatment by a third party member; participants and care-givers were blinded. The change in insulin dose (U kg(-1)day(-1)) over 48 months was chosen as primary endpoint and compared in 31 placebo-and 33 ChAglyCD3-treated patients. Adverse events were followed in 35 and 38 patients, respectively. RESULTS: Treatment with ChAglyCD3 delayed the rise in insulin requirements of patients with recent-onset diabetes and reduced its amplitude over 48 months (+0.09 vs +0.32 U kg(-1)day(-1) in the placebo group). Using multivariate analysis this effect was correlated with higher baseline residual beta cell function and a younger age. It was associated with better outcome variables in subgroups selected according to these variables. In the ChAglyCD3 subgroup with higher initial beta cell function, 0/11 patients became C-peptide-negative over 48 months vs 4/9 in the corresponding placebo subgroup. In the subgroup aged <27 years old, antibody treatment preserved initial beta cell function for 36 months (vs >80% decline within 24 months in the placebo subgroup <27 years old), resulted in lower HbA1c concentrations and tended to reduce glycaemic variability (p=0.08). No longterm adverse events were observed. CONCLUSIONS/INTERPRETATION: A 6 day ChAglyCD3 treatment can suppress the rise in insulin requirements of recent-onset type 1 diabetic patients over 48 months, depending on their age and initial residual beta cell function. In younger patients this effect is associated with reduced deterioration of metabolic variables. These observations help to define inclusion criteria for prevention trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT00627146 FUNDING: Center grants from the Juvenile Diabetes Research Foundation (4-2001-434, 4-2005-1327) and grants from the Belgian Fund for Scientific Research-Flanders and from Brussels Free University-VUB.

Relevance of cytotoxic alloreactivity under different immunosuppressive regimens in clinical islet cell transplantation.

Islet or beta cell transplantation provides a promising cure for type 1 diabetes patients, but insulin-independency decreases frequently over time. Immunosuppressive regimens are implemented attempting to cope with both auto- and alloimmunity after transplantation. We analysed the influence of different immunotherapies on autoreactive and alloreactive T cell patterns and transplant outcome. Patients receiving three different immunosuppressive regimens were analysed. All patients received anti-thymocyte globulin induction therapy. Twenty-one patients received tacrolimus-mycophenolate mofetil maintenance immunosuppression, whereas the other patients received tacrolimus-sirolimus (SIR, n = 5) or SIR only (n = 5). Cellular autoreactivity and alloreactivity (CTL precursor frequency) were measured ex vivo. Clinical outcome in the first 6 months after transplantation was correlated with immunological parameters. C-peptide levels were significantly different between the three groups studied (P = 0.01). We confirm that C-peptide production was correlated negatively with pretransplant cellular autoreactivity and low graft size (P = 0.001, P = 0.007 respectively). Combining all three therapies, cellular autoimmunity after transplantation was not associated with delayed insulin-independence or C-peptide production. In combined tacrolimus-SIR and SIR-treated patients, CTL alloreactivity was associated with less insulin independence and C-peptide production (P = 0.03). The percentage of donors to whom high CTLp frequencies were measured was lower in insulin-independent recipients (P = 0.03). In this cohort of islet cell graft recipients, clinical outcome in the first 6 months after transplantation correlates with the applied immunosuppressive regimen. An association exists between insulin-independence and lower incidence of CTL alloreactivity towards donor human leucocyte antigen. This observational study demonstrates the usefulness of monitoring T cell reactivity against islet allografts to correlate immune function with graft survival.

Allograft-specific cytokine profiles associate with clinical outcome after islet cell transplantation.

Islet cell transplantation can cure type 1 diabetes, but allograft rejection and recurrent autoimmunity may contribute to decreasing insulin independence over time. In this study we report the association of allograft-specific proliferative and cytokine profiles with clinical outcome. Peripheral blood mononuclear cells were obtained of 20 islet recipients. Cytokine values in mixed lymphocyte cultures (MLC) were determined using stimulator cells with graft-specific HLA class II. Qualitative and quantitative cytokine profiles were determined before and after islet transplantation, blinded from clinical outcome. Cytotoxic T Lymphocyte precursor (CTLp) assays were performed to determine HLA class I alloreactivity. Allograft-specific cytokine profiles were skewed toward a Th2 or regulatory (Treg) phenotype after transplantation in insulin-independent, but not in insulin-requiring recipients. IFNgamma/IL10 ratio and MLC proliferation decreased after transplantation in insulin-independent recipients (p = 0.006 and p = 0.01, respectively). Production of the Treg cytokine IL10 inversely correlated with proliferation in alloreactive MLC (p = 0.008) and CTLp (p = 0.005). Production of IL10 combined with low-MLC reactivity associated significantly with insulin independence. The significant correlation between allograft-specific cytokine profiles and clinical outcome may reflect the induction of immune regulation in successfully transplanted recipients. Islet donor-specific IL10 production correlates with low alloreactivity and superior islet function.

Identification of prediabetes in first-degree relatives at intermediate risk of type I diabetes.

Prevention trials of type I diabetes are limited by recruitment of individuals at high risk of the disease. We investigated whether demographic and biological characteristics can identify rapid progressors among first-degree relatives of known patients at intermediate (< 10%) 5-year risk. Diabetes-associated antibodies, random proinsulin : C-peptide (PI/C) ratio and HLA DQ genotype were determined (repeatedly) in 258 islet antibody-positive IA-2Antibody-negative (Abpos/IA-2Aneg) normoglycaemic first-degree relatives. During follow-up (median 81 months), 14 of 258 Abpos/IA-2Aneg relatives developed type I diabetes; 13 (93%) of them had persistent antibodies conferring a 12% [95% confidence interval (CI): 5-19%] 5-year risk of diabetes. In Abpos/IA-2Aneg relatives with persistent antibodies (n = 126), the presence of >/= 1 HLA DQ susceptibility haplotype in the absence of a protective haplotype (P = 0.033) and appearance on follow-up of a high PI/C ratio (P = 0.007) or IA-2A-positivity (P = 0.009) were identified as independent predictors of diabetes. In persistently antibody-positive relatives with HLA DQ risk a recurrently high PI/C ratio or development of IA-2A identified a subgroup (n = 32) comprising 10 of 13 (77%) prediabetic relatives and conferred a 35% (95% CI: 18-53%) 5-year risk. Under age 15 years, 5-year progression (95% CI) was 57% (30-84%) and sensitivity 62%. In the absence of IA-2A, the combination of antibody persistence, HLA DQ risk and elevated PI/C ratio or later development of IA-2A and young age defines a subgroup of relatives with a high risk of type I diabetes (>/= 35% in 5 years). Together with initially IA-2A-positive relatives these individuals qualify for standardized beta cell function tests in view of prevention trials.

Isolation of Penicillium chrysogenum PEX1 and PEX6 encoding AAA proteins involved in peroxisome biogenesis.

In Penicillium chrysogenum, key enzymes involved in the production of penicillin reside in peroxisomes. As a first step to understand the role of these organelles in penicillin biosynthesis, we set out to isolate the genes involved in peroxisome biogenesis. Here we report the cloning and characterization of P. chrysogenum PEX1 and PEX6, which encode proteins of the AAA family of ATPases. The second AAA module, which is essential for the function of Pex1p and Pex6p in peroxisome biogenesis, is highly conserved in both PcPexlp and PcPex6p. PcPEX1 and PcPEX6 contain three and two introns, respectively.

Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact.

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Deltapex1 or Deltapex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Deltapex4, Deltapex8 or Deltapex14 cells. Both proteins are loosely membrane-bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane-bound, but only partially co-localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity.

The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery.

We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisome-deficient mutant. The PEX4 translation product, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Deltapex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Deltapex4 cells. The PTS1 protein import defect in Deltapex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H. polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.

The Hansenula polymorpha PER9 gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity.

We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (Deltaper9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced Deltaper9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.

Peroxisomal remnants in peroxisome-deficient mutants of the yeast Hansenula polymorpha [corrected].

We have analyzed the presence of peroxisomal remnants ('ghosts') in three peroxisome-deficient (per) mutants of the yeast Hansenula polymorpha, namely delta per4, delta per5 and delta per10. Under peroxisome-inducing growth conditions peroxisomal membrane proteins (PMPs) were normally synthesized in cells of these mutants. In addition, these cells contained clusters of small membraneous vesicles, which were absent in cells grown under peroxisome-repressing growth conditions. These structures displayed typical peroxisomal properties in that they proliferated upon overproduction of Per8p, the H. polymorpha peroxisome proliferation factor. Moreover, in delta per4 and delta per5 these vesicles were susceptible to glucose-induced proteolytic degradation.

The Hansenula polymorpha PER3 gene is essential for the import of PTS1 proteins into the peroxisomal matrix.

PER genes are essential for the assembly of peroxisomes in Hansenula polymorpha. Here we describe the PER3 gene which was cloned by functional complementation of a H. polymorpha per3 mutant. The complementing PER3 gene encodes a protein of 569 amino acids (Per3p) with a calculated mass of 63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family and is located in both the cytosol and the peroxisomal matrix. Remarkably, Per3p does not contain a known targeting signal (PTS1 or PTS2). The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity). However, their function apparently cannot be interchanged since the P. pastoris PAS8 gene failed to functionally complement a H. polymorpha per3 disruption mutant. The per3 disruption mutant contained normal but small peroxisomes in which PTS2 proteins (both homologous and heterologous) were imported. Other matrix proteins (in particular PTS1 proteins) resided in the cytosol where they were normally assembled and active. We argue that Per3p is a component of the peroxisomal import machinery and most probably shuttles matrix proteins from the cytosol to the organellar matrix.