Predictive Modeling of Tacrolimus Dose Requirement Based on High-Throughput Genetic Screening.

Any biochemical reaction underlying drug metabolism depends on individual gene-drug interactions and on groups of genes interacting together. Based on a high-throughput genetic approach, we sought to identify a set of covariant single-nucleotide polymorphisms predictive of interindividual tacrolimus (Tac) dose requirement variability. Tac blood concentrations (Tac C0 ) of 229 kidney transplant recipients were repeatedly monitored after transplantation over 3 mo. Given the high dimension of the genomic data in comparison to the low number of observations and the high multicolinearity among the variables (gene variants), we developed an original predictive approach that integrates an ensemble variable-selection strategy to reinforce the stability of the variable-selection process and multivariate modeling. Our predictive models explained up to 70% of total variability in Tac C0 per dose with a maximum of 44 gene variants (p-value <0.001 with a permutation test). These models included molecular networks of drug metabolism with oxidoreductase activities and the multidrug-resistant ABCC8 transporter, which was found in the most stringent model. Finally, we identified an intronic variant of the gene encoding SLC28A3, a drug transporter, as a key gene involved in Tac metabolism, and we confirmed it in an independent validation cohort.

Hypogammaglobulinemia and risk of severe infection in kidney transplant recipients.

BACKGROUND: Recent data have outlined a link between hypogammaglobulinemia (HGG) and infection risk and suggested that HGG correction may decrease post-transplant infections. METHODS: We analyzed the risk factors of HGG and the relationship between HGG and the risk of severe infection in a cohort of 318 kidney transplant recipients (KTR) who were transplanted between 2003 and 2013. Immunoglobulin (Ig) concentration was measured prospectively at day 15 (D15), month 6 (M6), month 12 (M12), and month 24 (M24) post transplant. RESULTS: The prevalence of IgG HGG was 56% and 36.8% at D15 and M6, respectively. Age was the sole identified risk factors for D15 IgG HGG (odds ratio [OR] 1.02, P = 0.019). Risk factors for M6 IgG HGG were the presence of D15 IgG HGG (OR 6.41, P < 0.001) and treatment of acute rejection (OR 2.63, P = 0.014). Most infections occurred between D15 and M6 post transplant. Only age (hazard ratio 1.03, P < 0.001) was identified as a risk factor of infection between D15 and M6 post transplant. Survival free of infection (overall infections and bacterial or viral infections) did not differ significantly between patients with or without D15 IgG HGG. Only septicemia occurring between M6 and M12 post transplant was more frequently observed in patients with HGG. The low prevalence of severe HGG (<400 mg/dL) did not allow conclusions on the infectious risk associated with this patient subgroup. CONCLUSIONS: This study does not support the existence of a strong link between post-transplant HGG and the risk of severe infections in KTR. Correction of HGG to minimize the risk of severe infections in KTR is thus questionable and needs to be reevaluated in prospective studies.

Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses.

Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu(-/-) mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases.

Kidney transplant recipients carrying the CYP3A4*22 allelic variant have reduced tacrolimus clearance and often reach supratherapeutic tacrolimus concentrations.

CYP3A4*22 is an allelic variant of the cytochrome P450 3A4 associated with a decreased activity. Carriers of this polymorphism may require reduced tacrolimus (Tac) doses to reach the target residual concentrations (Co). We tested this hypothesis in a population of kidney transplant recipients extracted from a multicenter, prospective and randomized study. Among the 186 kidney transplant recipients included, 9.3% (18 patients) were heterozygous for the CYP3A4*22 genotype and none were homozygous (allele frequency of 4.8%). Ten days after transplantation (3 days after starting treatment with Tac), 11% of the CYP3A4*22 carriers were within the target range of Tac Co (10-15 ng/mL), whereas among the CYP3A4*1/*1 carriers, 40% were within the target range (p = 0.02, OR = 0.19 [0.03; 0.69]). The mean Tac Co at day 10 in the CYP3A4*1/*22 group was 23.5 ng/mL (16.6-30.9) compared with 15.1 ng/mL (14-16.3) in the CYP3A4*1/*1 group, p < 0.001. The Tac Co/dose significantly depended on the CYP3A4 genotype during the follow-up (random effects model, p < 0.001) with the corresponding equivalent dose for patients heterozygous for CYP3A4*22 being 0.67 [0.54; 0.84] times the dose for CYP3A4*1/*1 carriers. In conclusion, the CYP3A4*22 allelic variant is associated with a significantly altered Tac metabolism and carriers of this polymorphism often reach supratherapeutic concentrations.

Prognosis of vasculitis associated myelodysplasia.

Systemic and immune manifestations have been reported in patients with MDS. The correlation between immunological abnormalities and prognosis in myelodysplastic syndrome patients remains controversial. Most of the authors agree that the median survival in myelodysplastic syndrome is not related to the presence of systemic and immune manifestations, but only with the existence of a systemic vasculitis.

[Antineutrophil cytoplasmic autoantibodies in children: clinical features]. / Auto-anticorps anticytoplasme des polynucléaires neutrophiles en pédiatrie : caractéristiques cliniques.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) are usually considered as serological markers of vasculitis (microscopic polyangiitis, granulomatosis with polyangiitis, and Churg-Strauss syndrome), but they have also been described in other diseases. They are rarely observed in children. Therefore, this study aims to describe the clinical spectrum associated with positive ANCA in children. PATIENTS AND METHODS: All the children below 15 years of age, admitted to the Angers University Hospital between June 2004 and June 2010 presenting with an ANCA-positive test by indirect immunofluorescence were included in this study. The exhaustive list of ANCA-positive children was obtained from the immunology unit. Six antigenic targets were routinely tested by Elisa, i.e., proteinase 3, myeloperoxidase, bactericidal permeability increasing protein, cathepsin G, elastase, and lactoferrin. Clinical and biological data were retrospectively collected. RESULTS: Thirty-seven children were identified with positive ANCA in this 6-year period. None of the antigenic targets tested was found in 21 patients. The most frequent diseases associated were inflammatory bowel disease (n=10), infections (n=6), hematological disease (n=5), and juvenile idiopathic arthritis (JIA) (n=4). Patients with JIA presented with a predominance of antielastase antibodies. CONCLUSION: In contrast to the findings usually observed in adults, we obtained a wide spectrum of clinical entities associated with positive ANCA in this cohort. In children, the ANCA test has 2 advantages: to diagnose systemic vasculitis and to differentiate inflammatory bowel disease. Patients with JIA seemed to have more frequent antielastase antibodies: in the future, this finding should be further investigated in larger prospective studies.

The natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases.

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody.

Five-year results of a randomized trial comparing de novo sirolimus and cyclosporine in renal transplantation: the SPIESSER study.

Calcineurin inhibitors improve acute rejection rates and short-term graft survival in renal transplantation, but their continuous use may be deleterious. We evaluated the 5-year outcomes of sirolimus (SRL) versus cyclosporine (CsA) immunosuppressive treatment. This observational study was an extension of the SPIESSER study where deceased donor kidney transplant recipients were randomized before transplantation to a SRL- or CsA-based regimen and followed up 1 year. Data from 131 (63 SRL, 68 CsA) out of 133 patients living with a functional graft at 1 year were collected retrospectively at 5 years posttransplant. Seventy percent of CsA patients versus 54% of SRL patients were still on the allocated treatment at 5 years (p = 0.091), most discontinuations in each group being due to safety issues. In intent-to-treat, mean MDRD eGFR was higher with SRL: 54.2 versus 45.3 mL/min with CsA (p = 0.019); SRL advantage was greater in on-treatment analyses. There were no differences for patient survival (p = 0.873), graft survival (p = 0.121) and acute rejection (p = 0.284). Adverse events were more frequent with SRL (80% vs. 60%, p = 0.015). Results confirmed the high SRL discontinuation rate due to adverse events. Nevertheless, a benefit was evidenced on renal function in patients (more than 50%) still on treatment at 5 years.

A 5-year prospective follow-up study in essential cryofibrinogenemia patients.

INTRODUCTION: Cryofibrinogenemia may be essential, or secondary to diseases such as neoplasia, infection, thrombosis, and collagen vascular diseases. In a previous study, we reported the occurrence of neoplasia in some essential cryofibrinogenemia patients after a short period of follow-up. PURPOSE: We performed a prospective multi-center 5-year follow-up study in essential cryofibrinogenemia patients (2005-2009). RESULTS: 23 patients with essential cryofibrinogenemia were included, mean age 59 years (range: 33-79), 14 males. After a mean follow-up period of 24 months, 11/23 (47%) of cases that were initially diagnosed as essential cryofibrinogenemia were found to have an underlying lymphoma (6 T lymphoma and 5 B lymphoma). CONCLUSION: This prospective study suggests that some cases of cryofibrinogenemia that are initially considered as essential, may have underlying lymphoma. Thus, we further suggest that regular follow-up should be performed in patients with essential cryofibrinogenemia.

Systemic and immune manifestations in myelodysplasia: a multicenter retrospective study.

OBJECTIVE: The presence of systemic and/or immune manifestations in myelodysplasia has been currently reported. The influence of these manifestations on the natural outcome of myelodysplastic syndrome has to be considered. We present a multicenter retrospective study (2002-2009) of patients with myelodysplastic syndrome disclosing systemic and/or immune manifestations. METHODS: Forty-six patients with myelodysplasia presenting with systemic and/or immune manifestations were compared in terms of survival with 189 patients with myelodysplasia lacking these features. RESULTS: The clinical picture in these cases consisted of fever (13%), arthralgia or arthritis (13%), and cutaneous manifestations (67%). Four cases of systemic vasculitis have been reported in our series, and they have a worse prognosis. Immune anomalies were recorded in 29% of the cases, and the presence of cryoglobulins was also associated with a worse prognosis. CONCLUSION: A difference in survival between patients with myelodysplastic syndrome with systemic manifestations and patients lacking these manifestations has been observed in the presence of systemic vasculitis and/or cryoglobulins.

Successful rituximab therapy in a lupus patient with diffuse alveolar haemorrhage.

Diffuse alveolar haemorrhage (DAH) is a rare but life-threatening complication of systemic lupus erythematosus (SLE). Specific therapy is based on a heavy immunosuppressive treatment that usually associates corticosteroid and cyclophosphamide boluses and plasma exchange. Despite this treatment, an early mortality rate of 20-50% is reported in the literature. Immunosuppression-related complications are responsible for further mortality and morbidity. Rituximab, a specific anti-CD20 antigen B-cell antibody, has been used with success for the treatment of several refractory autoimmune disorders, but rarely for SLE-induced DAH. We report here the first case of SLE-induced DAH treated successfully with rituximab without cyclophosphamide administration in a patient intolerant to cyclophosphamide. We review the two other cases of SLE-induced DAH managed with rituximab as a part of the immunosuppressive regimen.

Optimization of initial tacrolimus dose using pharmacogenetic testing.

Retrospective studies have demonstrated that patients who are expressors of cytochrome P4503A5 (CYP3A5) require a higher tacrolimus dose to achieve a therapeutic trough concentration (C(0)). The aim of this study was to evaluate this effect prospectively by pretransplantation adaptation. We randomly assigned 280 renal transplant recipients to receive tacrolimus either according to CYP3A5 genotype or according to the standard daily regimen. The primary end point was the proportion of patients within the targeted C(0). Secondary end points included the number of dose modifications and the delay in achieving the targeted C(0). In the group receiving the adapted dose, a higher proportion of patients had values within the targeted C(0) at day 3 after initiation of tacrolimus (43.2% vs. 29.1%; P = 0.03); they required fewer dose modifications, and the targeted C(0) was achieved by 75% of these patients more rapidly. The clinical end points were similar in the two groups. Pharmacogenetic adaptation of the daily dose of tacrolimus is associated with improved achievement of the target C(0). Whether this improvement will affect clinical outcomes requires further evaluation.

Hypochloremia and hyponatremia as the initial presentation of cystic fibrosis in three adults.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Most diagnoses of CF are made during infancy or childhood, and are based on respiratory or digestive involvement. Initial extracellular dehydration leading to the diagnosis of CF is usual in infants but has only exceptionally been reported in adults. We describe three new adult cases of CF initially presenting with depletive hyponatremia and hypochloremia following exposure to heat. At first consultation, these patients had no symptoms suggestive of CF. One patient presented with a seizure induced by hyponatremia. The two other patients were siblings carrying a novel c.4434insA mutation in exon 24 of CFTR. Acute dehydration is a very rare initial manifestation of CF but may be life-threatening. The possibility of CF should not be ignored in cases of depletive hyponatremia, hypochloremia or hypokalemic metabolic alkalosis, even in otherwise healthy patients.

Endogenous PTX3 translocates at the membrane of late apoptotic human neutrophils and is involved in their engulfment by macrophages.

Neutrophils are short-lived innate immune cells that rapidly die by apoptosis. A rapid and efficient clearance of apoptotic cells is crucial to avoid autoimmunity. This process involves cell alterations, endocytic receptors expressed by phagocytic cells and soluble bridging molecules (opsonins) that facilitate internalization of apoptotic cells by phagocytes. Neutrophils constitutively express the prototypic long pentraxin PTX3 that binds to apoptotic cells and modulates their clearance. We thus evaluated whether endogenous PTX3 may interfere with the capture of apoptotic neutrophils. We observed that PTX3 accumulates in blebs at the surface of late apoptotic neutrophils, resulting from its active translocation from granules to the membrane. A neutralizing anti-PTX3 monoclonal Ab (mAb) inhibits the capture of late apoptotic neutrophils by macrophages. This study shows that intracellular PTX3 translocates at the surface of late apoptotic neutrophils and acts as an 'eat-me' molecule for their recognition and capture by macrophages.

Severe episodes of extra cellular dehydration: an atypical adult presentation of cystic fibrosis.

Cystic fibrosis (CF) is usually diagnosed during childhood by respiratory or gastro-intestinal symptoms. Hyponatremic hypochloremic dehydration with metabolic alkalosis is a rare but typical presentation of CF in infants. In contrast, only 3 cases have been described in adults. We report a case of CF in a 33-year-old Caucasian female presenting with a severe sodium and chloride depletion caused by inappropriate sweating. She experienced three episodes of severe dehydration before the diagnosis was suspected. Sweat chloride test was pathological and mild pulmonary involvement was found on CT scan. Delta F508 mutation and a rare mutation (3849+40 A/G) on the intron 19 of CFTR gene were found. Interestingly, our patient has a heterozygote twin sister, carrier of the same mutations of CFTR gene who also developed CF but with a different phenotype. We suspect modifier genes to be implicated in the differences observed between the two phenotypes. We discuss the physiopathology of electrolyte disturbance and review the other similar adults cases.

Antineutrophil cytoplasmic autoantibodies: how should the biologist manage them?

Antineutrophil cytoplasmic antibodies (ANCA) are directed against enzymes found in the granules of the polymorphonuclear (PMN) leukocytes. They are detected by indirect immunofluorescence microscopy assays on human ethanol fixed neutrophils. Three different fluorescence patterns can be distinguished: a cytoplasmic pattern (cANCA), a perinuclear pattern (pANCA), and an atypical pattern (aANCA). The use of other fixatives, e.g., formalin and methanol, allows differentiation between the pANCA and the antinuclear antibodies. ANCA specificity is determined by solid phase assays (ELISA, immunodot, and multiplex assay). ANCA with high titres and defined specificities (antiproteinase 3 [PR 3] or antimyeloperoxidase [MPO]) are proven to be good serological markers of active primary systemic vasculitis: c/PR 3-ANCA for Wegener's granulomatosis and p/MPO-ANCA for microscopic polyangiitis. The former have higher sensitivity and specificity for Wegener's granulomatosis than the latter for microscopic polyangiitis. ANCA with low titres and unknown specificity have been detected in a wide range of inflammatory and infectious diseases leading to a critical reappraisal of the diagnostic significance of ANCA testing. Physicians must keep in mind the possible occurrence of infectious diseases like subacute endocarditis that could be dramatically worsened by irrelevant immunosuppressive therapy. ANCA findings in certain manifestations, such as the pulmonary-renal syndrome in which massive pulmonary hemorrhage can quickly be life-threatening, warrant ANCA testing as an emergency test for patient care.

Clinical operational tolerance after kidney transplantation.

Induction of allograft-specific tolerance and the detection of a "tolerance" state in recipients under immunosuppression with long-term stable graft function are major challenges in transplantation. Clinical "operational tolerance," defined as stable and acceptable graft function without immunosuppression for years, is a rare event. There is no report on the clinical history of such patients. In this article, we report on the medical history of 10 kidney recipients who display an immunosuppressive drug-free "operational tolerance" for 9.4 +/- 5.2 years. Clinical factors that may favor such a tolerant state are underlined. Firstly, most of the patients interrupted immunosuppression over a long time period (until 4 years), which mimics the procedure of intentional immunosuppression weaning following liver transplantation. Secondly, donor age was younger (median 25 years) than the one of the general transplanted population, suggesting that graft quality is one of the conditions favoring "operational tolerance." Moreover, the "operationally tolerant" recipients may be 'low responders' to blood transfusions (PRA 6 +/- 5.4%, six blood transfusions). We also show that "operational tolerance" occurs in the presence of anti-donor class II antibodies, as assessed in two patients. Finally, two patients degraded their renal function 9 to 13 years after treatment withdrawal, however only one presented histological lesions of chronic rejection.

Lymphopenia in occupational pulmonary silicosis with or without autoimmune disease.

An increased prevalence of autoimmune diseases such as rheumatoid arthritis has been demonstrated in silica-exposed patients. The aim of this study was to determine the peripheral blood lymphocyte phenotype in a population of silicotic workers employed in the slate mines of the district. Silicosis was assessed in 58 patients according to the International Labor Office's criteria. Clinical and biological data including flow cytometric evaluation of the lymphocyte subsets were compared with those from 41 healthy volunteers. The silicotic patients had a higher prevalence of autoimmune diseases (6/58 versus 0/41: P < 0.05) and of elevated antinuclear antibody titres compared to the control group. A very significant decrease of total lymphocyte count (P < 0.001) involving B, T and Natural Killer cells was found in silicotic patients as compared with matched healthy volunteers. A significant increase in the percentage of activated T cells (12.3%) was observed in the silicotic group as compared to 6.5% in the control group (P = 5 x 10(-5)). Our results show that in silicotic patients, the absolute number of circulating lymphocytes is diminished with an increased proportion of activated T cells. Whether these findings could predispose to the development of autoimmune disorders is discussed.