Marcos López-Hoyos

Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients.

Background. Immunosuppression in renal transplantation, although manageable in the short-term, is a major hurdle for long-term graft survival. Recently, increased frequencies of CD4+CD25high regulatory T cells (Tregs) have been described as an additional mechanism that induces alloimmune tolerance. Methods. We assessed 64 renal transplant recipients with stable renal function for at least one year. Patients were divided into two groups according to the immunosuppression they were receiving at the moment of the study: one consisted of patients receiving rapamycin (Rapa) but not calcineurin inhibitors (CNI), and the other group received CNI but not Rapa. The Rapa group was further divided into three subgroups according to their previous experience with CNI: CNI-free, CNI withdrawal, and CNI conversion. Frequencies of blood Tregs were studied by flow cytometry after staining with monoclonal antibodies specific for different markers of Tregs. Results. Frequencies of CD4+ T cells with regulatory phenotype and function were significantly decreased in peripheral blood of renal transplant patients receiving CNI compared with those receiving Rapa. This effect was independent of an early exposure to CNI because the CNI-free patients in the Rapa group showed similar frequencies of Tregs to the CNI withdrawal and CNI conversion groups. Conclusions. CNI, but not Rapa, induce a decrease of circulating Tregs in stable renal transplant recipients. Thus, Rapa might be further explored in strategies using preservation of Tregs for transplant tolerance. Furthermore, quantification of blood Tregs may be a suitable tool to identify renal transplant recipients who may be candidates for reduced immunosuppression.

PLCG1 mutations in cutaneous T-cell lymphomas

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.

Aging is associated with circulating cytokine dysregulation

PURPOSE Aging is accompanied by a progressive increase in pro-inflammatory cytokine status. However, little is known about the development of age-dependent modifications in other circulating cytokines. The aim of this study was to investigate in vivo the influence of age on circulating cytokine production in healthy subjects (HC). METHODS Circulating cytokines were measured by CBA and ELISA in 73 HC. Intracellular cytokine production was assessed in CD3+ and CD14+ cells by flow cytometry. Production of cytokines in cell culture supernatants was also studied after polyclonal stimulation. RESULTS Subjects were divided into three different groups according to age: 28 young HC (<30 years, 26.2 ± 2.4), 24 middle age HC (30-60 years, 44.7 ± 8.4) and 21 elderly HC (>60 years, 70.6 ± 7.9). Age was positively correlated with the circulating levels of IL-12p70, IL-1β, TNFα, IL-6, and IL-10. Age had a negative correlation with circulating levels of IL-17. Besides, age was positively correlated with spontaneous intracellular expression of proinflammatory cytokines in circulating monocytes. No correlation was found with other intracellular cytokine expression or with the production of cytokines in cell culture supernatants after in vitro stimulation. Gender had a marginal effect on the circulating cytokine profile. CONCLUSION Aging has a significant impact on the production of circulating cytokines in healthy individuals. The circulating cytokine milieu may contribute to the development of age-restricted conditions.

Changes in the Serum Levels of Interleukin-17/ Interleukin-23 During Acute Rejection in Liver Transplantation

Interleukin‐23 (IL‐23) and T helper 17 (Th17) cells have been cast as major players in autoimmunity, but their role in transplantation immunity remains to be specified. The aim of our study was to investigate the time course of serum levels of IL‐23 and IL‐17 during hepatic allograft rejection. Serum levels of IL‐23 and IL‐17 were determined in 20 healthy subjects and 50 hepatic transplant recipients. These patients were divided into 2 groups: group I was composed of 15 patients with acute rejection, and group II was composed of 35 patients without acute rejection. Samples were collected on days 1 and 7 after liver transplantation and on the day of liver biopsy. The concentrations of IL‐23 were similar for the rejection group and nonrejection group at early postoperative times. We observed a significant increase in serum IL‐23 levels in the rejection group when a diagnosis of acute rejection had been established. Similarly to IL‐23, at the diagnosis of acute rejection, the concentration of IL‐17 was significantly higher in the rejection group versus the nonrejection group. The whole transplant group, including those with stable graft function, had higher serum levels of IL‐23 and IL‐17 than the controls during the entire postoperative period. In conclusion, IL‐23 and IL‐17 are up‐regulated during acute hepatic rejection. These findings suggest a role for Th17 cells in human liver allograft rejection. Liver Transpl 15:629–633, 2009.

Giant cell arteritis and polymyalgia rheumatica: Role of cytokines in the pathogenesis and implications for treatment

OBJECTIVE To summarize the contribution of cytokines to pathogenesis, clinical manifestations and prognosis of polymyalgia rheumatica (PMR) and giant cell arteritis (GCA). METHODS MEDLINE database search for studies published between 1980 and April 2008. RESULTS PMR and GCA are characterized by a hyperproduction of IL-6. The role of other circulating cytokines in their pathogenesis remains unclear. Cytokine mRNA in the arterial wall of GCA can distinguish different clinical subgroups of patients. The profile of T cell-derived cytokines in GCA suggests that it is a Th1-driven disease. The scarce number of studies makes difficult to evaluate the exact contribution of cytokine polymorphisms to their pathogenesis. Small studies have suggested the utility of TNF antagonists in patients with refractory PMR and GCA. However, these data have not been confirmed in controlled studies in patients with recent onset disease. CONCLUSION Further studies are needed to evaluate the role of circulating cytokines in PMR and GCA. The study of tissue cytokines has provided important insights into the mechanisms implicated in the local inflammatory response that occurs in GCA. The important advance in the knowledge of the role of cytokines in PMR and GCA will have clear implications for treatment.

ImmuKnow as a diagnostic tool for predicting infection and acute rejection in adult liver transplant recipients: A systematic review and meta‐analysis

Immune status monitoring of transplant recipients could identify patients at risk of acute rejection, infection, and cancer, which are important sources of morbidity and mortality in these patients. The ImmuKnow assay provides an objective assessment of the cellular immune function of immunosuppressed patients. Inconclusive results concerning the ability of the ImmuKnow test to predict acute rejection and infection have raised concerns about the predictive value of ImmuKnow in liver transplant recipients. We conducted a systematic literature review to identify studies published up to March 2012 that documented the use of ImmuKnow for monitoring immune function in liver transplant recipients. The study quality was assessed with the Quality Assessment of Diagnostic Accuracy Studies 2 score. We identified 5 studies analyzing ImmuKnow performance for infection and 5 studies analyzing ImmuKnow performance for acute rejection. The pooled sensitivity, specificity, positive likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curve were 83.8% [95% confidence interval (CI) = 78.5%‐88.3%], 75.3% (95% CI = 70.9%‐79.4%), 3.3 (95% CI = 2.8‐4.0), 14.6 (95% CI = 9.6‐22.3), and 0.824 ± 0.034, respectively, for infection and 65.6% (95% CI = 55.0%‐75.1%), 80.4% (95% CI = 76.4%‐83.9%), 3.4 (95% CI = 2.4‐4.7), 8.8 (95% CI = 3.1‐24.8), and 0.835 ± 0.060, respectively, for acute rejection. Heterogeneity was low for infection studies and high for acute rejection studies. In conclusion, the ImmuKnow test is a valid tool for determining the risk of further infection in adult liver transplant recipients. Significant heterogeneity across studies precludes the conclusion that ImmuKnow identifies liver transplant patients at risk for rejection. Liver Transpl 18:1245–1253, 2012.

Age and low levels of circulating vitamin D are associated with impaired innate immune function

This study investigated in vivo the influence of age and vitamin D status on innate immune function in HC. Serum 25OHD was measured in 71 HC. TLR expression on various subpopulations of PBMCs, as well as TLR function by stimulating PBMCs with specific ligands, was assessed by flow cytometry. Circulating cathelicidin levels were determined by ELISA. Serum 25OHD levels decreased with age, and there was a significant inverse correlation between 25OHD levels and age. There was a negative correlation between serum 25OHD levels and MFI expression of TLR7 on B cells, T cells, and monocytes. TLR7 function, addressed by in vitro stimulation with a specific agonist, was significantly correlated with serum 25OHD levels, and this was especially a result of the results in HC older than 60 years. MFI expression of TLR5 on T cells and TLR2 on monocytes was also negatively correlated with serum 25OHD levels. TLR1 (monocytes) and TLR2 (monocytes) expression was positively correlated with age. Furthermore, TLR4 and TLR8 function was negatively correlated with age. Circulating cathelicidin levels decreased with age and were positively correlated with 25OHD levels. Aging is accompanied by changes in expression and function of several TLRs. Serum 25OHD levels decrease with age and are also associated with a change in expression and defective function of certain TLRs, especially those involved in viral response.

Intracellular ATP concentrations of CD4 cells in kidney transplant patients with and without infection

Abstract:  In the field of organ transplantation, overimmunosuppression is associated with severe side effects, such as infection, drug toxicity, and cancer, whereas underimmunosuppression is associated with acute rejection. Intracellular adenosine triphosphate (iATP) concentration following CD4 cell activation provides an assessment of cellular immune function to help monitor the immune status of immunosuppressed patients. This assay has shown to be the first post‐transplant test related not only to the risk of acute rejection but also with the appearance of infection. The aim of our study was to compare the iATP concentrations of CD4 cells between healthy adults and kidney transplant recipients from a European population, analyzing the differences according to transplant clinical status. Samples from 81 kidney transplant patients who were admitted to our hospital over a nine‐month period were drawn. T‐cell activation was measured by determining the increase of iATP from CD4 cells. Results were compared with patient clinical status (rejection, infection, and stability). Three patients suffered an acute rejection episode and they were not included in the analysis (mean iATP concentration 247 ± 87 ng/mL). iATP concentrations differed significantly between stable and infected patients (313 ± 193 vs. 197 ± 114 ng/mL; p = 0.008). iATP concentration values were not related to the length of admission, age, peak and current panel reactive antibodies, mismatches, leukocytes, weight, creatinine, days after transplantation and blood levels of cyclosporin, tacrolimus, and sirolimus. This assay measures global immune responses of CD4 T cells from a whole‐blood sample, allowing for the assessment of the impact of immuno‐ suppressive drugs and of the patient’s underlying clinical conditions. This assay identifies transplant patients at risk for infection or rejection, providing information which can guide immunosuppressive therapy.

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice.

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) × C57BL/6)F1 mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW × C57BL/6)F1-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW × C57BL/6)F1-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.

Circulating cytokines in active polymyalgia rheumatica

Objective: To characterise the circulating cytokine profile and the cellular source of circulating cytokines in polymyalgia rheumatica (PMR). Methods: The study included 34 patients with active untreated PMR and 17 age-matched healthy controls (HC). Circulating cytokines were measured by cytometric bead array and ELISA. Intracellular cytokines were assessed in CD3+ and CD14+ cells by flow cytometry. Cytokines in cell culture supernatants were also determined after polyclonal stimulation of patients’ peripheral blood mononuclear cells. Results: Circulating levels of interleukin-6 (IL6) were significantly higher in subjects with active PMR than in HC. Corticosteroid (CS) treatment was followed by a decrease in the level of IL6. Intracellular cytokine staining showed that circulating monocytes did not produce higher amounts of proinflammatory cytokines in patients with PMR than in HC. There was a discordance between serum levels and cytokine-producing monocyte and T cells, and it was not possible to demonstrate a Th1 bias in the peripheral compartment. Conclusions: Active PMR is characterised by increased serum levels of IL6, but not of other proinflammatory cytokines, that are rapidly suppressed by CS treatment. As circulating monocytes do not show increased production of proinflammatory cytokines, IL6 may be mainly produced in the inflamed tissue. A study of the circulating cytokine profile and its cellular source may provide a clue to new therapeutic options.