High Pretransplant BAFF Levels and B-cell Subset Polarized towards a Memory Phenotype as Predictive Biomarkers for Antibody-Mediated Rejection.

Antibody-mediated rejection (AbMR) is one of the leading causes of graft loss in kidney transplantation and B cells play an important role in the development of it. A B-cell activating factor (BAFF) is a cytokine involved in B cell ontogeny. Here, we analyzed whether B cell maturation and the effect of B cell soluble factors, such as BAFF could be involved in AbMR. Serum BAFF levels and B and T cell subpopulations were analyzed 109 kidney transplant patients before transplantation and at 6 and 12 months after kidney transplantation. Pretransplant serum BAFF levels as well as memory B cell subpopulations were significantly higher in those patients who suffered clinical AbMR during the first 12 months after kidney transplantation. Similar results were observed in the prospective analysis of patients with subclinical antibody-mediated rejection detected in the surveillance biopsy performed at 12 months after kidney transplantation. A multivariate analysis confirmed the independent role of BAFF in the development of AbMR, irrespective of other classical variables. Pretransplant serum BAFF levels could be an important non-invasive biomarker for the prediction of the development of AbMR and posttransplant increased serum BAFF levels contribute to AbMR.

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells.

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.

Involvement of the intrinsic and extrinsic cell-death pathways in the induction of apoptosis of mature lymphocytes by the Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (LT) exhibits a broad range of immunomodulatory activities, including the induction of lymphocyte-programmed cell death. In previous studies, we have demonstrated that in vivo LT promotes apoptosis of immature T and B cells through the stimulation of endogenous glucocorticoids. In the present study, we show that the extrinsic cell-death pathway as well as the apoptosis-inducing factor do not participate in the LT-induced elimination of thymocytes. In contrast to developing lymphocytes, LT promotes the death of mature lymphocytes by both glucocorticoid- and Fas death receptor/Fas ligand-dependent mechanisms. However, the dependency of these mechanisms in the LT-induced cell-death activity seems to be different among CD4(+) and CD8(+) T cells. Altogether, our study shows that the same bacterial toxin can induce apoptosis of lymphoid cells through several mechanisms depending on the status of differentiation of these cells.

Is it possible to predict the evolution of IgAN? Validation of the IgA nephropathy progression calculator and its relationship with MEST-C score in our population. / ¿Es posible predecir la evolución de la nefropatia IgA? Validamos la calculadora de progresión de nefropatia IgA y su relación con Oxford score en nuestra población.

INTRODUCTION: IgA nephropathy (IgAN) is the most common and heterogeneous glomerular nephropathy. Several strategies have been used to determine the risk of progression to ESRD. We evaluate the prognostic significance and correlate the IgAN progression calculator (IgANPC) and the Oxford/MEST-C score in our population. MATERIAL AND METHODS: We performed a retrospective study of biopsied patients with diagnosis of IgA nephropathy from 1990 to 2015. We classified the biopsies using MEST-C score and we correlated the score to clinical evolution. We also calculated the risk of progression with the online IgANPC at the time of the biopsy. RESULTS: We analysed 48 biopsies, 83% of which were men with a mean age of 45 years at the time of the biopsy. Patients with a biopsy E1 according to MEST-C score had a higher IgANPC score than those with E0 (P=.021). The Pearson's correlation for the percentage of crescents and the IgANPC risk score was statistically significant (P=.014) with r=0.357. The percentage of patients with eGFR above 30 ml/min at 10 years was 100% for the low-risk group (group 1 of IgANPC), and 0% for the high-risk group (group 3), log rank P=0.001. The log rank comparison for variables of the MEST-C score, presented statistically significant results between E (0.036) and S (0.022) and the eGFR time<30 ml/min. A statistically significant relationship was also observed between T1 and eGFR<30 ml/min. The multivariate Cox regression analysis for IgANPC and eGFR<30 ml/min demonstrated a strong correlation (P=.016) between the risk group and eGFR <30 ml/min. CONCLUSION: In our study population, the IgANPC predicts the time to eGFR<30 ml/min, and adds information independent of the MEST. The MEST-C classification and IgANPC are useful and independent ÿolos for prognostic prediction, but more studies are needed to validate its use in the general population.

Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L.

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling pathways activated by EGFR and HER2 may help unravel novel therapeutic targets and molecular markers of response. Here, we show that treatment of breast cancer cell lines with blocking antibodies against EGFR (cetuximab) or HER2 (trastuzumab) promotes the specific induction of proapoptotic Bnip3L and chemosensitization. Moreover, we found that the Bnip3L gene is transcriptionally activated by FoxO3a. Trastuzumab-mediated induction of Bnip3L and nuclear translocation of FoxO3a was also shown in pleural effusion cells from a breast cancer patient. Transfection of breast cancer cells with constitutively active FoxO3a or with Bnip3L promotes sensitization to chemotherapy-induced apoptosis. On the contrary, blockade of Bnip3L expression by a small interfering RNA strategy significantly diminished the chemosensitizing effect of cetuximab. We found also an inverse correlation between EGFR and Bnip3L expression in surgical specimens from patients with breast cancer. Therefore, blockading EGFR or HER2 specifically up-regulates Bnip3L, which is required for chemosensitization of breast cancer cells. This novel pathway provides also the rationale for therapeutic strategies aimed to induce the expression of Bnip3L.

Within-Patient Variability in Tacrolimus Blood Levels Predicts Kidney Graft Loss and Donor-Specific Antibody Development.

BACKGROUND: Lack of adherence to immunosuppressive drugs is a risk factor for development of de novo donor-specific antibodies (dnDSA) and can contribute to antibody-mediated rejection and graft loss. Moreover, nonadherence is the main determinant of immunosuppressive drug level variability. High intrapatient variability of tacrolimus relates to a worse outcome in transplant recipients through unknown mechanisms. We hypothesized that a high within-patient variability of tacrolimus could increase the rate of dnDSA development and contribute to further death-censored graft loss (DCGL). METHODS: We included 310 adult renal transplants receiving twice-daily tacrolimus throughout their first posttransplant year, with (1) at least 3 blood trough levels available to calculate coefficient of variation (CV) from month 4 to 12, (2) graft survival longer than 1 year, and (3) absence of pretransplant DSA. The dnDSA were analyzed in sera at 1, 3, and 5 years and around 6 month before the last follow-up visit or graft loss by single-antigen beads. RESULTS: During the follow-up, 53 patients lost their graft excluding death. A total of 116 patients (37.4%) had a CV greater than 30% and 39 (12.6%) developed dnDSA. Coefficient of variation greater than 30% (hazards ratio, 2.613; 95% confidence interval, 1.361-5.016; P = 0.004) independently related to DCGL. Acute rejection, re-transplant and CV greater than 30% (hazards ratio, 2.925; 95% confidence interval, 1.473-5.807; P = 0.002) were the only variables related to dnDSA development by Cox regression analysis. CONCLUSIONS: Tacrolimus level variability is a strong risk factor for dnDSA development and DCGL. Variability must be added to the current monitoring of kidney transplant recipients due to its relationship with adherence and to graft outcome.

Critical role for DP5/Harakiri, a Bcl-2 homology domain 3-only Bcl-2 family member, in axotomy-induced neuronal cell death.

The survival of neurons is maintained primarily by neurotrophic factors that suppress the apoptotic program. Axotomy or removal of peripheral targets causes neuronal cell death, but the mechanisms involved in the induction of this type of cell death remain poorly understood. Here, we show that DP5/Harakiri, a Bcl-2 homology domain 3-only member of the Bcl-2 family, is induced in motoneurons after transection of the hypoglossal nerve in mice and in sympathetic neurons after nerve growth factor (NGF) withdrawal. To assess the role of DP5 in neuronal cell death, mutant mice deficient in DP5 were generated by gene targeting. DP5-/- mice were viable and exhibited normal postnatal development. Notably, motoneurons from DP5-/- mice were highly protected from cell death induced by resection of the hypoglossal nerve compared with motoneurons from DP5+/+ littermate mice. In addition, deficiency of DP5 in superior cervical ganglia (SCG) neurons resulted in delayed neuronal cell death triggered by NGF withdrawal. Analysis of SCG neurons from DP5-/- mice revealed increased preservation of mitochondrial membrane potential and reduced activation of caspase-3 compared with neurons from wild-type mice. These results indicate that DP5 plays an important role in neuronal cell death induced by axotomy and NGF deprivation through the regulation of mitochondrial function and caspase-3 activation.

Leflunomide derivative FK778 inhibits production of antibodies in an experimental model of alloreactive T-B cell interaction.

OBJECTIVES: The contribution of humoral immune response in allograft and xenograft rejection has been clearly demonstrated in recent years. For this reason, inhibition of alloantibody production has become essential in managing transplanted patients. Here, we assessed the effects of the leflunomide derivative FK778 (FK778) in the control of antibody production resulting from semiallogeneic cognate T-B-cell interactions. MATERIALS AND METHODS: BALB/c mice were tolerized at birth with semiallogeneic spleen cells from (BALB/c X C57BL/6) F1 mice, with or without overexpression of human bcl-2 transgene in B cells. These tolerized mice were treated with different dosages of FK778, either from birth, or from the third week of age, when autoantibody production was detected. The production of autoantibodies, used as markers of semiallogeneic cognate T-B - cell interactions, was evaluated at different time points during drug administration or after the interruption of treatment. RESULTS: FK778 treatment started at birth inhibited the production of semiallogeneic-driven antibodies in a dose-dependent manner. In addition, FK778 also reduced the levels of preformed circulating autoantibodies in adult mice, although the dosage required was 4 times higher than that used in neonates. However, the levels of IgG antibodies in these tolerized mice increased after FK778 withdrawal, indicating that FK778 failed to induce tolerance to semiallogeneic host CD4+ Th2 and/or donor B cells. CONCLUSIONS: Our results demonstrate the efficacy of FK778 in the control of antibody production resulting from semiallogeneic cognate T-B - cell interactions.

A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

Embryonic loss due to exposure to polycyclic aromatic hydrocarbons is mediated by Bax.

The high miscarriage rates observed in women smokers raises the possibility that chemicals in cigarette smoke could be detrimental to embryo development. Previous studies have established that polycyclic aromatic hydrocarbons (PAHs), transactivate the arylhydrocarbon receptor (AhR), leading to cell death. Herein we show that PAH exposure results in murine embryo cell death, acting as a potential mechanism underlying cigarette-smoking-induced pregnancy loss. Cell death was preceded by increases in Bax levels, activation of caspase-3 and decreased litter size. Chronic exposure of females to PAHs prior to conception impaired development, resulting in a higher number of resorptions. This embryonic loss could not be prevented by the disruption of Hrk, but was diminished in embryos lacking Bax. We conclude that exposure of early embryos to PAHs reduces the allocation of cells to the embryonic and placental lineages by inducing apoptosis in a Bax-dependent manner, thus compromising the developmental potential of exposed embryos.

Characterization of 8p21.3 chromosomal deletions in B-cell lymphoma: TRAIL-R1 and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes.

Deletions of chromosome 8p are a recurrent event in B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of a tumor suppressor gene. We have characterized these deletions using comparative genomic hybridization to microarrays, fluorescence in situ hybridization (FISH) mapping, DNA sequencing, and functional studies. A minimal deleted region (MDR) of 600 kb was defined in chromosome 8p21.3, with one mantle cell lymphoma cell line (Z138) exhibiting monoallelic deletion of 650 kb. The MDR extended from bacterial artificial chromosome (BAC) clones RP11-382J24 and RP11-109B10 and included the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene loci. Sequence analysis of the individual expressed genes within the MDR and DNA sequencing of the entire MDR in Z138 did not reveal any mutation. Gene expression analysis and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) showed down-regulation of TRAIL-R1 and TRAIL-R2 receptor genes as a consistent event in B-NHL with 8p21.3 loss. Epigenetic inactivation was excluded via promoter methylation analysis. In vitro studies showed that TRAIL-induced apoptosis was dependent on TRAIL-R1 and/or -R2 dosage in most tumors. Resistance to apoptosis of cell lines with 8p21.3 deletion was reversed by restoration of TRAIL-R1 or TRAIL-R2 expression by gene transfection. Our data suggest that TRAIL-R1 and TRAIL-R2 act as dosage-dependent tumor suppressor genes whose monoallelic deletion can impair TRAIL-induced apoptosis in B-cell lymphoma.

Genetic variation and activity of mouse Nod2, a susceptibility gene for Crohn's disease.

Genetic variation in human Nod2 has been associated with susceptibility to Crohn's disease. The mouse Nod2 locus is located at chromosome 8 and composed of 12 exons, 11 of which encode the Nod2 protein. Sequence analysis of Nod2 from 45 different strains of Mus musculus and Mus spretus revealed extensive polymorphism involving all exons of Nod2. Of the 140 polymorphic sites identified, 68 were located in the coding region, of which 28 created amino acid substitutions in Nod2. Expression of mouse Nod2 activated NF-kappaB and conferred responsiveness to bacterial components, an activity that was deficient in mutants corresponding to those associated with susceptibility to Crohn's disease. These studies demonstrate a conserved role for Nod2 in the response to bacterial components and suggest that selective evolutionary pressure exerted by pathogens may have contributed to the high level of variability of Nod2 sequences in both humans and mice.