JAK inhibitor treatment for inborn errors of JAK/STAT signaling: An ESID/EBMT-IEWP retrospective study.

BACKGROUND: Inborn errors of immunity (IEI) with dysregulated JAK/STAT signaling present with variable manifestations of immune dysregulation and infections. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but initially reported outcomes were poor. JAK inhibitors (JAKi) offer a targeted treatment option that may be an alternative or bridge to HSCT. However, data on their current use, treatment efficacy and adverse events are limited. OBJECTIVE: We evaluated the current off-label JAKi treatment experience for JAK/STAT inborn errors of immunity (IEI) among European Society for Immunodeficiencies (ESID)/European Society for Blood and Marrow Transplantation (EBMT) Inborn Errors Working Party (IEWP) centers. METHODS: We conducted a multicenter retrospective study on patients with a genetic disorder of hyperactive JAK/STAT signaling who received JAKi treatment for at least 3 months. RESULTS: Sixty-nine patients (72% children) were evaluated (45 STAT1 gain of function [GOF], 21 STAT3-GOF, 1 STAT5B-GOF, 1 suppressor of cytokine signaling 1 [aka SOCS1] loss of function, 1 JAK1-GOF). Ruxolitinib was the predominantly prescribed JAKi (80%). Overall, treatment resulted in improvement (partial or complete remission) of clinical symptoms in 87% of STAT1-GOF and in 90% of STAT3-GOF patients. We documented highly heterogeneous dosing and monitoring regimens. The response rate and time to response varied across different diseases and manifestations. Adverse events including infection and weight gain were frequent (38% of patients) but were mild (grade I-II) and transient in most patients. At last follow-up, 52 (74%) of 69 patients were still receiving JAKi treatment, and 11 patients eventually underwent HSCT after receipt of previous JAKi bridging therapy, with 91% overall survival. CONCLUSIONS: Our study suggests that JAKi may be highly effective to treat symptomatic JAK/STAT IEI patients. Prospective studies to define optimal JAKi dosing for the variable clinical presentations and age ranges should be pursued.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa3 oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa3 oxidase supercomplex (bcc-aa3 supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa3 supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa3 supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa3 supercomplex physically associate.

Tumor-derived TGF-ß inhibits mitochondrial respiration to suppress IFN-γ production by human CD4+ T cells.

Transforming growth factor-ß (TGF-ß) is produced by tumors, and increased amounts of this cytokine in the tumor microenvironment and serum are associated with poor patient survival. TGF-ß-mediated suppression of antitumor T cell responses contributes to tumor growth and survival. However, TGF-ß also has tumor-suppressive activity; thus, dissecting cell type-specific molecular effects may inform therapeutic strategies targeting this cytokine. Here, using human peripheral and tumor-associated lymphocytes, we investigated how tumor-derived TGF-ß suppresses a key antitumor function of CD4+ T cells, interferon-γ (IFN-γ) production. Suppression required the expression and phosphorylation of Smad proteins in the TGF-ß signaling pathway, but not their nuclear translocation, and depended on oxygen availability, suggesting a metabolic basis for these effects. Smad proteins were detected in the mitochondria of CD4+ T cells, where they were phosphorylated upon treatment with TGF-ß. Phosphorylated Smad proteins were also detected in the mitochondria of isolated tumor-associated lymphocytes. TGF-ß substantially impaired the ATP-coupled respiration of CD4+ T cells and specifically inhibited mitochondrial complex V (ATP synthase) activity. Last, inhibition of ATP synthase alone was sufficient to impair IFN-γ production by CD4+ T cells. These results, which have implications for human antitumor immunity, suggest that TGF-ß targets T cell metabolism directly, thus diminishing T cell function through metabolic paralysis.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Human regulatory T cells lack the cyclophosphamide-extruding transporter ABCB1 and are more susceptible to cyclophosphamide-induced apoptosis.

ATP-binding cassette (ABC) transporters, including ABC-transporter B1 (ABCB1), extrude drugs, metabolites, and other compounds (such as mitotracker green (MTG)) from cells. Susceptibility of CD4(+) regulatory T (Treg) cells to the ABCB1-substrate cyclophosphamide (CPA) has been reported. Here, we characterized ABCB1 expression and function in human CD4(+) T-cell subsets. Naïve, central memory, and effector-memory CD4(+) T cells, but not Treg cells, effluxed MTG in an ABCB1-dependent manner. In line with this, ABCB1 mRNA and protein was expressed by nonregulatory CD4(+) T-cell subsets, but not Treg cells. In vitro, the ABCB1-substrate CPA was cytotoxic for Treg cells at a 100-fold lower dose than for nonregulatory counterparts, and, inversely, verapamil, an inhibitor of ABC transporters, increased CPA-toxicity in nonregulatory CD4(+) T cells but not Treg cells. Thus, Treg cells lack expression of ABCB1, rendering them selectively susceptible to CPA. Our findings provide mechanistic support for therapeutic strategies using CPA to boost anti-tumor immunity by selectively depleting Treg cells.

Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch.

Antigen-experienced memory T cells acquire effector function with innate-like kinetics; however, the metabolic requirements of these cells are unknown. Here we show that rapid interferon-γ (IFN-γ) production of effector memory (EM) CD8(+) T cells, activated through stimulation mediated by the T cell antigen receptor (TCR) and the costimulatory receptor CD28 or through cognate interactions, was linked to increased glycolytic flux. EM CD8(+) T cells exhibited more glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity at early time points, before proliferation commenced, than did naive cells activated under similar conditions. CD28 signaling via the serine-threonine kinase Akt and the metabolic-checkpoint kinase mTORC2 was needed to sustain TCR-mediated immediate-early glycolysis. Unlike glycolysis in proliferating cells, immediate-early glycolysis in memory CD8(+) T cells was rapamycin insensitive. Thus, CD8(+) memory T cells have an Akt-dependent 'imprinted' glycolytic potential that is required for efficient immediate-early IFN-γ recall responses.

Mordedura canina: 140 casos em 6 meses / Dog bites: 140 cases in 6 months

O presente estudo é do tipo série prospectiva de casos e compreendeu 140 pacientes consecutivos atendidos inicialmente na emergência do Hospital do Trabalhador no período de maio a novembro de 2001. Oitenta e quatro por cento dos pacientes procediam da cidade de Curitiba; 65 por cento eram homens. A idade mais atingida foi entre 0 e 10 anos (25,7 por cento), sendo a mediana de 23 anos. Trinta e um por cento dos cäes tinham história anterior de ataque a outras pessoas. A face doi atingida em 31 pacientes (22,1 por cento) sendo 22 (71 por cento) destes, menores de 10 anos.Os membros superiores foram atingidos 102 vezes e o inferior 51. As crianças foram as mais atingidas, sendo o membro superior, membro inferior e face respectivamente os mais afetados. Quinze pacientes (10,7 por cento) necessitaram de internamento para tratamento cirúrgico, demonstrnado maior gravidade em nossa casuística do que o visto na literatura (4 por cento)