Mir142 loss unlocks IDH2R140-dependent leukemogenesis through antagonistic regulation of HOX genes.

AML is a genetically heterogeneous disease and understanding how different co-occurring mutations cooperate to drive leukemogenesis will be crucial for improving diagnostic and therapeutic options for patients. MIR142 mutations have been recurrently detected in IDH-mutated AML samples. Here, we have used a mouse model to investigate the interaction between these two mutations and demonstrate a striking synergy between Mir142 loss-of-function and IDH2R140Q, with only recipients of double mutant cells succumbing to leukemia. Transcriptomic analysis of the non-leukemic single and leukemic double mutant progenitors, isolated from these mice, suggested a novel mechanism of cooperation whereby Mir142 loss-of-function counteracts aberrant silencing of Hoxa cluster genes by IDH2R140Q. Our analysis suggests that IDH2R140Q is an incoherent oncogene, with both positive and negative impacts on leukemogenesis, which requires the action of cooperating mutations to alleviate repression of Hoxa genes in order to advance to leukemia. This model, therefore, provides a compelling rationale for understanding how different mutations cooperate to drive leukemogenesis and the context-dependent effects of oncogenic mutations.

Author Correction: Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell-related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group-namely, the tolerant recipients-were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model.

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility "conventional" (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora.

Helicobacter pylori induces in-vivo expansion of human regulatory T cells through stimulating interleukin-1ß production by dendritic cells.

Helicobacter pylori is one of the most common infections in the world. Despite inciting inflammation, immunological clearance of the pathogen is often incomplete. CD4(+) CD25(hi) forkhead box protein 3 (FoxP3(+)) regulatory T cells (T(regs)) are potent suppressors of different types of immune responses and have been implicated in limiting inflammatory responses to H. pylori. Investigating the influence of H. pylori on T(reg) function and proliferation, we found that H. pylori-stimulated dendritic cells (DCs) induced proliferation in T(regs) and impaired their suppressive capability. This effect was mediated by interleukin (IL)-1ß produced by H. pylori-stimulated DCs. These data correlated with in-vivo observations in which H. pylori(+) gastric mucosa contained more T(regs) in active cell division than uninfected stomachs. Inciting local proliferation of T(regs) and inhibiting their suppressive function may represent a mechanism for the chronic gastritis and carcinogenesis attributable to H. pylori.

Isolation and characterization of free haem from the shell gland of quail and hen.

Free haem was isolated from the shell gland of the quail, Coturnix coturnix japonica, and of the fowl, Galinus domesticus, and characterized by HPLC-ESI-MS/MS. Quantification by HPLC gave values of 1.17-1.40 nmol/mg quail shell gland protein for haem, 1.66-2.17 nmol/mg protein for protoporphyrin and 0.25-0.40 nmol/mg protein for biliverdin. Possible implications of this previously unreported finding are discussed but they are not considered incompatible with the conclusion that all eggshell pigments are endogenously synthesized in the oviduct system.

Relative resistance of human CD4(+) memory T cells to suppression by CD4(+) CD25(+) regulatory T cells.

Successful expansion of functional CD4(+) CD25(+) regulatory T cells (T(reg)) ex vivo under good manufacturing practice conditions has made T(reg) -cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T(reg) cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4(+) CD25(-) T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti-CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T(reg) cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T(reg) -cell therapy, it is important to define the effectiveness of T(reg) cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T(reg) cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T(reg) cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T(reg) cells.

Transcriptional regulation of the mucosal immune system mediated by T-bet.

The immune system faces the arduous task of defending the mucosal surfaces from invading pathogens, but must simultaneously repress responses against commensal organisms and other inert antigens that are abundant in the external environment, as inappropriate immune activation might expose the host to increased risk of autoimmunity. The behavior of individual immune cells is governed by the expression of transcription factors that are responsible for switching immune response genes on and off. T-bet (T-box expressed in T cells) has emerged as one of the key transcription factors responsible for controlling the fate of both innate and adaptive immune cells, and its expression in different immune cells found at mucosal surfaces is capable of dictating the critical balance between permitting robust host immunity and limiting susceptibility to autoimmunity and allergy.

Translational mini-review series on Th17 cells: development of mouse and human T helper 17 cells.

There has been a considerable amount of interest in the immunological community about new phenotypic subsets of CD4(+) T cells, particularly cells that produce the cytokine interleukin (IL)-17 [named T helper type 17 (Th17) cells]. While the initial discovery of Th17 cells and the pathways that controlled their development was in the mouse, recent attention has shifted to the existence of these cells and the relevant upstream cytokine signals in humans. While it is clear that CD4(+) T cells producing IL-17 exist in vivo, their relevance to disease pathogenesis is only just being understood. In this paper, we review the data regarding the generation of human Th17 cells in vitro and the evidence that this effector population is important in human disease states.

Translational medicine and the NIHR Biomedical Research Centre concept.

The realization of scientific discovery being delivered to patients for their clinical benefit is termed Translational Medicine. This requires the bridging of excellence in both basic scientific endeavour and clinical care. Whilst there is consensus that it is important to drive translation for the benefit of patient care, the mechanism whereby this is to be achieved is less clear. In this article, we describe a novel strategy for the realization of effective translation that encompasses capacity building, a flexible proof of concept in man and the creation of a translational faculty adjacent to clinical research facilities that forms the basis of our NIHR Comprehensive Biomedical Research Centre. The opportunity to deliver world-class biomedical research from within the UK has never been greater.

Branching dendrites with resonant membrane: a "sum-over-trips" approach.

Dendrites form the major components of neurons. They are complex branching structures that receive and process thousands of synaptic inputs from other neurons. It is well known that dendritic morphology plays an important role in the function of dendrites. Another important contribution to the response characteristics of a single neuron comes from the intrinsic resonant properties of dendritic membrane. In this paper we combine the effects of dendritic branching and resonant membrane dynamics by generalising the "sum-over-trips" approach (Abbott et al. in Biol Cybernetics 66, 49-60 1991). To illustrate how this formalism can shed light on the role of architecture and resonances in determining neuronal output we consider dual recording and reconstruction data from a rat CA1 hippocampal pyramidal cell. Specifically we explore the way in which an Ih current contributes to a voltage overshoot at the soma.

The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease.

Uncommitted (naive) murine CD4+ T helper cells (Thp) can be induced to differentiate towards T helper 1 (Th1), Th2, Th17 and regulatory (Treg) phenotypes according to the local cytokine milieu. This can be demonstrated most readily both in vitro and in vivo in murine CD4+ T cells. The presence of interleukin (IL)-12 [signalling through signal transduction and activator of transcription (STAT)-4] skews towards Th1, IL-4 (signalling through STAT-6) towards Th2, transforming growth factor (TGF)-beta towards Treg and IL-6 and TGF-beta towards Th17. The committed cells are characterized by expression of specific transcription factors, T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for Tregs and RORgammat for Th17 cells. Recently, it has been demonstrated that the skewing of murine Thp towards Th17 and Treg is mutually exclusive. Although human Thp can also be skewed towards Th1 and Th2 phenotypes there is as yet no direct evidence for the existence of discrete Th17 cells in humans nor of mutually antagonistic development of Th17 cells and Tregs. There is considerable evidence, however, both in humans and in mice for the importance of interferon (IFN)-gamma and IL-17 in the development and progression of inflammatory and autoimmune diseases (AD). Unexpectedly, some models of autoimmunity thought traditionally to be solely Th1-dependent have been demonstrated subsequently to have a non-redundant requirement for Th17 cells, notably experimental allergic encephalomyelitis and collagen-induced arthritis. In contrast, Tregs have anti-inflammatory properties and can cause quiescence of autoimmune diseases and prolongation of transplant function. As a result, it can be proposed that skewing of responses towards Th17 or Th1 and away from Treg may be responsible for the development and/or progression of AD or acute transplant rejection in humans. Blocking critical cytokines in vivo, notably IL-6, may result in a shift from a Th17 towards a regulatory phenotype and induce quiescence of AD or prevent transplant rejection. In this paper we review Th17/IL-17 and Treg biology and expand on this hypothesis.

Unexplained acidosis of malnutrition: a study by ion-exchange chromatography/mass spectrometry.

Keto-acidosis is usually associated with uncontrolled diabetes and typically poses few diagnostic problems when presenting as hyperglycaemia, metabolic acidosis and a high anion gap. An emaciated patient suffering from Duchenne Muscular Dystrophy and volume depletion presented with acidosis of unknown origin. Preliminary investigations appeared to rule out lactic acidosis, diabetic keto-acidosis and acidosis due to base loss. We have previously reported a technique utilizing liquid chromatography coupled to mass spectrometry (LC-MS) which can be used to characterize the underlying aetiology of acidosis and applied it to ultrafiltrate derived from a blood sample taken from this patient. The anion profile obtained on the chromatogram showed elevated levels of acetoacetate and hydroxybutyrate but no evidence of lactic acidosis, nor was the profile typical of that seen in 'unexplained' acidosis. We concluded that the patient was suffering from keto-acidosis associated with starvation and dehydration, the biochemical features being obscured by both the patient's chronic malnutrition and minimal muscle mass. A combination of enteral feeding and rehydration led to prompt resolution of the patient's metabolic acidosis.

Leptin prevents the fall in plasma osteocalcin during starvation in male mice.

Plasma osteocalcin, a marker of osteoblastic activity, is reduced in starvation, malnutrition, and anorexia nervosa, resulting in low bone turnover osteoporosis. Contradictory findings about the role of leptin as a link between nutritional status and bone physiology have been reported. We demonstrate that leptin-deficient ob/ob and leptin-resistant db/db male mice have increased plasma osteocalcin, and that in male ob/ob mice osteocalcin is not decreased by starvation, unlike control mice. Intraperitoneal leptin administration increased plasma osteocalcin in male ob/ob mice, and prevented its fall during 24h fasting and 5 days of food restriction in normal male mice. This effect may be mediated via actions on the hypothalamic-pituitary-testicular or -growth hormone axes, or a direct action on osteoblasts. These studies support the hypothesis that the fall in leptin during starvation and weight loss is responsible for the associated reduction in osteoblast activity, and suggest a role for leptin in regulating bone turnover.

Amino acid and peptide conjugates of protoporphyrin: preparation and analysis by high-performance liquid chromatography, high-performance liquid chromatography/electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Amino acid and peptide conjugates of protoporphyrin have been prepared by reacting protoporphyringen with cysteine, glutathione and peptides containing a free thiol group under acidic conditions. The conjugates were formed by the addition of the thioamino acids or peptides to the vinyl groups of protoporphyrin during the autoxidation of protoporphyinogen to protoporphyrin and is free-radical-mediated. The conjugates were separated by high-performance liquid chromatography (HPLC) and characterized by HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). All the conjugates formed were diconjugates consisting of diastereoisomers.

Chemical and analytical aspects of areca nut.

A capillary zone electrophoretic (CZE) method for the rapid analysis of the major alkaloids (arecoline and guavacoline) in areca nut extract is described. Areca nuts were pulverized and then extracted with water by sonication in a water bath. After centrifugation, the supernatant was analysed on a fused-silica capillary with 100 mM ammonium acetate-acetic acid (pH 4.6) as the running buffer at a voltage of 20 kV and temperature of 30 degrees C. The method is applicable to the analysis of alkaloids in the nut, commercial preparations (pan masala) and in the saliva of areca nut chewers.

Urothelial malignant disease and Chinese herbal nephropathy.

We have previously reported occurrence of a specific type of nephropathy due to ingestion of Chinese herbs (Chinese herbal nephropathy [CHN]) in two patients in the UK. These cases highlighted the role of aristolochic acid in causing this nephropathy, which was first described in a Belgian cohort. We now report development of invasive transitional cell carcinoma of the urinary tract associated with the presence of aristolochic acid-DNA adducts in one of these patients. This work clearly shows the carcinogenic potential of aristolochic acid in this new type of nephropathy.

Leptin potentiates experimental autoimmune encephalomyelitis in SJL female mice and confers susceptibility to males.

SJL (H-2s) female mice are more susceptible than males to experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin-derived peptides. The reasons for this sexual dimorphism are unclear, but may include such factors as sex-related differences in immune responsiveness, hormonal effects and sex-linked genetic factors. Recent evidence indicates that leptin modifies T cell immunity promoting T helper (Th) 1 pro-inflammatory immune responses. Circulating leptin levels show a marked sexual dimorphism, being higher in females than in males. In the present study, we investigated whether leptin treatment altered the course of relapsing-remitting EAE, induced by the proteolipid protein peptide (PLP(139-151)), in SJL susceptible females and EAE-resistant males. Administration of leptin to female SJL mice before or after disease onset significantly worsened the disease, with a concomitant increase in the PLP(139-151)-specific delayed-type hypersensitivity (DTH) reactivity and in vitro IFN-gamma secretion. Leptin treatment at priming with antigen or before disease onset rendered male SJL mice susceptible to EAE, with the appearance of PLP(139-151)-specific DTH reactivity and a switch from a Th2 to Th1 pattern of cytokine release. Our findings indicate that leptin administration to susceptible females resulted in a more severe disease, and that reduced leptin levels in male SJL mice may contribute to the gender-related differences in the induction phase of EAE.