Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W.

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl(-)/HCO(-) 3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.

Fragile TIM-4-expressing tissue resident macrophages are migratory and immunoregulatory.

Macrophages characterized as M2 and M2-like regulate immune responses associated with immune suppression and healing; however, the relationship of this macrophage subset to CD169+ tissue-resident macrophages and their contribution to shaping alloimmune responses is unknown. Here we identified a population of M2-like tissue-resident macrophages that express high levels of the phosphatidylserine receptor TIM-4 and CD169 (TIM-4hiCD169+). Labeling and tracking of TIM-4hiCD169+ macrophages in mice revealed that this population is a major subset of tissue-resident macrophages, homes to draining LNs following oxidative stress, exhibits an immunoregulatory and hypostimulatory phenotype that is maintained after migration to secondary lymphoid organs, favors preferential induction of antigen-stimulated Tregs, and is highly susceptible to apoptosis. Moreover, CD169+ tissue-resident macrophages were resistant to oxidative stress-induced apoptosis in mice lacking TIM-4. Compared with heart allografts from WT mice, Tim4-/- heart allografts survived much longer and were more easily tolerized by non-immunosuppressed recipients. Furthermore, Tim4-/- allograft survival was associated with the infiltration of Tregs into the graft. Together, our data provide evidence that M2-like TIM-4hiCD169+ tissue-resident macrophages are immunoregulatory and promote engraftment of cardiac allografts, but their influence is diminished by TIM-4-dependent programmed cell death.

TIM family proteins promote the lysosomal degradation of the nuclear receptor NUR77.

T cell immunoglobulin and mucin domain (TIM) proteins are cell-surface signaling receptors in T cells and scavenger receptors in antigen-presenting cells and kidney tubular epithelia. Here, we demonstrated a function for TIM proteins in mediating the degradation of NUR77, a nuclear receptor implicated in apoptosis and cell survival. TIM proteins interacted with and mediated the lysosomal degradation of NUR77 in a phosphoinositide 3-kinase-dependent pathway. We also showed dynamic cycling of TIM-1 to and from the cell surface through clathrin-dependent constitutive endocytosis. Blocking this process or mutating the phosphatidylserine-binding pocket in TIM-1 abrogated TIM-1-mediated degradation of NUR77. In an in vitro model of kidney injury, silencing TIM-1 increased NUR77 abundance and decreased epithelial cell survival. These results show that TIM proteins may affect immune cell function and the response of the kidney to injury.

OX40 signaling favors the induction of T(H)9 cells and airway inflammation.

The mechanisms that regulate the T(H)9 subset of helper T cells and diseases mediated by T(H)9 cells remain poorly defined. Here we found that the costimulatory receptor OX40 was a powerful inducer of T(H)9 cells in vitro and T(H)9 cell-dependent airway inflammation in vivo. In polarizing conditions based on transforming growth factor-ß (TGF-ß), ligation of OX40 inhibited the production of induced regulatory T cells and the T(H)17 subset of helper T cells and diverted CD4(+)Foxp3(-) T cells to a T(H)9 phenotype. Mechanistically, OX40 activated the ubiquitin ligase TRAF6, which triggered induction of the kinase NIK in CD4(+) T cells and the noncanonical transcription factor NF-κB pathway; this subsequently led to the generation of T(H)9 cells. Thus, our study identifies a previously unknown mechanism for the induction of T(H)9 cells and may have important clinical implications in allergic inflammation.

Introduction of modified cervical cardiac transplant model in mice.

OBJECTIVES: The murine cervical heterotopic heart transplant model was initially designed to test the immune response to third-party allografts, modified by cuff techniques. While cuff techniques simplify the execution of this procedure, cutting of the carotid artery and the external jugular vein alters the blood supply to central nervous system and makes it difficult to achieve long-term graft survival. In the present study, we describe modified techniques that preserve the continuity and function of blood vessels and improve transplant integrity. MATERIALS AND METHODS: The modified techniques in this study comprise the following aspects: (1) Preservation of the sternal head of the right sternocleidomastoid muscle, (2) use of the donor's intrathoracic inferior vena cava for anastomosis and (3) preservation of the function of the recipient's carotid artery and external jugular vein and thus, continuity of blood flow to the central nervous system. RESULTS: Stable, long-term, disease-free allograft survival has been achieved with syngeneic transplants (> 200 days), whereas allografts from fully major histocompatibility complex-mismatched donors were acutely rejected in a time similar to the traditional abdominal heterotopic heart transplant model (8.2 ± 1.3 vs 8.4 ± 1.4 days; P = .73 in the Mantel Cox test, and P = .61 in the Gehan-Breslow-Wilcoxon test). Similar alloresponses could be induced in these 2 models. CONCLUSIONS: It is possible and feasible to achieve long-term graft survival in the mouse cervical heart transplant model using the modified procedures described in the present study.

Orphan nuclear receptor Nur77 promotes acute kidney injury and renal epithelial apoptosis.

Nur77 and its family members Nurr1 and Nor-1 are inducible orphan nuclear receptors that orchestrate cellular responses to diverse extracellular signals. In epithelia, Nur77 can act as a potent proapoptotic molecule in response to cellular stress, suggesting a possible role for this nuclear receptor in the tissue response to injury. Here, we found that Nur77 promotes epithelial cell apoptosis after AKI. Injury of proximal tubular epithelial cells rapidly and strongly induced Nur77, Nor-1, and Nurr1 both in vitro and in vivo. After renal ischemia-reperfusion, Nurr77-deficient mice exhibited less apoptosis of tubular epithelial cells and better renal function than wild-type mice. Nur77-mediated renal injury involved a conformational change of Bcl2 and an increase in the protein levels of proapoptotic Bcl-xS. Ligand-activated retinoic acid receptors repressed Nur77 induction and function. Pretreatment of wild-type mice with retinoic acid before renal ischemia-reperfusion blunted the induction of Nur77, conferred protection of renal function, attenuated renal histologic injury, and reduced the expression of epithelial-derived proinflammatory cytokines. Retinoic acid also inhibited hypoxia-mediated induction of proinflammatory cytokines in cultured renal epithelial cells. Results obtained from proximal tubule cultures derived from Nur77-deficient mice suggested that the inhibition of Nur77 expression mediated the renoprotective effects of retinoic acid. In summary, Nur77 promotes epithelial apoptosis after ischemia-reperfusion injury, and retinoic acid-mediated inhibition of Nur77 expression is a promising therapeutic strategy for the prevention of AKI.

Cancer therapy via modulation of micro RNA levels: a promising future.

Micro RNAs (miRNAs) are a class of naturally occurring ∼ 22 nt long non-coding small RNA molecules that regulate the expression of a wide range of genes involved in development, growth, proliferation and apoptosis. miRNAs are evolutionarily conserved from plants to animals, and they regulate and fine-tune a diverse array of biological processes. Recently, they have been shown to act as either oncogenes or tumor suppressors in a wide variety of tumors. Here, we review the studies that document the role of miRNAs as key players in human cancer and the potential therapeutic modality of exploiting miRNAs for cancer prognosis and treatment.

Tim-1 signaling substitutes for conventional signal 1 and requires costimulation to induce T cell proliferation.

Differentiation and clonal expansion of Ag-activated naive T cells play a pivotal role in the adaptive immune response. T cell Ig mucin (Tim) proteins influence the activation and differentiation of T cells. Tim-3 and Tim-2 clearly regulate Th1 and Th2 responses, respectively, but the precise influence of Tim-1 on T cell activation remains to be determined. We now show that Tim-1 stimulation in vivo and in vitro induces polyclonal activation of T cells despite absence of a conventional TCR-dependent signal 1. In this model, Tim-1-induced proliferation is dependent on strong signal 2 costimulation provided by mature dendritic cells. Ligation of Tim-1 upon CD4(+) T cells with an agonist anti-Tim-1 mAb elicits a rise in free cytosolic calcium, calcineurin-dependent nuclear translocation of NF-AT, and transcription of IL-2. Because Tim-4, the Tim-1 ligand, is expressed by mature dendritic cells, we propose that interaction between Tim-1(+) T cells and Tim-4(+) dendritic cells might ensure optimal stimulation of T cells, when TCR-derived signals originating within an inflamed environment are weak or waning.

TIM-4 expressed on APCs induces T cell expansion and survival.

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.