CD40-mediated activation of NF-kappa B in airway epithelial cells

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.     

Patterns of initial recurrence and prognosis after sentinel lymph node biopsy and selective lymphadenectomy for melanoma

The histologic status of the sentinel lymph node is a highly significant prognostic factor for patients with clinically localized cutaneous melanoma. The patterns of initial treatment failure of patients with positive sentinel lymph node biopsy versus those with negative results have not been well described. The purpose of this study was to determine the relative prognostic importance of sentinel lymph node status and to compare patterns of initial treatment failure and prognosis of node-positive versus node-negative cutaneous melanoma patients staged by sentinel lymph node biopsy and selective lymphadenectomy. The authors reviewed the pertinent demographic and surgical data in a consecutive series of patients with cutaneous melanoma who underwent sentinel lymph node staging of nonpalpable regional nodes. Sentinel lymph node biopsy was performed using a combination of blue dye and radiolocalization. Patients with positive biopsy results underwent selective lymphadenectomy, whereas those with negative results were observed. Site(s) and date(s) of initial recurrence and death were determined, and disease-free and overall survival probabilities were compared between positive and negative groups using the log-rank test and multivariable Cox regression analysis. Between February of 1994 and August of 2000, 408 patients with melanoma underwent sentinel lymph node biopsy to stage 518 regional lymph node basins. Mean Breslow tumor thickness was 2.27 mm (range, 0.2 to 14.0 mm). Eighty-five patients (20.8 percent) had at least one histologically positive sentinel lymph node, and selective lymphadenectomy yielded additional positive lymph nodes in 18 of 84 patients (21.4 percent). Recurrences were noted in 70 patients (17 percent) at a median follow-up period of 31.4 months. Recurrences were more frequent in patients with positive biopsy results (36.5 percent) than in those with negative results (12.1 percent, p < 0.0001). Distant sites of initial recurrence were more likely in the positive group than in the negative group (71 percent versus 49 percent of recurrences, respectively; p = 0.06). The false-negative rate for sentinel lymph node staging was 4.5 percent and overall accuracy was 99 percent compared with clinical follow-up. Disease-free and overall survival correlated significantly with tumor thickness, ulceration, sentinel lymph node status, and the number of tumor-positive lymph nodes (two-sided p < 0.0001 for all comparisons). Multivariable analysis revealed that sentinel lymph node status (p = 0.003), tumor thickness (p = 0.016), ulceration (p = 0.006), and age (p = 0.003) were significant independent predictors of survival for the entire group. Tumor thickness and ulceration were significant predictors of recurrence and survival in sentinel node-negative patients but not in sentinel node-positive patients. Sentinel lymph node histology is possibly the most important negative predictor of early recurrence and survival in patients with American Joint Committee on Cancer stage I and II melanoma. The number of positive lymph nodes provides additional prognostic information. Although sentinel node-negative patients are a prognostically favorable group, various combinations of local and regional recurrences comprise the most common pattern of initial relapse after a negative sentinel lymph node biopsy result.     

Targeting B cells for the treatment of rheumatoid arthritis

The role of B cells in rheumatoid arthritis (RA) has been debated for decades. However, recent clinical trial data indicating that depletion of B cells in RA patients is of therapeutic benefit has validated the importance of this cell type in the pathogenesis of the disease. Elucidation of the molecular basis of B cell development and activation has allowed the identification of a number of possible therapeutic targets that are appealing for drug development. This review discusses briefly a number of these molecules and the rationale for targeting them for the treatment of RA.     

A defect in a novel ADAMTS family member is the cause of the belted white-spotting mutation

Several features of the pigment defect in belted (bt) mutant mice suggest that it occurs as a result of a defect in melanocyte development that is unique from those described for other classical white-spotting mutations. We report here that bt mice carry mutations in Adamts20, a novel member of the ADAMTS family of secreted metalloproteases. Adamts20 shows a highly dynamic pattern of expression in the developing embryo that generally precedes the appearance of melanoblasts in the same region, and is not expressed in the migrating cells themselves. Adamts20 shows remarkable homology with GON-1, an ADAMTS family protease required for distal tip cell migration in C. elegans. Our results suggest that the role of ADAMTS proteases in the regulation of cell migration has been conserved in mammalian development.     

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19⁺CD1d^highCD5⁺ B cells (CD1d^highCD5⁺ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1d^highCD5⁺ Breg cells as compared to age-matched WT control mice. CD1d^highCD5⁺ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4⁺ T cells. Interestingly, CD1d^highCD5⁺ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.     

Identification of the lipooligosaccharide biosynthetic gene cluster from Mycobacterium marinum

Lipooligosaccharides (LOSs) are antigenic glycolipids that are present in some species of Mycobacterium including the Canetti strain of M. tuberculosis. The core LOS structures from several mycobacterial organisms have been established, but the biosynthetic pathways of LOSs remain unknown. In this study, we describe two transposon insertion mutants of M. marinum that exhibit altered colony morphology. Cell wall analysis reveals that the MRS1271 mutant is defective in the synthesis of LOS-II, whereas the MRS1178 mutant accumulates an intermediate between LOS-I and -II. The genetic lesions were localized to two genes, MM2309 and MM2332. MM2309 encodes a UDP-glucose dehydrogenase that is involved in the synthesis of d-xylose. MM2332 is predicted to encode a decarboxylase. These two genes and a previously identified losA gene are localized in a gene cluster likely to be involved in the biosynthesis of LOSs. Our results also show that LOSs play an important role in sliding motility, biofilm formation, and infection of host macrophages. Taken together, our studies have identified, for the first time, a LOS biosynthetic locus. This is an important step in assessing the differential distribution of LOSs among Mycobacterium species and understanding the role of LOSs in mycobacterial virulence.     

Dimerization of inositol monophosphatase <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> SuhB is not constitutive,but induced by binding of the activator Mg<Superscript>2+</Superscript>

Background  

The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear.     

A novel series of IKKβ inhibitors part II: description of a potent and pharmacologically active series of analogs

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.