A study of HLA (Bg) on red cells and platelets by immunoblotting with monoclonal antibodies

HLA class I antigens (Bg) on red cells (RBCs) are expressed by some normal donors and by many patients with systemic lupus erythematosus (SLE). To identify the membrane components previously detected by hemagglutination with HLA class I-specific monoclonal antibodies (MoAbs), RBC membrane preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the HLA class I MoAbs. Two components were obtained that reacted with the MoAbs: a heavy chain of 45 kDa and a light chain termed beta2-microglobulin (beta2-M) of 11 kDa. The effect of chloroquine and acid elution in stripping HLA antigens is shown to be due to the removal of beta2-M, as only that component was detected in eluates from reactive RBCs. Neither antibody elution method affected the heavy chain expression assessed by immunoblotting. It is concluded that HLA class I antigens on RBCs are integral membrane components of the type normally found and wisely distributed on many nucleated cells. Platelets, which have stronger HLA class I antigen expression, were also studied, and their membrane preparations yielded heavy chain and beta2-M molecules; the effect of chloroquine treatment was harder to assess than that of acid elution, owing to the sensitivity with which both components are detected in immunoblotting. In eluates obtained from acid treatment only beta2-M is detected.     

Benefits of a school-based asthma treatment program in the absence of secondhand smoke exposure: results of a randomized clinical trial

BACKGROUND: Daily maintenance medications are recommended for all children with mild persistent to severe persistent asthma; however, poor adherence to these medications is common. OBJECTIVE: To evaluate the impact of school-based provision of inhaled corticosteroids on asthma severity among urban children with mild persistent to severe persistent asthma. DESIGN: Children aged 3 to 7 years with mild persistent to severe persistent asthma were identified at the start of the 2000-2001 and 2001-2002 school years in Rochester. Children were assigned randomly to a school-based care group (daily inhaled corticosteroids provided through the school) or a usual-care group (inhaled corticosteroids not given through school). MAIN OUTCOME MEASURE: Improvement in parent-reported symptom-free days. RESULTS: Of 242 eligible children, 184 were enrolled from 54 urban schools. Data for 180 children were available. Parents of children in the school-based care group had a greater improvement in quality of life compared with parents of children in the usual-care group (change score, 0.63 vs 0.24; P =.047); also, children in the school-based care group vs the usual-care group missed less school because of asthma (mean total days missed, 6.8 vs 8.8; P =.047) and experienced more symptom-free days during the early winter months (mean days per 2-week period, 9.2 vs 7.3; P =.02). A post hoc analysis revealed that all significant findings were produced by differences among children who were not exposed to secondhand smoke. Furthermore, among children not exposed to smoke, those in the school-based care group vs the usual-care group had more symptom-free days overall (11.5 vs 10.5; P =.046), had fewer days needing rescue medications (1.6 vs 2.3; P =.03), and were less likely to have had 3 or more acute visits for asthma (6 [13%] of 47 children vs 17 [31%] of 54 children; P =.03). CONCLUSIONS: School-based provision of inhaled corticosteroids significantly improved symptoms, quality of life, and absenteeism among urban children with mild persistent to severe persistent asthma. This effect was seen only among children not exposed to secondhand smoke.     

The salivary microbiota as a diagnostic indicator of oral cancer: A descriptive,non-randomized study of cancer-free and oral squamous cell carcinoma subjects

Background  

The purpose of the present investigation was to determine if the salivary counts of 40 common oral bacteria in subjects with an oral squamous cell carcinoma (OSCC) lesion would differ from those found in cancer-free (OSCC-free) controls.     

Cholesterol-enriched membrane microdomains are required for inducing host cell cytoskeleton rearrangements in response to attaching-effacing Escherichia coli

The diarrheal pathogens enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL56 and enteropathogenic Escherichia coli (EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells through attaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimate adherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal transduction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in the plasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence was assessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 caused characteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56- and E2348/69-induced cytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterol to depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced alpha-actinin mobilization. Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reduced following cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangements on skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. These findings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletal rearrangements following infection with attaching-effacing Escherichia coli. Differences in initial adherence indicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.     

Survival and differentiation of pituitary colony-forming cells in vivo

Growth hormone (GH) deficiency is a significant clinical problem, since growth hormone is essential for the regulation of growth, metabolism, and the cardiovascular system. Stem and progenitor cells have been identified in many adult tissues. Recently, our laboratory identified a cell type within the adult pituitary gland with stem cell-like properties, which we have termed pituitary colony-forming cells (PCFCs). Herein we investigate the ability of PCFCs to survive and differentiate in vivo. Enriched populations of PCFCs were transplanted into an in vivo microchamber model. Grafts were harvested at 6 weeks post-transplant and tested for surviving donor cells (LacZ(+)) or for differentiation (GH(+)). The results showed that donor cells survived in chambers (LacZ(+)) and underwent division (phosphohistone-H3-positive). Furthermore, grafted cells showed colocalization of LacZ and GH, suggesting differentiation. To confirm differentiation, donor cells were obtained from a GH-enhanced green fluorescent protein (eGFP) reporter transgenic mouse model that expressed eGFP under control of the GH promoter. Cells that were eGFP(-), that is, GH(-), were selected by fluorescence-activated cell sorting (FACS) and transplanted. After 6 weeks, eGFP(+)GH(+) cells were detected in grafts by immunostaining and by FACS analysis of digested grafts. In conclusion, PCFCs have the capacity to divide and differentiate into GH(+) cells in vivo. The vascularized tissue chamber model is an ideal model to investigate the environmental niche for PCFC expansion and differentiation and has the potential to be developed into a growth hormone-releasing organoid in vivo. Disclosure of potential conflicts of interest is found at the end of this article.