Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora.

Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.     

A rapid screening assay for detection of IgE-binding factors in humans

During the last few years studies in rats and mice have demonstrated IgE-binding factors, some of which have IgE-selective regulatory activities. This prompted us to develop a rapid, sensitive screening assay for measuring IgE-binding factors in humans. The principle of the assay is to measure the degree of inhibition of binding between anti-human IgE antibodies and human IgE. Thus, 200 pg IgE plus testing samples were added to each well precoated with anti-human IgE antiserum. After an overnight incubation, the wells were washed and radiolabeled anti-IgE antibodies were added to the wells. Under the optimum conditions, the assay can detect 10(-11)M anti-human IgE antibodies. With this assay, we have been able to detect IgE-binding factors in the supernatants of 2 human B cell lines which bear Fc receptors for IgE (FcR epsilon) on their surface membranes (e.g., WIL-2 and RPMI 8866), but not in the supernatants of DAUDI cells (a human cell line without FcR epsilon). Furthermore, the IgE-binding factors of WIL-2 cells were specifically adsorbed to, and eluted from, IgE-coupled Sepharose, but not BSA-Sepharose. These findings prove that the inhibition factors are indeed human IgE-binding factors, and that the assay described herein is a specific and sensitive screening assay for detecting human IgE-binding factors.     

The N-terminal flanking region of the invariant chain peptide augments the immunogenicity of a cryptic "self" epitope from a tumor-associated antigen

The N-terminal flanking region of the invariant chain peptide termed CLIP appears to have superagonistic properties interacting with the T cell receptor and the MHC class II molecule at or near the binding site for the bacterial superantigen Staphylococcal enterotoxin B (SEB). The present studies explored the hypothesis that the N-terminal segment of CLIP can augment the immunogenicity of cryptic "self" tumor-associated antigens. A chimeric construct of an MHC class II binding peptide from the c-erb oncogene (Her-2/neu) containing the N-terminal flanking region of CLIP elicited potent antitumor activity against a Her-2/neu-positive tumor in a rat model system. Comparatively, the unmodified parent peptide was ineffective. The induction of effective antitumor immunity, however, required presentation of the chimeric peptide construct on irradiated tumor cells or the peptide construct in concert with a Her-2/neu MHC class I-restricted peptide from Her-2/neu. As revealed by adoptive transfer studies, effective protective antitumor immunity in this setting required the CD4 T helper subset. Additionally, in vitro analysis revealed that immunization with the parent peptide resulted in a weak immune response to the unmodified peptide consisting of both type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-10) cytokine-producing cells analyzed by RT-PCR (qualitative and quantitative) and by limiting dilution assay. Comparatively, immunization with the chimeric construct elicited a potent immune response to the parent peptide with predominantly type 1 cytokine-producing cells. Taken together, the results suggest that immunization with the chimeric Her-2/neu peptide induced protective antitumor immunity. Associated with this immunization strategy was the enhancement of a type 1 cytokine response.     

Preparation of graded porous titanium coatings on titanium implant materials by plasma spraying

Graded porous titanium coatings have been deposited on titanium substrates for dental implants by plasma spraying in an argon atmosphere. X-ray diffraction (XRD), scanning electron microscopy (SEM), surface roughness measurement, and tensile strength tests were performed on graded porous coatings. The results showed that Ti(3)O(5) was formed in the outermost surface of the porous coatings due to oxidation. The graded porous coatings consisted of three layers. The outer layer was full of macropores with a surface roughness of approximately 100 microm. The diameter of many macropores reached and even surpassed 150 microm, which could be beneficial for tissue to grow into the coating. The middle layer consisted of a mixture of micropores and macropores. The inner layer was a very dense and tight interface layer that included mechanical, physical, and metallurgical bonding. In tensile strength tests, testing bars peeled off the coatings, because the adhesive agent fractured, but the coatings remained intact.     

Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.     

Altered patterns of migration of cytokine-producing T lymphocytes in skin-grafted naive or immune mice following in vivo administration of anti-VCAM-1 or -ICAM-1.

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen-specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule-1L: very late activation antigen-4 (VCAM-1:VLA-4) or intracellular adhesion molecule-1:lymphocyte function-associated antigen-3 (ICAM-1:LFA-1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumour necrosis factor-alpha, interferon-gamma and transforming growth factor-beta]. In addition, using limiting dilution analysis, we investigated the frequency of allo-specific and third-party reactive cells producing IL-2 and IL-4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti-LFA-1:ICAM-1 superior for naive mice compared with anti-VLA-4:VCAM-1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen-specific type-2 cytokine-producing cells.     

The dysregulated glomerular cell growth in Denys–Drash syndrome

While diffuse mesangial sclerosis is traditionally described as being the glomerulopathy of Denys–Drash syndrome (DDS), the podocyte proliferative lesions may be overlooked in these DDS cases. In the present study, an evolving process is extrapolated from a selected case of DDS that demonstrated glomerulopathy with conspicuous podocyte proliferation. The observation that podocytes express proliferation markers (Ki67, proliferating-cell nuclear antigen and topoisomerase II) in non-proliferative, mature-looking glomeruli suggests an initial pathogenic act to activate or to keep podocytes from quiescence. The subsequent proliferation of podocytes is in keeping with downregulation of WT1 and cyclin kinase inhibitors of p16 and p21. The emergence of cytokeratin-positive cells in glomeruli that show typical mesangial sclerosis implies elimination of podocytes and replacement with tubular and/or parietal epithelial cells. The final scene of evolving glomerulopathy displays apoptosis and expression of Fas-L and Bax in sclerotic mesangial lesions, which eventually end up with global sclerosis. This novel concept of DDS glomerulopathy implies complex molecular mechanisms involved in glomerular injury.     

Molecular epidemiological and clinical aspects of hepatitis D virus in a unique triple hepatitis viruses (B,C, D) endemic community in Taiwan

The molecular epidemiological and clinical aspects of hepatitis D virus (HDV) in a unique HBV, HCV, and HDV triple virus endemic community in southern Taiwan were investigated. A total of 2,909 residents aged 45 or older were screened for hepatitis B surface antigen (HBsAg), anti-HCV antibody, and anti-HDV antibody (specifically for HBsAg-positive carriers). Factors that might be associated with HDV infection, viral nucleic acid detection, and genotyping of HBV, HCV, and HDV were investigated. The prevalence of HBsAg and anti-HCV were 12.6% (366/2,909) and 41.6% (1,227/2,909), respectively. For HBsAg carriers, 15.3% (56/366) were positive for anti-HDV assay. Living in a higher endemic district of HCV infection (odds ratio [OR] = 3.2; 95% confidence interval [CI] = 1.7-6.3), male gender (OR = 1.9; 95% CI = 1.1-3.6) and co-infection with HCV (OR = 1.8; 95% CI = 1.0-3.3) were significantly independent factors associated with HDV infection. The detection rate of HDV RNA among anti-HDV-positive patients was only 12.7% (7/55). The mean HBV titer of triple infection group was significantly lower than in the HBV/HDV co-infection group (2.23 vs 3.05 in log(10), copies/ml, P = 0.046). HCV RNA detection among the triple infection group showed 47.4% (9/19) viremia rate and viral loads of 579,121 IU/ml in median (16,803-1,551,190 IU/ml). The prevalent genotype of HBV was type B (23/25); HCV was 1b (7/9) and HDV was IIa/IIb (4/4). Only the presence of HCV RNA predicted the presence of elevated ALT significantly (OR = 25.0; 95% CI = 3.39-184.6). In conclusion, the geographical aggregation of HDV infection paralleled that of HCV infection in this community. HCV suppressed the replication of HBV among triple vital infection patients. HBV and HDV lapsed into a remission or nonreplicative phase in most cases, and HCV acted as a dominant factor in triple viral-infected individuals. Only the presence of HCV RNA was associated with elevated ALT values, but not HBV or HDV.     

Primary giant cell malignant fibrous histiocytoma of the kidney with staghorn calculi

Malignant fibrous histiocytomas (MFH) as primary renal tumours are rare, with less than 50 cases described in the literature. We report a case of primary renal MFH of giant cell type in a 56-year-old man, who presented with bilateral dull flank pain, intermittent gross haematuria and body weight loss (6 kg in 3 months). Intravenous urography, computerized tomography (CT) and magnetic resonance image (MRI) showed right ureteral stones with mild hydronephrosis, and a solid mass at the lower pole of the left kidney associated with staghorn calculi, as well as tumour thrombi in the left renal vein and inferior vena cava. Left radical nephrectomy and evacuation of tumour thrombi from the left renal vein and inferior vena cava were performed. Histopathologic examination revealed malignant fibrous histiocytoma (MFH) of giant cell type. To the best of our knowledge, this is the first report of primary renal MFH associated with staghorn calculi.     

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma

Cancer metastasis involves distinct steps that depend on complicated tumor–host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.