Histones interact with anionic phospholipids with high avidity; its relevance for the binding of histone-antihistone immune complexes.

Antibodies recognizing anionic phospholipids have been described in systemic lupus erythematosus (SLE) and other autoimmune diseases. Recent studies have shown that some of these antibodies may recognize a cardiolipin-binding protein (apolipoprotein H) rather than phospholipids. A similar possibility is conceivable for other cardiolipin-binding proteins that are targets of autoantibodies. In this study we have addressed whether this might be the case for histones, a set of highly cationic and widely distributed proteins that react in a well known autoantibody system. Our results indicate that: (i) histones bind to anionic phospholipids (cardiolipin and phosphatidylserine) with high avidity, but not to zwitterionic phospholipids (phosphatidylcholine); (ii) monoclonal and polyclonal antihistone antibodies recognize histones bound to cardiolipin; (iii) the addition of histones to serum samples containing antihistone antibodies often enhances their anticardiolipin reactivity. In addition, we have found that antihistone-producing hybridomas derived from MRL-lpr mice may show anticardiolipin activity due to the presence of histones in the cell culture supernatants with the resultant formation of immune complexes. Taken together, the results suggest a potential role for histones in the anti-cardiolipin activity detected in sera containing antihistone antibodies. These histone-phospholipid interactions should be taken into account when evaluating the pathogenic effects of antihistone antibodies or other autoantibodies reacting with nuclear components (e.g. nucleosomes) containing histones.     

Gene therapy for pituitary tumors

Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.     

Cytomegalovirus-infected cell polypeptides immune-precipitated by sera from children with congenital and perinatal infections.

Congenital or perinatally acquired human cytomegalovirus (CMV) infections in children may be symptomatic or asymptomatic. In this study, we characterized the electrophoretic properties of CMV-infected cell polypeptides immune-precipitated by sera from children with different types of CMV infections from birth to 4 years of age. Sodium dodecyl sulfate-polyacrylamide gel analysis of immune precipitates formed with radiolabeled extracts of cells infected with CMV strain AD169 showed the following. (i) Electrophoretic profiles of CMV polypeptides immune-precipitated by sera from children with perinatal and congenital infections were similar. At least 11 polypeptides with apparent molecular weights of 150,000, 140,000, 110,000, 100,000, 74,000, 66,000, 50,000, 49,000, 34,000, 25,000, and 20,000 were precipitated. Antibody titer in anticomplement immunofluorescence tests and virus titer in urine correlated with the intensity of polypeptide profiles in autoradiograms. (ii) The initial immune response of children with symptomatic congenital infections was delayed as compared to that of children with asymptomatic congenital and perinatal CMV infections. Sera obtained serially from symptomatic children for years after birth continued to precipitate CMV polypeptides, whereas sera from children with subclinical congenital infections precipitated lesser amounts over time. (iii) Immune precipitates obtained with sera from CMV-infected patients and with monoclonal antibodies to CMV contained polypeptides with comparable electrophoretic and immunological properties.     

Monoclonal antibodies to human cytomegalovirus: three surface membrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants

Seventy-seven clones of hybridomas selected for reactivity by immunofluorescence with human cytomegalovirus (CMV)-infected cells were produced by fusing mouse myeloma cells with the spleen cells of mice immunized with CMV strain AD169. The clones were classified into seven groups on the basis of the electrophoretic properties of the polypeptides immune precipitated from extracts of CMV-infected cells. Studies on the three groups of monoclonal antibodies directed against CMV surface membrane antigens showed the following. Clones in each group were differentiated by immunoglobulin subclass, neutralizing activity, and reactivity with the antigenic domains of proteins exposed on the surface membranes of intact CMV-infected cells. Monoclonal antibodies in each group precipitated one slowly migrating protein and multiple faster migrating forms which shared antigenic determinants. The first group of monoclonal antibodies precipitated four glycosylated polypeptides with apparent molecular weights of 130,000, 110,000, 100,000, and 60,000. Monoclonal antibody CH51 of this group neutralized infectious virus but failed to react with antigenic domains on the surfaces of infected cells. The second group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of approximately 66,000, 55,000, 50,000, and 46,000. Monoclonal antibodies CH65 and CH134 in this group had neutralizing activity and reacted with antigenic domains of proteins exposed on the surface of CMV-infected cells. The third group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of 49,000, 48,000, 34,000, and 25,000. Serological analysis of 15 naturally occurring CMV strains with a panel of monoclonal antibodies to surface membrane proteins showed that the antigenic determinants reactive with the antibodies tested were conserved in all of the strains. Monoclonal antibodies to surface membrane proteins on CMV-infected cells may prove to be valuable reagents for identification of virus isolates.     

Expression of retinoblastoma protein in human growth hormone-secreting pituitary adenomas

The retinoblastoma gene (RB1) is a tumor-suppressor gene in chromosomal region 13q14.2. Its role in the pathogenesis of pituitary tumors has not been fully clarified. Some studies have shown that losses in this chromosomal region are related to aggressive tumor behavior, although the retinoblastoma protein (pRB) is still expressed. Conversely, lack of expression of pRB was observed in one fourth of GH-secreting pituitary adenomas (GH-tumors). In order to further study the expression of pRB in GH-tumors, we evaluated this protein in 49 tumors from patients with acromegaly (20 noninvasive, 25 invasive, and 4 with no information) and 8 normal pituitaries using immunohistochemistry (IHC). Nuclear staining for pRB ranged from 0 to 90% (median 40%) in the tumors and from 40 to 80% (median 58%) in normal pituitaries. In 10 tumors (20% of total) the adenomatous cells were negative (5 cases) or had very low labeling (5 cases) for pRB. Sixty three percent (31/49) of the tumors showed staining in 10–80% of the cells and in 16% (8/49) of the cases >80% of the adenomatous cells were positive for pRB. The expression of pRB was not different in invasive and noninvasive tumors. In conclusion, pRB is underexpressed in a subgroup of GH-tumors, and this may represent an early event in the pathogenesis of this tumor subtype.     

DADOS-Survey: an open-source application for CHERRIES-compliant Web surveys

Background  

The Internet has been increasingly utilized in biomedical research. From online searching for literature to data sharing, the Internet has emerged as a primary means of research for many physicians and scientists. As a result, Web-based surveys have been employed as an alternative to traditional, paper-based surveys. We describe DADOS-Survey, an open-source Web-survey application developed at our institution that, to the best of our knowledge, is the first to be compliant with the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). DADOS-Survey was designed with usability as a priority, allowing investigators to design and execute their own studies with minimal technical difficulties in doing so.     

Induction of HLA-DR expression on thyroid follicular cells by cytomegalovirus infection in vitro. Evidence for a dual mechanism of induction.

Cytomegalovirus (CMV) infection of primary cultures established from human thyroid nodular and normal (paranodular) tissues resulted in induction of human leukocyte antigen (HLA) DR expression on thyroid follicular cells (TFC), as detected by cell-surface immunofluorescence staining with monoclonal antibodies (MAb). Two distinct modalities of induction were observed. The first type occurred in cultures of normal tissue obtained from CMV-seropositive but not seronegative donors, was detected on 30% to 50% of the TFCs, even though the vast majority of these cells failed to show any morphologic or antigenic evidence of individual CMV infection, and was associated with production of gamma-interferon (gamma-IFN) in vitro. The induced molecules displayed the characteristic DR polypeptide profile on immunoprecipitation and electrophoretic analysis. These results demonstrate that CMV infection of normal thyroid cultures may induce DR expression on TFCs in the absence of pre-existing lymphoid infiltrates and suggest that the induction is the result of an in vitro response to CMV by previously sensitized immunocompetent cells present in these primary cultures. Such a response, associated with the release of gamma-IFN, would induce DR expression on neighboring uninfected cells. The second mode of induction occurred in all CMV-infected cultures, regardless of their tissue origin (nodular or normal) or the serologic status of the donors. Up to 50% of infected TFCs at a late stage of infection, having fully developed CMV antigen-positive intranuclear inclusions, also displayed the cell-surface DR-related determinant recognized by one of the four anti-DR MAbs used. This induction was restricted to TFCs, while CMV-infected fibroblastoid cells present in the monolayers were invariably negative. Induction by CMV of major histocompatibility class II antigens on human epithelial cells may have significant implications in the development of normal immune responses against local viral infection, the enhancement of alloimmune rejection of grafted organs, and the generation of organ-specific autoimmune responses.     

Heterologous expression of Trypanosoma cruzi trans-sialidase in Leishmania major enhances virulence

Earlier studies showed that mice primed for a few hours with the trans-sialidase (TS) of Trypanosoma cruzi, the agent of Chagas' disease, become highly susceptible to trypanosomal infection. These studies suggest that TS affects parasite virulence independent of antigenic stimulation. Potentially, TS could enhance or reduce the virulence of heterologous microbes depending on the mechanism of TS action and on the type of immune response elicited by the particular parasite. We tested this hypothesis by expressing heterologous TS in Leishmania major, a protozoan parasite that causes cutaneous leishmaniasis and lacks TS and the TS product alpha2-3-linked sialic acid. Leishmania cells transfected with a T. cruzi TS expression construct made high levels of active enzyme, which was present in the promastigotes and shed into the extracellular milieu. TS expression did not affect L. major binding to and entry into cultured macrophages or its tropism for macrophage infection in vivo. However, TS-expressing L. major exhibited elevated virulence in BALB/c mice, as determined by lesion progression, parasite numbers, and macro- and microscopic examination of cutaneous lesions. Several genetic tests proved that the enhanced virulence was directly attributable to TS expression. The results are consistent with TS functioning to sabotage the mouse immune system to confer a growth advantage on T. cruzi and transgenic L. major. These data suggest that heterologous expression of T. cruzi virulence factors in Leishmania may provide a new approach for dissecting their function in vivo.