Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry

Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell- mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ- specific autoimmune disease.      

Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium

Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17beta. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17beta, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17beta. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype.      

Macrophage tumor cell interaction is enhanced by C3 fragments.

We tested the capacity of Lewis Lung carcinoma cells (3LL) to activate the alternative pathway of complement and to bind the C3 fragments on the plasma membrane. C3 fragments were detected by cytofluorometry and by immunoblotting. In time, the fixed C3b molecules were further cleaved into iC3b. The presence of C3b/iC3b on the target enhanced the formation of conjugates with macrophages. In spite of increased contacts, macrophages from tumor bearing mice were not cytotoxic. Only preactivated macrophages, by in vivo treatment with Corynebacterium parvum, were shown to be cytotoxic; this function was potentiated when the target cells were opsonized with C3b/iC3b.     

Antibodies to HIV-1 gp41 recognize synthetic peptides of human IFN-alpha and IFN-beta

Based on our finding that a common epitope exists between HIV-1 gp41 and human type I interferons (IFN-alpha and IFN-beta), and increased levels of antibodies against human IFN-alpha and IFN-beta were observed in HIV-1-infected individuals, we tried to explain the mechanism of increased levels of antibodies. Mouse antisera recognizing HIV-1 recombinant soluble (rs) gp41 (aa 539-684) interacted with two synthetic peptides sequence-corresponding to the IFN-alpha/beta receptor binding site on human IFN-alpha and IFN-beta, while normal mouse serum (pooled normal sera) did not. The anti-rspg41 antisera after adsorption by IFN-beta sepharose column lost the activity of interaction with both synthetic peptides. In another experiment, rsgp41 could bind to sepharose column conjugated with anti-IFN-beta polyclonal antibodies (IgG). These results indicate that the common epitope on gp41 and type I interferons could induce antibodies recognizing the receptor binding site on IFN-alpha and IFN-beta, suggesting that increased levels of antibodies against IFN-alpha and IFN-beta in HIV-1-infected individuals could be induced by gp41.     

Prevalence and clinical manifestations of shiga toxin-producingEscherichia coli infections in Austrian children

The prevalence and clinical manifestations of infections associated with Shiga toxin-producingEscherichia coli (STEC) among Austrian children were assessed. Stool samples from 280 pediatric patients were analyzed by enzyme immunoassay (EIA) for the presence of free fecal Shiga toxin (Stx) 1 and 2, and by culture on sorbitol MacConkey agar. Specimens testing positive by the EIA were subjected to a cytotoxicity assay, polymerase chain reaction analysis, and a colony hybridization test. Direct culture on MacConkey agar demonstrated the presence of threeEscherichia coli O157:H7-positive stools. These were also positive by EIA and by the DNA-based methods. An additional six samples were positive by EIA, and in four of these, non-O157 STEC of serotypes O111H^–, O146: H^–, and O113:H53 could be isolated. Analysis of stools for a variety of enteric pathogens demonstrated that STEC was the third most common bacterial pathogen. The clinical manifestations of STEC infections were difficult to distinguish from those of infections caused by other enteric pathogens, as most patients presented with watery diarrhea. The median age of children with STEC infections was 27.6 months (range, 7 months to 5.75 years); children withSalmonella orCampylobacter infections were younger on average, while those withRotavirus infections were older. This study demonstrated that althoughEscherichia coli O157:H7 could be identified with the same sensitivity by both EIA or agar-based methods, the identification of non-O157 STEC strains was enhanced by the use of EIA followed by colony hybridization. Analysis of overnight cultures from 53 STEC isolates revealed that all strains producing Stx1, Stx2, or Stx2c reacted in the EIA. However, culture supernatants from Stx2e-producingEscherichia coli O101 were negative in the EIA. Despite this disadvantage, the EIA is easy to perform and time efficient and can be recommended as a screening test for non-O157 STEC in children with diarrhea.     

Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections.

To evaluate the performance of a serological test for human immunodeficiency virus type 1 (HIV-1) infections based on the use of a recombinant envelope gene-derived protein as the antigen, we caused expression of a 1.4-kilobase fragment of HIV.DNA that codes for the complete gp41 transmembrane protein in an Escherichia coli expression vector and used Western blots (WB; immunoblots) prepared with recombinant material (pEX-41) to detect antibodies to HIV-1. This test detected all 339 sera which were positive by a combination of conventional serodiagnostic assays and produced no false-positive results with 311 negative samples. Also no false-positive results were obtained with 20 sera from systemic lupus erythematosus patients which had high titers of cross-reactive autoantibodies. In six cases, the pEX-41 WB proved to be more sensitive than individual assays applied on their own, and in five cases it was even more sensitive than a combination of conventional assays. We tested 221 sera in both our pEX-41 WB and a commercially available recombinant enzyme immunoassay (EIA [Abbott]). The results were identical in 188 cases. A total of 27 sera containing antibodies to gp41 as demonstrated in the pEX-41 WB, as well as the Abbott recombinant EIA, had no antibodies to the recombinant core antigen as measured in the Abbott EIA. However, 25 of these sera did stain the 24-kilodalton band on a WB with purified virus. Six sera that were positive in all of the conventional confirmatory assays and reacted strongly with the pEX-41 WB did not recognize the surface antigen used in the Abbott recombinant EIA. We conclude that the use of WB prepared with recombinant-derived p41 offers a very sensitive and specific method to detect antibodies to HIV.     

Are animal models useful for understanding the pathophysiology of lysosomal storage disease?

Spontaneously occurring genetic lysosomal storage diseases are as rare in other mammalian species as in man. However, the advent of gene targeting technology has revolutionized the state of animal models of genetic diseases. Nearly all lysosomal storage diseases known in man have been duplicated in the mouse. The technology now allows, not only complete inactivation of endogenous genes, but also the introduction of essentially any type of mutation. These animal models can overcome many of the limitations inherent in studies of human patients - rarity of the disease, extremely complex genetic background and logistical and ethical constraints in the design and execution of experiments with human subjects. For example, genetic manipulations of germ cells or cross-breeding experiments between two mutants are readily feasible with animal models. Two major areas of the utility of animal models are the clarification of the pathophysiology/pathogenetic mechanism of disease and the exploration of therapeutic approaches. Examples of experiments using animal models of lysosomal storage disease are presented, primarily from studies undertaken in our own laboratory.
Conclusion : Animal models have proved invaluable in extending our knowledge of the lysosomal storage diseases and exploring potential therapies.