Smad4 overexpression causes germ cell ablation and leydig cell hyperplasia in transgenic mice

Members of the transforming growth factor-beta (TGF-beta) superfamily play a variety of important roles in testicular development and function. The tumor suppressor gene, Smad4, is a common mediator of TGF-beta, activin, and bone morphogenetic protein-mediated signaling pathways. To investigate the role of the Smad4 gene during testicular development and function, transgenic mice were generated using a Flag-tagged Smad4 gene driven by 180-bp fragment of the Mullerian inhibiting substance upstream promoter sequence. Three Smad4 transgenic founders (A, B, and G) were detected by Southern blot analysis; line B showed the highest expression of the Smad4 transgene and was further studied. The fertility in F1 generation (B) and F2 generation (BB) of the Smad4 transgenic mice was not impaired. However, in the F3 generation (B2x) all animals were impacted by the overexpression of the Smad4 transgene and two kinds of phenotypes were observed. In one group animals were completely infertile, while in the other group animals were fertile and sired the normal number of pups/litter. These groups are designated as infertile and fertile in the text. Histological evaluation of the testes from the infertile group showed variable degrees of Leydig cell hyperplasia, apoptosis of germ cells, spermatogenic arrest, seminiferous tubule degeneration, and infertility. In the fertile group, there was no apparent change in the histology of the testis except for a slight increase in the number of Leydig cells. Serum follicle-stimulating hormone levels in the adult animals of both groups of Smad4 transgenic male mice were not significantly different from normal littermates; however, testosterone levels in both groups were significantly (P < 0.05) increased. These results suggest that overexpression of Smad4 leads to testicular abnormalities and infertility supporting the hypothesis that the TGF-beta signaling pathways are carefully orchestrated during testicular development. In the absence of normal levels of Smad4 testicular function is compromised.     

Helicobacter pylori-Specific Immune Responses of Children: Implications for Future Vaccination Strategy

We analyzed the specific anti-Helicobacter pylori immunoglobulin G (IgG) antibody profile for a sample of 824 asymptomatic schoolchildren in southern Germany (mean age, 10.7 ± 0.65 years) with an H. pylori-specific IgG enzyme-linked immunosorbent assay and Western blot analysis. The prevalence of infection was 19.8% (95% confidence interval, 17.1 to 22.7%). The immunoresponses were characterized predominantly by antibodies against low-molecular-mass antigens of 14 and 29 kDa, with a significant difference between children of German and Turkish nationalities (P = 0.0012 and P < 0.0001, respectively).     

Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. USH is clinically and genetically heterogeneous with at least 11 chromosomal loci assigned to the three USH types (USH1A-G, USH2A-C, USH3A). Although the different USH types exhibit almost the same phenotype in human, the identified USH genes encode for proteins which belong to very different protein classes and families. We and others recently reported that the scaffold protein harmonin (USH1C-gene product) integrates all identified USH1 molecules in a USH1-protein network. Here, we investigated the relationship between the USH2 molecules and this USH1-protein network. We show a molecular interaction between the scaffold protein harmonin (USH1C) and the USH2A protein, VLGR1 (USH2C) and the candidate for USH2B, NBC3. We pinpoint these interactions to interactions between the PDZ1 domain of harmonin and the PDZ-binding motifs at the C-termini of the USH2 proteins and NBC3. We demonstrate that USH2A, VLGR1 and NBC3 are co-expressed with the USH1-protein harmonin in the synaptic terminals of both retinal photoreceptors and inner ear hair cells. In hair cells, these USH proteins are also localized in the signal uptaking stereocilia. Our data indicate that the USH2 proteins and NBC3 are further partners in the supramolecular USH-protein network in the retina and inner ear which shed new light on the function of USH2 proteins and the entire USH-protein network. These findings provide first evidence for a molecular linkage between the pathophysiology in USH1 and USH2. The organization of USH molecules in a mutual 'interactome' related to the disease can explain the common phenotype in USH.     

Role of CD40 antigen and interleukin-2 in T cell-dependent human B lymphocyte growth

In the present study, we examined the participation of CD40 ligand (L)-CD40 interaction in T cell-dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4⁺ cloned T cells activated with immobilized anti-CD3 antibody. The anti-CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD⁺ B cell was significantly less inhibited by anti-CD40 than that of sIgD^? cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL-2. This contrasts with the CD40 system where B cells are essentially responsive to IL-4 and IL-10 but not to IL-2 alone. Collectively, these data indicate that CD40L-CD40 interaction plays an important role in IL-2-mediated T cell-dependent B cell responses. However, the activation of a subset of sIgD⁺ cells may be independent of this interaction.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

Indications of the Protective Role of Natural Killer Cells in Human Cutaneous Leishmaniasis in an Area of Endemicity

The role of natural versus acquired immunity to Leishmania aethiopica infection in humans is the focus of our studies. We found in previous studies that mononuclear cells from nonexposed healthy Swedish donors responded to Leishmania antigen stimulation by proliferation and gamma interferon production. The main cell type responding was CD3⁻ CD16/56⁺ natural killer (NK) cells. These findings led us to suggest that the potential to produce a rapid, nonacquired NK cell response may be a protective phenotype. In order to test this hypothesis, an area in Ethiopia where Leishmania is endemic was selected, and peripheral blood mononuclear cells were obtained from individuals who had lived in the area most of their lives but had no evidence of past or present leishmaniasis. Their responses were compared with those of confirmed leishmaniasis patients from the same region with active lesions or cured leishmaniasis lesions. Cells from these donors were stimulated in vitro with L. aethiopica antigen. Responses were measured by proliferation, cytokine production, and phenotype analysis by fluorescence-activated cell sorting. The association of NRAMP1 alleles with the studied phenotype and susceptibility to L. aethiopica-induced leishmaniasis was also evaluated. The results show that Leishmania antigens can induce NK cell and CD8⁺-T-cell responses in vitro. This is clearly seen in proliferating cells from the cured (immune) individuals and the apparently protected controls from the area of endemicity. It contrasted with the reactivity of the patients, where some NK proliferation was coupled with enhanced CD4⁺-T-cell proliferation. We conclude from these observations that NK cells and CD8⁺ cells proliferating in response to Leishmania stimulation are involved in protection from and healing of (Ethiopian) cutaneous leishmaniasis; however, such mechanisms appear to be unrelated to the NRAMP1 host resistance gene.     

Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells

The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7⁺ dendritic cells to process antigen for presentation to CD4⁺ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen-presenting cells (APC) in stimulation cultures of major histocompatibility complex class II-restricted T cells. It was found that they induce antigen-dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells. Moreover, antigen presentation needs intracellular processing within endo- or lysosomes as chloroquine-treatment prevents T cell activation. Titration of APC numbers revealed that contaminating APC most likely did not account for antigen-specific T cell activation by DC. No evidence was found for release of antigenic peptides or for partial antigen processing possibly done by cell surface located enzymes on DC. In conclusion, these results indicate that freshly enriched DC are able to process antigens similarly to other APC.     

Interpretive criteria for susceptibility testing of CI-960 (PD127391, AM-1091), fleroxacin, lomefloxacin, and temafloxacin against Neisseria gonorrhoeae, including drug stability in GC agar medium.

CI-960, fleroxacin, lomefloxacin, and temafloxacin were tested against over 100 strains of Neisseria gonorrhoeae. Each organism was tested in triplicate by using agar dilution and disk diffusion methods recommended by the National Committee for Clinical Laboratory Standards. CI-960 was the most potent compound, with a MIC against 90% of the strains tested of 0.008 microgram/ml, and the least active was fleroxacin (MIC against 90% of strains, 0.12 microgram/ml). Only the susceptible interpretive category was recommended for the CI-960 tests as follows: 5-micrograms disk, greater than or equal to 39 mm (MIC correlate, less than or equal to 0.12 microgram/ml). Three interpretive categories were proposed for the other fluoroquinolones as follows: fleroxacin, 5-micrograms disk susceptible at greater than or equal to 33 mm (MIC correlate, less than or equal to 0.25 microgram/ml), intermediate at 28 to 32 mm (MIC correlate, 0.5 microgram/ml), and resistant at less than or equal to 27 mm (MIC correlate, greater than 0.5 microgram/ml); lomefloxacin, 10-micrograms disk susceptible at greater than or equal to 35 mm (MIC correlate, less than or equal to 0.12 microgram/ml), intermediate at 28 to 34 mm (MIC correlates, 0.25 to 0.5 microgram/ml), and resistant at less than or equal to 27 mm (MIC correlate, greater than 0.5 microgram/ml); and temafloxacin, 5-micrograms disk susceptible at greater than or equal to 36 mm (MIC correlate, less than or equal to 0.06 microgram/ml), intermediate at 28 to 35 mm (MIC correlates 0.12 to 0.25 microgram/ml), and resistant at less than or equal to 27 mm (greater than 0.25 microgram/ml). Interpretive agreement between disk diffusion results and the MICs was 100% for each agent, with the exception of lomefloxacin, which had a 0.9% minor error. All drugs were stable in GC agar medium for at least 21 days when stored at 2 to 5 degrees C.     

Effect of loteprednol etabonate nasal spray suspension on seasonal allergic rhinitis assessed by allergen challenge in an environmental exposure unit

BACKGROUND: Loteprednol etabonate (LE) is a novel soft steroid that was designed to improve the benefit/risk ratio of topical corticosteroid therapy. This study assesses the clinical efficacy and safety of three different doses of LE nasal spray in seasonal allergic rhinitis (SAR). METHODS: In this single-center, double-blind, placebo-controlled, parallel-group trial 165 subjects with SAR to grass pollen received daily single doses of either 100, 200, 400 microg LE nasal spray, or placebo for 14 days. The patients underwent three 4-h allergen challenges with grass pollen in an environmental exposure unit at a screening visit (baseline) and on days 7 and 14 of treatment. Standardized nasal symptom scores were obtained every 20 min. Nasal flow, nasal secretions, and FEV(1) were measured every hour during allergen challenges. RESULTS: After 14 days of treatment, patients who received 400 microg LE had significantly lower total nasal symptom scores compared with those receiving placebo (P = 0.007). LE400 reduced rhinorrhea, nasal congestion, nasal itching, the amount of nasal secretions, and improved nasal flow as compared with placebo (P < 0.05). LE100 and LE200 were not significantly different from placebo. All treatments were well tolerated. CONCLUSIONS: Loteprednol 400 microg once daily is superior to placebo and the only effective dose tested in improving nasal symptoms and objective parameters in patients with SAR.     

Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes

To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgGl. The CD40-Fc fusion protein was able to specifically bind to CD4⁺ and various CD8⁺ T cell clones activated with immobilized anti-CD3. The ¹²⁵I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4⁺ T cell clone (MT9) with an equilibrium dissociation constant (Ka) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of ≈ 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8⁺ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.