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.     

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.     

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.     

Interleukin-1 receptor antagonist attenuates airway hyperresponsiveness following exposure to ozone

The role of an interleukin (IL)-1 receptor antagonist (IL-1Ra) on the development of airway hyperresponsiveness (AHR) and airway inflammation following acute O(3) exposure in mice was investigated. Exposure of C57/BL6 mice to O(3) at a concentration of 2.0 ppm or filtered air for 3 h resulted in increases in airway responsiveness to inhaled methacholine (MCh) 8 and 16 h after the exposure, and an increase in neutrophils in the bronchoalveolar lavage (BAL) fluid. IL-1beta expression, assessed by gene microarray, was increased 2-fold 4 h after O(3) exposure, and returned to baseline levels by 24 h. Levels of IL-1beta in lung homogenates were also increased 8 h after O(3) exposure. Administration of (human) IL-1Ra before and after O(3) exposure prevented development of AHR and decreased BAL fluid neutrophilia. Increases in chemokine levels in lung homogenates, tumor necrosis factor-alpha, MIP-2, and keratinocyte chemoattractant following O(3) exposure were prevented by IL-1Ra. Inhalation of dexamethasone, an inhibitor of IL-1 production, blocked the development of AHR, BAL fluid neutrophilia, and decreased levels of IL-1 following O(3) exposure. In summary, acute exposure to O(3) induces AHR, neutrophilic inflammation, epithelial damage, and IL-1. An IL-1Ra effectively prevents the development of altered airway function, inflammation, and structural damage.     

Identification and characterization of a new variant of Shiga toxin 1 in Escherichia coli ONT:H19 of bovine origin

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx(1) gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx(1) from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx(2) by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods.     

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

bcl-2 transgene expression promotes survival and reduces proliferation of CD3-CD4-CD8- T cell progenitors

Proliferative expansion and apoptotic cell death play prominent roles in T cell development. The molecular control of cell cycle progression and apoptosis appear to be inter-connected since the Bcl-2 protein can inhibit apoptosis and slow cell cycle progression in cortical thymocytes and mature T cells, particularly during the transition from the quiescent state into the cell cycle. Here the impact of bcl-2 transgene expression on CD3-CD4-CD8- T cell progenitors was assessed. Bcl-2 enhanced the survival of these progenitors at all of the four major differentiation stages, CD25- CD44+ (pro-T1), CD25 + CD44+ (pro- T2), CD25 + CD44- (pro-T3) and CD25-CD44- (pro-T4). However, it reduced cell cycling and slowed turnover only in the pro-T4 subset. From an analysis of bcl-2 transgenic mice expressing a TCR transgene or bearing a mutation in the scid or rag-1 gene we conclude that Bcl-2 inhibits proliferation only of T cell progenitors that are activated via the pre- TCR, not those stimulated via c-Kit and the IL-7 receptor.      

The significance of angiogenesis in malignant pheochromocytomas

The purpose of this study was to investigate tumor angiogenesis in a series of benign and malignant pheochromocytomas and to determine whether there is a correlation between angiogenesis and the presence of distant metastases. In this study, the CD31 monoclonal antibody was selected to measure intratumoral microvessel density. Nineteen patients with malignant pheochromocytomas and nineteen patients with benign pheochromocytomas who underwent operation were studied. In order to quantify intratumoral microvessel density, the total number of pixels of CD31-positive reactivity was assessed and expressed as a percentage of the total tissue area in the analyzed field. Analysis of variance revealed a statistically significant correlation between malignancy and intratumoral microvessel density (p=0.0009). Although there was a considerable variability in the intratumoral microvessel density from tumor to tumor within both the benign and the malignant group, a percentage of more than 28.5% anti-CD31 stained area was found only in malignant tumors. In conclusion, this study shows that the mean intratumoral microvessel density in malignant pheochromocytomas is increased approximately two-fold as compared with benign tumors. However, the clinical significance of this prognostic marker is rather weak, because only 4 of the 19 malignant pheochromocytomas had microvesel density higher than this threshold of 28.5%.