Tadpoles fed supplemented diet with probiotic bacterium isolated from the intestinal tract of bullfrog Lithobates catesbeianus: Haematology, cell activity and electron microscopy.

The aim of this study is to select and isolate autochthonous bacteria with probiotic potential for use in a supplemented diet for bullfrog tadpoles, Lithobates catesbeianus. A total of 20 strains of lactic acid bacteria were isolated. Nine out of these were used in the following in vitro assays: antagonism against pathogenic bacteria (ANT), antimicrobial activity from extracellular compounds (MIC), tolerance to bile salts (TBS), pH reduction, protease production, sensitivity to antimicrobial tetracycline, cell viability, growth rate and doubling time. Using these data was defined an ideotype (ideal strain) based on the best results. Distances were estimated with the Mahalanobis (D2) test, and the best candidates, presenting the shortest ideotype distances, were considered to be used. The best strain was found to be Lactobacillus plantarum because it presented 10.00 ± 0.50 mm of ANT against Aeromonas hydrophila, 3.99 ± 0.01 of MIC independent of pathogenic bacteria, 85.07 ± 0.01 of TBS, 4.20 ± 0.02 of final pH, 17.67 ± 1.15 of protease production, 13.50 ± 2.00 sensitivity to antimicrobial tetracycline, 9.36 ± 0.04 of cell viability, 0.20 ± 0.00 of growth rate and 3.46 ± 0.00 doubling time. Therefore this probiotic candidate was then supplemented (2.045 ± 1.07 × 107 colony forming unities. g-1) into the diets of bullfrog tadpoles for a period of 42 days. At the end of the trial, samples of blood and intestines were collected to verify the haematological alterations and the intestinal morphology using transmission and scanning electron microscopy. Tadpoles fed the supplemented diet showed successful lactic acid bacterium colonisation, an increased number of circulating thrombocytes, monocytes, eosinophil and LG-PAS+ and also an increase in the length and density of intestinal microvilli. This study shows the feasibility of using probiotics isolated from farmed bullfrogs as a supplement in the diets of tadpoles, providing a promising alternative for modulating the health of these animals.

Exacerbation of DSS-induced colitis in mice lacking kinin B(1) receptors through compensatory up-regulation of kinin B(2) receptors: the role of tight junctions and intestinal homeostasis.

BACKGROUND AND PURPOSE: Kinins are pro-inflammatory peptides that are released during tissue injury, including that caused by inflammatory bowel disease. Herein, we assessed the role and underlying mechanisms through which the absence of kinin B(1) receptors exacerbates the development of dextran sulfate sodium (DSS)-induced colitis in mice. EXPERIMENTAL APPROACH: B(1) and B(2) receptor antagonists and B(1) receptor knockout mice (B1(-/-) ) were used to assess the involvement of B(1) and B(2) receptor signalling in a DSS-colitis. B(1) receptor, B(2) receptor, occludin and claudin-4 expression, cytokine levels and cell permeability were evaluated in colon from wild-type (WT) and B1(-/-) mice. KEY RESULTS: DSS-induced colitis was significantly exacerbated in B1(-/-) compared with WT mice. IL-1ß, IFN-γ, keratinocyte-derived chemokine and macrophage inflammatory protein-2 were markedly increased in the colon from DSS-treated B1(-/-) compared with DSS-treated WT mice. Treatment of WT mice with a selective B(1) receptor antagonist, DALBK or SSR240612, had no effect on DSS-induced colitis. Of note, B(2) receptor mRNA expression was significantly up-regulated in colonic tissue from the B1(-/-) mice after DSS administration. Moreover, treatment with a selective B(2) receptor antagonist prevented the exacerbation of colitis in B1(-/-) mice following DSS administration. The water- or DSS-treated B1(-/-) mice showed a decrease in occludin gene expression, which was partially prevented by the B(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: A loss of B(1) receptors markedly exacerbates the severity of DSS-induced colitis in mice. The increased susceptibility of B1(-/-) may be associated with compensatory overexpression of B(2) receptors, which, in turn, modulates tight junction expression.

Circumsporozoite gene of plasmodium cynomolgi (Gombak):cDNA cloning and expression of the repetitive circumsporozoite epitope.

We report the identification, sequence, and expression in Escherichia coli of the immunodominant epitope of the circumsporozoite (CS) gene of Plasmodium cynomolgi (Gombak), a simian malaria parasite. This epitope is encoded by a DNA sequence that is tandemly repeated 10 times in the cDNA clone. Subclones that contain and express only repeats and in variable number have been constructed. We show that the binding of a specific anti-CS protein monoclonal antibody correlates positively with the number of repeats in each subclone. The CS gene of another strain of P. cynomolgi (NIH) encodes an immunodominant epitope that is immunologically distinct from that of the Gombak strain. We present evidence that these two CS genes share extensive overall homology, although the nucleotide sequences that encode the epitopes appear to be unrelated.

Mechanisms of resistance to the intracellular protozoan Encephalitozoon cuniculi in mice.

Mechanisms of resistance to the obligate intracellular protozoan Encephalitozoon cuniculi were studied in BALB/c mice. Resistance to lethal disease was T cell-dependent because transfer of T-enriched, but not T-depleted, spleen cells from sensitized BALB/c donors would protect infected BALB/c-nu mice. A modified focus-forming assay was utilized to measure effects on E. cuniculi infectivity in vitro. The results show that antibodies exert an opsonization effect and may block parasite entry into nonphagocytic cells. No cytotoxic T cells were demonstrated. Supernatants from E. cuniculi-sensitized spleen cells incubated with E. cuniculi in vitro could induce adherent PEC to kill E. cuniculi.

Murine encephalitozoonosis model for studying the host-parasite relationship of a chronic infection.

Encephalitozoon cuniculi caused chronic nonlethal infections in euthymic BALB/cAnN mice, whereas athymic BALB/cAnN-nu mice died from infection. Specific, anamnestic, transferable, and acquired responses against E. cuniculi were expressed by infected euthymic mice. Resistance to lethal disease appears to be T-cell dependent. Immune serum failed to protect infected athymic mice, whereas the transfer of T-enriched spleen cells from E. cuniculi-sensitized euthymic donors prevented lethal E. cuniculi infections in athymic mice. These findings indicate that murine encephalitozoonosis may be an excellent system for studies of a chronic infection in an immunologically well-characterized host.

Status of the major epicardial coronary arteries 80 to 150 days after percutaneous transluminal coronary angioplasty. Analysis of 3 necropsy patients.

Certain clinical and necropsy cardiac findings are described in 3 men who had percutaneous transluminal coronary angioplasty (PTCA) of the left anterior descending (LAD) coronary artery 80, 90, and 150 days before sudden death. Each patient had a decrease in the mean transstenotic coronary gradient (17, 38, and 43 mm Hg) and an angiographic increase in the LAD luminal diameter (55, 60, and 65%). At necropsy, the LAD coronary artery in the area of the PTCA in each patient was narrowed 76 to 95% in cross-sectional area by atherosclerotic plaques. No cracks in plaques or other lesions which may have resulted from the PTCA procedure were identified histologically in the LAD coronary artery of any patient.

Autoradiographic studies of oral lichen planus.

3H cytidine autoradiographic labeling patterns have been categorized into those characteristic for neoplastic, preneoplastic, and normal cell types. Specific differences in the patterns of transcription (RNA synthesis) revealed that the histology of a particular lesion generally correlated with (1) the percentage of cells labeled with the isotope, (2) the number of grains per cell, and (3) their intracellular distribution. When these three criteria of labeling were applied to lesions of the oral mucosa, very interesting data were collected on cases of lichen planus. The benign histologic appearance of the oral lichen planus epithelium exhibited a preneoplastic-appearing autoradiogram, an observation which is of particular interest in view of the fact that a small number of patients with this disease develop intraoral squamous cell carcinoma.

Susceptibility of selected inbred strains of mice to Encephalitozoon cuniculi.

Selected inbred strains of mice were examined for susceptibility to peritoneal infections with Encephalitozoon cuniculi. Marked differences in susceptibility and resistance among mouse strains were not controlled by loci within the mouse major histocompatibility (H-2) complex, but resistance in BALB/c mice was influenced by a dominant gene(s) outside the H-2 complex. The lethality of peritoneal infections in hypothymic nude mice suggested that resistance to encephalitozoonosis is dependent on thymus-derived lymphocytes. Peritoneal infection with E. cuniculi was associated with slightly enhanced proliferative spleen cell responses to mitogens in BALB/c mice and significantly depressed responses in C57Bl/6 mice. Infection in BALB/c mice was accompanied by normal antibody responses to sheep erythrocyte immunogens, but C57Bl/6 mice had depressed antibody responses. The results indicate that the immune system plays a major role in determining the course of peritoneal infections with E. cuniculi and that encephalitozoonosis can modulate the host's immune system.

The effect of ecdysterone on nonhistone proteins in salivary gland chromosomes of Sciara coprophila.

Synthesis and transport of proteins to the cell nucleus during puff induction was studied in S. coprophila. Changes in grain distribution along chromosomes (L-methionine [35S] incorporation into protein) were correlated with puffs induced by ecdysterone in vitro; A pattern of specific labelling at the sites of incipient puffs was noted within 2 h after the addition of the hormone, i.e. grains on the chromosomes were in clusters, characteristic for this time point and not seen in the controls (where only non-specific labeling was noted 0-4 h). Characteristic chromosomal puffs appeared between 3-4 h after the addition of ecdysterone. It was concluded that during ecdysterone-induced puff formation in salivary gland chromosomes, proteins which had been previously synthesized were selectively transported from the cytoplasm to specific sites on the chromosomes.

Hyaline bodies: some observations on their ultrastructure.

The ultrastruct of hyaline bodies was examined. Their constant association with elements of squamous epithelium pointed to an epithelial component in their structure. Elements of red blood cells, blood vessels, and endothelium were not evi-ent in these hyaline bodies, thereby negating avascular origin. Due to ultrastrucural limitations, the exact composition of the structures could not be determined.

Hormone-induced alterations in nonhistone protein. Methylation and phosphorylation in Sciara coprophila.

Methylation and phosphorylation of chromosomal nonhistone protein (NHP) has been demonstrated in the salivary gland cells of diptera [5, 7] and implicated in the control of gene expression [35, 36]. Furthermore, hormones can stimulate methyl and phosphoryl side chain metabolism and thus enhance template activity. Salivary glands from late fourth instar female larvae of Sciara coprophila (cortisone-supplemented and normal diet) were incubated in 3H-uridine (10 muCi/ml), 3H-thymidine (10 muCi/ml), 3H-methyl-methionine (20 muCi/ml), 35S-methionine (10 muCi/ml) and 32P-orthophosphate (1 mc/ml), for varying time periods, to measure RNA synthesis, DNA synthesis, methylation, protein synthesis and phosphorylation, respectively. Following selective extraction of lipid, histone and nucleic acids, glands were prepared for light microscope autoradiography. A more specific labelling pattern, as well as increased grain number on particular bands, interbands and bulbs, was noted on chromosomes from cortisone-fed larvae incubated in 3H-methyl-methionine for 1 min when compared with larvae on the standard diet. Cortisone also increased RNA synthesis and nucleoprotein phosphorylation, but not DNA or protein synthesis. In summary, cortisone enhances the specificity and degree of NHP methylation and phosphorylation at discrete chromosomal loci, i.e. alterations in side chain metabolism which may be responsible for increased RNA synthesis.

Verruciform xanthoma. Further light and electron microscopic studies, with the addition of a third case.

The authors report their third case of verruciform xanthoma confirmed by electron microscopy and frozen-section lipid stains. Studies of the blood in this case revealed normal levels of triglycerides, cholesterol, and glucose, just as in the previous two cases reported. An ultrastructural search for etiologic factors with the use of material from all three cases resulted in the hypothesis that this lesion is initiated within the epithelium. The authors propose that epithelial entrapment with degeneration and accumulation of lipid may be the logical sequence of early events leading to the formation of foam cells.