Immunohistochemical localization of TNF-α and IL-4 in granulomas of immunocompetent and immunosuppressed New Zealand white rabbits infected with Encephalitozoon cuniculi.

Encephalitozoon cuniculi is a fungi-related, obligate, zoonotic, spore-forming intracellular eukaryotic microorganism. This emerging pathogen causes granulomas to form in the brain and kidneys of infected individuals. The objective of the current study was to detect the distribution of TNF-α- and IL-4-positive cells using immunohistochemistry within these granulomas in both infected immunocompetent (group A) and immunosuppressed (group B) New Zealand white rabbits. In the brain, labeled TNF-α immune cells were mainly located in the granuloma peripheries in group B. Granulomas examined in the kidneys of groups A and B were TNF-α positive, but were significantly different (p < 0.001) when compared with the brain. IL-4-producing immune cells in the brain and kidneys were disseminated within granulomas in groups A and B; however, no significant difference (p > 0.05), was observed. IL-4 positive cells were more numerous in brain sections of group B and differed significantly (p < 0.05) when compared with kidneys. Granulomas were not observed in control animals (groups C and D). In conclusion, we identified TNF-α positive cells in both the brain and kidneys of immunocompetent and immunosuppressed animals; IL-4 positive cells were numerous in the brains of immunosuppressed rabbits; however, in terms of percentage were numerous in the brains of immunocompetent rabbits. Immunosuppression appeared to stimulate a change in the cellular phenotype of Th1- to Th2-like granulomas in the brain and kidneys via an unknown mechanism. Expression of pro- and pre-inflammatory cytokines in microsporidian granulomas suggests a mechanism by which E. cuniculi evades the immune response, causing more severe disease. These results increase our understanding of TNF-α and IL-4-positive cells within the E. cuniculi granuloma microenvironment.

Mast cell density during initiation and progression of the local Shwartzman reaction.

OBJECTIVE AND DESIGN: To quantify the number of mast cells in the skin of rabbits during initiation and progression of the local Shwartzman reaction. MATERIALS: Thirty New Zealand rabbits were divided in two groups (n = 15/group). One group was subjected to the Shwartzman reaction and the other group served as control. Subsequently, animals were further subdivided in six groups of five animals each according to time of euthanasia. TREATMENTS: The local Shwartzman reaction was induced by two inoculations of Salmonella typhimurium lipopolysaccharide. Preparatory inoculation was given intradermally and, 24 h later, the provocative injection was administered intravenously. Controls were subjected to the same procedure but received saline. After provocative injection animals were killed at 1, 8, and 15 days. METHODS: Skin samples were fixed in Carnoy's solution and mast cells were identified employing a low pH toluidine blue stain. Numbers of mast cells were determined per square millimetre and, subsequently, those cells degranulated were identified and quantified to obtain absolute values. A Student's t test was initially used to compare Shwartzman versus controls at each time point. Subsequently, an ANOVA test employing a factorial experiment was used to assess a possible interaction between time of euthanasia and treatments. RESULTS: The values were transformed (natural logarithms) for appropriate statistical comparisons. Independent comparisons at each time point showed that Shwartzman groups had higher numbers of mast cells than controls at 1 and 8 days, but not at 15 days (5.71 +/- 1.00 Vs. 2.40 +/- 0.58, P < 0.005; 3.77 +/- 0.90 Vs. 2.33 +/- 0.56, P < 0. 025, and 2.61 +/- 0.25 Vs. 2.39 +/- 0.39, P > 0.05, respectively). Degranulated cells were numerous in Shwartzman groups, particularly at day 1 (3.48 +/- 0.78) and less obvious at day 8 (0.72 +/- 0.50), but were scarce by day 15 (-0.67 +/- 0.99) as well as controls (-0.68 +/- 0.91). The factorial experiment demonstrated that the Shwartzman reaction and time of euthanasia were independently significant (P < 0.005) but their interaction at day 1 was the major contributor (P < 0.005). Tukey's w pairwise comparisons of means confirmed that the Shwartzman group killed at day 1 was significantly different from the others (P < 0.05). CONCLUSIONS: Mast cells significantly increase in the early stages of the local Shwartzman reaction. Thus, mast cells are a highly dynamic cell population, which have a prominent role during the acute phase of this lipopolysaccharide-induced inflammatory reaction but not during healing.

Serologic survey in animals of 'Q' fever in Nuevo Leon.

The serological prevalence of Q fever in Mexico is unknown. A serological survey for Coxiella burnetii was undertaken on a randomly selected population of dairy cattle, beef cattle, goats and sheep flocks. Serological examination of animal sera for antibodies against Coxiella burnetii was carried out by the ELISA technique. The 28% of the dairy cattle and 10% of beef cattle examinated were antibody positive. Sera from goats and sheep also had antibodies against this rickettsia, 35% and 40% respectively.

Immunolocalization of cathepsin B in equine dyschondroplastic articular cartilage.

A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage of young horses. In normal articular cartilage (n = 6 animals), significant amounts of enzyme were detected only in hypertrophic chondrocytes in the deep zone. The enzyme was intracellular, located in the lysosomal granules. No extracellular matrix staining was observed. Levels of cathepsin B were increased slightly above normal in the deep zone in age-matched dyschondroplastic cartilage (n = 5 animals). The most striking finding, however, was the abundance of the enzyme in chondrocyte clonal clusters associated with the lesions. Cathepsin B levels were low in chondrocytes isolated from normal cartilage (n = 6), but increased progressively during serial subculture, reaching a maximum at passage 5-6. In contrast, primary cultures of dyschondroplastic chondrocytes (n = 3) expressed abundant cathepsin B.

Cellular heterogeneity in cathepsin D distribution in equine articular cartilage.

The distribution of cathepsin D in normal equine growth cartilage has been examined immunocytochemically using an antiserum raised against human cathepsin D. The cross-reactivity and specificity of the antiserum for equine cathepsin D was confirmed, and its lysosomal localisation was demonstrated in horse skin fibroblasts by confocal scanning microscopy. Cultured horse chondrocytes were heterogenous in their expression of cathepsin D. Heterogeneity of distribution of the enzyme was also seen in chondrocytes in cartilage from different anatomical sites. A high level of cathepsin D was observed in the deep layer of cartilage from the lateral trochlear ridge of the distal femur. Cathepsin D was absent in the hypertrophic zone of the distal radial growth plate.