[Influence of connective tissue differentiation on scar tissue formation in children]. / Vliyanie differentsiatsii soedinitel'noi tkani na formirovanie rubtsovoi tkani v detskom vozraste.

OBJECTIVE: To study the immunohistochemical features of various scar tissues in children without connective tissue pathology and with undifferentiated connective tissue dysplasia syndrome. MATERIAL AND METHODS: Tissue biopsy was performed in 217 children who underwent surgical treatment for various lesions, such as injuries, burns, as well as other procedures. There were 127 boys (58.5%) and 90 (41.5%) girls. The main group consisted of 98 (48.2%) children with scar tissue; group of UCTD syndrome - 65 (30.0%) children; control group - 43 (24.8%) patients without pathological scars. Histological examination of scar tissue and intact skin was carried out during primary or redo reconstructive surgery. Immunohistochemical study of antibodies against CD34, CD105, CD140b, PDGFs, COL types I, III and IV was performed. RESULTS: The study showed a quantitative characteristic of expression of COL type I in hypertrophic scar with predominance in the main group (77.5±5.4%; p<0.05), and decrease in COL type IV. Keloid form was associated with predominance of granulation tissue in all layers of dermis and high levels of all types of collagen. In the group of UCTD, COL type III prevailed in all pathological forms of the scar. We determined quantitative indicators of expression of vascularization factors (CD34; CD105) and fibroblastic activity (CD140b; PDGFs). CONCLUSION: Understanding the process of fibrinogenesis and analysis of stages of triggering mechanisms are essential for development of preventive algorithms. Individualized approach should be considered in the treatment. These studies are especially important in children with UCTD syndrome as high-risk group for pathological scarring. Thus, further research is required.

Vaccination of calves with Mycobacterium bovis Bacillus Calmette-Guerin reduces the frequency and severity of lesions of bovine tuberculosis under a natural transmission setting in Ethiopia.

Bovine tuberculosis (bTB) is highly prevalent in intensive dairy farms of the urban "milk-sheds" in Ethiopia, and vaccination could be a cost-effective disease control strategy. In the present study, the efficacy of Bacillus Calmette-Guerin (BCG) to protect against bTB was assessed in Holstein-Friesian calves in a natural transmission setting. Twenty-three 2-week-old calves were subcutaneously vaccinated with BCG Danish SSI strain 1331, and matched 26 calves were injected with placebo. Six weeks later, calves were introduced into a herd of M. bovis-infected animals (reactors) and kept in contact with them for 1 year. In vitro and in vivo immunological tests were performed to assess immune responses post-vaccination and during exposure. Successful vaccine uptake was confirmed by tuberculin skin test and IFN-γ responses in vaccinated calves. The kinetics of IFN-γ responses to early secretory antigen target 6 and culture filtrate protein 10 (ESAT6 and CFP10, respectively) and tuberculin skin test responses post-exposure suggested that the animals were infected early after being placed in contact with the infected herd as immunological signs of infection were measurable between 2 and 4 months post-initial exposure. Protection was determined by comparing gross and microscopic pathology and bacteriological burden between vaccinated and control calves. BCG vaccination reduced the proportions of tissues with visible pathology in vaccinates compared to control calves by 49% (p < .001) with 56%, 43%, 72%, and 38% reductions in the proportion of lesioned tisues in head, thoracic, abdominal lymph nodes, and lungs, respectively (p-values .029-.0001). In addition, the lesions were less severe grossly and microscopically in vaccinated calves than in non-vaccinated calves (p < .05). The reduction in the overall incidence rates of bTB was 23%, 28%, and 33% on the basis of the absence of gross pathology, M. bovis culture positivity, and histopathology, respectively, in vaccinated animals. In conclusion, BCG vaccination reduced the frequency and severity of the pathology of bTB significantly, which is likely to reduce onwards transmission of the disease.

[Structural changes of toothbrush bristles by brushing in patients with dental implants].

Lack of proper oral hygiene practices can lead to treatment failure in patients with implant-retained restorations. Structural changes of toothbrush bristles were studied using scanning electron microscopy and correlated with cleaning efficiency which was assessed at baseline and after 3 months of use of various toothbrushes types in 146 patients with implant-retained restorations. Oral hygiene was valued according to several indices (Approximal Plaque-Index (API), the Turesky index (PI), a modified superstructure plaque index Silness-Loe (PLIsk). Ultrasound toothbrush provided the best and the most efficient cleaning outcome in patients with implant-retained restorations. Scanning electron microscopy proved ultrasonic toothbrush bristles to be more resistant to abrasion during the three-month use.

Effect of skin testing and segregation on the prevalence of bovine tuberculosis, and molecular typing of Mycobacterium bovis, in Ethiopia.

In 2002, the prevalence of bovine tuberculosis (tb) among 500 cattle on Holeta Farm, near Addis Ababa, Ethiopia, was 48 per cent, and the farm was divided into positive and negative herds. After three consecutive rounds of skin testing and segregation of skin test-positive and -negative animals, the prevalence of bovine tb was reduced from 14 per cent to 1 per cent in the negative herd in a year. Spoligotyping of 41 isolates from 17 cows gave an identical and unique spoligotype pattern, which can be represented as the binary number 1100000101111110111111100010000000000100000, where 1 indicates the presence of a spacer and 0 represents a loss. This spoligotype pattern had not previously been reported on the Mycobacterium bovis spoligotype database, and it was therefore designated SB1176, Ethiopian M bovis strain 1 (EMbs1). The variable number tandem repeat (VNTR) profile of the strain was 5254(*)33.1, which differed from the VNTR profile of strains reported in Great Britain.

In vivo effects of chromium.

The production of reactive oxygen species on addition of hexavalent chromium (potassium dichromate, K2Cr2O7) to lung cells in culture was studied using flow cytometer analysis. A Coulter Epics Profile II flow cytometer was used to detect the formation of reactive oxygen species after K2Cr2O7 was added to A549 cells grown to confluence. The cells were loaded with the dye, 2',7'-dichlorofluorescein diacetate, after which cellular esterases removed the acetate groups and the dye was trapped intracellularly. Reactive oxygen species oxidized the dye, with resultant fluorescence. Increased doses of Cr(VI) caused increasing fluorescence (10-fold higher than background at 200 microM). Addition of Cr(III) compounds, as the picolinate or chloride, caused no increased fluorescence. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that three (as yet unidentified) spectral "signals" of the free radical type were formed on addition of 20, 50, 100, and 200 microM Cr(VI) to the A549 cells in suspension. Two other EPR "signals" with the characteristics of Cr(V) entities were seen at field values lower than the standard free radical value. Liver microsomes from male Sprague-Dawley rats treated intraperitoneally with K2Cr2O7 (130 mumole/kg every 48 hr for six treatments) had decreased activity of cytochromes P4503A1 and/or 3A2, and 2C11. Hepatic microsomes from treated female Sprague-Dawley rats, in contrast, had increased activities of these isozymes. Lung microsomes from male Sprague-Dawley rats had increased activity of P4502C11.

Differentiation of HL-60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle.

The human promyelocytic leukaemia cell line HL-60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP). Differentiation begins within 16-24 h of treatment and is associated with a time- and dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2 + M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL-60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2 + M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL-60 cells.

Distinct patterns of chemotactic peptide-induced calcium mobilization in differentiated myeloid leukemia cells and peripheral blood neutrophils.

Flow cytometry was used to compare intracellular calcium mobilization in mature neutrophilic granulocytes (PMN) with HL-60 promyelocytic leukemia cells induced to differentiate with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Using the calcium-specific probe indo-1 acetoxymethyl ester, we found that the ability of differentiating HL-60 cells to mobilize calcium in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) developed concomitantly with expression of receptors on the cells for this peptide. Mobilization of calcium in HL-60 cells, as well as in PMN, in response to fMLP was dose dependent and was related to the presence of calcium in the culture medium. In calcium-free medium, tenfold higher concentrations of fMLP were required to induce calcium mobilization than in calcium-containing medium. In HL-60 cells, calcium mobilization occurred more rapidly than in PMN and was independent of extracellular calcium. Furthermore, the response of HL-60 cells to fMLP was more prolonged than the response of PMN and was also of greater magnitude. Calcium mobilization in both HL-60 cells and PMN was partially inhibited by the calcium channel blocker, verapamil, but completely blocked by trimethylbenzoic acid 8-(diethylamino) octyl ester, an intracellular calcium antagonist. These results indicate that although both cell types mobilize calcium in response to fMLP, the characteristics of the responses are distinct. These differences may underlie distinct functional responses of PMN and differentiated HL-60 cells to fMLP.

Enhanced hydroperoxide production by peripheral blood leukocytes following exposure of murine epidermis to 12-O-tetradecanoylphorbol-13-acetate.

Previous studies have shown that the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates phagocytic leukocytes to produce reactive oxygen intermediates in vitro. The present studies focused on the production of reactive oxygen intermediates by peripheral blood leukocyte cell populations following in vivo exposure of murine epidermis to TPA. TPA induced a dose-dependent (0.2-20 micrograms) increase in polymorphonuclear cells (PMN) that appeared to be recruited from the marginal pools, while simultaneously decreasing the number of peripheral blood mononuclear cells. These alterations were detected as early as 2 h following topical application of TPA and persisted over a 21 day time period, using a twice-weekly TPA treatment schedule. The oxidation of 2',7'-dichlorofluorescin (DCFH) was used to examine the hydroperoxide production in peripheral blood PMN isolated from SENCAR mice treated with TPA. TPA stimulated a 2-fold increase in PMN-associated DCFH oxidation (645.4 +/- 118 fmol DCF) 4 h after topical application of 10 micrograms TPA when compared to PMN isolated from acetone-treated mice (339.0 +/- 35.8 fmol DCF). These observations suggest that topical application of TPA recruits PMN that are activated prior to their infiltration into the epidermis. Given the ability of these cells to migrate to local sites, they may serve as a primed cell population that significantly contributes to cutaneous alterations observed during acute and chronic inflammation following TPA exposure.

Distinct patterns of sulfated proteoglycan biosynthesis in human monocytes, granulocytes and myeloid leukemic cells.

The production of sulfated proteoglycans was compared in mature peripheral blood granulocytes and monocytes and HL-60 promyelocytic leukemia cells. We found that HL-60 cells synthesized 5-10 times more proteoglycans than peripheral blood leukocytes. Differentiation of HL-60 cells toward mature monocytes by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or towards granulocytes by treatment with retinoic acid or dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP) resulted in a small (20-30%) decrease in sulfated proteoglycan biosynthesis. Chondroitin sulfate was found to be the predominant proteoglycan produced by monocytes, PMN and undifferentiated HL-60 cells. Differentiated HL-60 cells produced chondroitin sulfate as well as sulfated proteoglycans sensitive to nitrous acid degradation. Similar results were observed in TPA, dbcAMP and retinoic acid differentiated HL-60 cells indicating that the changes in proteoglycan biosynthesis observed were independent of the developmental pathway. Using specific monoclonal antibodies and flow cytometry, we also found that HL-60 cells and monocytes produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate while PMN only produced chondroitin-4-sulfate. In addition, although there were no significant differences in antibody binding to undifferentiated and differentiated HL-60 cells, the tumor cells bound 5-20 times more of the antibodies than the peripheral blood leukocytes. Our data demonstrate that sulfated proteoglycan production by HL-60 cells is distinct from PMN and monocytes. In addition, the fact that differentiated HL-60 cells continue to synthesize larger amounts of the proteoglycans than the peripheral blood leukocytes indicates these cells have not completely matured.

Failure of F-Met-Leu-Phe to induce chemotaxis in differentiated promyelocytic (HL-60) leukemia cells.

Treatment of HL-60 promyelocytic leukemia cells with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) induced these cells to differentiate toward mature neutrophilic granulocytes (PMN). DbcAMP was found to produce a rapid time- and dose-dependent inhibition of HL-60 cell proliferation, reaching a maximum after 48 hr treatment of the cells with 250-500 microM. This was associated with morphologic alterations consistent with maturing PMN. Using immunofluorescence and flow cytometry, we found that dbcAMP-treated HL-60 cells expressed some, but not all, surface markers characteristic of mature phagocytes. Thus receptors for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP); the monocyte/granulocyte markers My-8, Mo-1, and Leu-15; as well as the monocyte markers Mo-2 and My-4 were identified on the cells. In contrast, dbcAMP-treated cells did not express the PMN-specific antigen DL1.2. We also found that dbcAMP-treated HL-60 cells produced hydrogen peroxide in response to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, the magnitude of this response was significantly less than that observed in mature PMN. In contrast, fMLP-stimulated levels of hydrogen peroxide production were comparable in both cell types. In addition, despite the fact that dbcAMP-treated cells expressed fMLP receptors, they did not exhibit directed migration toward this chemotactic peptide. Taken together, these data suggest that dbcAMP-induced differentiation of HL-60 cells is incomplete.

Differentiation of U-937 histiocytic lymphoma cells towards mature neutrophilic granulocytes by dibutyryl cyclic adenosine-3',5'-monophosphate.

Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to differentiate towards granulocytes. dBcAMP produced a dose- and time-dependent inhibition of U-937 cell growth reaching a maximum after 48-h treatment with 500 microM. At this concentration, dBcAMP had no effect on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a concomitant increase in cells in the G0/G1 phase. dBcAMP also induced the appearance of f-met-leu-phe receptors on U-937 cells as well as the ability to produce hydrogen peroxide and superoxide anion. These data suggest that dBcAMP-treated U-937 cells were functionally mature. Using specific monoclonal antibodies and flow cytometry, we found that differentiated U-937 cells expressed the monocytic/granulocytic surface markers MY8 and MAC-1, but not the monocyte specific markers MO2 or MY4. In addition, dBcAMP-treated U-937 cells did not stain for nonspecific esterase, displayed less HLA-DR antibody binding than undifferentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies indicate that differentiation of U-937 cells is not necessarily limited to the monocytic pathway of development.

Functional and biochemical properties of rat Kupffer cells and peritoneal macrophages.

Functional and biochemical techniques were used to further characterize heterogeneity between rat Kupffer cells and peritoneal macrophages. Both macrophage cell types were found to phagocytize antibody coated sheep red blood cells in a time-dependent manner. However, Kupffer cells were two to three times more phagocytic than were peritoneal macrophages. In contrast, the peritoneal cells released significantly more superoxide anion in response to the complement cleavage product, C5a and the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate, and produced more hydrogen peroxide than did the liver macrophages. Both cell types responded chemotactically to C5a. These results suggest that macrophages may develop specialized functions depending on the needs of their local environment. Using one and two dimensional SDS-polyacrylamide gel electrophoresis, we also compared the production of newly synthesized proteins by Kupffer cells and peritoneal macrophages. In general, the macrophages were found to produce similar types and numbers of proteins with some exceptions. These included proteins that were unique to peritoneal macrophages and other proteins observed only in Kupffer cells. The production of these proteins in liver macrophages did not appear to correlate with levels of functional activation, but may be more related to the tissue origin of the cells.