Perianal nodule in a young woman.

A young woman presented with a perianal nodular lesion, which was found to have histopathological findings of hidradenoma papilliferum. The anal skin is an uncommon location for this neoplasm.

Dermatomyositis panniculitis: a clinicopathological and immunohistochemical study of 18 cases.

BACKGROUND: Panniculitis occurring in dermatomyositis is uncommon, with only a few cases described in the literature, most of them as case reports. OBJECTIVE: This report describes the clinicopathological and immunohistochemical findings in a series of 18 patients with panniculitis associated with dermatomyositis. METHODS: In each patient, we collected the clinical data of the cutaneous lesions as well as the characteristic clinical and laboratory findings. A series of histopathologic findings was recorded in the biopsy of each patient. A panel of antibodies was used in some cases to investigate the immunophenotype of the infiltrate. Data of treatment and follow-up were also collected. RESULTS: Of the 18 patients, 13 were female and 5 were male, ranging in age from 13 to 74 years (median, 46.4 years). In addition to panniculitis, all patients presented pathognomonic cutaneous findings of DM and reported proximal muscle weakness prior to the diagnosis of panniculitis. Muscle biopsy was performed in 17 patients and MRI in one, all with the diagnosis of inflammatory myopathy. None of the patients presented any associated neoplasia. Panniculitis lesions were located in the upper or lower limbs. Histopathology showed a mostly lobular panniculitis with lymphocytes as the main component of the infiltrate. Most cases showed also numerous plasma cells and lymphocytes surrounding necrotic adipocytes (rimming) were frequently seen. Lymphocytic vasculitis and abundant mucin interstitially deposited between collagen bundles of the dermis were also frequent findings. Late-stage lesions showed hyaline necrosis of the fat lobule and calcification. Immunohistochemistry demonstrated that most lymphocytes of the infiltrate were T-helper lymphocytes, with some B lymphocytes in the lymphoid aggregates and small clusters of CD-123-positive plasmacytoid dendritic cells in the involved fat lobule. CONCLUSION: Panniculitis in dermatomyositis is rare. Histopathologic findings of panniculitis dermatomyositis are identical to those of lupus panniculitis. Therefore, the final diagnosis requires clinic-pathologic correlation.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Mutant Wars2 gene in spontaneously hypertensive rats impairs brown adipose tissue function and predisposes to visceral obesity.

Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms of metabolic disturbances. Recent linkage analysis in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), identified two closely linked quantitative trait loci (QTL) associated with glucose oxidation and glucose incorporation into BAT lipids in the vicinity of Wars2 (tryptophanyl tRNA synthetase 2 (mitochondrial)) gene on chromosome 2. The SHR harbors L53F WARS2 protein variant that was associated with reduced angiogenesis and Wars2 thus represents a prominent positional candidate gene. In the current study, we validated this candidate as a quantitative trait gene (QTG) using transgenic rescue experiment. SHR-Wars2 transgenic rats with wild type Wars2 gene when compared to SHR, showed more efficient mitochondrial proteosynthesis and increased mitochondrial respiration, which was associated with increased glucose oxidation and incorporation into BAT lipids, and with reduced weight of visceral fat. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance in muscle and white adipose tissue. In summary, these results demonstrate important role of Wars2 gene in regulating BAT function and consequently lipid and glucose metabolism.

Hepatotoxic effects of fenofibrate in spontaneously hypertensive rats expressing human C-reactive protein.

Dyslipidemia and inflammation play an important role in the pathogenesis of cardiovascular and liver disease. Fenofibrate has a well-known efficacy to reduce cholesterol and triglycerides. Combination with statins can ameliorate hypolipidemic and anti-inflammatory effects of fibrates. In the current study, we tested the anti-inflammatory and metabolic effects of fenofibrate alone and in combination with rosuvastatin in a model of inflammation and metabolic syndrome, using spontaneously hypertensive rats expressing the human C-reactive protein transgene (SHR-CRP transgenic rats). SHR-CRP rats treated with fenofibrate alone (100 mg/kg body weight) or in combination with rosuvastatin (20 mg/kg body weight) vs. SHR-CRP untreated controls showed increased levels of proinflammatory marker IL6, increased concentrations of ALT, AST and ALP, increased oxidative stress in the liver and necrotic changes of the liver. In addition, SHR-CRP rats treated with fenofibrate, or with fenofibrate combined with rosuvastatin vs. untreated controls, exhibited increased serum triglycerides and reduced HDL cholesterol, as well as reduced hepatic triglyceride, cholesterol and glycogen concentrations. These findings suggest that in the presence of high levels of human CRP, fenofibrate can induce liver damage even in combination with rosuvastatin. Accordingly, these results caution against the possible hepatotoxic effects of fenofibrate in patients with high levels of CRP.

Effects of transgenic expression of dopamine beta hydroxylase (Dbh) gene on blood pressure in spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and left ventricular hypertrophy. Catecholamines play an important role in the pathogenesis of both essential hypertension in humans and in the SHR. Recently, we obtained evidence that the SHR harbors a variant in the gene for dopamine beta hydroxylase (Dbh) that is associated with reduced adrenal expression of Dbh mRNA and reduced DBH enzymatic activity which correlated negatively with blood pressure. In the current study, we used a transgenic experiment to test the hypothesis that reduced Dbh expression predisposes the SHR to hypertension and that augmentation of Dbh expression would reduce blood pressure. We derived 2 new transgenic SHR-Dbh lines expressing Dbh cDNA under control of the Brown Norway (BN) wild type promoter. We found modestly increased adrenal expression of Dbh in transgenic rats versus SHR non-transgenic controls that was associated with reduced adrenal levels of dopamine and increased plasma levels of norepinephrine and epinephrine. The observed changes in catecholamine metabolism were associated with increased blood pressure and left ventricular mass in both transgenic lines. We did not observe any consistent changes in brainstem levels of catecholamines or of mRNA levels of Dbh in the transgenic strains. Contrary to our initial expections, these findings are consistent with the possibility that genetically determined decreases in adrenal expression and activity of DBH do not represent primary determinants of increased blood pressure in the SHR model.

Rosuvastatin ameliorates inflammation, renal fat accumulation, and kidney injury in transgenic spontaneously hypertensive rats expressing human C-reactive protein.

Recently, we derived "humanized" spontaneously hypertensive rats (SHR-CRP) in which transgenic expression of human CRP induces inflammation, oxidative stress, several features of metabolic syndrome and target organ injury. In addition, we found that rosuvastatin treatment of SHR-CRP transgenic rats can protect against pro-inflammatory effects of human CRP and also reduce cardiac inflammation and oxidative damage. In the current study, we tested the effects of rosuvastatin (5 mg/kg) on kidney injury in SHR-CRP males versus untreated SHR-CRP and SHR controls. All rats were fed a high sucrose diet. In SHR-CRP transgenic rats, treatment with rosuvastatin for 10 weeks, compared to untreated transgenic rats and SHR controls, was associated with significantly reduced systemic inflammation which was accompanied with activation of antioxidative enzymes in the kidney, lower renal fat accumulation, and with amelioration of histopathological changes in the kidney. These findings provide evidence that, in the presence of high CRP levels, rosuvastatin exhibits significant anti-inflammatory, anti-oxidative, and renoprotective effects.

Gender-related effects on substrate utilization and metabolic adaptation in hairless spontaneously hypertensive rat.

Cold exposure of rats leads to ameliorated glucose and triglyceride utilization with females displaying better adaptation to a cold environment. In the current study, we used hairless rats as a model of increased thermogenesis and analyzed gender-related effects on parameters of lipid and glucose metabolism in the spontaneously hypertensive (SHR) rats. Specifically, we compared hairless coisogenic SHR-Dsg4 males and females harboring mutant Dsg4 (desmoglein 4) gene versus their SHR wild type controls. Two way ANOVA showed significant Dsg4 genotype (hairless or wild type) x gender interaction effects on palmitate oxidation in brown adipose tissue (BAT), glucose incorporation into BAT determined by microPET, and glucose oxidation in skeletal muscles. In addition, we observed significant interaction effects on sensitivity of muscle tissue to insulin action when Dsg4 genotype affected these metabolic traits in males, but had little or no effects in females. Both wild type and hairless females and hairless males showed increased glucose incorporation and palmitate oxidation in BAT and higher tissue insulin sensitivity when compared to wild type males. These findings provide evidence for gender-related differences in metabolic adaptation required for increased thermogenesis. They are consistent with the hypothesis that increased glucose and palmitate utilization in BAT and muscle is associated with higher sensitivity of adipose and muscle tissues to insulin action.

Reactivities of a monoclonal antibody directed against an immunosuppressive molecule in boar seminal plasma.

Using a monoclonal antibody (LIS-4) to an immunosuppressive factor isolated from boar vesicular gland secretion it was determined that this gland secretes a tissue-specific immunosuppressive molecule that is absorbed onto the acrosome of spermatozoa during ejaculation. Absorption of the immunosuppressive molecule onto murine embryos at the 2-, 4-, 8-cell, morula and blastocyst stages in vitro was evaluated by indirect immunofluorescence. In vivo absorption was detected on the zona pellucida of murine embryos obtained from oviducts injected with the immunosuppressive molecule. Immunofluorescence revealed that the immunosuppressive molecule was not absorbed onto murine embryos after solubilization of the zona pellucida. There was no effect of the antibody to the immunosuppressive molecule on the ability of boar spermatozoa to penetrate the porcine zona pellucida.

Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension.

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.

Genetic analysis of "metabolic syndrome" in the spontaneously hypertensive rat.

In the current review, we summarize results of genetic analyses of "metabolic syndrome" in the spontaneously hypertensive rat (SHR). These results include (1) linkage analyses in the HXB/BXH recombinant inbred (RI) strains derived from SHR and Brown Norway (BN-Lx) strains which revealed quantitative trait loci (QTL) for hemodynamic and metabolic traits on several chromosomes, (2) genetic isolation of these putative QTL within differential chromosome segments of SHR.BN congenic strains, (3) detailed mapping of these QTL within limited chromosome segments of SHR.BN congenic sublines, (4) sequencing of selected positional candidate genes which revealed important mutations in the Cd36 and Srebp1 SHR genes, (5) functional tests of these candidate genes in SHR transgenic lines, and (6) integrated gene expression profiling and linkage mapping in RI strains which will be used to identify co-regulated genes and to determine co-segregation of transcriptional profiles with physiological and pathophysiological phenotypes.

The construction of different types of aggregates and chimaeric aggregates from individual blastomeres of mouse 8-cell embryos.

Different types of aggregates and chimaeric aggregates were constructed from individual 1/8-blastomeres of C57BL/6J and 129/Sv 8-cell embryos. Addition of phytohaemagglutinin into manipulation medium allowed us to construct large numbers of 4/8 to 9/8 aggregates and chimaeric aggregates exactly according to the design of the experiment. The developmental potential of reconstructed embryos was high. Over 93% of 4/8 to 8/8 aggregates and 90% of different types of 7/8 to 9/8 chimaeric aggregates formed blastocysts during 30 to 34 h of in vitro culture. All types of 4/8 to 8/8 aggregates and 7/8 to 9/8 chimaeric aggregates, in vitro cultured for 24 h, were able to undergo normal implantation after transfer into day-3 recipients. Live young were born after the transfer of morulae and blastocysts developed from 8/8 aggregates, and chimaeric morulae and blastocysts formed from different types of 7/8 to 9/8 chimaeric aggregates. All young born after the transfer of chimaeric embryos were overt C57BL/6J in equilibrium 129/Sv chimaeras.

Cryopreservation of mouse zona-free embryos and embryos exposed to biopsy and reconstruction by vitrification in microdrops.

Zona-free zygotes, 2-cell, 4-cell, and 8-cell embryos, morulae, blastocysts and embryos exposed to biopsy and reconstruction were cryopreserved in microdrops of vitrification medium expelled directly into liquid nitrogen. While none of the zygotes and 2-cell embryos survived storage in liquid nitrogen, 54% 4-cell and 97% 8-cell embryos, 99% morulae, 88% blastocysts, 98% biopsied and 100% reconstructed embryos were intact one hour after thawing. Developmental potential of cryopreserved zona-free embryos evaluated after 30 h of in vitro culture was high. Of the frozen embryos, 49% 4-cell and 92% 8-cell embryos, 95% morulae, 79% blastocysts, 84% biopsied and 90% reconstructed embryos were capable of further cleavage. After the transfer of zona-free 8-cell embryos, morulae, blastocysts, biopsied and reconstructed embryos into day-1 recipients - 4 (16%), 14 (56%), 3 (12%), 0 and 8 (32%) live foetuses were recorded, respectively.

Rapid thawing of rabbit embryos after storage at -196 degrees C.

Rabbit morulae frozen to -25 degrees C and transferred from this temperature to -196 degrees C were thawed at different rates. Maximum survival of the embryos was obtained at thawing rate of 650 degrees C/min. Slower and more rapid thawing decreased the survival. At thawing rates of 25 degrees C/min and 1 550 degrees C/min all morulae died. The ability of morulae to develop after storage at -196 degrees C and thawing at 650 degrees C/min was assayed by culture in vitro and transfer into the recipients. Of 241 morulae obtained after thawing, 197 (81.7%) continued normal development. Of 100 unfrozen morulae, 97 (97%) developed to the blastocyst stage. Of 41 frozen and 20 unfrozen embryos transferred into the recipients, 28 (68.3%) and 19 (95%), respectively, implanted. After transfer of 37 blastocysts obtained by culture of morulae thawed at 650 degrees C/min, 5 recipients have born 21 (56.8%) live offspring.

Factors influencing the results of transfers of rabbit embryos stored at -196 degrees C.

Rabbit embryos at the 8-cell and morula stages were frozen and stored at -196 degrees C for 2-200 days. After thawing the embryos were examined for their viability in vitro and in vivo. In vitro, 62.5% of frozen 8-cell embryos and 81.4% of frozen morulae developed to blastocysts. In the control group of unfrozen embryos, 93.2% 8-cell embryos and 92.4% morulae developed to the blastocyst stage. Culture permitted a more reliable elimination of the embryos damaged during freezing and thawing. Embryos were transferred into the reproductive tracts of the recipients either directly after thawing or after 24 h in culture. Synchronous transfers of frozen rabbit embryos were not successful. After asynchronous transfers of morulae and blastocysts into the oviducts, implantation was 31.8% and 42.9%, respectively. After transfer of blastocysts into the uterine horns of the recipients, 47.6% embryos implanted.

A simplified method for freezing mouse blastocysts.

Mouse blastocysts frozen slowly (0.3-0.5 degrees C min-1) to -25 degrees C and transferred to -196 degrees C survive long-term storage to the extent comparable to unfrozen blastocysts. After thawing at 450 and 650 degrees C min-1, 91 and 93% blastocysts, respectively, continued normal development under in vitro conditions. The length of equilibration at -25 degrees C before transfer to -196 degrees C had no marked effect on survival of blastocysts. It has been shown that blastocysts stored at -196 degrees C may be transferred into recipients immediately after thawing. The prerequisite of a normal development was the transfer into pseudopregnant females on day 2 of pseudo-pregnancy; 60.3% foetuses were found 14-15 days after transfer. Transfers of thawed blastocysts into pseudopregnant females on day 3 or day 4 of pseudopregnancy failed (3.9 and 0% foetuses, respectively). After transfer of unfrozen blastocysts into pseudopregnant females on day 3 of pseudopregnancy 58.7% foetuses were found. Transfers of unfrozen blastocysts into pseudopregnant females on day 2 and day 4 of pseudopregnancy were less effective (6.4 and 27.3% foetuses, respectively).

Protection of non-cellular investments of rabbit morulae stored at--196 degrees C.

The non-cellular investments were damaged in only 2.8% of rabbit morulae frozen/ thawed in plastic straws. Similar results (4.8%) were obtained with glass ampoules only when morulae were slowly frozen and slowly thawed in siliconized ampoules. Freezing/thawing by other methods and the use of untreated ampoules resulted in high incidence of embryos with damaged investments (41-61%). Morulae frozen/thawed by a method allowing an almost complete elimination of investment injury displayed high survival when slowly frozen and rapidly thawed (90.7%) while slow freezing and slow thawing decreased survival (74.5%). Morulae subjected to rapid freezing were for the first time successfully stored at--196 degrees C. After rapid thawing 59.6% morulae survived. The mechanism of damage to the non-cellular investments of rabbit embryos is discussed.

Activity and subcellular localization of proteolytic enzymes of boar and rabbit spermatozoa.

Comparison of the mode and time course of depolymerization of the gelatin by boar and rabbit spermatozoa obtained from the ejaculates and the female reproductive tract revealed that the proteolytic activity of the spermatozoa was not changed during their passage through the female reproductive tract. In all sperm samples the depolymerization started at the level of the equatorial segment, proceeded to the apical part and digested the substrate around the sperm head. The time course of depolymerization was also the same in all samples. Slight differences were caused by degeneration changes affecting the acrosomal region of the sperm head and accelerating the initial course of substrate depolymerization. The results were confirmed by investigating the state of the acrosomes of individual sperm samples using the silver proteinate method. It appeared that samples with greater numbers of spermatozoa with damaged acrosome begin to affect the substrate a few minutes earlier. The disadvantage of the methods used is that the release of the proteolytic enzymes from the sperm head is probably caused by postmortem degeneration changes of the spermatozoa.

"Two-step" freezing of individual blastomeres from mouse eight-cell embryos.

Eight-cell embryos recovered from superovulated C57BL/6J mice were disaggregated into individual 1/8 blastomeres. For cryopreservation of blastomeres a simple "two-step" technique based on direct transfer of blastomeres into the final concentration of DMSO (0.5 to 1.5 M) and exposure of plastic straws with blastomeres to -25 degrees C for 10 min before immersion into liquid nitrogen was used. The samples stored for 2 to 60 days were thawed at different temperature (20 to 80 degrees C) and intact 1/8 blastomeres were transferred into DMSO-free medium. The survival of blastomeres in individual groups varied from 52% to 90% according to the combination of DMSO concentration, exposure time to DMSO prior to freezing and temperature of thawing bath. Intact frozen-thawed 1/8 blastomeres were able to undergo cleavage and aggregates constructed from 4, 6, 7, and 8 blastomeres developed throughout preimplantation period. Of the aggregates constructed 1 to 2 h after thawing, 78% to 93% reached the blastocyst stage within 54 h of in vitro culture. Possible use of frozen-thawed 1/8 blastomeres in nuclear transplantation experiments and genetic manipulation on mammalian embryos is discussed.