Experimental mitochondrial myopathy induced by chronic intoxication by Senna occidentalis seeds.

Histochemical and electron microscopic studies of biceps femoris, pectoralis major and rectus femoris of chronically treated birds with seeds of the poisonous plant Senna occidentalis (0.2% external/internal tegment), were performed. The muscles had similar features of human mitochondrial myopathy as ragged-red fibers, cytochrome-oxidase negative fibers, and weak activity of the oxidative enzymes. Fibers with lipid storage were also present. Acid phosphatase activity in rare muscle fibers was also detected, and represents probably a secondary degenerative process. By electron microscopy, enlarged mitochondria with disrupted or excessively branched cristae were seen. The present study presents a new experimental model of mitochondrial myopathy that may be useful for the best knowledge of this group of diseases and for experimental trials of drugs that could reverse the mitochondrial impairment in the mitochondrial myopathies.

Detection of the 20-kDa virulence-associated antigen of Rhodococcus equi in malakoplakia-like lesion in pleural tissue obtained from an AIDS patient.

A malakoplakia-like lesion was detected in a pleural biopsy from an AIDS patient presenting clinical and radiologic features of pneumonia. Cultures of bronchoalveolar lavage and pleural fluid evidenced Rhodococcus equi as the causative agent of pleuro-pulmonary infection. Immunochemical characterization of the R. equi isolate showed the presence of a strain similar to the ATCC 33704 reference strain presenting the capsular antigen of serotype 4, and the intermediate virulence-associated antigen of 20-kDa. Histopathology of the patient's pleural biopsy showed plaques of macrophages interspersed with lymphocytes, and intracytoplasmic cocci and bacilli in macrophages, which were variably acid-fast positive. Immunohistochemistry of cocci, bacilli and their degradation products resulted strongly positive when stained with a mouse monoclonal antibody (MAb) produced against the 20-kDa antigen. This finding could have important implications for the pathogenicity of R. equi for human beings, since we do not know yet all the factors involved in the formation of malakoplakia. Indeed, the results obtained in the present study, taken together with the results obtained for pigs inoculated with R. equi strains of intermediate virulence (Madarame et al. 1998), raise the possibility that most strains presenting the 20-kDa antigen may be capable of inducing malakoplakia. If this hypothesis is confirmed by immunohistochemical analysis of human pulmonary malakoplakia cases due to R. equi, the detection of this antigen may be extremely helpful in the diagnosis and treatment of such patients. This is the first report of R. equi infection in human beings that suggests a relationship between pleural malakoplakia and the virulence-associated antigen of 20-kDa.

Cerebral amebiasis in the acquired immunodeficiency syndrome.

Rare cases of cerebral amebiasis have been described in AIDS patients. We report the case of a 46 year-old homosexual man with AIDS who developed an intermittent amnesia and a right palpebral ptosis. The cerebrospinal fluid contained 169 cells (75% lymphocytes). The patient died five days after hospitalization. Necropsy revealed thrombosis of small vessels of the periventricular regions as well as necrosis and hemorrhage of the periventricular tissue, cerebellum and brainstem. The inflammatory process was scarce and composed mainly of CD-68 positive macrophages. In these regions as well as in meninges there were many trophozoites of ameba of the Acanthamoeba group. Although cerebral amebiasis is rare even in AIDS, the clinician should be attentive to this diagnosis in patients with an insidious encephalitis and cerebral cognitive abnormalities, with or without focal motor signs.

Squamous intraepithelial lesions in cervical smears of human immunodeficiency virus-seropositive adolescents.

Various authors have reported a high rate of human papillomavirus (HPV) infection and HPV-related neoplasias in human immunodeficiency virus (HIV)-seropositive women. On the other hand, young women are most susceptible to cervical infection because of immaturity of the cervix, as it appears that HPV has more access to the basal cells of the differentiating epithelium. The purpose of the present work was to study cervical smears of 82 adolescent HIV-seropositive women (13-21 years of age) to search for cytological evidence of cervical intraepithelial neoplasias. Twenty-one cases showed characteristic features of HPV infection and squamous intraepithelial lesions (SIL; 25.6%). Sixteen cases aged from 17 to 21 years (mean age 19.5 years) had low-grade SIL (LSIL; 19.5%) and five cases aged from 18 to 21 years (mean age 20.2 years) had high-grade SIL (HSIL; 6.1%). There was no significant difference between the mean age of patients with LSIL and HSIL. Two cases had atypical squamous cells of undetermined significance (ASCUS). In the present work it was found that HIV-seropositive adolescents have a high risk for preneoplastic lesions of the cervix (25.6%) as well as a high incidence of more aggressive lesions (6.1% of HSIL) when compared to the general population of adolescents. As it can be assumed that, if the age of acquisition of the infection in both groups (in the general population and HIV-seropositive women) is the same, it is probable that HIV infection in adolescents not only increases the frequency of HPV infections but also facilitates the evolution to more aggressive preneoplastic lesions of the cervix due to HPV.

Mitochondrial metabolism impairment in muscle fibres of rats chronically intoxicated with Senna occidentalis seeds.

The chronic administration of S. occidentalis seeds was found to induce a mitochondrial myopathy in hens. This study was undertaken to determine if the chronic treatment with S. occidentalis seeds of rats (as a mammalian model) would induce a mitochondrial myopathy similar to those described in humans and to determine if the histological changes could be correlated with the amount of ingested seeds. Twenty-one days old rats were fed S. occidentalis seeds at different diet concentrations (1, 2, 3%). Rats fed 1% S. occidentalis seeds had only a few COX-negative muscle fibers in the pectoralis major muscle. Rats fed 3% Senna occidentalis seeds had a greater number of COX-negative fibers. Rats fed 2% had an intermediate number of COX-negative fibers. Activity of SDH and NADH-tr were decreased in rats of groups 2% and 3%. Our data indicate that a progressive mitochondrial metabolism impairment can be produced in rats fed S. occidentalis seeds and that this impairment can be correlated with the amount of ingested seeds.

Sarcoplasmic lipase and non-specific esterase inhibition in myofibers of rats intoxicated with the organophosphate isofenphos.

The expression of sarcoplasmic esterases, lipases as well as the lipid content in the myofibers of the diaphragm of rats intoxicated with the organophosphate isofenphos was studied. Lipid accumulation was documented at light, electron microsopic and by morphometric studies. The distribution of these lipid droplets was irregular and abundant in myofibers with numerous mitochondria (predominantly oxidative fibers). Histochemical inhibition of sarcoplasmic esterases and lipases was observed in the intoxicated animals. This sarcoplasmic inhibition of esterases occurs roughly in parallel to the inhibition of plasma cholinesterase activity. The inhibition of sarcoplasmic lipases may explain, at least partially, the accumulation of lipids. This inhibition probably makes difficult the use of lipids as fuel, especially in the oxidative fibers. In contrast to the small amount of muscle necrosis, (1.30+/-0.745), metabolic muscle impairment was intense and extensive, i.e., decreased activities of esterases and lipases in the sarcoplasm, that should contribute to muscle weakness. Therefore, because segmental necrosis was most prominent in oxidative fibers (and these fibers use lipids as the principal fuel and contain the greater amount of lipases in the sarcoplasm), it is possible that inhibition of activity of lipases is responsible for the segmental necrosis. Although the exact role of these metabolic changes is not known, it is possible that they contribute not only to the induction and evolution of muscle cell necrosis but also to the muscle weakness and clinical impairment of animals and humans in the acute intoxication by these compounds.

Salpingitis due to Entamoeba histolytica.

We describe the pathology of a unique case of Fallopian tube amebiasis, associated with hydrosalpinx, in a 21-year-old woman. She complained of lower abdominal pain, had a foul-smelling green vaginal discharge and fever during one week. There was a discrete increase in body temperature and a painful abdominal palpation at the lower right side, with signs of local peritoneal irritation. Pathological examination showed a marked dilatation of the fallopian tube and hydrosalpinx. Microscopic examination showed a poorly formed granuloma composed of large macrophages with many Entamoeba histolytica trophozoites inside the fallopian tube. Even though it is a rare disease the correct diagnosis of female genital tract amebiasis is of great importance for the indication of proper therapy.

The lysosomal enzymes acid phosphatase and cathepsin D in rats intoxicated with Senna occidentalis seeds.

Chronic administration of Senna occidentalis seeds induces an experimental toxic myopathy characterized by skeletal muscle fibers atrophy, decrease in histochemical activity of cytochrome oxidase, and increase of the acid phosphatase activity in muscle fibres at the light microscopic level. The mechanisms that lead to the increase of this lysosomal enzyme activity are not known and could be related to other biochemical disturbs than the mitochondrial function impairment. The main aim of the present study is to localize the acid phosphatase activity using a cytochemical method at transmission electron microscopy level and to quantify cathepsin D in muscle of rats chronically intoxicated with Senna occidentalis seeds by immunoblotting. Acid phosphatase was observed in lysosomes and over profiles of some organelles apparently not involved by lysosomal membrane. In addition immunoblotting demonstrated a decrease in the content of the precursor and of the mature form of cathepsin D in samples of muscles and liver of intoxicated animals. We concluded that there is a selective increase in acid phosphatase activity in muscle--and maybe in other tissues--of animals intoxicated with Senna occidentalis, that can be related to the skeletal muscle atrophy and the intense decrease in weight gain of these animals. Further studies should be performed to establish the mechanisms of selectivity in increase of lysosomal enzymes in different situations and pathological states.

Distribution of COX-negative mitochondria in myofibers of rats intoxicated with Senna occidentalis seeds.

We have described that administration of seeds or parts of the seed of Senna occidentalis (coffee senna) for long periods, induces histochemical changes in the skeletal muscles of hens and rats that are characteristic of a mitochondrial myopathy--as decrease of SDH and COX activity, with some COX negative fibers. In this experimental model of mitochondrial myopathy, as in many human mitochondrial diseases, there is a random distribution of COX negative fibers. Some fibers are completely COX negative while others are partially negative and others are completely positive. In the present work we have studied the distribution of COX negative mitochondria at transmission electron microscopy in skeletal muscle of rats in this experimental myopathy. In myofibers of intoxicated animals the expression of COX was heterogeneous. The histochemical reaction was observed in the internal membrane (more evident in mitochondrial cristae) of all mitochondria of some myofibers, while it was almost absent in other myofibers. In these myofibers the great part of the mitochondria were negative for COX reaction while other ones had a weak expression of this enzyme (dot or focal expression of COX). Our results indicated that the COX mitochondrial activity is heterogeneously impaired in myofibers of rats intoxicated with S. occidentalis. These abnormalities remember those observed in some types of human mitochondrial myopathies.

Muscle atrophy induced in broiler chicks by parts of Senna occidentalis seeds.

Senna occidentalis (formerly Cassia occidentalis) is a common contaminant of agricultural commodities. It is toxic to cattle and poultry, reportedly being responsible for skeletal myodegeneration in these animals. All parts of the plant present toxicity, but the seeds are the most toxic. The toxin(s) responsible for the myodegeneration have not been definitively identified, nor is it known which part of the seeds is most toxic. Intoxication by this plant leads to weight loss with considerable economic repercussions. The effects of the whole seed and of parts of S. occidentalis seeds (1% in commercial feed) were compared on the pectoralis major muscle of broiler chicks intoxicated from birth until 22 days of life. There were severe clinical signals and reduced body weight in birds that received the external tegment of the seed, whereas no adverse effects were observed in birds that received the whole seed or other parts of the seed. Histological and morphometric studies showed an intense muscle fibre atrophy (both type 1 and type 2 fibres were affected) in the group that received 1% external tegment. This study may be the first step to identifying the substance(s) involved in this pathological process.

Ki-67 expression in anal intraepithelial neoplasia in AIDS.

CONTEXT: AIDS is one of the most important risk factors for progression and recurrence of anogenital condyloma. In a previous work, we observed that patients with warts and high-grade AIN (HAIN) had recurrences more frequently than did patients with warts without AIN. The mechanisms of this increased incidence of high-grade lesions in AIDS are not known. OBJECTIVE: We studied the expression of the proliferative marker Ki-67 by immunohistochemical methods, in specimens of anal condyloma from HIV+ patients to clarify whether its expression can be associated to the grade of AIN. DESIGN: A retrospective study of histological specimens. SETTING: University referral unit. SAMPLE: 34 patients were divided into two groups: (1) condylomas with low grade AIN (LAIN), with 25 patients; and (2) condylomas with HAIN, with 9 patients. In this latter group we examined two areas: 2A (HAIN area) and 2B (LAIN area). MAIN MEASUREMENTS: The immunohistochemical reaction for Ki-67 was done on histological sections. Slices were lightly stained with hematoxylin, to help us in Ki-67 positive cell counting. The percentage of Ki-67 marked nuclei was calculated. We applied one-way variance analysis for statistics. RESULTS: The mean number of Ki-67 positive cells in group 1 was 19.68 +/- 10.99; in group 2 (area A) it was 46.73 +/- 10.409; and in area B it was 36.43 +/- 14.731. There were statistical differences between groups 1 and 2A and between groups 1 and 2B. Ki-67 positive cells predominated in the lower layer in LAIN. Positive Ki-67 cells were found in all layers in group 2A, and in group 2B they predominated in the two lower or in all layers of the epithelium. CONCLUSIONS: Our results suggest that LAIN areas (using routine staining techniques) in HAIN can have a biological behavior more similar to HAIN.

Non-isotopic in situ hybridization of HPV types in cervical intraepithelial lesions in patients with AIDS.

Human papilloma viruses (HPVs), particularly types 16 and 18 have a key role in the development of preneoplastic and neoplastic lesions of the uterine cervix. We studied, by non isotopic in situ hybridization using probes to HPV 6, 11, 16 and 18, cervical biopsies from AIDS patients with condilomata or cervical intraepithelial neoplasia. There were 32 biopsies which showed low-grade cervical intraepithelial neoplasia (Lo-CIN); 5 biopsies showed high-grade cervical intraepithelial neoplasia (Hi-CIN). Of 32 Lo-CIN biopsies, 18 (56.3%) were positive for HPV; 7 for HPV 6 and/or 11 (21.9%), 11 for HPV 16 and/or 18 (34.4%) and one for HPV 6 and 18. Of 5 Hi-CIN biopsies 3 were positive for HPV: one for HPV 6 and 2 for HPV 16 or 18. The total positivity was 56.8% (21/37). This result was similar to those obtained by various other authors studying the general population.

Skeletal muscle pathology in 2 siblings infected with Toxoplasma gondii.

Skeletal muscle can be the site of inflammatory diseases that lead to muscle weakness, pain, and increased myogenic serum enzymes. Most of these inflammatory myopathies are idiopathic. In some cases inflammatory myopathies are due to infectious agents. We describe the pathological aspects of muscle biopsies of 2 Brazilian siblings who acquired toxoplasmosis at the same time and in similar conditions. One developed a tetraplegia that was confirmed to be due to inflammatory myositis due to toxoplasma. The other developed myocarditis, with heart failure, without skeletal muscle weakness. In both cases many toxoplasma organisms were observed in the muscle biopsies, but in case 1 only was there an inflammatory myopathy with myofiber necrosis; the inflammatory cells were predominantly macrophages with some CD4+ cells and rare CD20+ cells. In case 1, expression of CD54 was observed in many inflammatory cells as well in endothelial cells, but only in endothelial cells in case 2. After treatment with clindamycin and corticosteroids both cases had only partial improvement, case 1 with a residual muscle weakness and case 2 with residual cardiac insufficiency (requiring digoxin). These cases show that the presence of the parasite in myofibers is not enough to induce an inflammatory myositis with muscle cell necrosis. This suggests that immunological disturbances may contribute to the development of inflammatory myositis due to toxoplasma.

Early expression of ubiquitin in myofibers of rats in organophosphate intoxication.

The degenerative process of the myofibers of the diaphragm of rats intoxicated with the organophosphate isofenphos, a compound that inhibits esterases, was studied at different intervals of intoxication. Early disorganization of the intermyofibrillar network and of the myofilaments, as well as dilatation of organelles, were observed by use of transmission electron microscopy. These changes precede macrophage invasion of the muscle fibers. Early expression of ubiquitin was observed in segments of muscle fibers by immunohistochemistry. Bands of polyubiquitin complexes in muscle homogenates were observed by immunoblotting. These bands disappeared in later stages of intoxication. A 42.5-kDa band corresponds to actin, as observed by immunoblotting using antisarcometric actin. This indicates relatively large amounts of polyubiquitin complex associated with sarcomeric actin in muscle fibers in early stages of intoxication. Based on these results it seems that actin is an important target in organophosphate-induced myofiber degradation and that the degradation of this protein-by the polyubiquitin pathway-may play an important role in the early disorganization of the sarcomere, as observed by electron microscopy. A possible role of the ubiquitin proteolytic pathway is that of trying to eliminate proteins modified in the early phases of muscle fiber degeneration, which is a necessary step for regeneration of the posterior segmental muscle.

Histologic peripheral nerve changes in rats induced by deltamethrin.

Synthetic pyrethroid insecticides have been used in the last two decades largely because of their high activity as an insecticide and low mammalian toxicity. Some studies have demonstrated that these products, especially compounds with an alpha-cyano group, are toxic to the mammalian central nervous system (CNS) in acute intoxications. However, morphological studies are scarce. In the present work the histopathologic changes of the sciatic and tibial nerves of rats submitted to acute intoxication with the cyanopyrethroid deltamethrin were studied. For 3 consecutive days male Wistar rats received by oral gavage deltamethrin at a dose of 45 mg/kg body wt. On the 4th day fragments of sciatic and tibial nerves were studied by transmission electron microscopy (TEM) and teasing of individual nerve fibers. In addition, another group of rats were allowed to recover until the 10th day. Teasing of nerves of animals sacrificed on the 4th day revealed myelin ovoids, which are indicative of axonal damage. TEM demonstrated rare degenerated axons completely filled with organelles, in particular mitochondria, and with electron-dense lamellar bodies that resemble myelin figures. In addition, great cytoplasmic vacuolization caused by proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus was observed in some Schwann cells. No lesion was found 7 days after discontinuation of the treatment (group2). Since these histologic changes are transitory and scarce, the question arises: Are they related to the changes in NA(+), K(+)-ATPase activity or Na(+) channels caused by pyrethroid compounds?

Mitochondrial myopathy in Senna occidentalis-seed-fed chicken.

Plants of the genus Senna (formerly Cassia) have been recognized as the cause of a natural and experimental syndrome of muscle degeneration frequently leading to death in animals. Histologically, it demonstrated skeletal and cardiac muscle necrosis, with floccular degeneration and proliferation of sarcolemmal nuclei. Recently, it was described as an experimental model of mitochondrial myopathy in hens chronically treated with Senna occidentalis. Currently, skeletal muscles of chicks intoxicated with seeds of the poisonous plant S. occidentalis were studied by histochemistry and electron microscopy. Since birth, the birds were fed ground dried seeds of this plant with a regular chicken ration at a dose of 4% for 11 days. Microscopic examination revealed, besides muscle-fiber atrophy, lipid storage in most fibers and a moderate amount of cytochrome oxidase-negative fibers. By electron microscopy, enlarged mitochondria with disrupted or excessively branched cristae were seen. This picture was characteristic of mitochondrial myopathy. These findings have hitherto remained unnoticed in skeletal muscle of young birds treated with S. occidentalis.

Toxic peripheral neuropathy of chicks fed Senna occidentalis seeds.

Plants of the genus Senna (formerly Cassia) are poisonous to livestock and other laboratory animals, leading to a syndrome of a widespread muscle degeneration, incoordination, recumbence, and death. The main histologic lesion is necrosis of skeletal muscle fibers. Recently, a mitochondrial myopathy with ragged-red and cytochrome oxidase (COX)-negative muscle fibers was recognized in hens chronically intoxicated with parts of seeds of S. occidentalis. The purpose of the present work was to investigate if there was peripheral nerve involvement in the acute intoxication of chicks with S. occidentalis seeds. Teasing of individual fibers revealed signs of extensive axonal damage with myelin ovoids. Ultrathin sections confirmed the axonal damage. Axons were filled with membranes, some residual disorganized filaments, and enlarged mitochondria. In some instances the axon disappeared and there was secondary degeneration of the myelin sheath. The present work is the first description of the neurotoxic effect of S. occidentalis intoxication. Future work should attempt to determine the mechanisms involved in this neuropathy.

Muscle degeneration in chicks caused by Senna occidentalis seeds.

Acute intoxication with Senna occidentalis seeds was studied in chicks. Seven-day-old chicks were fed ground dried seeds of this plant mixed with regular chicken ration at a concentration of 4% by weight for 15 days. Feed intake and body weight were markedly affected and a high level of lethality was observed. Necropsy examination of chicks from the experimental group revealed paleness and atrophy of thoracic muscles. Degenerative and necrotic fibres were observed in skeletal muscle by histological examination. Muscle histochemistry showed accumulation of lipids and numerous acid phosphatase-positive muscle fibres. Electron microscopy revealed atrophic muscle fibres, lipid storage, dilatation of the sarcoplasmic reticulum and abnormal mitochondria.

Nerve biopsy in patients with AIDS.

Patients with AIDS can present clinical involvement of the peripheral nervous system due to different causes. In the present work, it was studied the histopathological changes in sural nerve biopsy of fifteen patients with AIDS with this clinical involvement. It was observed the presence of a polyarteritis nodosa-like vasculitis of small arteries with fibrinoid necrosis in the sural nerve of 3 patients, one of them associated to polyradiculitis due to cytomegalovirus infection (CMV). Six patients presented mild axonal loss by light microscopy. Three other patients had a more important axonal neuropathy with myelin ovoids by teasing. By the electron microscopy in these patients were observed some fibers with axonal damage. The other three patients had normal sural nerves. We concluded that sural nerve biopsy may be important in peripheral neuropathies or myelo-radiculo-polyneuropathies in AIDS especially to search for nerve vasculitis, because it can change the therapeutic approach.

Protective effect of pralidoxime on muscle fiber necrosis induced by organophosphate compounds.

OBJECTIVE: To determine the protective effect of pralidoxime on muscle fiber necrosis induced by organophosphate acute intoxication in rats. DESIGN: Adult male Wistar rats were given oral organophosphate compounds dissolved in glycerol formal: dichlorvos, isofenphos, metamidophos, and diazinon. Half of the animals also received pralidoxime mesylate (20 mg/kg, intraperitoneal). Control animals received only the solvent. Twenty-four hours after treatment, the diaphragm muscle was collected for histological counts of necrotic muscle fibers in transverse sections. RESULTS: Metamidophos- and isofenphos-treated animals showed the highest percentage of necrotic muscle fibers: 1.66 +/- 1.112 and 1.34 +/- 0.320, respectively. Diazinon-treated animals had a lower percentage of necrotic fibers: 0.40 +/- 0.032 (p < 0.05) compared to the first 2 products, and dichlorvos-treated animals showed the smallest: 0.05 +/- 0.021 (p < 0.05) when compared to the other 3 products. Pralidoxime reduced necrotic fibers about 20 times in metamidophos-treated animals, 10 times in isofenphos-treated animals and 6 times in diazinon-treated animals. Pralidoxime administration did not increase plasma cholinesterase activity in any group, although symptoms were reduced. CONCLUSIONS: Oxime reduced diaphragmatic muscle necrosis in experimental organophosphate intoxication, despite little effect on plasma cholinesterase. Since respiratory insufficiency is an important cause of mortality and morbidity in organophosphate intoxications, early oxime administration may be particularly beneficial.