Anti-parietal cell antibodies in patients with autoimmune thyroid diseases.

BACKGROUND: Autoimmune thyroid disease (ATD) patients may have a higher prevalence of anti-parietal cell antibodies (APCA) than normal population. OBJECTIVE: To study the prevalence of APCA in a cohort of ATD patients to know its association with patient's clinical profile and gastrointestinal complaints. METHODS: APCA was sought for by indirect immunofluorescence test in 243 ATD patients: 136 (55.9%) with Graves' disease and 107 (44.0%) with Hashimoto's thyroiditis. A structured questionnaire for gastrointestinal symptoms, previous history of thrombosis, arthralgia and other autoimmune diseases in the patients and their families was applied. Positive and negative APCA individuals were compared. Positive patients were invited to perform upper gastrointestinal endoscopy and biopsy of duodenum segments. Sera from 100 healthy individuals from the same geographic area were used as controls. RESULTS: APCA was present in 20.1% (49/243) of ATD patients: 21.3% (29/136) in the Graves' sample and 18.6% (20/107) in the Hashimoto's sample (p = 0.61). Patients with positive APCA had more anemia (p = 0.03; OR = 2.89; 95% CI = 1.03-8.07) and less heartburn (p = 0.01; OR = 0.4; 95% CI = 0.20-0.83). Among the group of 49 APCA-positive patients, 24 agreed with upper endoscopy and it was found that 54.1% had atrophic gastritis. CONCLUSIONS: There is a high prevalence of positive APCA in ATD patients. APCA are more common in those with anemia and less common in those with complaints of heartburn. Almost half of positive APCA patients had atrophic gastritis.

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy.

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Distribution of a Ca2+ storing site in PtK2 cells during interphase and mitosis. An immunocytochemical study using an antibody against calreticulin.

To study the distribution of a major Ca(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase and mitosis, we prepared an affinity-purified polyclonal antibody against bovine liver calreticulin (CRT), a major Ca(2+)-binding protein of the endoplasmic reticulum (ER). Immunofluorescence microscopy and immunoperoxidase electron microscopy showed that the anti-CRT antibody labeled a continuous reticular network of the ER and the nuclear envelope in interphase PtK2 cells. The same PtK2 cells double-stained with DiOC6 (3) and the anti-CRT antibody revealed labeling of identical reticular membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These results confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in non-muscle cells. In mitotic cells, marked changes of the labeled structure began at prophase-prometaphase and persisted throughout all phases of mitosis. The cytoplasm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microscopy and immunoelectron microscopy demonstrated that the CRT-containing membranes changed to segmented tubuloreticular structures, which were concentrated around the mitotic spindle. The ER containing CRT could be responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during interphase.

Hepatitis C virus in long-term bone marrow transplant survivors.

The hepatitis C virus (HCV) infection may change the outcome of patients undergoing stem cell transplantation. This study aimed at determining the prevalence of the HCV antibody in patients who were alive 10 or more years after BMT, defining the annual progression rate of hepatic fibrosis in those patients, and identifying cases of cirrhosis among those who were positive for HCV antibody. Between 1979 and 1990, 259 patients had a bone marrow transplant, and 91 were alive in March 2000. Of those, 80 were included in the study after having been scanned for serum HCV antibodies. A total of 39 were positive (48.8%), one was indeterminate and 40 were negative (50%). The patients who were HCV positive or undetermined were called for a medical appointment and 22 (55%) attended. A total of 16 patients (72.7%) were male, the mean age was 37.8+/-9.2 years and all of them had had an allogeneic transplant. Of the 22 patients studied, 12 (54.5%) agreed to have a liver biopsy. Hepatic fibrosis was diagnosed in 10 patients. The hepatic fibrosis annual progression rate was 0.156 UF/year. Among the anti-HCV-positive patients assessed, three (13.6%) already had cirrhosis.

Invasive medullary thymoma associated with Myasthenia gravis: an unusual case.

Thymomas are tumors characterized by a remarkable morphological heterogeneity and variable clinical behavior. This tumor has unique clinical associations, most notably with hematological abnormalities and myasthenia gravis. According with the Müller-Hermelink criteria, there are significant differences between the histological types of thymomas and the association with myasthenia gravis. Among the different histological types, medullary thymoma is the least frequent variant associated with this autoimmune disease. In this report we describe a case of medullary thymoma presenting in a 71-year- old woman with a myasthenic syndrome.

[Acute cholestatic hepatitis induced by propylthiouracil. Case report]. / Hepatite aguda colestática pelo propiltiouracil. Relato de caso.

Propylthiouracil is widely used to treat patients with hyperthyroidism. However, propylthiouracil-induced hepatitis is an uncommon entity. The case of a 15-year-old boy treated with propylthiouracil for hyperthyroidism who developed a cholestatic acute hepatitis is reported. Viral, metabolic and autoimmune liver diseases were excluded and liver biopsy showed a pattern suggestive of drug-induced cholestatic hepatitis. After discontinuing the drug, there was a progressive resolution of symptoms and normalization of liver biochemical tests. Despite its rarity, patients receiving propylthiouracil are exposed to develop severe hepatotoxicity.

[Benign recurrent intrahepatic cholestasis: a seven-year follow-up report]. / Colestase intra-hepática recurrente benigna: seguimento de um caso por 7 anos.

Benign recurrent intrahepatic cholestasis is a rare autosomal recessive disorder characterized by repeated episodes of intense pruritus and jaundice. Patients are completely asymptomatic for months to years between symptomatic periods. We report a case of a patient with a 7-year history of benign recurrent intrahepatic cholestasis. During the follow-up period the patient has suffered three attacks of cholestasis, confirmed by biochemical tests and histological exam. Liver enzymes were normal between the cholestasis episodes. Despite multiple attacks of cholestasis, no permanent liver damage has occurred. Although the diagnosis of benign recurrent intrahepatic cholestasis is rare, it should be included in the evaluation of a patient with cholestasis. The patients should be reassured of the benign course of this disorder.

[Results of liver transplantation in patients with hepatocellular carcinoma]. / Resultados do transplante hepático em portadores de hepatocarcinoma.

BACKGROUND: Hepatocellular carcinoma is one of the most common malignancies worldwide. Liver transplantation has emerged as a good option for early-stage hepatocellular carcinoma yielding survival rates as good as for recipients without this type of tumor. OBJECTIVE: To assess the outcome of cirrhotic patients with hepatocellular carcinoma undergoing liver transplantation at the Liver Transplantation Service of the "Hospital de Clinicas", Federal University of Paraná, Curitiba, PR, Brazil. METHODS: Retrospective study of cirrhotic patients with hepatocellular carcinoma undergoing orthotopic liver transplantation at the mentioned Institution between September 1991 and September 2000. The diagnosis of hepatocellular carcinoma was established during the pretransplant workup in five patients and the tumor was an incidental finding in the native liver in three. The indication for liver transplantation was restricted to solitary tumor equal to or less than 5 cm or up to 3 nodules, with each nodule measuring less than 3 cm, and no evidence of vascular invasion or extrahepatic spread. Patient survival and evidence of tumoral recurrence posttransplant were evaluated. RESULTS: The most common cause for pretransplantation liver disease was hepatitis C virus (50%). On examination of the explanted liver, the majority of patients (6/8, 75%) had a single lesion; one patient had two nodules and one had a multifocal hepatocellular carcinoma found incidentally in the native liver. Tumor size ranged from 0.2 to 5.0 cm. All cases had neither vascular invasion nor linfonodal envolvement. All patients remained alive and free of tumor recurrence at the time of the study with a mean follow-up of 18.5 months (range, 5-29 months). CONCLUSION: Liver transplantation is a good therapeutic option for early stage hepatocellular carcinoma arising in cirrhotic patients. With proper selection, liver transplantation can offer excellent survival rates free of tumor recurrence.

Organization of calsequestrin-positive sarcoplasmic reticulum in rat cardiomyocytes in culture.

The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca2+ ions in muscle cells. Calsequestrin is a major Ca(2+)-storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin-positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine-phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross-striations at Z-lines. When newborn cardiomyocytes were isolated, calsequestrin-positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin-positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross-striations at the Z-lines. Double-labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin-positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin-positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR.

Switching of the dominant calcium sequestering protein during skeletal muscle differentiation.

A major Ca(2+)-storing protein in endoplasmic reticulum (ER) of non-muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca(2+)-binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti-calsequestrin, anti-calreticulin, anti-desmin, and anti-sarcomeric myosin antibodies and rhodamine-phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin-positive cells demonstrated the reticular staining of calreticulin, as did desmin-negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin-positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti-calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation.

Association of cytoplasmic dynein with manchette microtubules and spermatid nuclear envelope during spermiogenesis in rats.

During spermiogenesis, the shape of the spermatid nucleus, which is spherical, changes and it becomes the sperm head. A microtubular structure called a manchette is thought to be involved in this morphogenetic process. In this report, we demonstrate the localization of cytoplasmic dynein and manchette development by a double immunofluorescence technique using anti-bovine brain MAP 1C and anti-tubulin. Before step 6 of the Leblond and Clermont staging, the microtubules showed a fine reticular network, and the dynein staining was homogeneous. In step 6, the microtubular network was concentrated around the nucleus. The manchette developed in step 7 spermatids, and was fully formed, with a skirt-like appearance, covering the nuclear surface in step 8. Dynein fluorescence was associated with the microtubular manchette in steps 7-10. During these steps, the nucleus was protruded from the cytoplasm. In steps 11-13, the most active stages in nuclear shaping, the dynein was densely localized at the nuclear surface covered by the manchette. As the nucleus acquired a shape similar to the mature spermatozoon at step 14, the dynein fluorescence was localized only at the concave side of the nuclear caudal edge. The manchette became narrower and elongated. In step 15, the manchette extended into the elongated cytoplasm, diminishing during steps 16-18. The localization of the dynein was limited to the ventral aspect of the caudal head in these steps. There was little dynein fluorescence in mature spermatozoa. Immunoelectron microscopy showed positive reactions in the nuclear envelope and the inner region of the microtubular manchette. These observations suggest that cytoplasmic dynein, possibly bound to the nuclear envelope, and manchette microtubules are involved in the protrusion of the spermatid nucleus from the cytoplasm.

Alterations of expression and distribution of the Ca(2+)-storing proteins in endo/sarcoplasmic reticulum during differentiation of rat cardiomyocytes.

Calsequestrin (CS) is a Ca(2+)-storing protein present in the sarcoplasmic reticulum (SR) of muscle cells. Calreticulin (CR) is a functional homologue of CS in the endoplasmic reticulum (ER) of non-muscle cells. During skeletal muscle differentiation, the major Ca(2+)-storing protein switches from CR to CS. To study the regulation of CS and CR expression in cardiomyocytes and the morphological maturation of Ca(2+)-storing sites in the SR, we examined rat hearts at various developmental stages and cultured adult cardiomyocytes by Western blotting and immunocytochemical analyses. CR was expressed in 14-day-old fetal rat cardiomyocytes, but disappeared gradually by 1 week after birth. CR reappeared in dedifferentiated adult cardiomyocytes in long-term culture. On Western blots, the concentration of CS in the heart did not change during development. Immunostaining for CS in fetal or neonatal rat cardiomyocytes revealed as scattered dots. CS-positive structures increased with development, and the regular striated distribution of CS at Z lines was completed around 3 weeks after birth. These results indicated that (1) CR expression is downregulated during cardiac differentiation and upregulated during dedifferentiation, and (2) maturation of SR involves the organization of CS-positive structure after birth.

Sarcomatoid carcinoma of the urinary tract.

Sarcomatoid carcinoma is a rare variant of malignant tumor arising from the urinary tract. This tumor had been termed carcinosarcoma because of its carcinomatous and sarcomatous components. There is still some confusion in the terminology between true carcinosarcoma and sarcomatoid carcinoma; however, the latter is now regarded as primarily a malignant epithelial tumor with pseudosarcomatous transformation. Four cases of sarcomatoid carcinoma arising from the urinary tract are reported. The patients were a 77 year old female, and three males aged 62, 69 and 80 years. All but the eldest patient complained of gross hematuria. Surgical removal was performed in the younger three cases, and an autopsy was done in the remaining case. All the tumors were macroscopically polypoid. Histopathologic examination revealed fasciculated spindle-cell tumors with myxoid stroma or malignant fibrous histiocytoma-like spindle cell tumors. The epithelial nature was proven in these sarcomatous cells by immunohistochemical and/or electron-microscopic examinations. Only a small amount of squamous cell carcinoma components was also evident in the latter three cases. Although the younger three patients were alive at 44, 23 and 39 months' follow-up, respectively, constant careful monitoring is recommended.