The sensitivity of in situ hybridization for detection of human papillomavirus

We studied a sensitivity of HPV DNA detection by in situ hybridization method using 3H labeled HPV DNA. The materials were CaSki cells and SiHa cells which were derived from as a negative control. The total cellular DNAs extracted from these cell lines were estimated copy numbers of HPV 16 DNA using Southern blot hybridization. In our result, CaSki cell has 400 copies/cell, SiHa cell were appeared to have 1-5 copies/cell. Simultaneously these cells were fixed by periodate-buffered lysine-paraformaldehyde-glutaraldehyde (PLPG) and were detected HPV 16 DNA using in situ hybridization. We detected HPV 16 DNA in CaSki cells and SiHa cells by in situ hybridization also. We concluded that the sensitivity of our in situ hybridization technique is 1-5 copies/cell.     

Extrapleural solitary fibrous tumor: clinicopathologic study of 17 cases and molecular analysis of the p53 pathway

Solitary fibrous tumor (SFT) occurring at various extrapleural sites is sometimes difficult to diagnose because of its histologic variability. Although a solitary fibrous tumor is usually a slow-growing tumor with favorable prognosis, a small number of malignant cases have been reported. In the present study, we examined the clinical behavior, histologic, immunohistochemical and molecular features of 17 cases of extrapleural SFT. Four tumors were located in the pelvic cavity, two in the nasal cavity, two were confined to the pulmonary parenchyma, and there was one each in the meninges, kidney, mediastinum, retroperitoneum, temporal region, neck, groin, buttock and thigh. Histologically, all the tumors were characterized by the presence of areas consisting of a proliferation of bland spindle cells with variable amounts of thick, often hyalinized or keloid-like intercellular collagen bundles. Highly cellular areas were observed in three tumors, frequent mitoses in two, and cellular pleomorphism and tumor necrosis in one each. All 17 tumors showed immunoreactivity to CD34 and 15 (88%) to bcl-2 protein. The labeling indices of p53, mdm2 protein and Ki-67 were generally low. PCR-SSCP and a subsequent sequence analysis of the p53 gene disclosed point mutation at codon 161 in exon 5 in one of the 13 cases analyzed. According to follow-up information, none of the patients had developed local recurrence or distant metastasis. Our results suggest that most extrapleural SFTs behave in a benign fashion even in a higher histologic grade group, and it is difficult to predict their clinical outcome. Complete surgical excision in order to obtain clear margins and long-term follow-up is advisable for patients with an extrapleural SFT.     

Methylated 5'-terminal caps of adenovirus type 2 early mRNA: evidence for at least six 5'-termini.

The 5′-terminal caps of early adenovirus (Ad) 2 mRNA were isolated, fractionated, and compositionally analyzed. Cultured human KB cells were labeled with l-[methyl-³H]methi-onine and ³²Pi from 2 to 7 hr postinfection in the presence of cycloheximide. Labeled RNA was extracted from polysomes and fractionated into poly(A)⁺ and poly(A)^? molecules. Viral mRNA was isolated by hybridization to Ad2 DNA, then digested with RNase T2, and cap 1 [m⁷G(5′)ppp(5′)N^mpNp] and cap 2 [m⁷G(5′)ppp(5')N^mpN^mpNp] fractions were isolated by DEAE-Sephadex chromatography in 7 M urea. m⁷G was shown to be located at the 5′ termini of early Ad2 mRNA by periodate oxidation and β elimination. Cap 1 and cap 2 fractions were fractionated by electrophoresis on DEAE-cellulose paper. The cap 1 fraction separated into three major spots (a, b, and d), in order of increasing negative charge), and the cap 2 fraction into four major spots (a, b, c, and d). The methylated constituents of each spot were determined and used to deduce the number and composition of individual caps. The following results were obtained. Cap 1 structures: Spot a is m⁷GpppU^mpN1; b contains m⁷GpppU^mpN2 (N1 and N2 are different unmethylated nucleosides) and m⁷GpppA^mpN; c is m⁷GpppN^1mpN, m⁷Gpppm⁶A^mpN, and m⁷GpppG^mpN (N^1m is m₂⁶A^m or an unknown base-modified 2′-O-methyl nucleoside). Cap 2 structures: Spot a is m⁷Gppp(U^mp,N^1mp)N1; b contains at least two components, combinations of G^m, U^m, N^1m, and m⁶A^m; c contains at least two components, combinations of G^m, U^m, A^m, and m⁶A^m; d is at least two components containing combinations of C^m, A^m, and m⁶A^m. No significant differences in methylated constituents were detected in early mRNA repared from infected cells in the presence or absence of cycloheximide. Although we detect at least 13 caps, the minimum number of distinct 5′ termini is 6, because (i) N^1m could be m₂⁶A^m (2′-0-methyl N⁶,N⁶-dimethyl adenosine), (ii) m⁶A^m and A^m could be undermethylated precursors to m₂⁶A^m, and (iii) cap 1 structures could represent cap 2 structures without methylation of the penultimate nucleoside. Therefore there may be from 6 to 13 distinct Ad2 early mRNAs, which is interesting considering that only four general areas of the genome (each close to the 3′ end of each early gene block) appear to function as promoters [Berk, A. J., and Sharp, P. A. (1977). Cell12, 45–55; Evans, R. M., et al. (1977). Cell12, 733–739]. Analysis of KB cell mRNA resolved about 12 cap 1 and 10 cap 2 structures. All Ad2 mRNA cap spots resolved by electrophoresis corresponded in mobility and composition to major cap spots found in cell mRNA. Thus, the methylated caps of early mRNA are similar to those of the host cell. In contrast, most late Ad2 mRNA molecules have an identical cap sequence [Gelinas, R. E., and Robert, R. J. (1977). Cell11, 533–544].     

Mild emphysema: a novel method using formalin-fixed lungs for computed tomography and pathological analyses

We evaluated a novel method of computed tomography (CT) analysis using formalin-fixed lungs of autopsy cases with mild emphysema. Eight formalin-inflated lungs (FILs) obtained at autopsy were examined using CT after draining off the formalin and air inflation with an air pump, and subjected to pathological study including pathological scoring of emphysema and microscopic image analysis (MIA). Satisfactory CT examination was carried out within 5 h of lung fixation. The mean alveolar area determined by MIA correlated highly with the lung volume (r=0.845) and CT score (r=0.722). This method is simple compared with conventional polyethylene glycol fixation for CT and enables CT examination of resected lungs without anxiety about biohazards. Mild emphysema can be detected by MIA.     

Thyroid dysfunction and histologic correlation using thyroid needle biopsy specimens

In this article, we describe the usefulness of thyroid needle biopsy in the differential diagnosis of thyroid disorders revealing unusual thyroid function. Firstly, we describe the relationship between thyroid function and its histology in 601 cases of chronic thyroiditis. In the histologic group A, the majority of the cases showed a latent or overt hypothyroidism and in histologic group B, hyperthyroid, euthyroid and latent hypothyroid cases were found in nearly equal frequency, respectively. In histologic group C, most cases were in euthyroid and in histologic group D, most cases showed a hyperthyroidism. In the silent thyroiditis and postpartum thyroiditis, known to show a characteristic clinical and laboratory finding, the histologic features of thyroid gland were as follows: the observed characteristic histologic changes in both diseases were an extensive follicular destruction associated with chronic diffuse thyroiditis. These destructive changes disappeared in association with the clinical and laboratory recovery. In the patients with iodine-excess hypothyroidism, the thyroid glands also revealed characteristic histologic changes. A marked hyperplastic change of follicular cells and a lack of colloid material in the follicular lumen was the predominant histologic feature. Based on our personal experiences, thyroid needle biopsy should be recommended as a useful tool for differentiation of causes of hyper- or hypothyroidism.     

Multiple epithelial cysts of the spleen and on the splenic capsule, and high serum levels of CA19-9, CA125 and soluble IL-2 receptor

An 18-year-old woman with abdominal pain was diagnosed as having splenic cysts by computed tomography scan. She had high serum levels of CA19-9 (2886.8 U/mL; normal value, <35 U/mL), CA125 (131.1 U/mL; normal value, <35 U/mL) and soluble IL-2 receptor (1490 U/mL; normal range, 220-530 U/mL). The resected spleen weighed 1050 g, was 14 x 28 cm, and had more than 10 macroscopic cysts up to 10.3 x 9.5 cm. There were numerous microscopic cysts in the spleen and several on the splenic capsule. The levels of CA19-9 and CA125 in the cyst fluid were 2165550 U/mL and 160400 U/mL, respectively. After the surgery, the serum levels of the tumor markers decreased gradually. The inside of the largest cyst was mainly covered by granulation tissue with a focal lining of epithelial cells, and the other macroscopic cysts had stratified squamous epithelium. The microscopic splenic cysts and cysts on the splenic capsule were lined by either attenuated single-layered or multilayered epithelial cells. The lining epithelial cells of these cysts were positive for epithelial membrane antigen and cytokeratins. CA19-9 and CA125 were detected in the lining cells of the splenic cysts. In the present case, it is suspected that the splenic cysts were derived from the capsular lining cells that showed migration from the capsule or formed microcysts on the splenic capsule, as in the case of ovarian inclusion cysts.     

Immune responses of interferon gamma (IFN-gamma) knock out mice to repeated Haemaphysalis longicornis (Acari: Ixodidae) nymph infestations

To investigate the immunological mechanisms of acquired resistance to tick infestation, interferon gamma (IFN-gamma) deficient mice (IFN-gamma mice) were used to assess interleukin-4 (IL-4) and antibody production levels against tick salivary gland antigen on three successive infestations with Haemoaphysalis longicornis Neumann nymphs. The engorged body weight of the ticks decreased during the second and third infestations. Similar observations were noted in IFN-gamma+/+ mice. However, the engorged body weight of the ticks from IFN-gamma +/+ mice were considerably lower than those from IFN-gamma-/- mice. A marked increase in antibody production during the second and third infestations was observed indicating that IFN-gamma-/- mice could acquire immunological resistance against H. longicornis nymphs. Moreover, IL-4 levels were higher during the first and third infestations but decreased during the second infestation. IL-4 levels were significantly higher in IFN-gamma-/- mice than in IFN-gamma+/+ mice. We have shown here that the statistically significant high IL-4 levels observed in IFN-gamma-/- mice may be a result of type 2 helper cell (Th2) polarization. However, the apparently higher IL-4 levels during the first and third infestations and the notable decline during the second infestation suggest that other cytokines or factors in the host immune system may play a part in regulating IL-4 levels.     

Retroperitoneal mucinous cystadenoma

Primary retroperitoneal mucinous cystadenoma is an uncommon tumor found exclusively in women. Herein, we describe a patient who had resection of a large retroperitoneal cystic mass. Histologic, immunohistochemical, and electron microscopic examination of the lining epithelial cells showed features of mesothelial cells in addition to ovarian mucinous cystadenoma. These findings suggest that these tumors arise from inclusions of mesothelial cells and subsequent mucinous metaplasia of the lining cells to form a cystadenoma. Estrogen receptors may be implicated in tumor promotion, explaining the occurrence exclusively in women.     

Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis

We have recently reported the accumulation of oligoclonal activated T cells in the spontaneously developed autoimmune pancreatitis in aly/aly mouse. In this study, we examined the effects of FK506 in this mouse model in preventing autoimmune pancreatitis and investigated its action on calcium signalling apoptosis of alymphoplasia (aly) lymphocytes in vitro. Mice were treated with FK506 from 8 to 25 weeks of age. At the age of 15 weeks, minimal mononuclear cell infiltration was observed in the pancreas in both the FK506 treated group and the control group. Furthermore, a marked cell infiltration associated with destruction of acini and partial fatty changes were observed in 25-week-old control mice. In contrast, FK506 treated mice showed almost no tissue destruction or mononuclear cell infiltration at the age of 25 weeks. Furthermore, at 15 weeks of age, most mononuclear cells in FK506-treated mice were TUNEL positive, whereas only a few were positive in control mice. This augmentation of T cell apoptosis by FK506 was confirmed using naive splenocytes activated by PMA and ionomycin in vitro. Finally, a suppressive effect of FK506 on Bcl-2 production but not on Bax production was confirmed by Western blotting. This unique effect of FK506 on the augmentation of T cell apoptosis is probably one of the mechanisms explaining its beneficial effect on aly autoimmune pancreatitis.