Surfactant protein C G100S mutation causes familial pulmonary fibrosis in Japanese kindred.

Several mutations in the surfactant protein C (SP-C) gene (SFTPC) have been reported as causing familial pulmonary fibrosis (FPF). However, the genetic background and clinical features of FPF are still not fully understood. We identified one Japanese kindred, in which at least six individuals over three generations were diagnosed with pulmonary fibrosis. We examined the patients radiologically and histopathologically and sequenced their SFTPC and ABCA3 genes. We also established a cell line stably expressing the mutant gene. All the patients had similar radiological and histopathological characteristics. Their histopathological pattern was that of usual interstitial pneumonia, showing numerous fibroblastic foci even in areas without abnormal radiological findings on chest high-resolution computed tomography. No child had respiratory symptoms in the kindred. Sequencing of SFTPC showed a novel heterozygous mutation, c.298G>A (G100S), in the BRICHOS domain of proSP-C, which co-segregated with the disease. However, in the ABCA3 gene, no mutation was found. In vitro expression of the mutant gene revealed that several endoplasmic reticulum stress-related proteins were strongly expressed. The mutation increases endoplasmic reticulum stress and induces apoptotic cell death compared with wild-type SP-C in alveolar type II cells, supporting the significance of this mutation in the pathogenesis of pulmonary fibrosis.

Increased prevalence of Kaposi΄s sarcoma-associated herpesvirus in the Kaposi΄s sarcoma-endemic area of western Kenya in 1981-2000.

Kaposi΄s sarcoma (KS) had been endemic in Africa before the appearance of human immunodeficiency viruses (HIV) in 1985. Incidence of African KS has increased over the time and the risk of contracting KS become greater in HIV-positive as opposed to HIV-negative individuals. KS specimens were collected in 1981-2000 from 228 surgical cases originating from a KS-endemic area of Western Kenya and examined for Kaposi΄s sarcoma-associated herpesvirus (KSHV) by an immunoperoxidase assay. The results showed that the specimens from 1981-1985 (before the HIV epidemic) were KSHV-positive in 10.3% in contrast to the KSHV positivity of 50.1-63.5% in 1986-2000. The linear increase of KSHV positivity in 1981-2000 was statistically significant. The most plausible explanation for the increased prevalence of KSHV in KS cases is that the endemic KS has changed to the epidemic one.

Detection of Human papillomavirus and cellular regulators p16INK4a, p53, and NF-kappaB in penile cancer cases in Kenya.

Human papillomavirus (HPV) E6 and E7 gene products play a central role in the induction of benign proliferation and malignant transformation by interacting with several cellular regulatory proteins such as p53, p16(INK4a), and nuclear factor kappaB (NF-kappaB). In this study, HPV DNA was detected by in situ hybridization (ISH) and p53, p16(INK4a), and NF-kappaB by immunochemistry in 22 penile cancer cases in Kenya. HPV DNA was found in 68.2% of the cases. There was no difference in the p53- and p16(INK4a)-positivities in HPV DNA-positive and HPV DNA-negative cases. In HPV DNA-positive cases, the NF-kappaB positivity in the nucleus, cytoplasm, and nucleus and/or cytoplasm amounted to 73.3%, 93.3%, and 100%, respectively, while in HPV DNA-negative cases, a 28.7% NF-kappaB positivity of in the nucleus and/or cytoplasm was observed. It is concluded that NF-kappaB in penile cancer is expressed more frequently in the presence of HPV infection than in its absence.

Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice.

To elucidate the in vivo mechanisms involved in the impairment in pulmonary defence as the result of treatment with glucocorticoids, we established fatal pneumonia with bacteraemia in dexamethasone (DEX)-treated mice by means of an intratracheal challenge of Pseudomonas aeruginosa. An increased neutrophil influx was observed in bronchoalveolar lavage (BAL) fluids from both untreated and DEX-treated mice. The complete suppression of an inducible isoform of nitric oxide synthase (iNOS) mRNA expression and tumour necrosis factor alpha (TNF-alpha) production during the early phase of pneumonia, but not CXC chemokine production, were found in the case of the DEX-treated mice. An immunohistochemical study with a specific antibody also revealed negative staining for nitrotyrosine in the lung tissue of DEX-treated mice, while the formation of nitrotyrosine, which indirectly indicates the generation of peroxynitrite with a potent bactericidal activity, was detected clearly in the bronchial epithelium as well as alveolar phagocytic cells of lung tissue from untreated mice. Furthermore, an intraperitoneal administration of S-methyl-isothiourea (SMT), a potent inhibitor of NOS, significantly decreased the survival and increased bacterial density in the case of untreated mice. In contrast, no significant effects on the survival and bacterial density in the lung and blood were found as the result of treatment with SMT in DEX-treated mice. Collectively, a complete repression of iNOS gene expression and a lack of the generation of peroxynitrite as well as an inhibition of TNF-alpha production in the lung appeared to be responsible for the progression of the fatal pneumonia due to P. aeruginosa in DEX-treated mice.

Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice.

Hepatocyte growth factor (HGF) is postulated to play an important role in the repair of pulmonary epithelium in acute lung injury. To evaluate the role of HGF in bacterial pneumonia, the kinetics of HGF production and the cellular sources of HGF have been examined in the lungs of mice that had been intratracheally challenged with Pseudomonas aeruginosa. Neutrophil accumulation in the airway occurred immediately, reached a peak at 36 h, and then progressively declined by 14 d after infection. We found a biphasic pattern of HGF messenger RNA expression and protein synthesis in the lung after bacterial infection. The first peak for HGF production was found at 6 h after infection, and the primary source of HGF was shown to be bronchial epithelial cells. Interestingly, the second peak for HGF production, which was found around 48 to 72 h after infection, was closely associated with the increase in the percentage of alveolar macrophages (AMs) that became positive for myeloperoxidase, indicating phagocytosis of apoptotic neutrophils. The cellular source of the second peak was found to be AMs. Further, murine AMs which phagocytosed apoptotic neutrophils induced higher levels of HGF production in vitro. These results strongly indicate a novel mechanism of HGF production by AMs, which are phagocytosing apoptotic neutrophils, and the pivotal role of AMs in the healing and repair of damaged pulmonary epithelium through the production of HGF.

Role of cytokine-induced neutrophil chemoattractant-2 (CINC-2) alpha in a rat model of chronic bronchopulmonary infections with Pseudomonas aeruginosa.

In order to investigate the role of the cytokine-induced neutrophil chemoattractant (CINC) in chronic bronchopulmonary infection, we developed a rat model of bronchopulmonary infection with Pseudomonas aeruginosa by using the agar bead method, and determined the kinetics of bacterial and cell number, as well as the concentrations of CINC-1, CINC-2, and CINC-3 in bronchoalveolar lavage (BAL) fluids in this model. The bacterial number in the lung rapidly increased from days 1 to 4, and declined 14 days after challenge. Neutrophil number in BAL fluid increased up to one day after challenge, and then slowly decreased during 14 days post-challenge. Among the CINCs, the local production of CINC-2 alpha sharply increased at day 1 and then decreased until day 4 post-challenge, while the local production of CINC-1 slightly increased at day 1 post-challenge. Neither CINC-2 beta nor CINC-3 were detected during the entire course of the infection. Increased CINC-2 mRNA expression in the lung tissue after challenge was associated with CINC-2 alpha production in BAL fluid. Moreover, an immunohistochemical study demonstrated the localization of CINC-1 and CINC-2 alpha primarily in alveolar macrophages and, to a much lesser extent, in bronchial epithelium of infected lung tissues, whereas CINC-2 beta and CINC-3 were not detected. When anti-CINC-1 or anti-CINC-2 alpha polyclonal antibodies were used for neutralizing neutrophil chemotactic activities in BAL fluids, the anti-CINC-2 alpha antibody inhibited 70% of the chemotactic activity in BAL fluids from infected rats at day 1 after challenge. No inhibition was observed by anti-CINC-1 antibody. These data indicate that CINC-2 alpha, which is produced by alveolar macrophages and bronchial epithelial cells, plays a pivotal role in neutrophil accumulation in the airway of a rat model of chronic bronchopulmonary infection with P. aeruginosa.

Cloning of the gene of beta-N-acetylglucosaminidase from Lactobacillus casei ATCC 27092 and characterization of the enzyme expressed in Escherichia coli.

The gene encoding beta-N-acetylglucosaminidase (GlcNAcase) of Lactobacillus casei ATCC 27092 was cloned and expressed in Escherichia coli. The gene consisted of 1581 nucleotides, and had a promoter, Shine-Dalgarno, and rho-independent type transcription termination sequences typical in bacteria. The protein deduced from the sequence consisted of 526 amino acids, and had a putative signal peptide of 14 amino acids and 5 possible asparagine-linked glycosylation sites. A conserved sequence was homologous to the 12 other hexosaminidases from different origins. The recombinant GlcNAcase (r-GlcNAcase) purified from the transformed E. coli had a MW of 39 kDa and lacked oligosaccharide chains. The isoelectric point and the optimum pH for the activity of r-GlcNAcase were similar to those of original GlcNAcases (o-GlcNAcase). However, the thermal stability was lower, and sensitivity to Cd2+, Fe2+, Cu2+ and sodium dodecyl sulfate (SDS) was higher than those of o-GlcNAcases, suggesting that the oligosaccharide moieties of the enzyme contribute to their stability. The Km value for p-nitrophenyl-N-acetyl-beta-1,4-D-glucosamine (PNP-GlcNAc) of r-GlcNAcase (6.4 microM) implied that the affinity of r-GlcNAcase for the substrate was 200-fold higher than that of the original ones.

Impairment of endotoxin-induced macrophage inflammatory protein 2 gene expression in alveolar macrophages in streptozotocin-induced diabetes in mice.

To elucidate the mechanism of the high incidence of lower respiratory tract infections in patients with diabetes mellitus, we investigated the kinetics of production of macrophage inflammatory protein 2 (MIP-2), an important mediator of lung neutrophil recruitment, using mice with streptozotocin-induced diabetes. Intratracheal challenge with 1 mg of lipopolysaccharide (LPS), an endotoxin, per kg of body weight resulted in a time-dependent increase in the levels of MIP-2 protein in bronchoalveolar lavage (BAL) fluid, with the peak concentration (49.4 +/- 13 ng/ml) occurring at 3 h and significant neutrophil accumulation becoming apparent by 3 h in normal mice. In diabetic mice, the peak level of MIP-2 protein in BAL fluid did not occur until 6 h and was reduced to 21.9 +/- 10 ng/ml. Immunohistochemical studies using anti-MIP-2 antibody confirmed that the main cellular source of MIP-2 in the lung after LPS challenge was alveolar macrophages (AMs) in normal mice. The lungs in diabetic mice, however, showed no AMs staining for MIP-2 within 3 h after LPS challenge. PCR analysis using whole-lung RNA showed a time-dependent increase in MIP-2 mRNA levels after LPS instillation. The level of MIP-2 mRNA in diabetic mice was markedly decreased compared to that in normal mice. Our results indicate that impairment of MIP-2 mRNA expression in the AMs in diabetic mice resulted in delayed neutrophil recruitment in the lungs, and this may explain the development and progression of pulmonary infection in diabetes mellitus.

[A fatal case of streptococcal toxic shock-like syndrome probably caused by acupuncture].

A 41-year-old male received acupuncture in the right shoulder for the sake of arthralgia. Three days after acupuncture he was admitted due to severe epigastralgia. Erythematous change and swelling were observed around the right shoulder. A study by magnetic resonance showed an increased signal intensity in a portion of the right subscapular muscle. Four hours after admission he became hypotensive. The erythematous and necrotic change in the right shoulder skin rapidly spread. Excisional debridement in the right lateral chest wall was immediately done. However, the patient died one day after admission despite administration of a high-dose ampicillin and other supportive therapies. Bacteriological and histological examinations confirmed severe streptococcal myositis. This is a case report of toxic shock-like syndrome probably caused by acupuncture.

Purification and Properties of Three ß-N-Acetylglucosaminidases from Lactobacillus casei ATCC 27092.

Three ß-N-acetylglucosaminidases, GlcNAcase A, B, and C, were purified from the culture fluid of Lactobacillus casei ATCC 27092, and the molecular weights of these enzymes were estimated to be 54,000, 51,000, and 44,000, respectively, by SDS-PAGE. The production of these GlcNAcases was accelerated by the addition of N-acetylglucosamine to the culture. These enzymes had pIs of about 5.2, an optimum pH of 5.0-5.5, and an optimum temperature of 37-40°C. The K m values of GlcNAcase A, B, and C for p-nitrophenyl-ß-N-acetylglucosamine were 0.85, 1.30, and 1.04 mM and those for p-nitrophenyl-ß-N-acetylgalactosamine were 39.6, 57.7, and 60.8 mM, respectively.

B-cell antigen marker expression in nasopharyngeal carcinoma.

Immunohistochemical analysis was carried out to examine the characteristics of nasopharyngeal carcinoma (NPC) using 38 biopsy cases obtained from southern China. These cases were divided into 3 groups according to their predominant pattern associated with cell and tissue differentiation which is based on World Health Organization (WHO) classification as follows: 6 cases of squamous cell carcinoma (16%), 25 cases of differentiated non-keratinizing carcinoma (66%), 7 cases of undifferentiated carcinoma (18%). All tumor tissues reacted with MB-1, but they did not react with L26 (CD20), 4KB5 (CD45R), MT-1, and leukocyte common antigen (LCA). Keratin and epithelial membrane antigen (EMA) as epithelial markers focally stained NPC tissues in all cases. Carcinoembryonic antigen (CEA)-positive staining was detected in 7 (28%) of the 25 cases of differentiated non-keratinizing carcinoma and in 3 (43%) of the 7 cases of undifferentiated carcinoma; thus, of 38 cases, 10 (26%) were CEA-positive. On the other hand, squamous cell carcinoma cases did not react with CEA. These NPC tissues did not react with S-100 protein, alpha-1-antichymotrypsin (ACT), lysozyme, vimentin, and desmin. Therefore, it is concluded that some cases of NPC are difficult to distinguish from malignant lymphoma. In certain cases, NPC may be distinguished from malignant lymphoma, using immunohistochemical methods for the detection of MB-1, keratin, EMA, and LCA. Specifically, this evidence suggests that MB-1 may be useful as a tumor marker of NPC. Moreover, the CEA reaction to NPC may be related to the cell differentiation.

Long-term trend of decreasing frequency of hepatitis B virus infection.

Statistical and histopathological analysis was carried out to determine whether hepatitis B surface antigen (HBsAg) with the autopsy diagnosis of hepatitis, cirrhosis, and hepatocellular carcinoma declined during 1964-1991. In this study, the liver specimens from 673 autopsy cases at Nagasaki University Hospital were used, and these materials were stained for HBsAg with immunoperoxidase method. For statistical calculation, we used Cochran's chi-square test with one degree of freedom for linear trend for proportions. We investigated HBsAg positive rates for the four time periods, 1964-1970, 1971-1977, 1978-1984, and 1985-1991, and for the diagnosis of hepatitis, cirrhosis, and hepatocellular carcinoma. The decreasing linear trend of HBsAg positivity with time was significant for hepatocellular carcinoma and cirrhosis.