Transforming growth factor-beta(2) polymorphisms are associated with childhood atopic asthma.

BACKGROUND: Transforming growth factor (TGF)-beta plays an important role in the regulation of airway inflammation and remodelling in asthma. Recent studies suggest that TGF-beta(2) is a predominant isoform expressed in severe asthma and it is also associated with airway remodelling. OBJECTIVE: To determine whether the polymorphisms in TGF-beta(2) are associated with childhood atopic bronchial asthma in a Japanese population. METHODS: We identified a total of eight polymorphisms and characterized the linkage disequilibrium (LD) mapping of the gene. Three variants in the promoter and 3'UTR were genotyped, and we conducted an association study of TGF-beta(2) (childhood atopic asthma n=297, normal controls n=555). An association analysis of these variants and an expression and functional analysis were performed. RESULTS: 3'UTR 94862T >A was found to be significantly associated with the risk of childhood atopic asthma (P=0.00041). The -109-->ACAA ins promoter variant was also associated with the risk of childhood atopic asthma (P=0.0037). TGF-beta(2) expression was observed in both the normal and asthmatic bronchial epithelium, and both real-time PCR and an ELISA showed a significant basal and TGF-beta(1)-induced TGF-beta(2) expression in the bronchial epithelial cell line BEAS2B. Furthermore, the promoter variant -109-->ACAA ins increased the TGF-beta(2) promoter-reporter activity in BEAS2B cells. CONCLUSIONS: Our data suggest that TGF-beta(2) may therefore be involved in the development of childhood atopic asthma by means of functional genetic polymorphism. The polymorphisms in TGF-beta(2) may become important information for asthma susceptibility in children.

Allergen activates peripheral blood eosinophil nuclear factor-kappaB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-alpha and interleukin-8.

BACKGROUND: Allergic inflammation is characterized by the influx and activation of eosinophils. Cytokines generated by both resident and infiltrating cells are responsible for the initiation and maintenance of this pathogenesis. This study focuses on allergen-induced activation of eosinophil NF-kappaB and generation of granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-alpha, and IL-8. METHODS: Peripheral blood eosinophils were enriched to >99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NF-kappaB activation by 10 microg/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NF-kappaB (NF-kappaBa). The authenticity of NF-kappaB was confirmed by Western blot. Cytokine production was assessed both by immuno-staining of eosinophils and by assay of cytokines in the cell supernatant. RESULTS: Activation of peripheral blood eosinophils from atopic, but not non-atopic, donors induced activation of NF-kappaB, which peaked at 4 h and was accompanied by a decline in IkappaB-alpha. The activation of authentic NF-kappaB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NF-kappaB. Immunofluorescent confocal microscopy demonstrated localization of NF-kappaBa to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NF-kappaB inhibitors, calpain inhibitor 1 (10 microm), pentoxifylline (0.5 mm), pyrrolidine dithiocarbamate (PDTC, 10 microm) or gliotoxin (1 pg/mL) reduced the generation of GM-CSF, TNF-alpha and IL-8 in parallel with their inhibition of NF-kappaB. CONCLUSIONS: HDM allergen activates human eosinophil NF-kappaB leading to the production of the cytokines GM-CSF, TNF-alpha and IL-8. We speculate that a role for eosinophil NF-kappaB-dependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.

[Evaluation of an HIV screening assay kit for the combined detection of anti-HIV-1/2 antibodies and HIV p24 antigen].

We have evaluated a new HIV screening assay kit (Genscreen HIV Ag-Ab) for the HIV antigen-antibody combined test by comparing with two HIV antigen-antibody combined assay kits (VIDAS HIV DUO, Enzygnost HIV integral). Genscreen HIV Ag-Ab is a microwell plate enzyme immunoassay for the detection of HIV infection, based on the detection of anti-HIV-1/2 antibodies and HIV p24 antigen in human serum or plasma. In this study, 90 samples of HIV-1 antibody positive sera and 670 samples of HIV negative sera were examined. The sensitivity was 100% and the specificity was 99.7%. All of HIV-1 group M sera (subtypes A to G and B/D), HIV-1 group O sera and HIV-2 sera in worldwide HIV performance panel-302 were positive with Genscreen HIV Ag-Ab. Ten commercially available HIV-1 seroconversion panels were tested to evaluate sensitivity of three HIV antigen-antibody combined assay kits. Genscreen HIV Ag-Ab detected infection at the same bleeds as VIDAS HIV DUO in 8 of 10 seroconversion panels and 1 to 2 bleeds earlier than Enzygnost HIV integral in 5 of 10 seroconversion panels. However, VIDAS HIV DUO indicated false negative on 5th bleed in panel BB (PRA952). The result of the specimen was positive on 3rd bleed, equivocal on 4th bleed, negative on 5th bleed and again positive on 6th bleed. All of these specimens were positive by Genscreen HIV Ag-Ab. Therefore, Genscreen HIV Ag-Ab that shorten the window period is a useful and reliable for HIV screening test, especially in case of primary infection.

Scattered fat invasion: an indicator for poor prognosis in premenopausal, and for positive estrogen receptor in postmenopausal breast cancer patients.

BACKGROUND: Intracrine mechanisms which accelerate cancer cell growth are becoming well known in breast cancer research. In this study, microscopic invasive patterns of breast cancer cells into surrounding stromal tissues were evaluated. PATIENTS AND METHODS: 174 patients (110 patients < or = 50, and 64 > or = 60 years) underwent total or partial mastectomy from 1985 to 1993. In these patients the microscopic invasive pattern (especially scattered invasion into fat, SIF), influence on hormone receptor status, survival, and adjuvant therapy effectiveness were studied. RESULTS: A correlation was shown between being SIF- and estrogen receptor-positive only in older (> or = 60 years) patients (chi(2) test, p = 0.058). In younger patients, SIF was also related to a poorer prognosis (log rank test, p = 0. 0004). These results were also confirmed by multivariate analysis. Adjuvant therapy activity was influenced by SIF status in premenopausal patients. CONCLUSION: In older patients, SIF may be related to an intracrine supply of estrogen. In younger patients, SIF seems to work by a different mechanism for survival. These results also suggest some influence of SIF on adjuvant chemo-endocrine therapy results.

[HIV-1 (human immunodeficiency virus type 1) mutant strain associated with zidovudine resistance detected from a Japanese patient with primary HIV-1 infection].

Sequences of HIV-1 pol gene were determined by direct sequencing from a Japanese patient with primary HIV-1 infection. The patient did not receive antiretroviral therapies. However we observed a HIV-1 mutant strain associated with zidovudine (ZDV) resistance. The patient had both the codon 70 and the codon 215 amino acid substitutions in the RT region. Our data indicated that the patient was infected with a HIV-1 mutant strain associated with ZDV resistance and this is the first report in Japan.

[Gastrointestinal diseases associated with HIV infection].

A clinical studies were carried out on gastrointestinal diseases associated with HIV infection. During the 6 years between January 1993 and December 1998, 71 HIV infected cases visited to Yokohama Municipal Citizen's hospital, and 26 of them developed gastrointestinal complications during the course of their illness. They consisted of 24 males and 2 females, with the mean age of 44.7 years and the medial value of 42.5 years. Of the 26 patients, 21 were Japanese, and the remaining 5 were Southeast Asian. The mean CD4 count was 143/microliter and the medial value was 32/microliter at the time of development of complications. Gastrointestinal complications were esophageal candidiasis in 6 patients, cytomegalovirus (CMV) gastritis and gastric Kaposi's sarcoma in 1 patient each, amebiasis in 8 patients, infectious colitis in 11 patients, and asymptomatic pathogen carriers in 3 patients. Esophageal and gastric complications were common in patients with low count of CD4, and endoscopy was useful for diagnosis. Amebiasis developed even in patients with normal CD4 and was common in males with experience in homosexual contact. It seems that homosexual contact acquire not only HIV infection but also Entamoeba histolytica through sexual contact. Protozoan and acid-fast bacteria were detected at high rate in patients with infectious colitis and asymptomatic pathogen carriers. Besides food-born infections, imported infections were seen in foreign and Japanese patients who had traveled abroad. The gastrointestinal diseases associated with HIV infections for the most part were opportunistic infections or tumors but imported, food-born, and sexually transmitted infections were also observed. It seems necessary to take into consideration of varying background of patients in the treatment of gastrointestinal diseases associated with HIV infections.

Defective degranulation and calcium mobilization of bone-marrow derived mast cells from Xid and Btk-deficient mice.

Regulation of adhesion and degranulation of mast cells plays an important role in allergy and inflammation. We investigated a possible role of Bruton's tyrosine kinase (Btk) in the regulation of adhesion and degranulation by using bone marrow-derived mast cells from X-linked immunodeficiency (Xid) and Btk-deficient mice. Cross-linking of the high affinity IgE receptor (Fc epsilonRI) and steel factor (SLF) induced indistinguishable adhesive responses of mast cells to fibronectin in kinetics, and these adhesive responses were comparable among wild type, Xid, and Btk-deficient mast cells. Cross-linking of Fc epsilonRI, but not SLF triggered degranulation of bone marrow-derived mast cells. However, Fc epsilonRI-induced degranulation was impaired in Xid and Btk-deficient mast cells. Calcium influx induced by Fc epsilonRI cross-linking and SLF were also reduced in Xid and Btk-deficient mast cells. Degranulation and calcium influx were reduced more severely in Btk-deficient than in Xid mast cells. Consistently, cross-linking Fc epsilonRI and SLF augmented Btk kinase activities transiently. Inositol triphosphate (IP3) production was also severely reduced in Btk-deficient mast cells, indicating Btk play a critical role of Fc epsilonRI-induced IP3 production. The differential sensitivity of wortmannin on calcium influx in wild type and Xid mast cells suggested that the activation of phosphatidylinositol 3 kinase (PI 3-kinase) was required in calcium influx. Furthermore, abnormal secretory granules with translucent contents and variable in size were observed both in Xid and Btk-deficient mast cells. Our study demonstrated a critical role of Btk in regulating intracellular calcium and granule exocytosis.

A novel member of the Ig superfamily, RPE7, expressed on the retinal pigment epithelial cell and Müller cell of the bovine retina.

The retinal pigment epithelium of the vertebrate eye plays a major part in the maintenance of ocular function. To identify molecules involved in the exertion of their physiological functions, monoclonal antibodies against bovine retinal pigment epithelial cells were made. Analysis by immunofluoresence and immunoelectron microscopy showed that one of the monoclonal antibodies named anti-RPE7 stained the cell surface of retinal pigment epithelial cells and Müller cells. The anti-RPE7 antibody was revealed to recognize molecules of 45-55 kDa by Western blot analysis. Molecular cloning of the RPE7 cDNA and sequence analysis of the amino acids revealed that protein RPE7 belonged to the Ig superfamily. The high homology of RPE7 with metalloproteinase inducer suggests that the protein RPE7 might play a role in the matrix (interphotoreceptor matrix and basement membranes) remodeling as well as in retinal pigment epithelial cell migration under pathological conditions.

Asthma, adenosine, mast cells and theophylline.

Many clinical trials have suggested that theophylline has anti-inflammatory properties in the treatment of bronchial asthma. Proposed mechanisms of theophylline inhibition include phosphodiesterase inhibition, an adenosine receptor antagonist, the increase of circulating adrenaline, mediator antagonist and inhibition of calcium ion influx. Further to these observations we report on the inhibition by theophylline of NF-kappaB, a key transcription factor found in human purified mast cells, which plays a role in the transcription of TNF alpha, GM-CSF, and IL-8 within this cell. The suppression of NF-kappaB activation, indicates that theophylline, in addition to its bronchodilator activities, has the potential for anti-inflammatory activity.

[Correlation between apoptotic index, bcl-2 protein expression and progression and prognosis in breast carcinoma].

Apoptosis is considered to play a critical role in tumorigenesis. It has been shown that apoptosis is controlled by both pro-oncogenes bcl-2, c-myc and tumor-suppressor genes p53. We determined the apoptotic index (AI) on light microscopy and detected immunohistochemically the expression of bcl-2 and p53 in patients with breast cancer. The correlations between AI and clinicopathological factors, bcl-2 and p53 were also analyzed. Our results showed that bcl-2 expression was down-regulated in the process from normal breast epithelial cells to intraductal carcinoma and from intraductal carcinoma to invasive carcinoma. We failed to detect p53 protein in normal breast epithelial cells, and p53 positivity was 24% and 30% in intraductal and invasive cancer tissues, respectively. Moreover, AI was significantly associated with histological grade, mitotic index, and bcl-2 and p53 expression. In univariate analyses, lower AI and bcl-2 expression was significantly predictive of a better prognosis for both disease-free survival and overall survival. These results suggest that apoptosis and apoptosis-related gene (bcl-2, p53) are related to progression and prognosis in breast cancer.

[A case of imported paratyphoid fever associated with Giardia lamblia and Isospora belli infection].

We report a case of imported paratyphoid fever associated with Giardia lamblia and Isospora belli infection. The patient was a 23-year-old Japanese female with complaints of high grade fever and diarrhea after 10 days traveling to Nepal. Salmonella Paratyphi A was isolated from the blood and fecal cultures on admission and Ciprofloxacin of 200 mg tid was administered for 14 days. Fecal examination revealed cysts of G. lamblia at the same time and metronidazole of 250 mg tid for 7 days was effective for their eradication. During the follow-up studies oocysts of I. belli were found and cotrimoxazole of 960 mg bid for 10 days was effective for their eradication. Fecal examinations on parasites which is rare in Japan such as I. belli are recommended to the cases returning from tropical areas.

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

Sensitization primes platelet-activating factor (PAF)-induced accumulation of eosinophils in mouse skin lesions: contribution of cytokines to the response.

We have examined the hypothesis that cytokines mediate the enhanced responsiveness of eosinophils to PAF in sensitized mouse skin. PAF (10 ng per site) resulted in a considerable degree of eosinophil accumulation in ovalbumin (OA)-sensitized mice but not in non-sensitized mice. Intradermal preadministration of cytokines (IL-5, IL-3 and GM-CSF) also significantly enhanced PAF-induced migration of eosinophils in a dose-dependent manner. The relative potency with which these cytokines primed cell migration was IL-5 greater than IL-3 greater than GM-CSF, however, each cytokine alone showed no direct effect. We conclude that the sensitization or the exogenous application of cytokines is capable of augmenting PAF-induced eosinophil migration in mice in vivo, and the cytokines thus elicited by sensitization may contribute to the extensive recruitment of inflammatory cells in allergic diseases.

[Effects of sulbactam/cefoperazone in patients with respiratory infections in aged and/or with underlying respiratory diseases].

Sulbactam/cefoperazone (SBT/CPZ), a new antibacterial drug, was administered to 14 cases with respiratory infections for a duration of 5 approximately 13 days at a daily dose of 4 g. Diagnoses of these patients were 7 respiratory tract infections, and 7 bronchopneumonias. The underlying diseases were chronic pulmonary emphysema in 6 cases, bronchial asthma in 2 cases, and one each of bronchiectasis, diffuse panbronchiolitis and lung cancer with bronchoesophageal fistula. All patients had underlying respiratory diseases and/or were more than 70 years old. The rate of clinical efficacy was 78.6%. The incidence of penicillinase production by isolated bacteria was 18.2% and that of cephalosporinase was 63.6%. SBT/CPZ was expected to be more effective than CPZ alone in 3 cases judging from the susceptibility of the bacterial strains concerned, to antibiotics. No side effects were observed. We conclude that SBT/CPZ is useful in the treatment of respiratory infections of patients of advanced age and/or with underlying respiratory diseases.

Ocellar system of the insect: comparison of dorsal ocellus and lateral ocellus.

The ocellar receptor and neuropil in the dorsal ocellar system of the cockroach and the lateral ocellar system of the swallowtail butterfly larva have been compared. In the dorsal ocellus of the cockroach more than 10,000 retinular cells each with a rhabdomere are piled up one upon another without a specific pattern in the ocellar capsule. Retinular axons synapse exclusively with branches of four thick second order neurons in the lower region of the capsule. Dark granule-containing neurons, originating in the brain, also synapse with the second order neurons in the ocellar neuropil. The ocellar nerve is comprised of four thick second order processes (ca. 6-16 micron) and 20-40 thin processes (ca. 1-5 micron), which include retinular axons, collaterals of thick processes, possible third order processes and unidentified processes. Synapses occur against these processes in the ocellar nerve and in the extreme proximal region of the ocellar neuropil. Six lateral ocelli occur on both sides of head in the butterfly larva. Each ocellus contains seven retinular cells, which are grouped into three distal cells and four proximal cells, or four distal cells, two peripheral cells and one central cell by the position of their rhabdomeres. Retinular axons extend to the brain, and enter the distal optic neuropil. There are six neuropils, each of which contains the seven axons from one ocellus and a dozen or so second order processes. Of seven retinular axons, four occur peripherally and the remaining three occur centrally in the neuropil. All axons synapse with fine branches of interneurons. Second order processes together with at least three central retinular axons extend to the second optic neuropil, but the peripheral axons could not be followed there. The highly organized receptors and the neuropil in the lateral ocellar system suggest a complexly integrated function. An ocellar nerve and proximal ocellar neuropil appear like an extension of the CNS, and direct connections of retinular axons with interneurons in the CNS appear characteristic of the dorsal ocellus. This difference may be related to the distribution of the two ocellar systems: the dorsal ocelli co-exist with the compound eyes of the adult, whereas the lateral ocelli are the only photoreceptor organs of the larval insect.