Influence of retinoids on skin fibroblasts metabolism in vitro

The most dangerous environmental factor for our skin condition is ultraviolet light radiation. Chronic exposition to ultraviolet light can induce epidermal atrophy, keratosis, depigmentation and dysplasia. In the dermis, UV light causes dramatic up-regulation of extracellular matrix-degrading enzymes. Matrix metalloproteinases (MMPs) are engaged in collagen, elastin and other extracellular matrix components degradation. In addition, to increase level of destructive enzymes, UV light has been shown to decrease collagen production. As a consequence of UV impact on skin, it shows signs of aging including loss of tone and elasticity, increased skin fragility, blood vessels weakness and wrinkles. The most dangerous effect of UV on skin is an increased risk of melanoma and other skin cancers. Retinoids are well known antiaging agents. For many years this vitamin has been used for the prevention and treatment of photoaging. Retinoids abolish cellular atypia, increase compacting of the stratum corneum and reduce skin hyperpigmentation caused by sun light. Recent evidence suggests that retinoids also play a role in the prevention of aging, because of its inhibitory effects on metalloproteinases expression. The aim of this study was to examine if all-trans-retinoic acid (ATRA) effects MMP-1, MMP-2, MMP-3 and MMP-14 gene expression in fibroblasts cultured in vitro.     

Detection of TT virus in lymph node biopsies of B-cell lymphoma and Hodgkin's disease,and its association with EBV infection

Human TT virus (TTV) recently isolated from the serum of a patient with post-transfusion hepatitis does seem to have only hepatopathic effect. The virus can also infect the serum, peripheral blood mononuclear cells (PBMC) and bone marrow cells (BMC ). Additional evidence has indicated that TTV is also present in the serum of people with hematopoietic malignancies. A significant increase in the incidence of lymphoma has recently been observed worldwide. We have investigated the presence of TTV DNA in lymph node biopsies of Italian patients affected with the most common lymphoma types in Western Countries: follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and nodular sclerosis Hodgkin's disease (NS-HD). The possible role of a co-infection with Epstein-Barr virus (EBV) has also been investigated. DNA was extracted from 73 paraffin-embedded and 38 snap-frozen tissue specimens. From these, only 67 samples (29 paraffin-embedded and 38 snap-frozen tissues) from a total of 56 patients, were suitable for PCR analysis. TTV and EBV were detected by PCR using primers from two different conserved region in TTV and EBV genomes respectively. TTV DNA was detected in 30.0-50.0% of FL, 30.8% of DLBCL and 30.0-50.0% of NS-HD cases, depending on the primers used. All cases of non-specific reactive lymphoid hyperplasia (RLH), used as a putative control, were negative. The two major TTV genotypes circulating in Italy (G1 and G2) were detected in the analysed lymphoid neoplasms. EBV DNA was detected in 40.0% of FL, in 72.7%of DLBCL, in 80.0% of SN-HD and in 40.0% of RLH cases. EBV co-infection was found in 90% of TTV positive cases. The in situ hybridization assay was performed in TTV positive frozen samples. The significant prevalence of TTV DNA in lymphocytes circulating in the lymph nodes of both B-cell lymphomas and HD reported herewith suggests an implication of TTV infection in the development of these lymphoproliferative disorders.     

Induction of apoptosis in human lung fibroblasts and peripheral lymphocytes in vitro by Shosaiko-to derived phenolic metabolites

Shosaiko-to is a Kampo medicine used for the treatment of chronic hepatitis in Japan. Lately, over 200 cases of interstitial pneumonia have been reported resulting from Shosaiko-to therapy, and the number of cases increased when patients were administrated interferon (IFN)-alpha at the same time. However, the mechanisms of this Shosaiko-to implicated interstitial pneumonia are not fully understood. In this study, we examined by flow cytometry analysis the in vitro effects of 7 phenolic compounds (lignans and flavonoids), which were detected from human urine after administration of Shosaiko-to, and IFN-alpha on inducing apoptosis in human lung fibroblasts and peripheral blood mononuclear cells (PBMCs). Among the 7 compounds, baicalein and medicarpin (10 microg/ml) showed significant apoptosis-inducing effects on human PBMCs. In human lung fibroblasts, medicarpin exhibited a significantly higher activity to induce apoptosis compared to the control, and the percentage of cells undergoing apoptosis showed time- and dose-dependent increases. Baicalein (0.1 and 1 microg/ml), liquiritigenin (10 microg/ml) and davidigenin (10 microg/ml) also showed significant effects after 96 h treatment. Whereas, baicalin, oroxylin A and wogonin did not show any effect on inducing apoptosis in PBMCs and fibroblasts. Baicalein and medicarpin significantly inhibited the growth and reduced the viability of lung fibroblasts. IFN-alpha had no apoptosis-inducing effect, and it did not show synergistic interaction with any of the compounds derived from Shosaiko-to on inducing apoptosis in both human lung fibroblasts and PBMCs. These results suggested that phenolic compounds found in human post-administrative urine of Shosaiko-to, especially baicalein and medicarpin, exhibited a direct effect on human lung fibroblasts and immune cells to induce apoptosis.     

Relevance of in vitro tests to in vivo acute skin inflammation: potential in vitro applications of skin keratome slices, neutrophils, fibroblasts, mast cells and macrophages

In vitro tests on cells or keratome slices of skin may reproduce, or indirectly reflect, the first event in acute inflammation, the cytotoxic action of an irritant on epithelial/epidermal cells. Keratome slices of human or animal skin release enzymes, show histochemical changes and demonstrate increased or decreased utilization of isotope-labelled amino acids when exposed to chemicals, including surfactants, or bacterial toxins (Clostridium perfringens). The correlation with in vivo change is good for weak irritants and moderate to poor for strong irritants. Corrosive substances destroy the ability of the tissue to respond. Similar results have been obtained in tests on fibroblast cultures without or with an agar-keratin barrier. Neutrophils, or their separated granules, and mast cells have limited application in the prediction of chemical irritancy. The relevance and limitations of in vitro toxicological predictive tests are assessed in terms of the in vivo feature reflected by the in vitro test, the range of chemicals active in vitro compared to the in vivo responses, the ability to discriminate between intensities of reactions and the need for standards to compare results in different laboratories.     

In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants.

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have investigated the use of human skin fibroblasts for in vitro screening for skin toxicity. To assess the cytotoxic effects of surfactants, cell viability was measured by the NRU (neutral red uptake) assay and AB (Alamar blue) assay as in vitro methods. The skin irritation potential of surfactants by human skin patch test was assessed as in vivo methods. The close relationship was found between AB assay with human skin fibroblasts and human patch test (r=0.867). There was a relatively good agreement between the NRU and in vivo patch test (r=0.648). These results suggest that AB and NRU assay using cultured human fibroblast could be predictable methods for the irritancy of various surfactants in human.     

Enhancement of murine susceptibility to oral lactate dehydrogenase-elevating virus infection by non-steroidal anti-inflammatory agents, and antagonism by misoprostol

The murine lactate dehydrogenase-elevating virus (LDV) was used to study the effects of prostaglandin-acting agents on mucosal resistance to virus infection. Mice treated with non-steroidal anti-inflammatory drugs (NSAIDs) prior to oral exposure to LDV demonstrated a reduction in the mucosal barrier to LDV infection. Histological studies indicated that these NSAID effects were not a result of gross or microscopic tissue damage. The effects of two NSAIDs, indomethacin and diclofenac, were inhibited by co-treatment of mice with misoprostol, a synthetic PGE1 analog. The ability of misoprostol to modulate NSAID effects was not due to direct antiviral activity or to actions on LDV-permissive macrophages. These results show that the mammalian mucosal barrier to virus infection is prostaglandin-sensitive, and provide a model for the study of resistance to viral infection.     

Recognition of transformed cells by autologous lymphocytes: a review.

The studies on the T cell response against EBV carrying B cells demonstrated its high power in vivo. Malignancies with phenotypes similar to the in vitro transformed B cells occur only in severely immunosuppressed patients. Presently the goal of the studies is the identification of the antigens recognised by T lymphocytes and their association with the various HLA alleles. The studies on human sarcomas and carcinomas still struggle with the demonstration of specific T cell responses and the characterisation of the target molecules on the tumor cells. The main goals are the possibilities to readminister tumor reactive T cells for therapeutic purposes and the use of in vitro assays for guidance of the design and schedule for immunotherapy protocols.     

格尔德霉素体内外抑制单纯疱疹病毒1型的复制

格尔德霉素 ( GA)是一种抗生素 ,它的作用靶点是热休克蛋白 Hsp90 N末端的 ATP/ ADP结合位点。在我们对抗病毒的抗生素筛选中 ,发现格尔德霉素显著抗单纯疱疹病毒 1型 ( HSV- 1)。体外在 Vero细胞内 GA显著抑制 HSV- 1的复制 ,IC50 为0 .0 93μmol/ L,GA对 Vero细胞的毒性 CC50 为 35 0 μmol/ L,治疗指数可达 376 3。HSV- 1腹腔 ( ip)感染 1h后腹腔 ( ip)注射 GA( 0 .0 93～ 0 .37mg/ kg)可以将存活率增加到 6 7%～ 10 0 % ,皮下 ( sc)给药 ( 0 .37mg/ kg)的存活率为 4 3.8% ,都明显高于生理盐水对照组 ( ipP<0 .0 0 1,sc P<0 .0 5 )。GA对小白鼠的急性 L D50 为 15 .5 mg/ kg( ip)。格尔德霉素不影响病毒的吸附、穿入。由于 GA在体内外都能抑制单纯疱疹病毒 1型 ,GA可以成为新的抗单纯疱疹病毒感染的药物     

格尔德霉素体内外抑制单纯疱疹病毒l型的复制

格尔德霉素(GA)是一种抗生素,它的作用靶点是热休克蛋白Hsp90 N末端的ATP/ADP结合位点.在我们对抗病毒的抗生素筛选中,发现格尔德霉素显著抗单纯疱疹病毒1型(HSV一1).体外在Vero细胞内GA显著抑制HSv-1的复制,IC50为0,093μmol/L,GA对Vero细胞的毒性CC50为350μmol/L,治疗指数可达3763.HSV-1腹腔(ip)感染1h后腹腔(ip)注射GA(0.093～0.37mg/kg)可以将存活率增加到67%～100%,皮下(sc)给药(0.37mg/kg)的存活率为43.8%,都明显高于生理盐水对照组(ipP＜0.001.scP＜0.05).GA对小白鼠的急性LD50为15.5mg/kg(ip).格尔德霉素不影响病毒的吸附、穿人.由于GA在体内外都能抑制单纯疱疹病毒1型,GA可以成为新的抗单纯疱疹病毒感染的药物.     

Derivatives of amphotericin inhibit infection with human immunodeficiency virus in vitro by different modes of action

Three water-soluble derivatives of amphotericin B were tested for inhibition of HIV infection in vitro. The compounds amphotericin B methyl ester (AME) and N-(N'-(2-(4'-methylmorpholinio)ethyl)N"-cyclohexyl guanyl) amphotericin B methyl ester (MCG) inhibited HIV infection by 50% at 1 microgram/ml; N-(N'-(3-dimethylaminopropyl)N"-ethyl guanyl) amphotericin B (DAPEG) did so at 5-11 micrograms/ml. While the virus-inhibitory effect of AME was due to an interaction with target lymphocytes, the effect of MCG was due to a direct anti-viral action. AME increased the potential of infected cells to fuse with uninfected cells, but MCG had no significant effect on cell fusion. All compounds had a lower cellular toxicity than amphotericin B and were not toxic at concentrations below 20 micrograms/ml.     

Neurological disease in patients with human immunodeficiency virus infection.

We followed prospectively all patients with HIV infection admitted to the infectious diseases ward at Auckland Hospital over a seven month period. Neurological manifestations of HIV infection were the primary reason for admission in 18 of the 55 patients (33%). Diagnoses were usually presumptive, based on history, clinical findings, radiological appearances and response to empirical therapy. Eight patients had cerebral toxoplasmosis, three primary cerebral lymphoma, two cytomegalovirus retinitis, two HIV neuropathy, one cryptococcal meningitis, one HIV encephalopathy, and one HIV meningitis. Another patient with HIV infection was admitted to the neurology ward at Auckland Hospital with HIV myelopathy during the same seven month period. The median survival of the patients treated for presumptive toxoplasmosis was 7.5 months. Only two patients had not developed AIDS, one having HIV meningitis and the other HIV myelopathy, and in both, symptoms resolved spontaneously with no relapse at one year follow up. The spectrum of neurological manifestations of HIV infection is wide. Investigations to determine the most likely diagnosis are indicated and specific therapy may lead to both excellent palliation and prolonged survival.     

尿激酶体外溶凝量效与时效的实验观察

目的观察尿激酶(UK)体外溶凝的最佳量效和时效,以指导临床合理有效应用UK。方法应用自制的脑出血体外实验模型(参照临床10%设计),采用5×5拉丁方,分别加入0.2mLUK溶液,分别含有UK剂量(1000、2000、4000、10000、20000U),用药时机(抽血后4、6、8、16、24h)和溶凝时间(加UK后1、2、3、4、6h),观察UK对体外血肿溶凝的量效和时效。结果UK剂量对溶凝体积的差异有统计学意义(F=3.917,P<0.05),1000U(5000U/mL)组溶凝质量(230.90mg)明显低于4000U(311.39mg)溶凝质量,以及20000U(100000U/mL)溶凝质量(302.29mg),而后2组间的差异无统计学意义。用药时机和溶凝时间对溶凝体积的差异均无统计学意义(F=0.75,P>0.05;F=1.26,P>0.05)。结论本研究提示,UK溶凝的最佳有效剂量为4000U(20000U/mL),延迟24h用药不影响溶凝效果,溶凝1h足以发挥作用。     

Palmitate increases the susceptibility of cells to drug-induced toxicity: an in vitro method to identify drugs with potential contraindications in patients with metabolic disease

Fatty acids are an important source of energy. Excessive energy intake results in elevated levels of free fatty acids that are thought to be the pathogenic factors causing metabolic disorders such as dyslipidemia, obesity, insulin resistance, diabetes, and fatty liver. Underlying metabolic disorders have been suggested to be a predisposing factor for drug-induced liver injury. The steadily expanding population with metabolic disease may pose a higher risk for drug-induced toxicity. In order to understand the interaction of free fatty acids and drug-induced toxicity at the cellular level, we explored whether the saturated free fatty acid palmitate could modulate drug-induced cytotoxicity in HepG2 cells. A number of drugs known to induce hepatotoxicity in humans were selected to test this hypothesis. Drugs without reported hepatotoxicity were also tested to evaluate the specificity of the palmitate-induced effects. We demonstrate that palmitate, at sublethal concentrations, was able to potentiate the cytotoxicity and/or apoptosis induced by some but not all drugs tested. The palmitate and drug coincubation potentiated toxicity, which when combined with the plasma maximum concentration (C (max)), allowed us to identify idiosyncratic toxic drugs that were not flagged in previously deployed cytotoxicity assays. Our data suggest that treatment of cells with palmitate improves the sensitivity to detect compounds with risk of inducing idiosyncratic liver toxicity. Furthermore, this assay may be used to identify compounds that have higher safety risks in a population with metabolic syndrome.     

Borna病病毒与精神分裂症的关系研究

摘要目的探讨贵州省部分地区博尔纳病病毒（Bornadiseocevicus）在精神分裂症患者中的感染情况及与疾病发生的相关性。方法采用巢式逆转录荧光定量PCR法对来自于贵州省部分地区（遵义、毕节、铜仁）的30例精神分裂症（SCH）患者及100例健康人的外周血液单核细胞（PB—MC）中BDVp24基因片段进行检测,对阳性产物进行纯化、克隆、测序,用软件对所测定的序列结果进行同源性分析。结果精神分裂症患者中BDVp24基因片段阳性率为6．7％（2／30）;健康体检者中未检出,二者检出率比较无统计学差异（P〉0．05）。精神分裂症患者测序结果与GenBank提供的strainV毒株、C6BV毒株、He80／FR病毒株进行比较,突变率均为3％,均有3个位点出现一致性沉寂突变。与1766毒株进行比较,突变率为2％,有2个位点出现一致性沉寂突变,即与1766毒株的亲缘关系最近。结论本研究发现30例精神分裂症患者中检出BDVp24基因片段阳性2例,而健康人中未检出,说明Borna病病毒感染可能与精神分裂症有关,两组问BDV检出率无统计学差异,可能与所采标本地区较局限或标本量少有关。     

硒化壳聚糖促进体外培养皮肤成纤维细胞增殖

目的研究硒化壳聚糖对体外培养的皮肤成纤维细胞增殖的影响。方法用不同剂量（25、50、100、200、400mg/L）硒化壳聚糖作用于成纤维细胞,光镜下观察药物对细胞形态的影响;MTT法、3H-TdR掺入法、细胞生长动力学研究用于检测药物对细胞增殖影响,LDH漏出率检测药物对细胞增殖及损伤的影响,并以等体积细胞培养液处理为对照组。结果各药物浓度组处理细胞后,细胞形态未见明显改变,均可使细胞吸光度值增加,细胞倍增时间缩短,促进表皮细胞对3H-TdR的掺入（P〈0.05）,降低LDH漏出率（P〈0.05）。结论硒化壳聚糖对体外培养皮肤成纤维细胞增殖具有促进作用。     

Mouse models of dengue virus infection and disease

Dengue virus (DENV) causes the most significant mosquito-borne viral disease in the world in terms of illness, death, and economic cost, due to the lack of an approved vaccine or antiviral. Infections with one of the four serotypes of DENV (DENV1-4) can result in diseases ranging from an acute, self-limiting febrile illness (dengue fever, DF) to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), yet exactly how viral and host factors contribute to the severe disease is unknown. Clinical observations have provided information on DENV pathogenesis, but the lack of an adequate animal model has hindered research on this important human pathogen. A mouse model is ideal for investigating host-pathogen interactions due to the immunological tools available, however, wild-type mice are resistant to DENV-induced disease. Therefore, the mouse models for DENV infection developed to date include infection of severely immunocompromised mice, non-physiologic routes of infection, and mouse-human chimeras, which all have their limitations. An inbred mouse model in which mice develop signs of human DENV-induced disease is needed to investigate the contribution of various immune components to protection and pathogenesis of DENV infections, and to test the efficacy of DENV vaccines and antivirals.