Surface Accessibility of the 70-Kilodalton Chlamydia trachomatis Heat Shock Protein following Reduction of Outer Membrane Protein Disulfide Bonds

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.     

Avirulence of Candida albicans CaHK1 Mutants in a Murine Model of Hematogenously Disseminated Candidiasis

Deletion of both alleles of the Candida albicans CaHK1 gene, which causes cells to flocculate when grown at pH 7.5, a pH comparable to that of mammalian blood, abolishes the ability of the yeast to establish a successful infection in a murine model of hematogenously disseminated candidiasis. Within 72 h all mice inoculated with the parental C. albicans strain had died. The mice infected with either the heterozygote or revertant strain, either of which harbors only one functional CaHK1 allele, also succumbed to the infection, although survivors were observed for up to 16 days postinfection. However, mice inoculated with the Deltacahk1 null strain survived for the course of the infection. These results indicate that CaHK1 is required for the virulence of C. albicans in a murine model of hematogenously disseminated candidiasis. In contrast, CaHK1 is not required for the virulence of C. albicans in a rat model of vaginal candidiasis.     

细胞外热休克蛋白70及其生物学功能

热休克蛋白(HSP)一直被描述为一种细胞内蛋白质在胞内发挥一系列生物学作用,参与细胞内蛋白质的折叠、装配、降解和修复过程。近年来,有研究发现HSP70能被主动释放到胞外,成为细胞外HSP70(extracellular HSP70,eHSP70),但关于其功能尚不清楚。循环HSP70水平与多种疾病进程密切相关。有研究发现暴露于物理和心理刺激源作用下,HSP70可能通过脂筏或Exosome介导的分泌途径释放到细胞外,作为一种生物活性因子影响多种疾病的进程。     

Regulation of the Physiological Functions of Human Dendritic Cells by Recombinant Heat Shock Protein Hsp70

Dendritic cells (DC) are the most important antigen-presenting cells in the body. They are the target of action of various vaccines and dendritic cells have been used as the basis for developing cellular antitumor and antiviral vaccines, i.e., DC vaccines. At the same time, dendritic cells may provide a suitable model for studies of the activity and mechanisms of action of different immunotherapeutic formulations. One aspect of the optimization of the use of dendritic cells for inducing antigen-specific immune responses relates to the use of heat shock proteins (Hsp), particularly Hsp70. This protein can be used to introduce protein antigens into dendritic cells and to control the activity of dendritic cells. Important aspects of achieving these aims include knowledge of dendritic cell physiology and the characteristics of the interaction of Hsp70 and its complexes with antigens with dendritic cells of different levels of differentiation. Human recombinant Hsp70 was found not only to deliver antigens to dendritic cells, but also to regulate the activity of mature dendritic cells and to optimize the induction of antigen-specific cellular immune responses.     

Silencing of Heat Shock Protein 70 Expression Enhances Radiotherapy Efficacy and Inhibits Cell Invasion in Endometrial Cancer Cell Line

Aim

To investigate the role of heat shock proteins 70 (HSP70) in radiosensitivity and invasiveness of endometrial cancer in vitro.

Methods

HSP70 expression was silenced in relatively radioresistant, well-differentiated human endometrial cancer cell line ISK, using small interference RNA method, or by HSP70 overexpression after transfecting a HSP70-expressing vector. The effect of HSP70 on ISK cell line response to irradiation was evaluated. The surviving fraction was measured using colony-formation assay. Apoptosis was detected by flow cytometry and HSP70 expression was determined by quantitative real-time polymerase chain reaction, western-blot, and/or immuocytochemistry. Cell invasiveness was measured using transwell invasion assay.

Results

HSP70 silencing caused a significant increase in irradiation-induced cell killing in comparison with control cells, with an enhancement factor of 1.27, and in the percentage of apoptotic cells (14.22% vs 6.74%, P = 0.021). After 4 Gy irradiation, mean ± standard deviation survival fraction in ISK cells was reduced to 0.32 ± 0.04 in comparison with control values but in ISK/siRNA-HSP70 cells the survival fraction was higher and amounted to 0.51 ± 0.08 (P = 0.026). Silencing HSP70 significantly inhibited cell invasion before and after irradiation (106 ± 19 vs 219 ± 18 and 119 ± 16 vs 256 ± 31, P = 0.007). On the contrary, ectopic overexpression of HSP70 attenuated irradiation-induced apoptosis (7.15% vs 4.08%, P = 0.043) and induced more ISK/HSP70 cells invaded through the filters than mock-infected cells before and after irradiation (274 ± 21 vs 194 ± 16 before irradiation, and 298 ± 24 vs 227 ± 19 after irradiation, respectively, P = 0.032).

Conclusion

Disruption of HSP70-induced cytoprotection during irradiation enhances therapeutic effect of irradiation, which makes HSP70 a promising target in the research of endometrial cancer.Endometrial cancer is one of the most common gynecologic malignancies worldwide, with radiation therapy as a very important treatment option (1). However, the efficacy of radiation therapy is often limited by radioresistance, ie, diminished susceptibility of the irradiated cells to undergo apoptosis. In our previous studies, we found that human heat shock protein 70 (HSP70) was significantly overexpressed in radioresistant endometrial cancer cells line ISK compared with relatively radiosensitive cells after irradiation (2). This suggested that irradiation-induced HSP70 content may play an important role in the development of radioresistance.The HSP70 family contains at least 8 homologous chaperone proteins (3). Endoplasmatic reticulum and mitochondria have their specific HSP70 proteins, whereas the remaining 6 family members reside mainly in the cytosol and nucleus. HSP70B'' is the major human isoform in the HSP70 family that is strictly stress-inducible and therefore available to function only in stressed cells (4). Phylogenetic analysis indicated that the HSP70B’ protein sequence was most closely related to another major inducible human HSP70 – HSP72. HSP70B'' and HSP72 together play a role in cell survival after proteotoxic stress. Inducible HSP70 are up-regulated in the majority of human tumors and are believed to have an important role in cell proliferation, tumor growth, and cancer invasiveness (5-8). Overexpression of HSP70 in human tumors is associated with poor prognosis and poor response to radiation therapy (9,10). Some in vitro studies suggested that the viability of cells pretreated with exogenous HSP70 before radiation was indeed protected and that transfection of cells with a siRNA designed to interfere with HSP70 synthesis increased the irradiation-induced apoptosis (11,12). Despite this, the role of HSP70 in endometrial cancer is largely unknown. Understanding of HSP70s function in endometrial cancer may provide clues to the development of radioresistance and offer alternatives for clinical treatment. Meanwhile, it is well known that metastasis is highly dependent on tumor cell invasion. Previous studies reported that overexpression of HSP70 induced the expression of matrix metallopeptidase 9 (MMP-9) and thereby improved the cell invasiveness (13). In the present study, we investigated the role of HSP70 in radiosensitivity and invasiveness of a well defined endometrial cancer cell in vitro.     

Circulating Heat Shock Protein 70 in Health,Aging and Disease

Background

Heat shock proteins (Hsp) are ubiquitously synthesised in virtually all species and it is hypothesised that they might have beneficial health effects. Recent studies have identified circulating Hsp as an important mediator in inflammation - the effects of low-grade inflammation in the aging process are overwhelming. While much is known about intracellular Hsp70, scant data exist on circulating Hsp70 in the aging context. Therefore, the objectives of this study were to investigate the effect of age and disease on circulating Hsp70 and, in particular, to evaluate the association between circulating Hsp70 and inflammatory parameters.

Results

Serum Hsp70, Interleukin (IL) -10, IL-6 and Tumor Necrosis Factor (TNF) alpha concentrations were determined in 90 hospitalised geriatric patients (aged 83 ± 6 years) and in 200 community-dwelling control subjects (100 elderly, aged 74 ± 5 years, and 100 young, aged 23 ± 3 years). In the community-dwelling elderly, serum Hsp70 and IL-10 concentrations were significantly lower and IL-6 was significantly higher when compared to healthy young control subjects. Elderly patients presenting inflammation (CRP serum levels ≥5 mg/L) showed significantly (p = 0.007) higher Hsp70 values; and Hsp70 correlated positively (p < 0.001) with IL-6 and CRP, but not with TNF-alpha or IL-10. A significant association was also noted between Hsp70 levels and the degree of dependency and cognitive decline in geriatric patients.

Conclusions

The present data provide new evidence that serum concentration of Hsp70 decreases with age in a normal population. Our study also shows that higher levels of Hsp70 are associated with inflammation and frailty in elderly patients.

     

A Histoplasma capsulatum-Specific IgG1 Isotype Monoclonal Antibody,H1C,to a 70-Kilodalton Cell Surface Protein Is Not Protective in Murine Histoplasmosis

Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.Histoplasma capsulatum is a dimorphic fungal pathogen that is the causative agent of histoplasmosis. After Candida species, H. capsulatum is the most common causative agent of systemic mycoses in North America and either the first- or second-most-common cause in Central and South America (4, 7). We previously identified an IgG1 isotype monoclonal antibody (MAb), H1C, that reacts with a 70-kDa H. capsulatum cell wall antigen (6). The MAb is highly specific for H. capsulatum, as it did not react with yeast cell antigens from related microbes. An inhibition enzyme-linked immunosorbent assay (ELISA) system using H1C is also highly specific for the detection of antigenemia and antigenuria in patients with histoplasmosis (6). Furthermore, the inhibition ELISA may be useful for monitoring treatment responses in patients with histoplasmosis (5).We have shown that MAbs to the H. capsulatum cell surface proteins histone 2B (H2B) and heat shock protein 60 (Hsp60) can alter the intracellular fate of the fungus, modify the inflammatory response to infection, and prolong the survival of lethally infected mice (8, 9). In particular, we demonstrated the marked protective efficacy of IgG1 and IgG2a MAbs to Hsp60. In the present study, we assessed the capacity of H1C to modify host-pathogen interactions.H1C was purified from cell culture supernatants using protein G-agarose beads (Pierce Biotechnology) according to the manufacturer''s instructions. Aliquots of H1C were screened to ensure the absence of endotoxin with the Limulus amebocyte lysate assay (BioWhittaker Inc.). An irrelevant, isotype-matched mouse IgG1 MAb (SouthernBiotech) was used as a control for all experiments. H. capsulatum G217B was used as the reference strain for all the studies (gift from G. Deepe, University of Cincinnati). H. capsulatum yeast cells were grown in Ham''s F-12 medium at 37°C with rotary shaking and collected as described previously (9). Enumeration of the yeast cells was accomplished with a hemacytometer.Immunofluorescence and immunoblotting were performed as described previously (8), except that H1C labeled with Alexa 488 was used in lieu of MAbs to Hsp60. Fluorescence analysis revealed that H1C diffusely labeled the fungal cell surface in a punctuate manner (Fig. ​(Fig.11 A), and H1C reacted with a 70-kDa protein, as shown by immunoblotting (Fig. ​(Fig.1B).1B). A whole-cell H. capsulatum yeast ELISA was also used to examine the binding of H1C to H. capsulatum, as described previously (9). Significant binding occurred at concentrations >1 μg/ml H1C (Fig. ​(Fig.1C).1C). Hence, H1C readily interacted with the 70-kDa protein at the H. capsulatum cell surface.Open in a separate windowFIG. 1.MAb H1C binds to a 70-kDa protein on the cell surface of H. capsulatum. (A) Immunofluorescence analysis demonstrates that H1C diffusely reacts with the fungal cell surface. (B) H1C reacts with a 70-kDa protein in cell wall extracts from H. capsulatum. (C) Yeast cell ELISA demonstrates the reactivity of H1C with intact fungal cells. Experiments were repeated at least three times, with consistent results.Phagocytosis and killing assays were accomplished as described by confocal microscopy and fluorescence-activated cell sorting (9) using J774.16 macrophagelike cells. H1C increased the association of H. capsulatum with the J774.16 cells (Fig. ​(Fig.22 A). Although increases in yeast cell attachment occurred at 25 and 50 μg/ml of H1C (Fig. ​(Fig.2B),2B), phagocytosis of H. capsulatum increased at all the concentrations tested (Fig. ​(Fig.2C).2C). Further, H. capsulatum opsonized with H1C had significantly increased intracellular survival after 24 h compared to controls (Fig. ​(Fig.2D).2D). GraphPad Prism version 5.00 for Windows (GraphPad Software) was used for statistical analyses. Kruskal-Wallis nonparametrical tests were used for one-way analysis of variance to compare differences between groups, and individual comparisons of groups were performed with the Bonferroni posttest.Open in a separate windowFIG. 2.MAb H1C alters H. capsulatum-macrophage interactions. (A) H1C alters H. capsulatum association with J774.16 macrophage-like phagocytosis. H1C opsonization affects H. capsulatum attachment (B) and phagocytosis (C). (D) H1C-opsonized H. capsulatum has significantly improved intracellular survival. (E) Opsonization of H. capsulatum with H1C reduces nitric oxide production. (F) Opsonization of H. capsulatum with H1C increases macrophage death. Macrophage toxicity data are at 2 h of coculture, and similar results were present at 4 h. For each graph, three independent experiments were performed, with each condition tested in triplicate, except for the nitric oxide experiments, which were performed twice with triplicates. Graphs were generated with pooled data from the experiments. The bars are the mean values, with error bars representing the standard deviations. *, P < 0.05, which indicates a comparison between the condition and H. capsulatum in the absence of antibody. “Hc” represents yeast without antibody in the presence of macrophages, “irrelevant” indicates an IgG1 isotype control antibody with yeast cells and macrophages, and the concentrations listed represent the specific amounts of H1C MAb present with H. capsulatum yeast and macrophages.Since intracellular growth increased in the presence of H1C, we examined the production of nitric oxide and superoxide by J774.16 cells cocultured with H. capsulatum exposed to H1C, irrelevant antibody, or phosphate-buffered saline (PBS), as described previously (10), and also assessed J774.16 viability. Nitric oxide formation was assessed with a commercial Griess reagent kit (Promega), and superoxide dismutase activity was quantified with a kit (Cayman Chemical). H1C-opsonized H. capsulatum induced significantly less nitric oxide release than controls at concentrations ≥25 μg/ml after 2 h of coculture (Fig. ​(Fig.2E).2E). In contrast, there were no differences in superoxide radicals (data not shown). J774.16 viability after exposure to H. capsulatum was measured using an MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay (Sigma-Aldrich). H1C concentrations ≥25 μg/ml in the presence of H. capsulatum yeast cells resulted in a dose-dependent toxicity on the J774.16 cells at 2 h of coculture (Fig. ​(Fig.2D).2D). H1C alone in the presence of the J774.16 cells had no effect on the macrophages (data not shown). Given the damage to the macrophages caused by the opsonized H. capsulatum yeast cells, it is possible that the reduction in nitric oxide was a direct effect of the reduction in viable macrophages.C57BL/6 mice (6 to 8 weeks old, female; NCI) were used for survival studies, which were approved by the Animal Institute Committee of the Albert Einstein College of Medicine. C57BL/6 mice were injected intraperitoneally with 250 μg of H1C, an isotype-matched control MAb, or PBS. Two hours later, the mice were intranasally challenged with either 5 × 10⁶ CFU (sublethal dose) or 1.25 × 10⁷ CFU (lethal dose for survival studies) of H. capsulatum yeast. The mice were closely monitored. CFU studies revealed that there was a slight trend toward an increase in numbers of CFU at day 3 after infection in animals given H1C, but the differences at days 3 and 7 in the fungal burdens between H1C-treated animals and controls were not significant (Fig. ​(Fig.33 A). Lethally challenged mice receiving H1C died at day 10 ± 1, whereas control mice died between days 10 and 12, which results are not statistically different by Kaplan-Meier analysis (P > 0.05). We also tested the efficacy of 100 and 500 μg of H1C in the lethal-challenge model, but these doses similarly failed to alter survival (data not shown).Open in a separate windowFIG. 3.Pretreatment with intraperitoneal injections of MAb H1C. A dose of 100 μg does not alter the pulmonary fungal burden in sublethally infected mice (A) or prolong survival in mice receiving lethal challenge (B). CFU studies were preformed with three mice per condition for each day assessed. Survival studies were performed using groups of 10 mice, and the experiment was repeated, with similar results.MAbs have been shown to protect against several fungal diseases, including candidiasis, cryptococcosis, aspergillosis, and pneumocystosis (2, 3). We have previously demonstrated that IgM, IgG2a, and IgG1 MAbs to H. capsulatum H2B or Hsp60 modify histoplasmosis (8, 9), but an IgG2b to Hsp60 is disease enhancing (8, 9). Hence, this report presents the second nonprotective MAb to H. capsulatum. Isotype has been shown to influence the outcome of cryptococcosis with human and murine MAbs to the major polysaccharide of Cryptococcus neoformans; in addition, a recent study showed that human IgG1-treated mice had accelerated death (1). Our findings do not rule out the possibility that a protective antibody to the 70-kDa protein could be generated, either one with a different isotype or one corresponding to a different epitope on the protein. Notably, although we found no differences in fungal burden or survival in our murine infection model, our results nevertheless suggest a possible role for the 70-kDa molecule in the virulence of the fungus, since H1C-opsonized H. capsulatum had increased intracellular survival, modified nitric oxide production, and increased macrophage damage. Our data further demonstrate the complexities involved in antibody-pathogen interactions.(The data in this paper are from a thesis to be submitted by A. J. Guimarães in partial fulfillment of the requirements for a Ph.D. degree from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY.)     

热休克蛋白70对未成熟心肌细胞功能的影响

目的探讨热休克蛋白70(HSP70)对未成熟心肌细胞功能的影响。方法健康新生长耳大白兔随机分为2组。对照组(C):腹腔注射生理盐水0.4ml,注射后24h取离体心脏,常规建立Langendorff离体心脏灌注模型,灌注15min转为工作心15min后停灌45min,恢复灌注15min改为工作心30min。实验组(E):腹腔注射重酒石酸去甲肾上腺素,24h后取离体心脏,方法同对照组。测定心肌细胞中HSP70含量及相关生化指标并作统计学处理比较。结果HSP70含量E组明显高于C组;E组ATP含量、SOD活性方面均优于C组(P<0.01),MDA含量、CK、LDH漏出率方面均低于C组(P<0.01),心肌线粒体Ca2+-ATPase活性、[ATP]m均优于C组(P<0.01),心肌细胞内Ca2+含量、心肌线粒体Ca2+含量低于C组(P<0.01)。结论HSP70对缺血再灌注未成熟心肌细胞功能具有明显的保护作用。     

Effect of Recombinant Heat Shock Protein 70 of Mycobacterial Origin on Cytotoxic Activity and Immunophenotype of Human Peripheral Blood Mononuclear Leukocytes

Recombinant heat shock protein of mycobacterial origin with a molecular weight 70 kDa in concentration 0.1 μg/ml in vitro activates cytotoxic activity of mononuclear lymphocytes from peripheral blood of healthy donors against K-562 human erythroblastic leukemia cells sensitive to natural killers. In other concentrations (1.0 μg/ml and 10 μg/ml) this heat shock protein did not signifi cantly affect functional activity of mononuclear leukocytes. Expression of CD4, CD25, CD16 and CD56 on membrane of mononuclear leukocytes was also studied.     

Antibodies to 60-Kilodalton Heat Shock Protein and Outer Membrane Protein 2 of Chlamydia pneumoniae in Patients with Coronary Heart Disease

Evidence linking Chlamydia pneumoniae infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native C. pneumoniae proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-C. pneumoniae antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for C. pneumoniae infection, which would be of potential usefulness for its specificity and nonsubjective nature.     

Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 10³ purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection.The obligate intracellular protozoan parasites of the genus Cryptosporidium cause the diarrheal illness termed cryptosporidiosis (24). Approximately 30% of adults in high-income countries and nearly all adults in resource-poor countries have serologic evidence of prior infection with this organism. However, only a small minority of people have ever been diagnosed with clinical disease. This is thought to stem from the underutilization and poor sensitivities of current diagnostic tests. For example, in the outbreak in Milwaukee, WI, in which over 400,000 people developed diarrhea associated with contamination of the water supply with Cryptosporidium parasites, fewer than 1,000 cases were confirmed on the basis of stool examination (although nearly all individuals tested developed antibodies to the organism) (3). Studies employing sensitive assays (e.g., PCR) have greatly increased the ability to recognize the burden of cryptosporidiosis. For example, Cryptosporidium DNA was found in the stools of 20% of South African children with diarrhea and three-fourths of Ugandan AIDS patients with diarrhea (18, 22).The detection of oocysts in stool samples has served as the traditional basis for diagnosis. The organisms are usually overlooked by direct examination in the absence of special stains. Acid-fast stains (including modified the Ziehl-Neelsen and auramine O stains) or fluorescent dyes coupled to monoclonal antibodies (for the immunofluorescent-antibody test [IFAT]) are used in the standard assays used in clinical laboratories. The sensitivity of stool examination by acid-fast staining remains poor and requires an oocyst concentration of over 500,000 per ml in formed stools (23), with fewer cases being detected by acid-fast staining than by fluorescent methods (1, 12). IFAT has been reported to be up to 10 times more sensitive than acid-fast staining (5, 7, 10). Currently, the IFAT staining method is the “gold standard” for the microscopic examination of infected stool samples (2).Microscopy-based methods are increasingly being replaced with techniques that rely on molecular recognition for the specific targeting of the pathogens of interest. The enzyme-linked immunosorbent assay (13) and PCR (16) techniques have further improved the sensitivity and specificity of detection compared to those obtained by microscopic examination. However, these newer methods have limited applicability to point-of-care use or low-resource settings due to reagent and instrumentation costs, infrastructure needs, and the need for operator training. Rapid diagnostic tests for Cryptosporidium parasites, such as lateral-flow assays (8), have not been proven to be reliable alternatives to the existing methods.Recently, a simple colorimetric detection method that relies on the distance-dependent optical properties of oligonucleotide-functionalized gold nanoparticles has been described (6). This strategy offers a simple readout (a color change that can be visually monitored) and utilizes low-cost and robust reagents that have promising potential for point-of-care molecular diagnostics. Previous work has demonstrated that the color change associated with the target-induced formation of oligonucleotide-gold aggregates can be effectively monitored by spotting the solution on a glass slide illuminated via total internal reflection (20). The limit of detection of this approach for the detection of purified DNA from methicillin (meticillin)-resistant Staphylococcus aureus was previously shown to be <10⁵ bacteria (20).In the study described here, we demonstrate the detection of Cryptosporidium parvum oocysts using oligonucleotide-functionalized gold nanoparticles targeted against heat shock protein 70 (HSP70) DNA and RNA and this novel detection strategy. One advantage of targeting HSP70 is the ability to induce mRNA amplification by the use of a simple heating process that does not involve more complicated PCR-based amplification. We hypothesize that simple amplification (via the heat shock process) of targets present at very low copies (low zeptomole concentrations) in biological samples will facilitate the detection of the desired targets that are present at levels within the limit of detection of the gold aggregation assay.     

热休克蛋白70对离体心脏心肌间质的保护作用

目的探讨热休克蛋白70（HSP70）对大鼠离体心脏心肌间质的影响。方法Wistar大鼠16只，分为2组：对照组（C，n=8），腹腔注射生理盐水0．4ml，24h后取离体心脏灌注HTK心脏保护液，4℃保存3h后建立Langendorff灌注模型，灌注KH液2h；实验组（E，n=8），腹腔注射重酒石酸去甲肾上腺素，24h后取离体心脏，方法同对照组。以心肌细胞中HSP70含量、血流动力学指标、心肌组织羟脯氨酸（HP）、内皮索（ET）含量和心肌超微结构等作为观察指标。结果HSP70含量E组与C组比较明显增高；E组心功能恢复方面优于C组（P〈0．05），HP含量优于C组（P〈0．01），ET含量低于C组（P〈0．01），心肌超微结构损伤较C组明显减轻。结论HSP 70对供心心肌间质具有保护作用。     

Limited Role of Secreted Aspartyl Proteinases Sap1 to Sap6 in Candida albicans Virulence and Host Immune Response in Murine Hematogenously Disseminated Candidiasis

Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap-null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a Δsap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans-infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.The polymorphic yeast Candida albicans is an important opportunistic human pathogen causing infections that range from superficial mucosal lesions to life-threatening systemic disease. It is by far the most common cause of fungal invasive infections, which could be attributed to the little immunosuppression required to predispose an individual to invasive Candida infections (39). Host physical barriers and immune system integrity are crucial factors in controlling the establishment of infection. However, the high adaptability of C. albicans to different host niches, by the expression of appropriate sets of virulence-related genes, is also a determinant (19, 51). Several of these virulence attributes may participate in and influence the infective process, depending on the site and stage of invasion and on the nature of the host response (37). The secretion of hydrolytic enzymes during infection is required as a virulence attribute to aid adhesion, invasion, and the destruction of host immune factors, in addition to nutrient acquisition (21). Among these enzymes, secreted aspartyl proteinases (Sap), encoded by a 10-member gene family (SAP1 to SAP10) have been the most extensively studied (35). The 10 SAP genes that compose this family can be divided into subfamilies based on amino acid sequence homology alignments (SAP1 to SAP3, SAP4 to SAP6, SAP9, and SAP10). These genes exhibit differential expression profiles at different stages and sites of infection (33, 35, 46, 49) and have been linked with the virulence of the fungus since their discovery (10, 27, 48).The contribution of the SAP1 to SAP3, SAP4 to SAP6, SAP7, and SAP9 and SAP10 genes to virulence in different models of infection has been studied by using sap-null mutant strains (1, 13, 16, 20, 23, 25, 26, 29, 34, 43, 54). The subfamily consisting of the SAP4 to SAP6 genes, in particular, was shown to contribute significantly to C. albicans virulence in models of acute systemic candidiasis, murine peritonitis, and Candida gastrointestinal infection (16, 25, 43). These genes are expressed mainly during hypha formation (22, 34), and SAP5 in particular was found to be upregulated at all time points after either intravenous (i.v.) or intraperitoneal (i.p.) infection of mice (44, 50, 57).Hube et al. (20) previously reported that Δsap1, Δsap2, and Δsap3 null mutants displayed attenuated virulence in models of acute systemic candidiasis. The triple deletion of SAP4 to SAP6 resulted in a more marked impact on C. albicans virulence in similar experimental settings, suggesting an important role for these hypha-related genes in the establishment of disseminated candidiasis (43). These mutant strains were generated from auxotrophic laboratory strain CAI4 with the most common method used for disrupting genes in C. albicans, the Ura-blaster technique (17). The use of the URA3 marker for mutant construction in C. albicans can lead to a misinterpretation of the results in mutant virulence studies (5, 8, 11, 28). Although this can now be overcome by the integration of URA3 at the ENO1 (52) or RPS10 (8) locus, the mutant strains used in earlier studies did not share a site of URA3 integration. Therefore, it is conceivable that the Ura status could have influenced the results, and thus, the attenuated nature of these mutants during acute systemic candidiasis remains to be confirmed unequivocally (38). Therefore, in this study, we have used a set of Δsap null mutants constructed, as described previously by Lermann and Morschhäuser (29), from prototrophic wild-type (WT) strain SC5314 using the SAT1-flipping strategy (41) to readdress the importance of the SAP1 to SAP6 genes in a murine model of hematogenously disseminated candidiasis. In addition, we analyzed the histopathology of several organs and aspects of the immune response elicited in the spleens and kidneys of BALB/c mice infected with the wild-type strain and a sap-null mutant lacking SAP4 to SAP6.     

热休克蛋白70在大鼠心肌缺血再灌注过程中的表达及其意义

目的 :探讨热休克蛋白 70 (HSP70 )在心肌缺血再灌注损伤中的表达及其意义。方法 :结扎 /松解SD大鼠左冠状动脉前降支 ,建立大鼠缺血 0min、3 0min ,再灌注 60min、12 0min动物模型。免疫组化技术和图像分析技术检测心肌细胞内HSP70、Bcl 2 /Bax基因的蛋白表达 ,硫代巴比妥酸 (thiobarbituricacid ,TBA)法测心肌组织丙二醛 (MDA)含量 ,优化紫外分光光度法测血清肌酸激酶 (CK)活性。结果 :( 1)HSP70于缺血 0min即有表达 ,再灌注 60min达高峰 ,之后下降。 ( 2 )Bcl 2蛋白再灌注组表达较缺血组明显降低 (P <0 .0 5 )。( 3 )Bax蛋白表达、MDA含量、CK活性于缺血 3 0min开始升高直至再灌注 12 0min。结论 :HSP70的异常表达与心肌缺血再灌注损伤的发生有关 ,且可能是缺血再灌注损伤细胞发展、恶化的重要标志。     

Heat Shock Protein HSP70 Increases the Resistance of Cortical Cells to Glutamate Excitotoxicity

Preincubation of cultured slices of the olfactory cortex of rat brain with heat shock protein in a concentration of 1 μg/ml protected the pre- and postsynaptic mechanisms of glutamatergic synaptic transmission from glutamate excitotoxicity (50 mM) inducing blockade of excitatory postsynaptic function and reducing presynaptic processes. It was hypothesized that heat shock protein protects AMPA and NMDA receptor-mediated processes. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 7, pp. 4–8, July, 2005     

Effectiveness of a Vaccine Composed of Heat-Killed Candida albicans and a Novel Mucosal Adjuvant, LT(R192G), against Systemic Candidiasis

The incidence of fungal infections caused by the opportunistic yeast Candida albicans has increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenic Escherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 × 10⁷ HK-CA per dose, alone or in conjunction with 10 μg of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 10⁴, 10⁵, or 10⁶ viable C. albicans to assess protection as measured by survival and/or culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 10⁴ C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels of C. albicans compared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 10⁵ live C. albicans as well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killed C. albicans in conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.     

HSP90以及IL-6在系统型红斑狼疮发病中的作用

目的研究系统型红斑狼疮(systemiclupuserythematosus,SLE)患者外周血单个核细胞(PBMC)中热休克蛋白90(HSP90)、血清中白细胞介素6(IL6)的水平,探讨与活动期SLE的相关性。方法应用Western印迹检测20例SLE活动期患者(A组)与20例正常人(B组)PBMC中HSP90的表达,并利用酶联免疫法(ELISA)检测其血清中IL6水平。结果①A组HSP90表达水平为0.47±0.05,B组为0.18±0.07,差异有统计学意义(P<0.01);②血清中IL6水平在A组[(32.97±8.65)pg/ml]显著高于B组[(10.46±4.33)pg/ml](P<0.05);③A组血清中IL6水平与PBMC中HSP90表达呈正相关(r=0.61,P<0.01),且与SLE疾病活动指数(SLEDAI)正相关(r=0.48,P<0.01)。结论IL6可作为病情活动监测指标,IL6与HSP90的异常高表达可能在SLE发病机制中起重要作用。