kD Heat Shock Protein Research Articles

Extracellular 70-kd heat shock protein in mid-trimester amniotic fluid and its effect on cytokine production by ex vivo–cultured amniotic fluid cells

The 70-kd heat shock protein is released from cells in response to stress and functions as a regulator of innate immunity. We hypothesized that 70-kd heat shock protein in mid-trimester amniotic fluid might regulate local immune system activation. Amniotic fluid that was obtained from 200 women who underwent amniocentesis at 15 to 19 weeks of gestation was tested by enzyme-linked immunosorbent assay for 70-kd heat shock protein, tumor necrosis factor-alpha, and interleukin-1beta and -6. The amniotic fluid cellular fraction also was evaluated for Mycoplasma hominis by gene amplification. Whole amniotic fluids were incubated ex vivo in medium alone or medium that contained peptidoglycan, a TLR2 ligand, or lipopolysaccharide, a TLR4 ligand. After 24 hours, the supernatants were collected and assayed for 70-kd heat shock protein. The influence of exogenous 70-kd heat shock protein on tumor necrosis factor-alpha and interleukin-1beta and -6 production by whole amniotic fluid was assessed similarly. The 70-kd heat shock protein was detected in all amniotic fluids with a median (range) of 11.5 ng/mL (1.2-76.7). The intra-amniotic 70-kd heat shock protein concentration was correlated positively only with amniotic fluid tumor necrosis factor-alpha levels (P = .0002). Detection of M hominis was associated with an increased 70-kd heat shock protein concentration (median, 17.2 ng/mL; P = .01). The addition of peptidoglycan resulted in a stimulation of 70-kd heat shock protein production, and exogenous 70-kd heat shock protein stimulated the release of tumor necrosis factor-alpha by amniotic fluid cells. The 70-kd heat shock protein is released from cells in mid-trimester amniotic fluid as a consequence of TLR2 stimulation and potentiates tumor necrosis factor-alpha production.

Levels of HSP70 in HIV+ patients in different viroimmunological states

The aim of the study was to establish an association between the viroimmunological status of HIV positive patients and their levels of the 70-kD heat shock protein (Hsp70). The longitudinal retrospective case study involved 61 patients at the University Hospital in Granada (Spain) from 1999 to 2002. Twenty-five patients were viroimmunologically stable, while the other 36 patients had suffered virological failure. A minimum of three blood samples were taken at intervals of at least 3 months for the patients who were stable virologically and immunologically, whereas four samples were taken for the virological failure group: two previous to the onset of virological failure, a third corresponding to the time of virological failure, and a fourth at least 3 months after remission of virological failure. Blood samples were also obtained from 20 healthy control subjects; Hsp70 levels in all were determined by enzyme immunoassay. The mean concentration of Hsp70 was 145.4 ng/ml in the HIV-infected patients as opposed to 72.1 ng/ml in the controls. While the viroimmunologically stable group showed levels similar to those of the controls (66.5 ng/ml), the mean value of Hsp70 in the virological failure group was nearly four times as high (249.1 ng/ml), yet this difference was not statistically significant. The patients treated with reverse transcriptase inhibitors were found to have significantly higher levels of Hsp70 than the other subjects. The transformed variable Hsp70/CD4(+) presents less variability than the Hsp70 value itself, giving a higher degree of statistical significance, and may be considered a useful parameter for diagnostic, prognostic, and therapeutic management of HIV positive patients.

Identification of Mycobacterium marinum 65 kD heat shock protein gene by polymerase chain reaction restriction analysis from lesions of swimming pool granuloma

Nontuberculous mycobacterium (NTM) had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum (M. marinum) are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction (PCR) was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein (hsp) 65 kD gene. The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis (PRA) identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.

Mycobacterial 70 kD heat-shock protein is an effective subunit vaccine against bovine paratuberculosis

Paratuberculosis is a chronic granulomatous inflammation of the small intestine of cattle and other ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). The disease can be found in ruminant herds worldwide, causing substantial economic losses at farm level due to premature culling and production losses. In previous studies, it has been shown that immune responses to recombinant MAP Hsp70 proteins were predominantly cell mediated. As protective immunity to the intracellular mycobacterial pathogens is thought to be cell-mediated in origin, we have studied the use of a recombinant MAP Hsp70 as a subunit vaccine in cattle experimentally infected with MAP. The results of the current study demonstrate that recombinant MAP Hsp70 can be successfully used as a subunit vaccine against bovine paratuberculosis, significantly reducing shedding of bacteria in feces during the first 2 years following experimental infection.

Epithelial cells and adipose cells both have their own temporal profile in 72-kd heat-shock protein expression determining their tolerance for ischaemia

Seventy-two-kd heat-shock protein (HSP72) is one of the stress markers induced in cells under stress, such as in the case of ischaemia. Recent studies have suggested that HSP72 is a 'molecular chaperone' to protect cells from various kinds of stress, and that the temporal profile of HSP72 induction is related to ischaemic vulnerability. In this study, we attempted to analyse the temporal profiles of HSP72 induction in epithelial and adipose cells in skin flaps after various periods of transient ischaemia, and we investigated the reason why there were differences in ischaemic tolerance between these cells. We used the abdominal skin flap of Wister rats, which were divided into three groups: the sham control group (n=27), the 2-h ischaemia group (n=25), and the 8-h ischaemia group (n=25). At periods of 8, 24, 48, 96 h, and 7 days after reperfusion, we examined them for any histological changes and performed immunostaining for HSP72 (n=5, each time point). Two animals in the sham control group were sacrificed to harvest the samples immediately after the skin flaps were elevated. As a result, the epithelial cells in all groups revealed positive for HSP72 through the time course, regardless of the ischaemic stresses, and they were alive at 7 days. In the adipose cells, the cells in the sham control group revealed no immunoreactivity after the reperfusion, and they had no change at 7 days. In the 2-h ischaemia group, the adipose cells gradually increased the reactivity for HSP72; consequently they survived beyond 7 days. In the 8-h ischaemia group, the reactivity for HSP72 gradually decreased; consequently they played out a delayed cell death at 7 days. We concluded that these differences of HSP72 expression were related to the cellular vulnerability to ischaemia.

Upregulation of HSP-72 in a Human Renal Allograft during Reflow

Many types of renal injury, which are of potential relevance in transplantation medicine (such as extreme temperatures, ischemia, cytotoxic agents and cyclosporine), are known to induce the cellular stress response. Recently, we reported the first data on the 72-kD heat shock protein (HSP-72), a key player of the mammalian stress response, in a large number of nonperfused human donor kidneys (1). HSP-72 expression at both protein and mRNA levels remained low in the allografts at the time of engraftment regardless of pretransplant insults and posttransplant outcome. These data disqualified HSP-72 as a marker for allograft outcome in the clinical setting and suggested that constitutive HSP-72 expression does not play a role in graft protection. Herein, we report the findings on HSP-72 expression in a 10.5-year-old boy whose renal allograft was biopsied during early posttransplant reperfusion. His primary disease was chronic glomerulonephritis; the transplantation was performed after 12 months of peritoneal dialysis. The 52-year-old donor had died because of a cerebral insult. The creatinine at the time of explantation was 0.6 mg/dl, the donor was on low doses of pressors, cold ischemic time was 16 hours, and the mismatch was 2-2-0. The immunosuppressive regimen consisted of cyclosporine A, steroids, and mycophenolate mofetil. A routine donor kidney biopsy was performed immediately before engraftment, and a clinically indicated repeat biopsy was performed at 18 hours thereafter because of unexplained primary nonfunction. Immunohistochemical analysis of HSP-72 expression before and after engraftment is shown in Fig. 1.FIGURE 1.: HSP-72 expression in pre-transplant biopsy (upper panel) versus early re-perfusion (lower panel).Pretransplant biopsy demonstrated only weak and focal HSP-72 expression among different tubular segments (and thus failed to predict the complicated course). In strict contrast, the allograft biopsy during early reperfusion exhibited obvious overexpression of HSP-72 and thus suggested HSP-mediated cellular processes severe enough to produce a relevant in vivo stress response in the renal allograft. HSP-72 expression in the nonperfused donor kidney might have been insufficiently induced until the engraftment, and/or might have been prevented or even degraded during cold storage, despite significant expression at the time of harvest (2). At present, material from nonperfused organs constitutes the basis for most studies on posttransplant risk assessment. Data like ours should fuel discussions on strategies of early posttransplant biopsy procurement to improve clinical applicability of biomarkers. More importantly, this case provides the first direct evidence for the involvement of HSP-72 mediated cellular repair mechanisms in human renal allografts during reperfusion. These data corroborate a broad body of experimental evidence and support the potential of HSP-72 as a cytoprotective tool (3). Based on the recent finding of low pretransplant HSP-72 expression in the majority of donor kidneys, therapeutic interventions such as specific ex-vivo treatment of organs during cold storage fostering overexpression (and/or preventing degradation) of HSP-72 can be considered as an attractive concept in human transplant medicine. Regina Vargha Department of Pediatrics Medical University of Vienna Vienna, Austria Heinz Regele Department of Clinical Pathology Medical University of Vienna Vienna, Austria Christoph Aufrecht Department of Pediatrics Medical University of Vienna Vienna, Austria

The expression of the 27-kd heat shock protein in keratinization disorders: an immunohistological study

In human skin, the 27-kd heat shock protein (hsp27), a member of the small hsp family, is expressed mainly in the upper epidermal layers. Hsp27 functions as a molecular chaperone and is involved in the regulation of cell growth and differentiation. According to experimental evidence, epidermal hsp27, through its chaperone function, might play a role in the assembly of keratin filaments and the cornified cell envelope. This study was conducted to assess the expression pattern of hsp27 in a panel of different ichthyoses. Twelve hereditary and acquired skin diseases associated with an ichthyotic phenotype and 2 corresponding mouse models were investigated by immunohistochemistry on formalin-fixed paraffin-embedded tissue, using a monoclonal antibody specific for hsp27. In ichthyosis vulgaris, lamellar ichthyosis, Sjögren-Larsson syndrome, Netherton syndrome, and acquired ichthyosiform skin condition, the pattern of hsp27 expression resembles healthy human skin. Hsp27 expression was reduced in bullous ichthyosiform erythroderma and annular epidermolytic ichthyosis, and absent in X-linked recessive ichthyosis (1/3 patients) and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome (1/1). In X-linked dominant chondrodysplasia, 3 small samples are completely negative and 2 larger samples show a pattern resembling random X inactivation. In the mouse models, tattered and bare patches, representing the murine analogues to X-linked dominant chondrodysplasia and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome, expression of hsp25 (the murine homologue of hsp27) also showed lyonization, demonstrating a clear-cut link between hsp27 expression and underlying molecular pathology. Our results show that loss of hsp27 is a rare event in human epidermis that is associated with specific genetic defects. Among the cases described here, these defects are either in suprabasal keratins or in enzymes involved in cholesterol biosynthesis. The expression and chaperone function of hsp27 might be modified by low/absent epidermal cholesterol and aberrant substrates (ie, keratins) resulting in protein misfolding, dyskeratosis, and thus contribute to the ichthyotic phenotype.

Elevated expression of the Brn‐3a and Brn‐3b transcription factors in systemic lupus erythematosus correlates with antibodies to Brn‐3 and overexpression of Hsp90

Important developmental and antiapoptotic roles have been described for the Brn-3 family of transcription factors in mammalian cells. Following a report of pathogenic autoantibody-inducing T cell reactivity to the Brn-3 transcription factors in murine lupus, we undertook this study to investigate serum levels of antibodies to Brn-3 and levels of expression of Brn-3 in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). Serum and PBMC samples were obtained from 87 SLE patients and 30 normal control subjects. Serum antibodies to the Brn-3a and Brn-3b transcription factors were measured by enzyme-linked immunosorbent assay. Levels of Brn-3a and Brn-3b messenger RNA (mRNA) in PBMCs were measured by reverse transcription and real-time quantitative polymerase chain reaction. Elevated serum levels of antibodies to Brn-3a and Brn-3b were found in 43% and 32%, respectively, of SLE patients. This elevation paralleled enhanced expression of Brn-3a and Brn-3b in PBMCs of 44% and 31%, respectively, of SLE patients. Furthermore, we observed a significant correlation (P = 0.002) between elevated levels of anti-Brn-3b antibodies and elevated levels of Brn-3b mRNA in individual patients. A preliminary analysis of possible target genes for Brn-3a and Brn-3b revealed a significant correlation (P = 0.01) between the level of Brn-3a mRNA and the level of Hsp90 protein (90-kd heat-shock protein, which is overexpressed in SLE) in PBMCs of SLE patients. In addition, we observed that overexpression of Brn-3a and Brn-3b in cultured cells enhanced expression of Hsp90 protein and transcription of Hsp90 promoter-reporter constructs. Finally, we observed an association between elevated levels of Brn-3a mRNA and active SLE (P = 0.002). Expression of both Brn-3a and Brn-3b was found to be enhanced in SLE, and this correlated with enhanced levels of autoantibodies to these proteins and with the previously reported overexpression of Hsp90, which was shown to be a novel gene regulated by Brn-3a and Brn-3b. The overexpression of Brn-3a correlated with active disease, suggesting that it may play a role in the disease process via its targeting by the immune system and its ability to induce the expression of specific genes.

Phase I and Pharmacologic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients With Solid Tumors

To determine the clinical toxicities of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) given as a 1-hour infusion daily for 5 days every 3 weeks. Nineteen patients received 17-AAG over six dose levels (10 to 56 mg/m(2)) using an accelerated titration scheme. Drug levels of 17-AAG were determined by high-performance liquid chromatography. Biologic effects of 17-AAG were monitored by changes in the content of target proteins by immunoblot analysis of lysates prepared from peripheral-blood mononuclear cells. Toxicity was acceptable at doses up to 28 mg/m(2). The cohort was expanded to three patients at 40 mg/m(2) because a second occurrence of grade 2 hepatic transaminitis occurred. Two of six assessable patients who received 56 mg/m(2) had reversible, grade 3 hepatic transaminitis. Five additional patients were enrolled at 40 mg/m(2); none had dose-limiting toxicity. The maximum plasma concentrations (C(max)) of 17-AAG at 40 and 56 mg/m(2) were 1,724 and 2,046 ng/mL, respectively; the average plasma exposures (AUC) were 2,809 and 6,708 hours.ng/mL, respectively. Less than 3% of the daily dose was excreted into the urine. Clearance did not correlate with body-surface area. Possible biologic activity was suggested by apparent increased protein content of either glucose-related 78 kd protein or heat shock protein 70 with >/= 14 mg/m(2) and decreased protein content of either Lck or Raf1 with >/= 28 mg/m(2) of 17-AAG. 17-AAG 40 mg/m(2) (median dose, 70 mg) was well tolerated when given daily for 5 days every 3 weeks.

Association between vaginal 70-kd heat shock protein, interleukin-1 receptor antagonist, and microbial flora in mid trimester pregnant women

This study investigated the association among the inducible 70-kd heat shock protein, cytokines, and microbial flora in the vagina in mid trimester pregnant women and subsequent preterm delivery. Vaginal samples from 205 pregnant women, which were collected at 18 to 22 weeks of gestation, were analyzed for qualitative and quantitative vaginal microflora and for 70-kd heat shock protein, interleukin-1beta, interleukin-1 receptor antagonist, and tumor necrosis factor-alpha by enzyme-linked immunosorbent assay. Pregnancy outcome data were obtained subsequently. The 70-kd heat shock protein was detected in 38 vaginal samples (18.5%). Its presence was associated with elevated vaginal pH, a diagnosis of bacterial vaginosis, and elevated interleukin-1 receptor antagonist levels (P < .001). Among women with bacterial vaginosis, 70-kd heat shock protein-positive subjects had a >80% increase in median vaginal concentration of interleukin-1 receptor antagonist (P < .05). Vaginal 70-kd heat shock protein expression is associated with the down-regulation of the proinflammatory immune response to abnormal vaginal flora in mid trimester pregnant women.

Alteration in Gene Expression Profile by Full-Length Hepatitis B Virus Genome

Persistent expression of hepatitis B virus (HBV) proteins is thought to be involved in virus-related hepatocarcinogenesis. Here, we compared the gene expression profile of cells persistently expressing the full-length HBV with that of negative control cells to comprehensively investigate virus-mediated changes in the gene expression of the host cells. RNA samples from both virus-expressing and negative control cells were used for the DNA array assay. DNA array assay and subsequent corroboration assays revealed that expression of 14 of 1,176 genes (1.2%) was altered in response to virus expression. The upregulated genes included CD44, high mobility group protein-I, thymosin beta-10 and 27-kD heat shock protein, while the downregulated genes included NM23-H1, all of which are thought to be associated with the development or progression of carcinoma in the liver or other organs. Furthermore, virus expression resulted in the decrease of two apoptosis-inducing molecules, caspase-3 and BAX, which may also contribute to carcinogenesis through prolonged survival of the host cell. Thus, expression of the virus genome caused carcinogenesis-related changes in host cell gene expression. HBV expression may change the host cell to a malignant phenotype through alterations in the expression levels of a set of genes.

Hypoxia Modulates Expression of the 70-kD Heat Shock Protein and Reduces Leishmania Infection in Macrophages

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O₂, 5% CO₂, and balanced N₂) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected with Leishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity.

The 70kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer

The 70 kD heat shock protein (HSP70) plays essential cellular roles in mediating intra-cellular protein folding and protecting cells from proteotoxic stress. This study has examined the role of HSP70 in the expression of apoptosis in prostate carcinoma cells. Apoptosis was negatively correlated with HSP70 expression in PC-3 cells heat shocked in vivo. Further experiments carried out on an in vitro reconstituted system with isolated nuclei and cytoplasm from PC-3 cells showed that purified HSP70 directly inhibits apoptosis in a dose-dependant manner. Therefore, the potential role of depletion of intracellular HSP70 was examined as a means of inducing apoptosis in PC-3 cancer cells. Depletion of HSP70 by two independent strategies, either with anti-sense oligonucleotides directed against HSP70 mRNA or with the bioflavinoid drug quercetin, led to apoptosis in the absence of stress. In addition, quercetin pre-treatment synergistically enhanced apoptosis in combination with heat shock. Thus, HSP70 plays a physiological role in tumour cells as an inhibitor of apoptosis occurring both spontaneously and after stress and is a potential target for apoptosis-based cancer therapy.

Hypoxia modulates expression of the 70-kD heat shock protein and reducesLeishmania infection in macrophages

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O2, 5% CO2, and balanced N2) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected with Leishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity.

Inhibition of adjuvant‐induced arthritis by DNA vaccination with the 70‐kd or the 90‐kd human heat‐shock protein: Immune cross‐regulation with the 60‐kd heat‐shock protein

Adjuvant arthritis can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt). The mycobacterial 65-kd heat-shock protein (Hsp65) is targeted by arthritogenic T cells. However, Hsp65 and the mycobacterial 71-kd heat-shock protein are also recognized by T cells that can down-regulate adjuvant-induced arthritis (AIA). We have recently demonstrated that vaccination with human Hsp60 DNA inhibits AIA. The present study was undertaken to analyze the role of the T cell responses to self HSP molecules other than Hsp60 in the control of AIA. Lewis rats were immunized with DNA vaccines coding for human Hsp70 or Hsp90 (Hsp70 plasmid [pHsp70] or pHsp90), and AIA was induced. The T cell response to Mt, Hsp60, Hsp70, and Hsp90 (proliferation and cytokine release) was studied, and the T cell response to Hsp60 was mapped with overlapping peptides. The Hsp70 or Hsp90 DNA vaccines shifted the arthritogenic T cell response from a Th1 to a Th2/3 phenotype and inhibited AIA. We detected immune crosstalk between Hsp70/90 and Hsp60: both the Hsp70 and Hsp90 DNA vaccines induced Hsp60-specific T cell responses. Similarly, DNA vaccination with Hsp60 induced Hsp70-specific T cell immunity. Epitope mapping studies revealed that Hsp60-specific T cells induced by pHsp70 vaccination reacted with known regulatory Hsp60 epitopes. T cell immunity to Hsp70 and to Hsp90, like Hsp60-specific immunity, can modulate the arthritogenic response in AIA. In addition, our results suggest that the regulatory mechanisms induced by Hsp60, Hsp70, and Hsp90 are reinforced by an immune network that connects their reactivities.

Low levels of antibodies against E. coli and mycobacterial 65kDa heat shock proteins in patients with inflammatory bowel disease

The aim of the present study was to support and extend our initial observation, where we found low levels of antibodies against mycobacterial 65kD heat shock proteins in patients with inflammatory bowel disease (IBD). For this purpose we tested a new group of 124 patients with IBD, and beside measuring antibodies to Mycobacterium bovis 65kD heat shock protein (Hsp65) and human 60kD heat shock protein (Hsp60) as described previously, we also determined IgG antibody levels to Hsp65 from E. coli, called GroEL. seventy-four patients with Crohn's disease (CD) (30 males, 44 females, 33 (27-45) years old, median (interquartile range)) and 50 patients with ulcerative colitis (UC) (22 males, 28 females, 38 (30-50) years old) were involved in the study. 110 healthy subjects (34 males, 76 females, 47 (37-53) years old) served as controls. Study subjects were consecutive patients referred to an IBD center for complex treatment of the disease. Methods and statistical analysis: The amounts of IgG-type antibodies reacting with proteins of the chaperonin 60 family were assessed by ELISA. Since the antibody levels to heat-shock proteins as variables were not normally distributed, non-parametric Mann-Whitney test and Dunn post hoc test were used for group comparisons. Median levels of anti-GroEL (7,5 (3,5-18,3)) and anti-Hsp65 (4,8 (2,1-7,85)) were significantly (GroEL p = 0,008; and Hsp65 p < 0,001) lower in the IBD patients than in the healthy subjects (GroEL: 10,0 (5,4-31,0); Hsp65: 7,04 (4,66-12,77)). However this difference was found to be restricted to the CD patients (GroEL: 7,5 (3,7-14,2); p < 0,05; Hsp65: 4,35 (1,90-6,94); p < 0,001). We did not find difference in the concentration of anti-human Hsp60 IgG levels between patients (Hsp60: 45,5 (24,9-69,0)) and healthy controls (38,4 (21,6-69,4). Regarding the serum concentrations of each antibody tested there was no significant difference between the active and inactive stage of disease. Our present findings support conclusion of our previous work, antibody levels not only for Mycobacterium bovis hsp65 but for E. coli GroEl were found to be decreased as well. In contrast no changes in the concentrations of human anti-hsp60 antibodies were observed. These findings indicate that production of antibodies to 65 kDa bacterial heat shock proteins is selectively impaired in IBD.

Polymorphisms in the tumor necrosis factor-α gene at position −308 and the inducible 70 kd heat shock protein gene at position +1267 in multifetal pregnancies and preterm premature rupture of fetal membranes

The purpose of this study was to determine the relationship between preterm premature rupture of membranes, tumor necrosis factor-alpha, and heat shock protein-70 gene polymorphisms in multifetal gestations. Buccal swabs from 101 mother-neonate pairs of multifetal pregnancies were tested for single nucleotide polymorphisms at position -308 of the tumor necrosis factor-alpha gene and +1267 of the heat shock protein-70 gene. Pregnancy outcome data were obtained subsequently. Tumor necrosis factor-alpha allele 2 carriage by the first-born occurred in 10 of 27 pregnancies (37.0%) that resulted in preterm premature rupture of membranes compared with 6 of 67 pregnancies (9.0%) without preterm premature rupture of membranes ( P = .002). The allele frequency of tumor necrosis factor-alpha allele 2 and heat shock protein-70 allele 2 in the first born was higher in pregnancies that were complicated by preterm premature rupture of membranes (18.5% vs 4.5%; P = .003; and 57.7% vs 41.3%; P = .04, respectively). There was no relationship between tumor necrosis factor-alpha allele 2 or heat shock protein-70 allele 2 carriage by the second fetus or mother and preterm premature rupture of membranes. Tumor necrosis factor-alpha allele 2 and/or heat shock protein-70 allele 2 carriage by the first-born fetus is associated with preterm premature rupture of membranes in multifetal pregnancies.

Interferon-alpha-inducible proteins are novel autoantigens in murine lupus.

To investigate the spectrum of B cell autoimmunity in the recently described anti-CD1-autoreactive T cell receptor (TCR)-transgenic murine lupus-like (CD1 lupus-like) model. Lethally irradiated BALB/c/nu/nu mice were injected intravenously with donor BALB/c bone marrow and spleen cells expressing TCRalpha and TCRbeta transgenes that recognize CD1d. Sera from adoptive host animals that developed lupus (i.e., CD1 lupus mice) were collected at serial time points and analyzed by Western blotting and immunoprecipitation, using protein extracts prepared from NIH3T3 mouse fibroblasts and EL-4 lymphocytes, respectively. Sera obtained from older animals in several models of spontaneous lupus (NZB/NZW, MRL++, and MRL/lpr mice), unmanipulated BALB/c/nu/nu mice, and normal BALB/c mice were used as controls. Analyses demonstrated that the prominent targets of autoantibodies in the CD1 lupus-like model are interferon-alpha (IFNalpha)-inducible antigens. Biochemical and serologic characterizations identified one antigen as belonging to the interferon-inducible 202 (Ifi202) subfamily of proteins within the Ifi200 family, and a second antigen as a member of the 70-kd heat-shock protein family. Autoantibodies directed against these antigens were rapidly produced at an early stage of disease. Anti-p50 autoantibodies were present in sera from 7 (78%) of 9 CD1 lupus mice that developed severe kidney disease. IFNalpha-inducible proteins represent a novel class of autoantigens in murine lupus, and the findings suggest additional roles for IFNalpha in this disease. Since Ifi202 autoantigens are encoded by the murine non-major histocompatibility complex lupus-susceptibility gene locus Ifi202, these data provide a link between recent advances in lupus genetics and the formation of autoantibodies.

Heat‐shock induced nuclear retention and recycling inhibition of importin α

Heat-shock induces a strong stress response and modifies all aspects of cellular physiology, which involves dynamic changes in the nucleocytoplasmic distributions of a variety of proteins. Many distinct nucleocytoplasmic transport pathways exist in eukaryotic cells, but how a particular transport pathway is regulated under different cellular conditions remains elusive. The finding of this study indicate that conventional nuclear import, which is mediated by importin alpha/beta, is down-regulated, while the nuclear import of 70 kD heat-shock cognate protein is up-regulated in heat-shock cells. Among the factors involved in the mediation of the conventional nuclear import, significant levels of importin alpha accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin alpha with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin alpha becomes less mobile and its nucleocytoplasmic recycling is impaired in heat-shock cells. These data coincided well with biochemical and cytological studies. Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin alpha, resulting in the suppression of conventional nuclear import. This suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock.

Myocyte protection by 10 kD heat shock protein (Hsp10) involves the mobile loop and attenuation of the Ras GTP-ase pathway.

Heat shock proteins (hsp), hsp60 and hsp10, are involved in the folding of imported mitochondrial proteins and the refolding of denatured proteins after stress. We examined whether hsp10 can reduce myocyte death by its mitochondrial function or by interacting with cytoplasmic signaling pathways. Overexpression of hsp10 by adenoviral infection decreased myocyte death induced by hydrogen peroxide, sodium cyanide, and simulated ischemia and reoxygenation (SI/RO). We generated an adenoviral vector coding for a temperature-sensitive mutant hsp10 protein (P34H), incapable of cooperatively refolding denatured malate dehydrogenase with hsp60. Overexpression of the hsp10 mutant potentiated SI/RO-induced myocyte death. Analysis of electron transport chain function revealed increased Complex I capacity with hsp10 overexpression, whereas hsp10(P34H) overexpression decreased Complex II capacity. Hsp10 overexpression preserved both Complex I and II function after SI/RO. Examination of the Ras GTP-ase signaling pathway indicated that inhibition of Ras was required for protection by hsp10. Constitutive activation of Ras abolished the effects afforded by hsp10 and hsp10(P34H). Hsp10 overexpression inactivated Raf, ERK, and p90Ribosomal kinase (p90RSK) before and after SI/RO. Our results suggest that complex mechanisms are involved in the protection by hsp10 against SI/RO-induced myocyte death. This mechanism may involve the hsp10 mobile loop and attenuation of the Ras GTP-ase signaling pathway.