Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway

Background Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Objective Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. Methods We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. Results We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Conclusions and Clinical Relevance Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen-and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Molecular diversity of Primula apennina and phylogeny of Primula Subsection Euauricula

Primula apennina Widmer is endemic to the North Apennines (Italy). ISSR were used to detect the genetic diversity within and among six populations representative of the species distribution range. High levels of genetic diversity were revealed both at population (PPB = 75.92%, HS = 0.204, Hpop = 0.319) and at species level (PPB = 96.95%, HT = 0.242, Hsp = 0.381). Nei gene diversity statistics (15.7%), Shannon diversity index (16.3%) and AMOVA (14%) detected a moderate level of interpopulation diversity. Principal coordinate and bayesian analyses clustered the populations in three major groups along a geographic gradient. The correlation between genetic and geographic distances was positive (Mantel test, r = 0.232). All together, these analyses revealed a weak but significant spatial genetic structure in P. apennina, with gene flow acting as a homogenizing force that prevents a stronger differentiation of populations. Conservation measures are suggested based on the observed pattern of genetic variability. P. apennina belongs to Primula subsect. Euauricula which includes 15 species distributed on the whole Alps and Apennines. A phylogenetic analysis was carried out using AFLP markers in order both to clarify the relationships among the species of subsection Euauricula that remained unresolved in previous works and to make some hypoteses on their evolutive dynamics. NJ, PCO and BAPS analyses strongly confirmed the monophyly of P. subsect. Euauricula and all the species form strongly supported clades. NJ tree topology suggested a simultaneous fragmentations of ancestral species in a large number of isolated populations that survived in refugia along the unglaciated margins of the Alps in response to the Pleistocene climatic oscillations.

Untersuchung der Modulation adaptiver Immunantworten in Abhängigkeit der Reifung dendritischer Zellen

Im ersten Teil dieser Arbeit wurde die Bildung von DALIS während der Reifung von dendritischen Zellen untersucht. Dabei konnte festgestellt werden, dass die DALIS-Bildung mit dem Aktivierungszustand der Zelle eng verknüpft ist. Es konnten verschiedene Hitzeschockproteine in Zusammenhang mit der DaLIS-Bildung gebracht werden und ein Molekül der Siganlgebung identifiziert werden. Im zweiten Teil der Arbeit wurde die Veränderung von dendritischen Zellen in Anwesenheit von regulatorischen T-Zellen untersucht. Hierbei zeigte sich, dass zwischen den Zellen Gap Junctions entstehen, durch die cAMP von den regulatorischen T-Zellen auf die dendritischen Zellen übertragen werden kann. Weiter konnte gezeigt werden, dass regulatorische T-Zellen IL-10 sezernieren und so die dendritischen Zellen inhibieren.

Non-thermal emission in High Frequency Peaked blazars towards the Square Kilometer Array era

In this thesis work we will explore and discuss the properties of the gamma-ray sources included in the first Fermi-LAT catalog of sources above 10 GeV (1FHL), by considering both blazars and the non negligible fraction of still unassociated gamma-ray sources (UGS, 13%). We perform a statistical analysis of a complete sample of hard gamma-ray sources, included in the 1FHL catalog, mostly composed of HSP blazars, and we present new VLBI observations of the faintest members of the sample. The new VLBI data, complemented by an extensive search of the archives for brighter sources, are essential to gather a sample as large as possible for the assessment of the significance of the correlation between radio and very high energy (E>100 GeV) emission bands. After the characterization of the statistical properties of HSP blazars and UGS, we use a complementary approach, by focusing on an intensive multi-frequency observing VLBI and gamma-ray campaign carried out for one of the most remarkable and closest HSP blazar Markarian 421.

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

Proteomic changes in the rat brain induced by homogenous irradiation and by the bystander effect resulting from high energy synchrotron X-ray microbeams

Abstract Purpose: To further evaluate the use of microbeam irradiation (MBI) as a potential means of non-invasive brain tumor treatment by investigating the induction of a bystander effect in non-irradiated tissue. Methods: Adult rats were irradiated with 35 or 350 Gy at the European Synchotron Research Facility (ESRF), using homogenous (broad beam) irradiation (HI) or a high energy microbeam delivered to the right brain hemisphere only. The proteome of the frontal lobes were then analyzed using two-dimensional electrophoresis (2-DE) and mass spectrometry. Results: HI resulted in proteomic responses indicative of tumourigenesis; increased albumin, aconitase and triosphosphate isomerase (TPI), and decreased dihydrolipoyldehydrogenase (DLD). The MBI bystander effect proteomic changes were indicative of reactive oxygen species mediated apoptosis; reduced TPI, prohibitin and tubulin and increased glial fibrillary acidic protein (GFAP). These potentially anti-tumourigenic apoptotic proteomic changes are also associated with neurodegeneration. However the bystander effect also increased heat shock protein (HSP) 71 turnover. HSP 71 is known to protect against all of the neurological disorders characterized by the bystander effect proteome changes. Conclusions: These results indicate that the collective interaction of these MBI-induced bystander effect proteins and their mediation by HSP 71, may confer a protective effect which now warrants additional experimental attention.

Brain atrophy in pure and complicated hereditary spastic paraparesis: a quantitative 3D MRI study

Hereditary spastic paraparesis (HSP) is a heterogeneous group of neurodegenerative disorders with progressive lower limb spasticity, categorized into pure (p-HSP) and complicated forms (c-HSP). The purpose of this study was to evaluate if brain volumes in HSP were altered compared with a control population. Brain volumes were determined in patients suffering from HSP, including both p-HSP (n = 21) and c-HSP type (n = 12), and 30 age-matched healthy controls, using brain parenchymal fractions (BPF) calculated from 3D MRI data in an observer-independent procedure. In addition, the tissue segments of grey and white matter were analysed separately. In HSP patients, BPF were significantly reduced compared with controls both for the whole patient group (P < 0.001) and for both subgroups, indicating considerable brain atrophy. In contrast to controls who showed a decline of brain volumes with age, this physiological phenomenon was less pronounced in HSP. Therefore, global brain parenchyma reduction, involving both grey and white matter, seems to be a feature in both subtypes of HSP. Atrophy was more pronounced in c-HSP, consistent with the more severe phenotype including extramotor involvement. Thus, global brain atrophy, detected by MRI-based brain volume quantification, is a biological marker in HSP subtypes.

Proteomic alterations in heat shock protein 27 and identification of phosphoproteins in ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve

Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.

Diffusion tensor imaging of two unrelated Chinese men with hereditary spastic paraplegia associated with thin corpus callosum

Hereditary spastic paraplegia (HSP) associated with thin corpus callosum is a rare autosomal recessive neurodegenerative disorder characterized by an abnormally thin corpus callosum, normal motor development, slowly progressive spastic paraparesis and cognitive deterioration. To investigate and localize abnormalities in the brains of two Chinese patients with HSP-TCC, with mutations in the spatacsin gene. Diffusion tensor imaging (DTI) was used to determine the mean diffusion (MD) and fractional anisotropy (FA) in the brains of the patients in comparison to 20 healthy subjects. Voxel-based analysis (VBA) of both the diffusion and anisotropy values were performed using statistical parametric mapping (SPM). Significant changes with MD increase and FA reduction were found in the already known lesions including the corpus callosum, cerebellum and thalamus. In addition, changes were also found in regions that appear to be normal in conventional MRI, such as the brain stem, internal capsule, cingulum and subcortical white matter including superior longitudinal fascicle and inferior longitudinal fascicle. Neither increase in FA nor reduction in MD was detected in the brain. Our study provides clear in vivo MR imaging evidence of a more widespread brain involvement of HSP-TCC. MD is more sensitive than FA in detecting lesions in thalamus and subcortical white matter, suggesting that MD may be a better marker of the disease progression.

Immunohistochemical evaluation of heat shock protein expression in normal canine nerve and peripheral nerve sheath tumours

Abnormal expression of heat shock proteins (HSPs) has been observed in many human neoplasms and such expression has prognostic, predictive and therapeutic implications. The aim of this study was to evaluate immunohistochemically the expression of HSP 27, HSP 32 and HSP 90 in normal canine peripheral nerves and in four benign and 15 malignant canine peripheral nerve sheath tumours (PNSTs). In normal nerve, all of the HSPs were detected in axons, epineurial fibroblasts and scattered Schwann cell bodies. Cytoplasmic expression of HSP 27 was more widespread and intense in benign PNSTs compared with malignant PNSTs (P <0.05). Widespread and intense nuclear expression of HSP 32 was also associated with benign tumours (P <0.01), while high HSP 90 immunoreactivity was detected in all tumours, suggesting that HSP 90 might represent a new therapeutic target.

Association between HSP90 and Her2 in gastric and gastroesophageal carcinomas

BACKGROUND Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas. MATERIAL AND METHODS Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters (UICC pTNM category, tumor grading) and survival. RESULTS Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis (p=0.02; p=0.004; p=0.802). Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases. CONCLUSION Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment.

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Effect of combined drought and heat stress on heat shock proteins in wheat varieties

Introduction: The re sponse of crop plants ex posed on drought or heat shock is related to de crease in the synthesis of normal proteins, accompanied by increased translation of heat shock proteins (HSPs). Though drought and heat stress have been studied individually, little is known about their combined effect on plants. Methods: The wheat (Triticum aestivum L.) varieties (Katya-tolerant, Sadovo or Mladka-susceptible) were potted in soil. Eight-day-old plants were ex posed to with drawing water for seven days. Heat shock was realized in growth chamber at 40 °C for 6h. A combination of drought and heat shock was per formed by subjecting drought-stressed plants to heat shock treatment. Expression of HSPs in the first leaf of wheat varieties was analyzed by SDS electrophoresis and immunoblotting. Polyclonal antibodies against HSP20, HSP60, HSP110 and mononclonal antibodies against HSP70 were used to distinguish the mentioned HSPs. Results: The leaf relative water content (RWC), which indicated the level of plant dehydration decreased significantly (34 %) under drought stressed conditions The electrolyte leakage of ions (EL), representing the level of the cell membrane stability in creased mark edly (68 %), especially under combination of drought and heat. Maximum EL was ob served in drought susceptible varieties Sadovo and Mladka. Drought and heat shock combination in the wheat plants resulted in the induction of specific HSPs. Conclusions: Our results demonstrate that the response of the wheat plants to a combination of drought and heat stress is different from the response of plants to each of these stresses applied separately. Induction of synergetic effect on HSP expression in case of combination between drought and heat was discussed in the case of two contrasting wheat varieties.

Increased expression of heat shock protein 90 in keratinocytes and mast cells in patients with psoriasis.

BACKGROUND Psoriasis is a chronic inflammatory skin disease and various stress factors mediate inflammation. Heat shock protein (HSP) 90 plays an important role in cell survival; cytokine signaling, such as interleukin-17 receptor signaling; and immune responses. OBJECTIVE We sought to elucidate protein expression and distribution of HSP90 in psoriasis. METHODS HSP90 expression and its cellular source were analyzed on normal-appearing, nonlesional, lesional, and ustekinumab-treated psoriatic skin using immunohistochemistry and double immunofluorescence. RESULTS HSP90α, the inducible isoform of HSP90, was significantly up-regulated in epidermal keratinocytes and mast cells of lesional skin and down-regulated after ustekinumab therapy. LIMITATIONS There was a limited sample size. CONCLUSIONS HSP90 from keratinocytes and mast cells is a key regulator of psoriatic inflammation and HSP90 inhibitors may represent a novel therapeutic approach to the disease.