Effects of intracellular and extracellular heat shock proteins on anti-tumor immune responses

The goal of this study was to investigate the function of the heat shock protein 70 family members, expressed in tumors under physiological and stress conditions and to dissect their role in tumor immune recognition as a function of intra- versus extracellular location. Another goal was to investigate whether heat-treatment at clinically relevant thermal doses affects the immunophenotype of a given tumor, as defined by tumor cell sensitivity to immune effector cells. For these questions, the human melanoma system was selected because it is well characterized with regards to tumor-associated antigens, like tyrosinase and Melan-A/MART-1, their epitopes and restriction elements for MHC class I presentation. In the first part of the study the focus specifically was on the time-temperature dependent effects of heat exposure. Two different thermal doses (41,8°C/120 minutes and 45°C/22 minutes) were selected that mimic the heterogeneity of the achieved temperature distribution within the tumor and the time-temperature dependent changes were determined in: a) antigen expression (tyrosinase and Melan-A/MART-1) at the protein and mRNA level; b) expression of the inducible HSP70 and the constitutive HSC70; c) processing and presentation of tyrosinase and MART-1 via MHC class I; d) susceptibility of melanoma cell lines to cytotoxic T lymphocytes like CD8+ T cells, LAK and NK cells. It was demonstrated that HSP70 and antigen expression display distinct expression and kinetics that reflect the thermohistory of the cells, i.e. exposure to high or low thermal doses. Immunologically, a low thermal dose did not alter immune recognition of the cells despite the fact that intracellular HSP70 and tyrosinase protein were upregulated. High thermal dose induced a pleiotropy of effects, including stronger upregulation of HSP70 and tyrosinase protein but downregulation of tyrosinase at mRNA level. Concordant with reduced HLA-A2 surface expression and tyrosinase mRNA levels, immune recognition of the heat-treated cells was initially reduced, but pretreatment levels were restored after 72 hours of recovery. The observation that tumor cells treated with temperatures below the breakpoint temperature maintain an immunological homeostasis during the heat shock response is of critical importance for the clinical application of hyperthermia in the treatment of tumors. In the second part of the study, the ability of HSP70 to cross-present a naturally expressed human tumor antigen, tyrosinase, that is of low immunigenicity, a situation that more closely resembles the patient situation was investigated. It was demonstrated that HSP70-peptide complexes (HSP70-PC) purified from tyrosinase-positive (HSP70-PC/tyr+) but not from tyrosinase-negative (HSP70-PC/tyr-) melanoma cells deliver the tyrosinase antigen to immature DCs for MHC class I restricted T cell recognition. T cell stimulation by HSP70-PC/tyr+ incubated with immature DCs with was very efficient even without additional DC maturation signals (e.g. exogenous TNF-?) demonstrating the ability of tumor-derived HSP70-PC to act as a chaperone for peptides and a signal for DC maturation. HSP70-PC in exerting both functions on DCs, delivering antigens and maturing DCs, ensures that the peptides that are delivered to the DCs are presented in an immunogenic context optimal for T cell stimulation. In conlusion, induction of intracellular heat shock proteins (HSPs) by heat does not interfere with the tumor immune recognition and when HSPs are expressed extracellularly they acquire immunostimulatory properties. These observations open new perspectives for the application of hyperthermia in combination with HSP-based vaccine in the treatment of solid tumors

Transcription Pattern of Neurotrophic Factors and Their Receptors in Adult Zebrafish Spinal Cord

In vertebrates, neurotrophins and their receptors play a fundamental role in the central and peripheral nervous systems. Several studies reported that each neurotrophin/receptor signalling pathway can perform various functions during axon development, neuronal growth, and plasticity. Previous investigations in some fish species have identified neurotrophins and their receptors in the spinal cord under physiological conditions and after injuries, highlighting their potential role during regeneration. In our study, for the first time, we used an excellent animal model, the zebrafish (Danio rerio), to compare the mRNA localization patterns of neurotrophins and receptors in the spinal cord. We quantified the levels of mRNA using qPCR, and identified the transcription pattern of each neurotrophin/receptor pathway via in situ hybridization. Our data show that ngf/trka are the most transcribed members in the adult zebrafish spinal cord

Differential nickel-induced responses of olfactory sensory neuron populations in zebrafish

The olfactory epithelium of fish includes three main types of olfactory sensory neurons (OSNs). Whereas ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs) are common to all vertebrates, a third, smaller group, the crypt cells, is exclusive for fish. Dissolved pollutants reach OSNs, thus resulting in impairment of the olfactory function with possible neurobehavioral damages, and nickel represents a diffuse olfactory toxicant. We studied the effects of three sublethal Ni2+ concentrations on the different OSN populations of zebrafish that is a widely used biological model. We applied image analysis with cell count and quantification of histochemically-detected markers of the different types of OSNs. The present study shows clear evidence of a differential responses of OSN populations to treatments. Densitometric values for G\u3b1 olf, a marker of cOSNs, decreased compared to control and showed a concentration-dependent effect in the ventral half of the olfactory rosette. The densitometric analysis of TRPC2, a marker of mOSNs, revealed a statistically significant reduction compared to control, smaller than the decrease for G\u3b1 olf and without concentration-dependent effects. After exposure, olfactory epithelium stained with anti-calretinin, a marker of c- and mOSNs, revealed a decrease in thickness while the sensory area appeared unchanged. The thickness reduction together with increased densitometric values for HuC/D, a marker of mature and immature neurons, suggests that the decrements in G\u3b1 olf and TRPC2 immunostaining may depend on cell death. However, reductions in the number of apical processes and of antigen expression could be a further explanation. We hypothesize that cOSNs are more sensitive than mOSNs to Ni2+ exposure. Difference between subpopulations of OSNs or differences in water flux throughout the olfactory cavity could account for the greater susceptibility of the OSNs located in the ventral half of the olfactory rosette. Cell count of anti-TrkA immunopositive cells reveals that Ni2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram

Analysis of clasp2 Transcription Pattern in Male Germ Cells during Spermatogenesis: A Comparative Study in Zebrafish (Danio rerio) and Guppy (Poecilia reticulata)

Cytoplasmic linker-associated protein-2 (CLASP2) is a member of the CLIP-associating proteins (CLASPs) family involved in the structure and function of microtubules and Golgi apparatus. Several studies performed using different mammalian and non-mammalian model organisms reported that CLASP2 controls microtubule dynamics and the organization of microtubule networks. In Drosophila and mice, an important role of CLASP2 during the development of germ cell lines has been uncovered. However, no study has clearly defined its role during fish germ cell differentiation. In the present study, we used two excellent aquatic animal models among teleost fish: zebrafish (Danio rerio) and guppy (Poecilia reticulata). Using qPCR, we found that the clasp2 transcript level is significantly high in the testis of both fish. Then, by in situ hybridization, we localized the clasp2 transcript in the spermatozoa of zebrafish and the spermatozeugmata of guppy. Our data suggest a potential role for this gene in the last stage of spermiogenesis in fis

A Novel t(8;14)(q24;q11) Rearranged Human Cell Line as a Model for Mechanistic and Drug Discovery Studies of NOTCH1-Independent Human T-Cell Leukemia

MYC-translocated T-lineage acute lymphoblastic leukemia (T-ALL) is a rare subgroup of T-ALL associated with CDKN2A/B deletions, PTEN inactivation, and absence of NOTCH1 or FBXW7 mutations. This subtype of T-ALL has been associated with induction failure and aggressive disease. Identification of drug targets and mechanistic insights for this disease are still limited. Here, we established a human NOTCH1-independent MYC-translocated T-ALL cell line that maintains the genetic and phenotypic characteristics of the parental leukemic clone at diagnosis. The University of Padua T-cell acute lymphoblastic leukemia 13 (UP-ALL13) cell line has all the main features of the above described MYC-translocated T-ALL. Interestingly, UP-ALL13 was found to harbor a heterozygous R882H DNMT3A mutation typically found in myeloid leukemia. Chromatin immunoprecipitation coupled with high-throughput sequencing for histone H3 lysine 27 (H3K27) acetylation revealed numerous putative super-enhancers near key transcription factors, including MYC, MYB, and LEF1. Marked cytotoxicity was found following bromodomain-containing protein 4 (BRD4) inhibition with AZD5153, suggesting a strict dependency of this particular subtype of T-ALL on the activity of super-enhancers. Altogether, this cell line may be a useful model system for dissecting the signaling pathways implicated in NOTCH1-independent T-ALL and for the screening of targeted anti-leukemia agents specific for this T-ALL subgroup

Raman spectroscopy as a tool to investigate the structure and electronic properties of carbon-atom wires

Graphene, nanotubes and other carbon nanostructures have shown potential as candidates for advanced technological applications due to the different coordination of carbon atoms and to the possibility of π-conjugation. In this context, atomic-scale wires comprised of sp-hybridized carbon atoms represent ideal 1D systems to potentially downscale devices to the atomic level. Carbon-atom wires (CAWs) can be arranged in two possible structures: a sequence of double bonds (cumulenes), resulting in a 1D metal, or an alternating sequence of single–triple bonds (polyynes), expected to show semiconducting properties. The electronic and optical properties of CAWs can be finely tuned by controlling the wire length (i.e., the number of carbon atoms) and the type of termination (e.g., atom, molecular group or nanostructure). Although linear, sp-hybridized carbon systems are still considered elusive and unstable materials, a number of nanostructures consisting of sp-carbon wires have been produced and characterized to date. In this short review, we present the main CAW synthesis techniques and stabilization strategies and we discuss the current status of the understanding of their structural, electronic and vibrational properties with particular attention to how these properties are related to one another. We focus on the use of vibrational spectroscopy to provide information on the structural and electronic properties of the system (e.g., determination of wire length). Moreover, by employing Raman spectroscopy and surface enhanced Raman scattering in combination with the support of first principles calculations, we show that a detailed understanding of the charge transfer between CAWs and metal nanoparticles may open the possibility to tune the electronic structure from alternating to equalized bonds

Characteristics of patients affecting the duration of positivity at SARS-CoV-2: a cohort analysis of the first wave of epidemic in Italy

OBJECTIVES: to investigate the characteristics of patients affecting the duration of positivity test by RT-PCR in the population of Piedmont, a Region of North-West of Italy.DESIGN: observational cohort study.SETTING AND PARTICIPANTS: from the administrative database of the regional SARS-CoV-2 surveillance system, a cohort of all patients who tested positive by a RT-PCR assay to SARS-CoV-2 occurring from 22.02.2020 to 30.09.2020 in the Piedmont Region (N. 29,292) was obtained. The cohort has been linked to the hospital discharge database and to the vital statistics database.MAIN OUTCOMES MEASURES: outcome of the study was the risk of non negativization, estimated by fitting Generalizing Estimating Equation model (GEE), a longitudinal model which consider for each subject several records collected on fixed time intervals 15, 30, 45 or 60+ days from the first positive test. Negativization was defined as the condition in which two consecutive samples taken from the patient at least 24 hours apart were negative for the presence of SARS-CoV-2.RESULTS: the median duration of positive RT-PCR was 27 days. A higher median of days until positive persistence was observed in people over 80 (34 days, IQR 25-49), female (28 days, IQR 18-40), symptomatic patients (28 days, IQR 1940), hospitalized people (32 days, IQR 21-44), patients with Charlson's index >0 (34 days, IQR 23-49), patients host of elderly nursing homes (37 days, IQR 25-51). In the GEE multivariable model, the variables associated to the non negativization at all times intervals were: older age (at 15th day: class 65+, OR 2.56, 95%CI 2.39-2.74), female gender (at 15th day: OR 1.12, 95%CI 1.06-1.18), and to be hospitalized for COVID-19 (at 15th day: OR 1.38, 95%CI 1.29-1.48). The presence of comorbidities and of symptoms were associate with the non negativization at 15th day (respectively, class 4+: OR 1.29, 95%CI 1.08-1.56 and symptoms: OR 1.20, 95%CI 1.13-1.27), but not at 45th day.CONCLUSIONS: older age, female gender, presence of comorbidities and severity of disease (proxy hospitalization for COVID-19) were risk factors for non negativization at all times intervals. The presence of symptoms was a risk factors for the non negativization after 2 weeks from the first diagnosis and not at 45th day. Using a longitudinal model for the analysis of the dataset, it is possible to compare the weight of the variables included in the model at different times and correct an overestimation of the attributable risk after the first considered time interval

Evaluation of vestibular function in patients affected by obstructive sleep apnea performing functional head impulse test (fHIT)

Purpose: Obstructive sleep apnoea (OSA) is a common disease with significantly related complications. Since a connection between the vestibular nucleus and sleep regulator pathways has been demonstrated, vestibular evaluation in OSA patients was partially studied and none used functional head impulse test (fHIT) for this purpose. This paper aimed at evaluating the vestibular function in patients affected by OSA using fHIT, selecting patients who did not present any other related to cardiovascular, neurological, or metabolic diseases. Patients and Methods: Patients enrolled had a diagnosis of OSA by polysomnography type III and were cataloged according to American Association of Sleep Medicine criteria. Each patient underwent fHIT. Statistical significance was set at 0.05. Results: A total of 85 patients were enrolled in the study of which 50 had a diagnosis of OSA and were included in the case group, while 35 belonged to the control group. In 88.6% of subjects of the case group was evidenced a vestibular impairment with a substantial difference between the two study groups (p<0.05). Conclusion: The results show that the incidence of vestibular lesions in patients with obstructive sleep apnoea is underestimated and that fHIT can identify these lesions early