Potential Immunotherapy for Prostate Cancer: Recombinant Bacille Calmette-Guerin Expressing Prostate Specific Molecules

Purpose: The emergence of prostate cancer as a major health issue and the absence of curative treatment for metastatic disease requires the development of new treatment modalities. Prostate specific antigen (PSA) and prostate-specific membrane antigen (PSMA) are possible targets for prostate cancer immunotherapy. We have previously shown that PSA and PSMA can be expressed in recombinant bacille Calmette-Guérin (BCG) strains.Methods: The in vivo immunogenicity of the prostate-specific proteins produced by this recombinant BCG strain were examined by detection of specific antibody responses and delayed-type hypersensitivity (DTH) responses in mice vaccinated with these strains. These immune responses were compared with those of control mice vaccinated with phosphate buffered saline diluent or soluble PSA or PSMA.Results: Mice vaccinated with rBCG-PSA developed low levels of anti-PSA antibodies and strong DTH to PSA.  Mice vaccinated with rBCG-PSMA developed strong DTH to PSMA and no anti-PSMA antibodies. Conclusions: We conclude that recombinant BCG expressing PSA or PSMA induce strong cellular immune responses to these antigens. We propose that the innate adjuvant capacity of BCG could help stimulate a specific immune response against prostate-specific proteins produced by the bacteria, which in turn, could lead to the eradication of undetected metastatic prostate cancer cells in post-surgical patients

Improving diagnosis for rare diseases: the experience of the Italian undiagnosed Rare diseases network

Background For a number of persons with rare diseases (RDs) a definite diagnosis remains undiscovered with relevant physical, psychological and social consequences. Undiagnosed RDs (URDs) require other than specialised clinical centres, outstanding molecular investigations, common protocols and dedicated actions at national and international levels; thus, many "Undiagnosed RDs programs" have been gradually developed on the grounds of a well-structured multidisciplinary approach. Methods The Italian Undiagnosed Rare Diseases Network (IURDN) was established in 2016 to improve the level of diagnosis of persons with URD living in Italy. Six Italian Centres of Expertise represented the network. The National Centre for Rare Diseases at the Istituto Superiore di Sanita coordinates the whole project. The software PhenoTips was used to collect the information of the clinical cases. Results One hundred and ten cases were analysed between March 2016 and June 2019. The age of onset of the diseases ranged from prenatal age to 51 years. Conditions were predominantly sporadic; almost all patients had multiple organs involvements. A total of 13/71 family cases were characterized by WES; in some families more than one individual was affected, so leading to 20/71 individuals investigated. Disease causing variants were identified in two cases and were associated to previously undescribed phenotypes. In 5 cases, new candidate genes were identified, although confirmatory tests are pending. In three families, investigations were not completed due to the scarce compliance of members and molecular investigations were temporary suspended. Finally, three cases (one familial) remain still unsolved. Twelve undiagnosed clinical cases were then selected to be shared at International level through PhenomeCentral in accordance to the UDNI statement. Conclusions Our results showed a molecular diagnostic yield of 53,8%; this value is comparable to the diagnostic rates reported in other international studies. Cases collected were also pooled with those collected by UDNI International Network. This represents a unique example of global initiative aimed at sharing and validating knowledge and experience in this field. IURDN is a multidisciplinary and useful initiative linking National and International efforts aimed at making timely and appropriate diagnoses in RD patients who still do not have a confirmed diagnosis even after a long time

PET/CT Imaging of c-Myc Transgenic Mice Identifies the Genotoxic N-Nitroso-Diethylamine as Carcinogen in a Short-Term Cancer Bioassay

Background: More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay. Methodology/Principal Findings: mCT and 18 F-FDG mPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced mCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis. Conclusions/Significance: The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgeni

Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs

RD-Connect, NeurOmics and EURenOmics: collaborative European initiative for rare diseases

Functional Genomics of Muscle, Nerve and Brain Disorder

C4 nephritic factor in patients with immune-complex-mediated membranoproliferative glomerulonephritis and C3-glomerulopathy

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Discordant phenotype in monozygotic twins with renal coloboma syndrome and a PAX2 mutation

Background: Renal coloboma syndrome (RCS) is a highly variable syndrome characterized by renal and ocular abnormalities. It is associated in about 50 % of cases with mutations of PAX2, a gene encoding a transcription factor required during development. Case-Diagnosis/Treatment: The case study involves two monozygotic twin sisters with RCS showing highly discordant phenotypes. Twin 1 was antenatally diagnosed with multiple cysts in the right kidney. She had complicated vacuum-assisted delivery with acute renal failure. She developed proteinuria at age 4 years, followed by a progressive rise in serum creatinine requiring renal replacement therapy at age 22. No ocular abnormalities have been detected. Twin 2 experienced rapidly reversible acute renal failure without renal morphological abnormalities at birth. At age 2 years, complete visual acuity loss of the left eye secondary to an optic disc coloboma was diagnosed. No significant events occurred until the age of 20, when clinical proteinuria was detected. Proteinuria remission was obtained by multidrug treatment. In both patients, a novel de novo mutation of PAX2 was detected, which leads to the substitution of a highly conserved cysteine (p.C52Y). Conclusions: The patients described provide an extreme example of clinical variability in RCS. The role of environmental, genetic, and epigenetic factors is discussed

Glutamate AMPA receptor subunit 1 gene (GRIA1) and DSM-IV-TR schizophrenia: a pilot case-control association study in an Italian sample

Glutamatergic dysfunction is one of the major hypotheses for the pathogenesis of schizophrenia. The GRIA1 gene encodes for one (GluR1) of the four (GluR1–4) ionotropic AMPA receptor subunits. GRIA1 is a good candidate gene for susceptibility to schizophrenia since it maps in 5q33, a region where the presence of susceptibility loci has been suggested by independent genome-wide scans and because its expression has been found to be decreased in the brain of someschizophrenia patients. We present data from a case-control association study on the Italian population with eight polymorphisms spanning the whole GRIA1 gene. Single-locus analysis revealed a significantly different allele distribution in cases and in controls of two SNPs (rs707176, 0.41 vs. 0.31, P¼0.009; rs2963944, 0.41 vs. 0.30, P¼0.007), and one microsatellite (rs10631988, allele 9: 0.40 vs. 0.29, P¼0.004). Haplotype analysis showed an increased frequency of a specific haplotype for these markers (C09CC, 0.39 vs. 0.28, P¼0.009). Therefore our data indicate that GRIA1 may be involved in susceptibility to DSM-IV-TR schizophrenia

Association study and mutational screening of SYNGR1 as a candidate susceptibility gene for schizophrenia

Synaptogyrin 1 (SYNGR1) is a transmembrane protein of neurotransmitter-containing vesicle. Recently, suggestive association between SYNGR1 intragenic polymorphisms and schizophrenia has been reported in the Indian population. Furthermore, some rare nucleotide changes with a potential pathogenic effect have been found in Indian and Chinese schizophrenia patients. In this study, we have performed an association study and a resequencing analysis in an Italian sample. METHODS: Eight polymorphisms of the SYNGR1 gene were typed in a case-control sample consisting of 274 patients and 335 controls. In parallel, a mutational screening covering all SYNGR1 exons was conducted. RESULTS: Evidence of association has been found for rs715505 (P = 0.028), a marker already reported to be associated with the disease. Resequencing analysis revealed two novel polymorphisms and several rare variants (13 of 16 as new variants), some of which might have relevance for gene expression and function. CONCLUSION: The results of our association study support a contribution of SYNGR1 to schizophrenia susceptibility. In addition, the resequencing analysis evidenced mutations with a potential functional role at the mRNA and/or protein level. Of particular interest is the p.isoc:S26G missense mutation identified in six patients (0.011) and three controls (0.004) which might be involved in the elimination of a potential protein kinase C phosphorylation site