Epitope Context and Reshaping of Activated T Helper Cell Repertoire

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST—peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor Vβ usage and fine specificities of selected T cells.     

Neurotrophin-like immunoreactivity in the human pre-term newborn, infant, and adult cerebellum

The immunohistochemical occurrence of the neurotrophin (NT) proteins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3) is shown in the pre-term newborn, infant, and adult human post-mortem cerebellum. The NT-like immunoreactive structures were unevenly distributed and showed regional differences among cerebellar lobules and folia. NGF-, NT-4-, and NT-3-positive neuronal perikarya were observed in all specimens examined. At variance with the other neurotrophins, the BDNF antiserum labelled neuronal cell bodies only in newborn life and infancy, as well as extensive nerve fibre systems, whose density increased with age. The NT-antibodies, tested by Western blot on human cerebellum homogenates, revealed immunoreactive bands corresponding to proteins of heterogenous molecular weight. The results obtained provide a first demonstration of the tissue localization of the NTs in the human cerebellum from perinatal to adult age, thus suggesting their involvement in the development, differentiation and maintenance of the cerebellar connectivity. Codistribution of the four NTs or sets of them was observed in cortical and deep nuclei neurons. Multiple trophic roles for NTs, encompassing the classic target-derived and local mechanisms of support, are envisaged as significant in development, differentiation, and maintenance of the human cerebellar connectivity.     

Investigations on the Amidine System in Amidinobenzisothiazole Derivatives

The amino/imino equilibrium is studied in free bases and hydrochloride salts of amidinobenzisothiazole derivatives by IR and ¹H-NMR spectroscopy. The X-ray crystallographic analysis of two of these compounds shows that the free base exists as amino form whereas the hydrochloride as imino form.     

A comprehensive in vitro characterization of pancreatic ductal carcinoma cell line biological behavior and its correlation with the structural and genetic profile

There are a large number of stable pancreatic ductal carcinoma cell lines (PDCL) that are used by researchers worldwide. Detailed data about their differentiation status and genetic alterations are present in the literature, but a systematic correlation with cell biological behavior is often lacking. PDCL (n=12) were clustered by source of tumor cell (ascites, primary tumor, metastasis), and the data of functional cell biology were correlated with the reported structural and genetic profiles. Major histocompatibility complex expression, chemosensitivity and aneuploidia appeared to be related to the source of PDCL, and proliferative capacity appeared to be related to the grade of differentiation. No correlation between genetic/structural features of PDCL and biological behavior was found. All the cell lines appeared generally insensitive to in vitro treatment with 5-fluorouracil and showed variable degrees of susceptibility to gemcitabine, raltitrexed and oxaliplatin. All the PDCL showed resistance to Fas-mediated apoptosis but were significantly sensitive to the pro-apoptotic effect of inflammatory cytokines [interleukin (IL)-1, tumor necrosis factor (TNF) and interferon ]. PDCL were characterized for the secretion of several factors relevant to the tumor-immune cross talk. Vascular endothelial growth factor, CCL2, CCL5 and transforming growth factor were the factors most frequently released; less frequent was the secretion of CXCL8, CCL22, IL-6 and sporadically CXCL12, IL-10 and hepatocyte growth factor. The cytokines IL-1 and TNF were always undetectable. In conclusion, a clear correlation between structural/genetic features and function could not be detected, suggesting the weakness of a morphological classification for the in vitro studies of pancreatic cancer.     

Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/ micro l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.     

Early phylogenetic estimate of the effective reproduction number of SARS-CoV-2

To reconstruct the evolutionary dynamics of the 2019 novel-coronavirus recently causing an outbreak in Wuhan, China, 52 SARS-CoV-2 genomes available on 4 February 2020 at Global Initiative on Sharing All Influenza Data were analyzed. The two models used to estimate the reproduction number (coalescent-based exponential growth and a birth-death skyline method) indicated an estimated mean evolutionary rate of 7.8 × 10⁻⁴ subs/site/year (range, 1.1 × 10⁻⁴-15 × 10⁻⁴) and a mean tMRCA of the tree root of 73 days. The estimated R value was 2.6 (range, 2.1-5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing.     

Juvenile sudden death in a family with polymorphic ventricular arrhythmias caused by a novel RyR2 gene mutation: evidence of specific morphological substrates

We report on a family with a history of sudden death and effort-induced polymorphic ventricular arrhythmias. The index case was a 17-year-old boy who died suddenly and at postmortem had evidence of fibrofatty replacement in the right ventricular free wall, consistent with arrhythmogenic right ventricular cardiomyopathy, as well as calcium phosphate deposits within the myocytes. A molecular genetics investigation carried out in the paraffin-embedded myocardium of the subject and in blood samples of family members disclosed a missense mutation in exon 3 (230C-->T; A77V) of the cardiac ryanodine receptor type 2 gene. The carriers showed effort-induced polymorphic ventricular tachycardia in the setting of normal resting electrocardiogram and trivial echocardiographic abnormalities, consistent with catecholaminergic polymorphic ventricular tachycardia. The observation of both arrhythmogenic right ventricular cardiomyopathy type 2 and catecholaminergic polymorphic ventricular tachycardia in the same family suggests that the two entities might correspond to different degrees of phenotypic expression of the same disease. This experience underscores the importance of a precise autopsy diagnosis in the case of sudden cardiac death, including molecular genetics, and the mission of pathologists to guide further clinical investigation of family members.