Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Maturation signatures of conventional dendritic cell subtypes in COVID-19 suggest direct viral sensing.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, which negatively affects T-cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T-cell responses limit disease severity. Understanding how cDCs cope with SARS-CoV-2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures, with the upregulation of IFN-stimulated genes and IL-6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti-apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS-CoV-2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T-cell activation.

Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

Diagnostic Criteria for Commotio Cordis Caused by Violent Attack: Review of the Literature.

The diagnosis of lethal commotio cordis (CC) is really complex. The forensic pathologist's task is even more relevant when he/she has to explain a CC diagnosis caused by an assault in a trial. The purpose of this study was to analyze the literature on lethal CC as a result of violent attacks and identify relevant parameters that may help in the diagnosis. A review of the relevant articles was performed. Fifty-two cases of CC caused by violent attacks were identified. The collected data allowed to confirm the following literature's criteria for CC diagnosis in case of assaults: witnessed occurrence of a blunt, nonpenetrating blow to the chest preceding cardiovascular collapse; absence of structural damage to the sternum, ribs, or heart itself; and absence of any underlying cardiovascular abnormalities (such as other causes of sudden death). Regarding the assessment of the third criterion, the authors suggest that the pathologist should always specify the scientific autopsy guidelines that he/she used to differentiate CC from the other causes of sudden death. In addition, the authors highlight the importance of a multidisciplinary approach for a correct interpretation of clinical, autopsy, and laboratory findings.

An Intestinal Th17 Subset is Associated with Inflammation in Crohn's Disease and Activated by Adherent-invasive Escherichia coli.

IFNγ-producing ex-Th17 cells ['Th1/17'] were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterised a novel, potentially colitogenic subset of Th17 cells in the intestine of patients with Crohn's disease [CD]. Human Th17 cells expressing CCR5 ['pTh17'] co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17 cells. pTh17 cells, but not Th1/17 cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5[-]Th17 cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17 cells in the intestine. Importantly, intestinal pTh17 cells were selectively activated by adherent-invasive Escherichia coli [AIEC], but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from dendritic cells [DC]. Intestinal CCR5+Th1/17 cells responded instead to cytomegalovirus and were reduced in ulcerative colitis [UC], suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17 cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role.

Immunosuppressant Treatment in Rheumatic Musculoskeletal Diseases Does Not Inhibit Elicitation of Humoral Response to SARS-CoV-2 Infection and Preserves Effector Immune Cell Populations.

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections.

Persistence of SARS-CoV-2 RNA in post-mortem swab 35 days after death: A case report.

Post-mortem swabs for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA detection have been recommended by several Scientific Committees and Institutions as a standard procedure for post-mortem assessment of potential Coronavirus Disease-19 (COVID-19) related deaths. To date there is no data about the SARS-CoV-2 RNA detectability period in human bodies after death. The present case documents the persistence of SARS-CoV-2 RNA in the upper respiratory tract 35-days after death. Post-mortem swabs could be used as a valuable tool in preventive evaluation of the risks-benefits ratio associated with autopsy execution. SARS-CoV-2 RNA post-mortem detection could have a key diagnostic role in deaths lacking medical assistance, unattended deaths, and patients with multiple comorbidities. Based on the present report, staged post-mortem swabs should be performed even after a long post-mortem interval.

Integrated longitudinal immunophenotypic, transcriptional and repertoire analyses delineate immune responses in COVID-19 patients.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

Self-harm Risk Among Adolescents and the Phenomenon of the "Blue Whale Challenge": Case Series and Review of the Literature.

The "Blue Whale Challenge" is a dangerous Internet phenomenon. As per media reports, it involves a series of life-threatening tasks imposed by a "curator" to "players," who must fulfill the whole list, and it ends with the suicide of the player. The authors report the data of five suspected cases of "Blue Whales" managed from January 2016 to December 2017 by the staff of a unit (the "Bambi Unit" of the Pediatric Hospital "Regina Margherita" of Turin, Italy) that is dedicated to the evaluation of suspected abused children. Then, they analyzed this data in the light of the literature regarding self-harm. This comparison highlights the role of the Internet in the spreading of self-harm behavior among vulnerable adolescents who are characterized by epidemiological, psychological, psychiatric, social, and cultural risk factors. In conclusion, the authors suggest a multidisciplinary and specialized approach in the evaluation of adolescents who committed self-harm activities.

Juvenile Facial Growth and Mimicry: A Preliminary 3D Study.

Studies focused on facial development during childhood have been conducted by means of 3D technology to provide modifications of anthropometric parameters. Facial mobility was also considered. This study proposed a 3D approach to facial growth changes. Facial surface data of 6 subjects were acquired in T1 (age 7-14 years) and after 7 years (T2), in rest position, and during voluntary movements, by a 3D laser scanner. Linear and angular measurements on rest position scans at T1 and T2 were compared. Each mimic scan was superimposed with the corresponding rest scan. Displacement of significant anthropometric points was measured for each facial gesture and at T1 and T2 statistically compared. Vertical measurements were those most influenced by aging. Some measurements of central facial area were consistent over time. The pattern of soft tissues displacement for each expression was consistent in T1 and T2. These results may be helpful for missing children identification.

Child Sexual Abuse Perpetrated by Women: Case Series and Review of the Literature.

The literature on child sexual abuse (CSA) perpetrated by female sexual offenders (FSOs) is exiguous, and many studies have focused on judicial databases. The present retrospective study, instead, analyzed clinical and judicial data of a group of both victims and alleged FSOs, to additionally include women who have not been convicted by the criminal justice system, but who hold strong clinical suspicions of being perpetrators of CSA. The medical records and the Court files of 11 children and their eight suspected FSOs have been collected and critically reviewed in light of the literature to date. This approach allowed for a deeper understanding of the relationship between child and FSO. The authors hypothesize that the victims' severe psychopathological outcomes were a result of a failure to develop appropriate attachments with their prospective caregivers, which could have been damaged by the pathological relationship with FSOs, who were the victims' caregivers.

Staged crime scene determination by handling physical and digital evidence: Reports and review of the literature.

Geberth in 2006 stated that "staging is a conscious criminal action on the part of an offender to thwart an investigation." In the present paper two crime scenes staged by handling digital evidence are reported. The first case involves a 50-year-old woman who had been living with the offender for three years before he murdered her at the end of their relationship. He staged the scene as a sex-related crime committed by an unknown perpetrator. The second case concerns a young woman who was found dead in Southern Italy in January 2004 with a gunshot on the forehead. The boyfriend, responsible for the murder, had staged the crime scene as a suicide. Three years earlier in Germany, he had also murdered the victim's mother. In both cases, the correlation of physical and digital forensic evidence was crucial in the definition of the manner of death.

Facial Mobility after Maxilla-Mandibular Advancement in Patients with Severe Obstructive Sleep Apnea Syndrome: A Three-Dimensional Study.

Introduction. The functional results of surgery in terms of facial mobility are key elements in the treatment of patients. Little is actually known about changes in facial mobility following surgical treatment with maxillomandibular advancement (MMA). Objectives. The three-dimensional (3D) methods study of basic facial movements in typical OSAS patients treated with MMA was the topic of the present research. Materials and Methods. Ten patients affected by severe obstructive sleep apnea syndrome (OSAS) were engaged for the study. Their facial surface data was acquired using a 3D laser scanner one week before (T1) and 12 months after (T2) orthognathic surgery. The facial movements were frowning, grimace, smiling, and lip purse. They were described in terms of surface and landmark displacements (mm). The mean landmark displacement was calculated for right and left sides of the face, at T1 and at T2. Results. One year after surgery, facial movements were similar to presurgical registrations. No modifications of symmetry were present. Conclusions. Despite the skeletal maxilla-mandible expansion, orthognathic surgical treatment (MMA) of OSAS patients does not seem to modify facial mobility. Only an enhancement of amplitude in smiling and knitting brows was observed. These results could have reliable medical and surgical applications.

De novo transcriptome profiling of highly purified human lymphocytes primary cells.

To help better understand the role of long noncoding RNAs in the human immune system, we recently generated a comprehensive RNA-seq data set using 63 RNA samples from 13 subsets of T (CD4(+) naive, CD4(+) TH1, CD4(+) TH2, CD4(+) TH17, CD4(+) Treg, CD4(+) TCM, CD4(+) TEM, CD8(+) TCM, CD8(+) TEM, CD8(+) naive) and B (B naive, B memory, B CD5(+)) lymphocytes. There were five biological replicates for each subset except for CD8(+) TCM and B CD5(+) populations that included 4 replicates. RNA-Seq data were generated by an Illumina HiScanSQ sequencer using the TruSeq v3 Cluster kit. 2.192 billion of paired-ends reads, 2×100 bp, were sequenced and after filtering a total of about 1.7 billion reads were mapped. Using different de novo transcriptome reconstruction techniques over 500 previously unknown lincRNAs were identified. The current data set could be exploited to drive the functional characterization of lincRNAs, identify novel genes and regulatory networks associated with specific cells subsets of the human immune system.

Identification of new hematopoietic cell subsets with a polyclonal antibody library specific for neglected proteins.

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.