The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8⁺ T Cells in Response to a Lethal Poxvirus Infection.

CD8(+) T cells and NK cells protect from viral infections by killing virally infected cells and secreting interferon-γ. Several inhibitory receptors limit the magnitude and duration of these anti-viral responses. NKG2A, which is encoded by Klrc1, is a lectin-like inhibitory receptor that is expressed as a heterodimer with CD94 on NK cells and activated CD8(+) T cells. Previous studies on the impact of CD94/NKG2A heterodimers on anti-viral responses have yielded contrasting results and the in vivo function of NKG2A remains unclear. Here, we generated Klrc1(-/-) mice and found that NKG2A is selectively required for resistance to ectromelia virus (ECTV). NKG2A functions intrinsically within ECTV-specific CD8(+) T cells to limit excessive activation, prevent apoptosis, and preserve the specific CD8(+) T cell response. Thus, although inhibitory receptors often cause T cell exhaustion and viral spreading during chronic viral infections, NKG2A optimizes CD8(+) T cell responses during an acute poxvirus infection.

Loss of function mutations in essential genes cause embryonic lethality in pigs.

Lethal recessive alleles cause pre- or postnatal death in homozygous affected individuals, reducing fertility. Especially in small size domestic and wild populations, those alleles might be exposed by inbreeding, caused by matings between related parents that inherited the same recessive lethal allele from a common ancestor. In this study we report five relatively common (up to 13.4% carrier frequency) recessive lethal haplotypes in two commercial pig populations. The lethal haplotypes have a large effect on carrier-by-carrier matings, decreasing litter sizes by 15.1 to 21.6%. The causal mutations are of different type including two splice-site variants (affecting POLR1B and TADA2A genes), one frameshift (URB1), and one missense (PNKP) variant, resulting in a complete loss-of-function of these essential genes. The recessive lethal alleles affect up to 2.9% of the litters within a single population and are responsible for the death of 0.52% of the total population of embryos. Moreover, we provide compelling evidence that the identified embryonic lethal alleles contribute to the observed heterosis effect for fertility (i.e. larger litters in crossbred offspring). Together, this work marks specific recessive lethal variation describing its functional consequences at the molecular, phenotypic, and population level, providing a unique model to better understand fertility and heterosis in livestock.

Screening for depression in cancer patients receiving radiotherapy: Feasibility and identification of effective tools in the NRG Oncology RTOG 0841 trial.

BACKGROUND: Brief tools are needed to screen oncology outpatients for depressive symptoms. METHODS: Patients starting radiotherapy for the first diagnosis of any tumor completed distress screening tools, including the 9-item Patient Health Questionnaire (PHQ-9), the 2-item Patient Health Questionnaire (PHQ-2), the National Comprehensive Cancer Network Distress Thermometer (NCCN-DT), and the Hopkins Symptom Checklist (HSCL) (25-item version). Patients exceeding validated cutoff scores and a systematic sample of patients whose screening was negative completed the Structured Clinical Interview for DSM-IV (SCID) mood disorder modules via telephone. RESULTS: Four hundred sixty-three patients from 35 community-based radiation oncology sites and 2 academic radiation oncology sites were recruited. Sixty-six percent of the 455 eligible patients (n = 299) were women, and the eligible patients had breast (45%), gastrointestinal (11%), lung (10%), gynecologic (6%), or other cancers (27%). Seventy-five (16.5%) exceeded screening cutoffs for depressive symptoms. Forty-two of these patients completed the SCID. Another 37 patients whose screening was negative completed the SCID. Among the 79 patients completing the SCID, 8 (10.1%) met the criteria for major depression, 2 (2.5%) met the criteria for dysthymia, and 6 (7.6%) met the criteria for an adjustment disorder. The PHQ-2 demonstrated good psychometric properties for screening for mood disorders with a cutoff score of ≥3 (receiver operating characteristic area under the curve [AUC], 0.83) and was comparable to the PHQ-9 ( > 9; AUC = 0.85). The NCCN-DT did not detect depression (AUC = 0.59). CONCLUSIONS: The PHQ-2 demonstrated good psychometric properties for screening for mood disorders, which were equivalent to the PHQ-9 and superior to the NCCN-DT. These findings support using the PHQ-2 to identify patients in need of further assessment for depression, which has a low prevalence but is a clinically significant comorbidity. These findings could inform the implementation of distress screening accreditation standards. Cancer 2017;123:485-493. © 2016 American Cancer Society.

Immune functions encoded by the natural killer gene complex.

There has been marked progress in our understanding of the role of natural killer (NK) cells in immune responses, mainly due to the identification of NK-cell receptors and their ligands. The genes encoding many NK-cell receptors are located in the NK-gene complex (NKC). Here, we review the properties of NKC-encoded receptors, and provide a genomic and conceptual framework for an insight into NK-cell function and biology.

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

[Benefit of skull vibration-induced nystagmus test in occupational medicine]. / Intérêt du test de vibration osseux vestibulaire en médecine du travail.

BACKGROUND: Nystagmus induced by vibrations (NIV), has been optimized by the present authors this last decade. The skull vibration-induced nystagmus test (SVINT) can be designated as a high-frequency global "vestibular Weber test" and can be considered as an office-based examination to detect vestibular asymmetry. The aim of this study is to define the tolerance of the SVINT as well as its comparison to the simplified caloric test of Veits (CTV) in normal workers during the pre-employment visit at the occupational medicine center. MATERIAL AND METHODS: The vestibular function has been evaluated by the SVINT and the CTV in 87 healthy workers. The tolerance of the two procedures has been evaluated by a 4-items questionnaire (nausea, vomiting, sweating, asthenia). RESULTS: The caloric test was normal in each worker. The SVINT was positive in one patient who had a partial unilateral vestibular dysfunction related to trauma. The mean duration of the procedure was 15 min for CTV and 1 min for SVINT. Side effects (nausea, vomiting, sweating, asthenia) were present in 50% of the workers following CTV and in only one patient after SVINT. The SVINT demonstrated significantly less side effects for each item (p < 0.0001). CONCLUSIONS: SVINT is a valid, rapid, low-cost clinical screening test and does not cause patient discomfort. It is suggested that this test which explores vestibular high frequencies and is not modified by vestibular compensation is useful for the diagnostic screening of workers' vestibular dysfunction, when combined with other vestibular tests and complements the CTV.

Expression of calpastatin hcast 3-25 and activity of the calpain/calpastatin system in human glioblastoma stem cells: possible involvement of hcast 3-25 in cell differentiation.

Glioblastoma (GBM) is the most malignant brain tumor, characterized by cell heterogeneity comprising stem cells (GSCs) responsible for aggressiveness. The calpain/calpastatin (calp/cast) proteolytic system is involved in critical physiological processes and cancer progression. In this work we showed the expression profile of hcast 3-25 (a Type III calpastatin variant devoid of inhibitory units) and the members of the system in several patient-derived GSCs exploring the relationship between hcast 3-25 and activation/activity of calpains. Each GSC shows a peculiar calp/cast mRNA and protein expression pattern, and hcast 3-25 is the least expressed. Differentiation promotes upregulation of all the calp/cast system components except hcast 3-25 mRNA, which increased or decreased depending on individual GSC culture. Transfection of hcast 3-25-V5 into two selected GSCs indicated that hcast 3-25 effectively associates with calpains, supporting the digestion of selected calpain targets. Hcast 3-25 possibly affects the stem state promoting a differentiated, less aggressive phenotype.

Chromatin targeting of the RNF12/RLIM E3 ubiquitin ligase controls transcriptional responses.

Protein ubiquitylation regulates key biological processes including transcription. This is exemplified by the E3 ubiquitin ligase RNF12/RLIM, which controls developmental gene expression by ubiquitylating the REX1 transcription factor and is mutated in an X-linked intellectual disability disorder. However, the precise mechanisms by which ubiquitylation drives specific transcriptional responses are not known. Here, we show that RNF12 is recruited to specific genomic locations via a consensus sequence motif, which enables co-localisation with REX1 substrate at gene promoters. Surprisingly, RNF12 chromatin recruitment is achieved via a non-catalytic basic region and comprises a previously unappreciated N-terminal autoinhibitory mechanism. Furthermore, RNF12 chromatin targeting is critical for REX1 ubiquitylation and downstream RNF12-dependent gene regulation. Our results demonstrate a key role for chromatin in regulation of the RNF12-REX1 axis and provide insight into mechanisms by which protein ubiquitylation enables programming of gene expression.

Occupational risk factors for sinonasal inverted papilloma: a case-control study.

OBJECTIVES: Sinonasal inverted papilloma (IP) is an uncommon benign tumour characterised by frequent recurrence and, in approximately 10% of cases, by neoplastic transformation. IP aetiology is unknown but human papillomavirus is detectable in about one quarter of tumours. As some occupational hazards have been reported to be possible risk factors for IP, the aim of this study was to assess risk for sinonasal IP associated with prior exposure to suspected occupational risk factors for sinonasal malignancies. METHODS: Between 1996 and 2007, data on incident cases were collected from hospitals throughout the Piedmont region of Italy by the regional sinonasal cancer registry. A questionnaire on occupational history, completed by 127 cases and 337 hospital controls, was used to assign previous exposure to a list of 17 occupational hazards. The relationship between IP and cumulative exposure to these hazards was explored using unconditional logistic regression to statistically adjust for age, sex, area of residence, smoking and co-exposures. RESULTS: The risk of IP was significantly increased for ever exposure to welding fumes (OR 2.14) and organic solvents (OR 2.11) after controlling for potential confounders. For organic solvents only, a significant association with continuous cumulative exposure and a significant trend in risk across ordered cumulative exposure categories was found. CONCLUSIONS: The present study revealed a significant association and a significant dose-response relationship between occupational exposure to organic solvents and IP. The lack of a dose-response relationship for welding fumes suggests that the observed association with ever exposure should be interpreted with caution.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

[Proposal of entering the mastoid vibration test in health surveillance of workers exposed to industrial solvents]. / Proposta di inserimento del test vibratorio mastoideo nella sorveglianza sanitaria di lavoratori esposti a solventi organici industriali.

In view of the use of solvents and consequently the number of exposed subjects, health surveillance should include instrumental exams to highlight at an early stage alterations of health resulting from exposure. We evaluated 40 workers with chronic exposure to low-level xylene, toluene, fluorene and anthracene, showing that vestibular system was altered in a fair percentage of cases, compared to a control group characterized by 40 workers of the same age not exposed to solvents. The two groups were simultaneously exposed to noise. It is concluded that occupational exposure to industrial organic solvents may lead to chronic alteration of the vestibular system with possible alterations also on work capacity even below the established limits. Second, an assessment of the vestibular system should be introduced in the health surveillance of workers exposed, in which, in our opinion, the mastoid vibration test is a diagnostic tool absolutely valid and simple to perform.

Synthesis and investigation of the trypanocidal potential of novel 1,2,3-triazole-selenide hybrids.

Chagas Disease is caused by the protozoan Trypanosoma cruzi and is considered a tropical neglected disease by the World Health Organization (WHO). The main drugs used in the therapy of the disease are obsolete and, as a result, it still kills millions of people every year. Therefore, the development of new drugs is urgent, as is the research reported in this article, in which new triazole selenides were synthesized through a simple methodology and to evaluate their potential against T. cruzi, through a combination of in vitro and in silico assays. With the combination of two molecular scaffolds already known for this activity, sixteen new hybrid compounds were obtained, showing yields ranging from 40 to 90%, and their biological potentials were tested. Two of the evaluated hybrids showed potent trypanocidal activity (11m and 11n), comparable to the positive control benznidazole. Density functional theory (DFT) studies were correlated with cyclic voltammetry assays to investigate the LUMO energy, which demonstrated a correlation with the observed trypanocidal activity. These results are promising, considering 11m and 11n as hit compounds in the development of new antichagasic drugs.

Psychological Treatment for Patients Receiving Radiation: Results of NRG Oncology/RTOG 0841.

PURPOSE: NRG/RTOG 0841 assessed the feasibility of a depression screening procedure in patients receiving radiation therapy (RT). As a secondary endpoint, availability and barriers to psychosocial care data were collected in hopes of providing recommendations for improved psychosocial care among patients receiving RT. METHODS AND MATERIALS: Patients starting RT were prospectively recruited and assessed with self-reported distress screening tools. Patients exceeding a validated cutoff and a sample of patients who screened negative received the Structured Clinical Interview for DSM-IV (SCID) mood disorder modules via telephone. During that SCID evaluation, patients completed a validated scale ranking interview on barriers to psychosocial care and interest in various psychosocial intervention modalities. RESULTS: A total of 463 patients from 35 community-based and 2 academic RT oncology sites were recruited. Of the 455 eligible, 75 (16%) exceeded screening cutoffs for depressive symptoms. From this group, 78 patients completed the SCID; most were female (76%), white (88%), and had breast cancer (55%). Overall, the most common barriers to treatment, regardless of insurance, were costs (58%), daily responsibilities (44%), and physical health symptoms (38%). Patients from RT facilities without mental health services were significantly more likely to report difficulty with physical health problems, specifically serious illness and walking, compared with those treated at RT facilities with services (P = .013 and P = .039, respectively). Overall, there was interest in obtaining psychosocial services with face-to-face counseling at the cancer center and printed educational materials as the most commonly preferred interventions. Patients with difficult barriers to psychosocial interventions were significantly less interested in support away from the cancer center (P = .016), telephone and Internet counseling (P = .0062 &P = .011), and Internet support (P = .0048). CONCLUSION: Radiation oncology patients are interested in obtaining psychosocial services but face barriers to access to mental health services including cost, debilitating symptoms, and time constraints that prevent adequate care.

SPEN is required for Xist upregulation during initiation of X chromosome inactivation.

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

Transcriptome analysis of the zebrafish atoh7-/- Mutant, lakritz, highlights Atoh7-dependent genetic networks with potential implications for human eye diseases.

Expression of the bHLH transcription protein Atoh7 is a crucial factor conferring competence to retinal progenitor cells for the development of retinal ganglion cells. Several studies have emerged establishing ATOH7 as a retinal disease gene. Remarkably, such studies uncovered ATOH7 variants associated with global eye defects including optic nerve hypoplasia, microphthalmia, retinal vascular disorders, and glaucoma. The complex genetic networks and cellular decisions arising downstream of atoh7 expression, and how their dysregulation cause development of such disease traits remains unknown. To begin to understand such Atoh7-dependent events in vivo, we performed transcriptome analysis of wild-type and atoh7 mutant (lakritz) zebrafish embryos at the onset of retinal ganglion cell differentiation. We investigated in silico interplays of atoh7 and other disease-related genes and pathways. By network reconstruction analysis of differentially expressed genes, we identified gene clusters enriched in retinal development, cell cycle, chromatin remodeling, stress response, and Wnt pathways. By weighted gene coexpression network, we identified coexpression modules affected by the mutation and enriched in retina development genes tightly connected to atoh7. We established the groundwork whereby Atoh7-linked cellular and molecular processes can be investigated in the dynamic multi-tissue environment of the developing normal and diseased vertebrate eye.

A case-control study on occupational risk factors for sino-nasal cancer.

OBJECTIVES: Sino-nasal cancer has been consistently associated with exposure to wood dust, leather dust, nickel and chromium compounds; for other occupational hazards, the findings are somewhat mixed. The aim of this study was to investigate the risk of sino-nasal epithelial cancer (SNEC) by histological type with prior exposure to suspected occupational risk factors and, in particular, those in metalworking. METHODS: Between 1996 and 2000, incident cases were collected on a monthly basis from hospitals throughout the Piedmont region of Italy by the regional Sino-nasal Cancer Registry. A questionnaire on occupational history, completed by 113 cases and 336 hospital controls, was used to assign exposure to occupational hazards. The relationship between SNEC and cumulative exposure to these hazards was explored using unconditional logistic regression to statistically adjust for age, sex, smoking and co-exposures, allowing for a 10-year latency period. RESULTS: The risk of adenocarcinoma was significantly increased with ever-exposure to wood dust (odds ratio; OR = 58.6), and to leather dust (OR = 32.8) and organic solvents (OR = 4.3) after controlling for wood dust, whereas ever-exposure to welding fumes (OR = 3.7) and arsenic (OR = 4.4) significantly increased the risk for squamous cell carcinoma. For each of these hazards, a significant increasing trend in risk across ordered cumulative exposure categories was found and, except for arsenic, a significantly increased risk with ever-exposure at low intensity. Treating cumulative exposure on a continuous scale, a significant effect of textile dusts was also observed for adenocarcinoma. For a mixed group of other histological types, a significant association was found with wood dust and organic solvents. CONCLUSIONS: Some occupational risk factors for SNEC were confirmed, and dose-response relationships were observed for other hazards that merit further investigation. The high risk for adenocarcinoma with low-intensity exposure to wood dust lends support for a reduction in the occupational threshold value.

Vagal paragangliomas: two case reports.

Two uncommon cases of paragangliomas arising from the vagus nerve are described. The first patient underwent surgery for suspected carotid body tumour. In the second patient, computed tomography scan and digital angiography allowed a correct pre-operative diagnosis to be made. These cases confirm the prevalence of vagal paragangliomas in female sex and middle age, and the possibility of multiple similar tumours in the same patient. Histological benign features, absence of neurological symptoms, of local invasion or intracranial extension confirm the frequent benign behaviour of these neoplasms. Lack of catecholamine secretion confirms the low incidence of functioning tumours. Contrast computed tomography and digital angiography still remain the gold standard reliable instruments for diagnosis despite the success of magnetic resonance imaging, magnetic resonance angiography and octreotide scintigraphy to detect head and neck paragangliomas. A transcervical approach, without mandibulotomy, is suitable too for large tumours but complete removal, with sparing of involved segments of the vagus nerve, is rarely possible. Post-operative neurological morbidity is still an unsolved issue and, therefore, rehabilitation of deglutition and phonation is an integral part of management.

Expression of epidermal growth factor receptor and transforming growth factor alpha in human larynx carcinoma.

Altered expression of growth factors and growth factor receptors is frequently described in human tumors and human tumor cell lines. This further supports the hypothesis that oncogenesis is due to the subversion of mitogen-responsive pathways. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF alpha) in 13 larynx carcinomas and 2 carcinomas of the oral cavity. We found receptor overexpression in 7 out of 15 tumors at mRNA and/or protein level but low expression in the majority of the normal adjacent tissues. TGF alpha was expressed only in 1 case, but no tyrosine kinase activity of the receptor was detected by antiphosphotyrosine antibody.

Angiogenesis: prognostic significance in laryngeal cancer.

The aim of this study was to evaluate the role of angiogenesis in the progression of laryngeal squamous cell carcinoma (LSCC). We correlated disease-free survival with microvessel count (MC) in the hot spot areas of 97 randomly selected caucasian males with LSCC followed for 60 to 90 months after surgery with or without radiotherapy. The results obtained indicate that: a) MC higher than 130 microvessels/mm2 is a cut-off value that distinguished patients who relapsed during the follow up period; b) multivariated analysis indicates that MC (p < 0.00001) is an independent predictor of disease free-survival; c) multivariated analysis selectively done on cases with relapse demonstrates that MC correlates with the presence of metastasis (or/and M) with local relapse (T). We suggest that MC is useful in the assessment of prognosis in LSCC and probably will permit selection of patients that could benefit from anti-angiogenic therapy associated with chemotherapy and/or radiotherapy.

Specific lymphokine responses in cervical nodes of patients with laryngeal cancer.

The role of lymphokines in modulating the immune response in patients suffering from neoplastic disease is still controversial. Recent studies indicate that in patients with head and neck cancer, a decrease in LIF (leukocyte migration inhibiting factor) production is usually present in advanced disease. In this study, the authors investigate: the LIF production in lymphocytes derived from the peripheral blood and cervical nodes of patients with laryngeal carcinoma by using an autologous pattern (i.e., for each patient, the specific LIF production was challenged by means of an autologous cell extract derived from his own tumor); the influence of histologically confirmed cervical node metastases on LIF production. Our results indicate that: There is a significant decrease in LIF production in patients with histologically proven cervical node metastases as compared to patients with no metastatic foci. The decrease of LIF production is related to the presence of mononuclear adherent cells. When the latter are removed, there is a significant reversal of specific LIF suppression. The possible meaning of the data is discussed.