A pleiotropy scan to discover new susceptibility loci for pancreatic ductal adenocarcinoma.

Pleiotropic variants (i.e., genetic polymorphisms influencing more than one phenotype) are often associated with cancer risk. A scan of pleiotropic variants was successfully conducted ten years ago in relation to pancreatic ductal adenocarcinoma susceptibility. However, in the last decade, genetic association studies performed on several human traits have greatly increased the number of known pleiotropic variants. Based on the hypothesis that variants already associated with a least one trait have a higher probability of association with other traits, 61,052 variants reported to be associated by at least one genome wide association study (GWAS) with at least one human trait were tested in the present study consisting of two phases (discovery and validation), comprising a total of 16,055 pancreatic ductal adenocarcinoma (PDAC) cases and 212,149 controls. The meta-analysis of the two phases showed two loci (10q21.1-rs4948550 (P=6.52×10-5) and 7q36.3-rs288762 (P=3.03×10-5) potentially associated with PDAC risk. 10q21.1-rs4948550 shows a high degree of pleiotropy and it is also associated with colorectal cancer risk while 7q36.3-rs288762 is situated 28,558 base pairs upstream of the Sonic Hedgehog (SHH) gene, which is involved in the cell differentiation process and PDAC etiopathogenesis. In conclusion, none of the single nucleotide polymorphisms (SNPs) showed a formally statistically significant association after correction for multiple testing. However, given their pleiotropic nature and association with various human traits including colorectal cancer, the two SNPs showing the best associations with PDAC risk merit further investigation through fine mapping and ad hoc functional studies.

Cochlear implantation in single-sided deafness: a single-center experience of 138 cases.

OBJECTIVES: Assess the outcomes in patients who underwent cochlear implant (CI) for single-sided deafness (SSD). METHODS: All patients affected by SSD who underwent CI at Gruppo Otologico, Piacenza, from October 2012 to May 2022 with at least 6 months of follow-up were selected in the study group. The analysis included subjective and objective measures performed pre-operative and up to 24 months after surgery. Hearing threshold on both sides was evaluated with a weighted four-frequency average (PTA [0.5 kHz + 1 kHz + 2 kHz + 4 kHz]/4) on pure tone audiometry and speech audiometry (Speech Discrimination Score, SDS). The Speech Spatial and Qualities of Hearing scale (SSQ Questionnaire) for binaural hearing benefits and sound localization, the Tinnitus Handicap Inventory Questionnaire (THI) and Dizziness Handicap Inventory Questionnaire (DHI) were used for subjective assessment. RESULTS: Data from 138 patients, 69 males and 69 females, (mean age 49 years, range 17-77 years) underwent CI for SSD were examined. Single-sided hearing deprivation average before undergoing CI surgery was 2.5 years (range 3 months-35 years). There was a significant reduction of THI and DHI scores compared to pre-operative scores alongside a referred improvement in social, physical, and emotional well-being through the SSQ questionnaire. CONCLUSIONS: To the best of our knowledge, this paper descried the largest cohort of SSD who underwent CI in a single institution. According to our findings CI in patients affected by SSD represents a valuable tool for an overall improvement of tinnitus and dizziness but also quality of life, after at least 6 months of follow-up. Further studies are desirable to improve rehabilitation pathways and possibly set new standards of care of this condition.

Technical details for a robot-assisted hand-sewn esophago-gastric anastomosis during minimally invasive Ivor Lewis esophagectomy.

BACKGROUND: Minimally invasive Ivor Lewis esophagectomy (MIILE) provides better outcomes than open techniques, particularly in terms of post-operative recovery and pulmonary complications. However, in addition to requiring advanced technical skills, thoracoscopic access makes it hard to perform esophagogastric anastomosis safely, and the reported rates of anastomotic leak vary from 5 to 16%. Several minimally invasive esophago-gastric anastomotic techniques have been described, but to date strong evidence to support one technique over the others is still lacking. We herein report the technical details and preliminary results of a new robot-assisted hand-sewn esophago-gastric anastomosis technique. METHODS: From January 2018 to December 2020, 12 cases of laparoscopic/thoracoscopic Ivor Lewis esophagectomy with robot-assisted hand-sewn esophago-gastric anastomosis were performed. The gastric conduit was prepared and tailored taking care of vascularization with a complete resection of the gastric fundus. The anastomosis consisted of a robot-assisted, hand-sewn four layers of absorbable monofilament running barbed suture (V-lock). The posterior outer layer incorporated the gastric and esophageal staple lines. RESULTS: The post-operative course was uneventful in nine cases. Two patients developed chyloperitoneum, one patient a Sars-Cov-2 infection, and one patient a late anastomotic stricture. In all cases, there were no anastomotic leaks or delayed gastric conduit emptying. The median post-operative stay was 13 days (min 7, max 37 days); the longest in-hospital stay was recorded in patients who developed chyloperitoneum. CONCLUSION: Despite the small series, we believe that our technique looks to be promising, safe, and reproducible. Some key points may be useful to guarantee a low complications rate after MIILE, particularly regarding anastomotic leaks and delayed emptying: the resection of the gastric fundus, the use of robot assistance, the incorporation of the staple lines in the posterior aspect of the anastomosis, and the use of barbed suture. Further cases are needed to validate the preliminary, but very encouraging, results.

One year of surgical mask testing at the University of Bologna labs: Lessons learned from data analysis.

The outbreak of SARS-CoV-2 pandemic highlighted the worldwide lack of surgical masks and personal protective equipment, which represent the main defense available against respiratory diseases as COVID-19. At the time, masks shortage was dramatic in Italy, the first European country seriously hit by the pandemic: aiming to address the emergency and to support the Italian industrial reconversion to the production of surgical masks, a multidisciplinary team of the University of Bologna organized a laboratory to test surgical masks according to European regulations. The group, driven by the expertise of chemical engineers, microbiologists, and occupational physicians, set-up the test lines to perform all the functional tests required. The laboratory started its activity on late March 2020, and as of the end of December of the same year 435 surgical mask prototypes were tested, with only 42 masks compliant to the European standard. From the analysis of the materials used, as well as of the production methods, it was found that a compliant surgical mask is most likely composed of three layers, a central meltblown filtration layer and two external spunbond comfort layers. An increase in the material thickness (grammage), or in the number of layers, does not improve the filtration efficiency, but leads to poor breathability, indicating that filtration depends not only on pure size exclusion, but other mechanisms are taking place (driven by electrostatic charge). The study critically reviewed the European standard procedures, identifying the weak aspects; among the others, the control of aerosol droplet size during the bacterial filtration test results to be crucial, since it can change the classification of a mask when its performance lies near to the limiting values of 95 or 98%.

Probiotics for prevention of atopic diseases in infants: systematic review and meta-analysis.

Growing evidence underlines the pivotal role of infant gut colonization in the development of the immune system. The possibility to modify gut colonization through probiotic supplementation in childhood might prevent atopic diseases. The aim of the present systematic review and meta-analysis was to evaluate the effect of probiotic supplementation during pregnancy and early infancy in preventing atopic diseases. PubMed, Embase and Cochrane Library were searched for randomized controlled trials evaluating the use of probiotics during pregnancy or early infancy for prevention of allergic diseases. Fixed-effect models were used, and random-effects models where significant heterogeneity was present. Results were expressed as risk ratio (RR) with 95% confidence interval (CI). Seventeen studies, reporting data from 4755 children (2381 in the probiotic group and 2374 in the control group), were included in the meta-analysis. Infants treated with probiotics had a significantly lower RR for eczema compared to controls (RR 0.78 [95% CI: 0.69-0.89], P = 0.0003), especially those supplemented with a mixture of probiotics (RR 0.54 [95% CI: 0.43-0.68], P < 0.00001). No significant difference in terms of prevention of asthma (RR 0.99 [95% CI: 0.77-1.27], P = 0.95), wheezing (RR 1.02 [95% CI: 0.89-1.17], P = 0.76) or rhinoconjunctivitis (RR 0.91 [95% CI: 0.67-1.23], P = 0.53) was documented. The results of the present meta-analysis show that probiotic supplementation prevents infantile eczema, thus suggesting a new potential indication for probiotic use in pregnancy and infancy.

LH, progesterone, and TSH can stimulate aldosterone in vitro: a study on normal adrenal cortex and aldosterone producing adenoma.

Endocrine factors different from ACTH or angiotensin II can stimulate aldosterone secretion and have a role in the pathophysiology of hyperaldosteronism. Aldosterone may increase in luteotropic/progestogenic and in hypothyroid states; LH and, occasionally, TSH receptors have been detected in normal adrenal cortex and aldosterone-producing adenoma. The aim of the study was to compare adrenal contents of LH and TSH receptors between normal cortex and aldosterone-producing adenoma and to evaluate the ability of LH, its product progesterone, and TSH to stimulate aldosterone secretion in vitro from primary adrenocortical cells. Surgical aldosterone-producing adenoma fragments from 19 patients and adrenal cortex fragments from 10 kidney donors were used for Western blotting and cell cultures. LH (n=26), TSH (n=19) and progesterone (n=8) receptor proteins were investigated; LH receptor-mRNA was also tested in 8 samples. Aldosterone responses in vitro to LH, progesterone, and TSH stimulation were assayed. LH and TSH receptors were more expressed in adenoma than normal cortex (p<0.01, p<0.05, respectively); progesterone receptor was observed in 6/8 samples. Aldosterone increased after in vitro stimulation with LH (5/12 adenoma, 1/7 normal cells), progesterone (4/5 adenoma, 5/6 normal cells), and TSH (3/5 adenoma and 3/5 normal cells). LH and TSH receptors were more expressed in aldosterone producing adenoma than normal adrenal cortex. LH, progesterone, and TSH can stimulate aldosterone in vitro. Similar mechanisms could participate in vivo in the aldosterone increase in lutheotropic, progestogenic, or hypothyroid states and may exist in both normal adrenal cortex and adenoma in responsive individuals.

Mitochondrial DNA lineages of Italian Giara and Sarcidano horses.

Giara and Sarcidano are 2 of the 15 extant native Italian horse breeds with limited dispersal capability that originated from a larger number of individuals. The 2 breeds live in two distinct isolated locations on the island of Sardinia. To determine the genetic structure and evolutionary history of these 2 Sardinian breeds, the first hypervariable segment of the mitochondrial DNA (mtDNA) was sequenced and analyzed in 40 Giara and Sarcidano horses and compared with publicly available mtDNA data from 43 Old World breeds. Four different analyses, including genetic distance, analysis of molecular variance, haplotype sharing, and clustering methods, were used to study the genetic relationships between the Sardinian and other horse breeds. The analyses yielded similar results, and the FST values indicated that a high percentage of the total genetic variation was explained by between-breed differences. Consistent with their distinct phenotypes and geographic isolation, the two Sardinian breeds were shown to consist of 2 distinct gene pools that had no gene flow between them. Giara horses were clearly separated from the other breeds examined and showed traces of ancient separation from horses of other breeds that share the same mitochondrial lineage. On the other hand, the data from the Sarcidano horses fit well with variation among breeds from the Iberian Peninsula and North-West Europe: genetic relationships among Sarcidano and the other breeds are consistent with the documented history of this breed.

Efficacy and safety of Lactobacillus plantarum P 17630 strain soft vaginal capsule in vaginal candidiasis: a randomized non-inferiority clinical trial.

OBJECTIVE: To investigate the non-inferiority of efficacy and tolerability of Lactobacillus plantarum P 17630 soft vaginal capsules compared to the antifungal therapy with miconazole nitrate 400 mg soft vaginal capsules in patients with symptomatic vulvovaginal infection due to Candida. PATIENTS AND METHODS: Adult women with vulvovaginal candidiasis were randomized to either L. plantarum P17630 100,000,000 CFU soft vaginal capsules by vaginal route each day for 3 or 6 consecutive days or miconazole nitrate 400 mg soft vaginal capsule. Visual Analog Scale (VAS) scores for vaginitis symptoms were used, and vaginal fluid interleukin 6 (IL6) was dosed. The study was registered in EudraCT database (code LPP17630-C-018; number: 2018-003095-12). RESULTS: 200 patients were included in the study. The mean VAS scores for vaginitis symptoms were progressively reduced in both treatment groups at each visit, without significant difference between groups (p>0.05 for each symptom, at each time point). The efficacy of L. plantarum and the reference medicinal product was maintained at follow-up (day 21). The mean concentration of IL-6 decreased from visit 1 to visit 3 in both groups without a significant difference (p>0.05). No adverse events were reported. CONCLUSIONS: L. plantarum P17630 100,000,000 CFU soft vaginal capsules are effective and safe for treating vaginal candidiasis without the concomitant use of an antifungal product, which rules out the risk of antimicrobic resistance. The long-term effect on vaginal microflora may add the possibility of reducing the risk of recurrences.

Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy.

Inhibition of cerebral amyloid beta-protein deposition seems to be an important target for Alzheimer's disease therapy. Amyloidogenesis could be inhibited by short synthetic peptides designed as beta-sheet breakers. Here we demonstrate a 5-residue peptide that inhibits amyloid beta-protein fibrillogenesis, disassembles preformed fibrils in vitro and prevents neuronal death induced by fibrils in cell culture. In addition, the beta-sheet breaker peptide significantly reduces amyloid beta-protein deposition in vivo and completely blocks the formation of amyloid fibrils in a rat brain model of amyloidosis. These findings may provide the basis for a new therapeutic approach to prevent amyloidosis in Alzheimer's disease.

A critical evaluation of the factors affecting the survival and persistence of beneficial bacteria in healthy adults.

The efficacy of a probiotic depends on its ability to survive and persist in the digestive tract. Regulatory agencies around the world recommend minimum dosages in order for a product to be termed a probiotic. However, the effect of dosage on the survival of the bacteria in the gut - the primary objective of probiotic administration - has not been critically evaluated. We performed a systematic literature review to assess the available data on the survival rate, during gastrointestinal transit, of probiotic bacteria that were orally administered to healthy adults. We also evaluated the persistence of the administered strain(s) after discontinuation of treatment and the potential role played by the food matrix in which probiotics have been administered. From a regulatory perspective, the profile of the target population is key to establishing the efficacy of probiotics. Therefore, we focussed on subjects without disease conditions. We evaluated 17 studies of single strains and 13 studies of multi-strain products, which reported survival and persistence outcomes. Persistence in the gut and recovery from stool were strain dependent. When the administered dose was higher than 1010 cfu/day, the probiotic could be recovered from stool regardless of the strain used. Treatment duration did not affect faecal recovery. Thus, dosage recommendations for probiotics by regulatory agencies are lower than that required for a strain to survive, persist and be efficacious in the gut.

Analysis of tumour microstructure estimation from conventional diffusion MRI and application to skull-base chordoma.

Skull-base chordoma (SBC) is a rare tumour whose molecular and radiological characteristics are still being investigated. In neuro-oncology microstructural imaging techniques, like diffusion-weighted MRI (DW-MRI), have been widely investigated, with the apparent diffusion coefficient (ADC) being one of the most used DW-MRI parameters due to its ease of acquisition and computation. ADC is a potential biomarker without a clear link to microstructure. The aim of this work was to derive microstructural information from conventional ADC, showing its potential for the characterisation of skull-base chordomas. Sixteen patients affected by SBC, who underwent conventional DW-MRI were retrospectively selected. From mono-exponential fits of DW-MRI, ADC maps were estimated using different sets of b-values. DW-MRI signals were simulated from synthetic substrates , which mimic the cellular packing of a tumour tissue with well-defined microstructural features. Starting from a published method, an error-driven procedure was evaluated to improve the estimates of microstructural parameters obtained through the simulated signals. A quantitative description of the tumour microstructure was then obtained from the DW-MRI images. This allowed successfully differentiating patients according to histologically-verified cell proliferation information.Clinical Relevance - The impact on cancer management derives from the expected improvement of radiation treatment quality tailored to a patient-specific non-invasive description of tumour microstructure.

Focal adhesion kinase inhibition synergizes with nab-paclitaxel to target pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a very lethal disease, with minimal therapeutic options. Aberrant tyrosine kinase activity influences tumor growth and is regulated by phosphorylation. We investigated phosphorylated kinases as target in PDAC. METHODS: Mass spectrometry-based phosphotyrosine proteomic analysis on PDAC cell lines was used to evaluate active kinases. Pathway analysis and inferred kinase activity analysis was performed to identify novel targets. Subsequently, we investigated targeting of focal adhesion kinase (FAK) in vitro with drug perturbations in combination with chemotherapeutics used against PDAC. Tyrosine phosphoproteomics upon treatment was performed to evaluate signaling. An orthotopic model of PDAC was used to evaluate the combination of defactinib with nab-paclitaxel. RESULTS: PDAC cell lines portrayed high activity of multiple receptor tyrosine kinases to various degree. The non-receptor kinase, FAK, was identified in all cell lines by our phosphotyrosine proteomic screen and pathway analysis. Targeting of this kinase with defactinib validated reduced phosphorylation profiles. Additionally, FAK inhibition had anti-proliferative and anti-migratory effects. Combination with (nab-)paclitaxel had a synergistic effect on cell proliferation in vitro and reduced tumor growth in vivo. CONCLUSIONS: Our study shows high phosphorylation of several oncogenic receptor tyrosine kinases in PDAC cells and validated FAK inhibition as potential synergistic target with Nab-paclitaxel against this devastating disease.

P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities.

Human CD3-16+56+ natural killer (NK) cells have been shown to display a clonally distributed ability to recognize major histocompatibility complex (MHC) class I alleles. Opposite to T lymphocytes, in NK cells, specific recognition of MHC class I molecules appears to induce inhibition of cytolytic activity and, thus, to protect target cells. Since a precise correlation has been established between the expression of the NK-specific GL183 and EB6 surface molecules (belonging to the novel p58 molecular family) and the specificity of NK clones, we analyzed whether p58 molecules could function as receptors for MHC in human NK cells. NK clones displaying the previously defined "specificity 2" and characterized by the GL183+EB6+ phenotype, specifically recognize the Cw3 allele and thus fail to lyse the Fc gamma R+ P815 target cells transfected with Cw3. On the other hand, NK clones displaying "specificity 1" and expressing the GL183-EB6+ phenotype failed to lyse Cw4+ target cells. Addition of the F(ab')2 fragments of either GL183 or EB6 mAb as well as the XA141 mAb of IgM isotype (specific for the EB6 molecules) completely restored the lysis of Cw3-transfected P815 cells by the Cw3-specific NK clones EX2 and EX4. Similarly, both the entire EB6 mAb, its F(ab')2 fragment and the XA141 mAb reconstituted the lysis of C1R, a Fc gamma R- target cell expressing Cw4 as the only serologically detected class I antigen. Thus, it appears that masking of different members of p58 molecules prevents recognition of "protective" MHC class I alleles and thus the delivering of inhibitory signals. Further support to the concept that p58 molecules represent a NK receptor delivering a negative signal was provided by experiments in which the entire anti-p58 mAbs (of IgG isotype) could inhibit the lysis of unprotected Fc gamma R+ P815 target cells, thus mimicking the inhibitory effect of MHC class I molecules.

Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells.

The natural killer (NK) cell-specific p58 molecules EB6 and GL183 have been shown to represent the putative surface receptors for two distinct groups of human histocompatibility leukocyte antigen (HLA) C alleles. Interaction between p58 receptors and class I molecules expressed on target cells results in inhibition of the NK-mediated cytolytic activity and thus in target cell protection. In the present study, we show that EB6 molecules may also act as receptors mediating NK cell triggering. Activatory EB6 molecules were found to be confined only to certain donors. Moreover, in these donors, only a fraction of EB6+ NK clones expressed the activatory form of EB6 molecules, while the remaining clones expressed the conventional inhibitory form. Biochemical analysis of the activatory EB6 molecules revealed a molecular mass of approximately 50 kD (p50), thus differing from the 58-kD inhibitory form. This difference was not due to differential glycosylation of the same protein, as revealed by deglycosylation experiments of isolated EB6 molecules. Treatment of purified p58 or p50/EB6 molecules with proteolytic enzymes, including V8-protease, chymotrypsin, and papain, showed only minor differences in the resulting peptides. Treatment with pepsin followed by two-dimensional peptide mapping demonstrated that, although the majority of peptides migrated in identical positions, differences between the two forms could be detected for at least one major peptide. Anti-EB6 monoclonal antibody (mAb)-mediated cross-linking of p50 molecules was required to trigger the cytolytic activity and the intracellular calcium ([Ca+2]i) increases in appropriate NK clones. Likewise, mAb-mediated cross linking of the p58 EB6 molecules was needed to inhibit the cytolytic activity; however, in this case, no [Ca+2]i increases could be detected. In NK clones expressing the inhibitory p58 EB6 receptors, soluble anti-EB6 mAb prevented recognition of protective Cw4 molecules and reconstituted target cell lysis. In contrast, in clones expressing the activatory p50/EB6 receptor, EB6 masking frequently resulted in partial inhibition of the cytolytic activity against Cw4+ target cells. Therefore, it appears that NK clones expressing the p50/EB6 receptors are induced to lyse Cw4+ target cells upon specific interaction with Cw4 molecules. This concept was further substantiated by experiments in which target cells were represented by the HLA-negative LCL721.221 cell line transfected with the Cw4 allele. Phenotypic and functional analysis of a large number of NK clones showed that clones expressing the activatory p50/EB6 molecules consistently coexpressed inhibitory receptors for other HLA class I alleles.(ABSTRACT TRUNCATED AT 400 WORDS)

p46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity.

NKp46 has been shown to represent a novel, natural killer (NK) cell-specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3zeta chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.

In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors.

Purified CD3-4- thymocytes were obtained by depletion of CD3+ and CD4+ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20-40% of thymic CD16+ cells coexpressed CD1, while approximately half were cyCD3+. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3-4- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3-4- or CD3-4-16- thymocytes in the presence of irradiated H9 cells resulted in large proportions (approximately 50%) of CD16+ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16+ cyCD3+; CD16+ cyCD3-; CD16- cyCD3+; and CD16- cyCD3-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-gamma and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16+ cells expressed transcripts for CD16 and for CD3 epsilon (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long, 1988. J. Immunol. 140:1685.) and zeta chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Lond.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3- CD16+ NK cells may arise from immature thymocytes.

Human natural killer cell receptors for HLA-class I molecules. Evidence that the Kp43 (CD94) molecule functions as receptor for HLA-B alleles.

GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58-negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-specific clones, monoclonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7+ target cells by B7-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross-linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK-mediated cytolytic activity. Indirect immunofluorescence and FACS analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7-specific as well as in most p58+ NK clones; and (c) were brightly expressed as in the p58+ clone ET34 (GL183-/EB6+, Cw4-specific). Functional analysis revealed that Kp43 functioned as an inhibitory receptor only in NK clones displaying bright fluorescence. These studies also indicate that some NK clones (e.g., the ET34) can coexpress two distinct receptors (p58 and Kp43) for different class I alleles (Cw4 and B7). Finally, we show that Kp43 molecules function as receptors only for some HLA-B alleles and that still undefined receptor(s) must exist for other HLA-B alleles including B27.

Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

Variation within the CLEC16A gene shows consistent disease association with both multiple sclerosis and type 1 diabetes in Sardinia.

Variation within intron 19 of the CLEC16A (KIAA0350) gene region was recently found to be unequivocally associated with type 1 diabetes (T1D) in genome-wide association (GWA) studies in Northern European populations. A variant in intron 22 that is nearly independent of the intron 19 variant showed suggestive evidence of association with multiple sclerosis (MS). Here, we genotyped the rs725613 polymorphism, representative of the earlier reported associations with T1D within CLEC16A, in 1037 T1D cases, 1498 MS cases and 1706 matched controls, all from the founder, autoimmunity-prone Sardinian population. In these Sardinian samples, allele A of rs725613 is positively associated not only with T1D (odds ratio=1.15, P one-tail=5.1 x 10(-3)) but also, and with a comparable effect size, with MS (odds ratio=1.21, P one-tail 6.7 x 10(-5)). Taken together these data provide evidence of joint disease association in T1D and MS within CLEC16A and underline a shared disease pathway.