Predictors of germline status for hereditary melanoma: 5 years of multi-gene panel testing within the Italian Melanoma Intergroup.

BACKGROUND: The incidence of cutaneous melanoma is increasing in Italy, in parallel with the implementation of gene panels. Therefore, a revision of national genetic assessment criteria for hereditary melanoma may be needed. The aim of this study was to identify predictors of susceptibility variants in the largest prospective cohort of Italian high-risk melanoma cases studied to date. MATERIALS AND METHODS: From 25 Italian centers, we recruited 1044 family members and germline sequenced 940 cutaneous melanoma index cases through a shared gene panel, which included the following genes: CDKN2A, CDK4, BAP1, POT1, ACD, TERF2IP, MITF and ATM. We assessed detection rate according to familial status, region of origin, number of melanomas and presence and type of non-melanoma tumors. RESULTS: The overall detection rate was 9.47% (5.53% analyzing CDKN2A alone), ranging from 5.14% in sporadic multiple melanoma cases (spoMPM) with two cutaneous melanomas to 13.9% in familial cases with at least three affected members. Three or more cutaneous melanomas in spoMPM cases, pancreatic cancer and region of origin predicted germline status [odds ratio (OR) = 3.23, 3.15, 2.43, P < 0.05]. Conversely, age > 60 years was a negative independent predictor (OR = 0.13, P = 0.008), and was the age category with the lowest detection rate, especially for CDKN2A. Detection rate was 19% when cutaneous melanoma and pancreatic cancer clustered together. CONCLUSIONS: Gene panel doubled the detection rate given by CDKN2A alone. National genetic testing criteria may need a revision, especially regarding age cut-off (60) in the absence of strong family history, pancreatic cancer and/or a high number of cutaneous melanomas.

Cellular consequences of small supernumerary marker chromosome derived from chromosome 12: mosaicism in daughter and father

Constitutional genomic imbalances are known to cause malformations, disabilities, neurodevelopmental delay, and dysmorphia and can lead to dysfunctions in the cell cycle. In extremely rare genetic conditions such as small supernumerary marker chromosomes (sSMC), it is important to understand the cellular consequences of this extra marker, as well the factors that contribute to their maintenance or elimination through successive cell cycles and phenotypic impact. The study of chromosomal mosaicism provides a natural model to characterize the effect of aneuploidy on genome stability and compare cells with the same genetic background and environment exposure, but differing in the presence of sSMC. Here, we report the functional characterization of different cell lines from two familial patients with mosaic sSMC derived from chromosome 12. We performed studies of proliferation dynamics, stability, and variability of these cells using fluorescent in situ hybridization (FISH), sister chromatid exchanges (SCE), and conventional staining. We also quantified the telomere-related genomic instability of sSMC cells using 3D telomeric profile analysis by quantitative-FISH. sSMC cells exhibited differences in the cell cycle dynamics compared to normal cells. First, the sSMC cells exhibited lower proliferation index and higher frequency of SCE than normal cells, associated with a higher level of chromosomal instability. Second, sSMC cells exhibited more telomeric-related genomic instability. Lastly, the differences of sSMC cells distribution among tissues could explain different phenotypic repercussions observed in patients. These results will help in our understanding of the sSMC stability, maintenance during cell cycle, and the cell cycle variables involved in the different phenotypic manifestations.

Evolución de pacientes con bronquiolitis tratados con cánulas de alto flujo de oxígeno que ingresan a la unidad de cuidados intensivos / Outcome of patients with bronchiolitis treated with high-flow oxygen nasal cannulas admitted to the intensive care unit

Objetivo: Describir la evolución en la Unidad de Cuidados Intensivos Pediátricos (UCIP) de los pacientes con bronquiolitis, tratados inicialmente con cánula nasal de alto flujo de oxígeno (CAFO) en la Unidad Emergencias. Determinar factores predisponentes de ingreso a ventilación no invasiva (VNI) o invasiva con intubación orotraqueal (TET). Métodos: Trabajo descriptivo retrospectivo por revisión de historias clínicas. Se incluyeron todos los pacientes menores de 2 años de edad con diagnóstico de bronquiolitis, sin comorbilidades, que ingresaron a UCIP polivalente luego de haber sido previamente tratados con CAFO en la Unidad de Emergencias entre los meses de Agosto 2017 y Agosto 2019. Resultados: Se evaluaron 145 pacientes. La mediana de edad fue de 4,4 meses (RIC 2-6 meses), con una mediana de tiempo desde el inicio de los síntomas hasta la consulta de 45,4 hs (RIC 24-72). La mediana del score de TAL modificado al ingreso a UCIP 8,4 (RIC 8-9). El 98,6% requirió asistencia respiratoria mecánica (ARM), en el grupo VNI 52,4% (75) y en el grupo TET 47,5% (68). El rescate de germen fue en 60% de los casos virus sincicial respiratorio (VSR). El 5,5% fueron co-infecciones. De los pacientes con rescate positivo para VSR, el 52,9% (46) requirieron VNI y 47,1% (41) TET. Los pacientes estudiados permanecieron en CAFO una mediana de 20 hs previo al ingreso a UCIP (RIC: 6-24). Aquellos que estuvieron en VNI con una mediana de 23,3 hs (RIC 6-24) y los que requirieron TET 17 hs (RIC 6-21). La mortalidad evidenciada en el grupo TET fue de 1,38% correspondiente a 2 pacientes, donde también se encontró un 7,5% de complicaciones. La mediana de días de internación en UCIP fue de 8,6 (5-11) para la totalidad de la población estudiada siendo 5,2 (4-6) para los pacientes en VNI y 12 días (9-14) para los pacientes en TET. Conclusiones: Casi la totalidad de pacientes tratados con CAFO en la Unidad Emergencias que requirieron pasar a UCI necesitaron ARM. Ni el score de TAL ni la cantidad de horas de CAFO previo al ingreso en UCI nos permitieron diferenciar los pacientes del grupo VNI de aquellos del grupo TET (AU)

Objective: To describe outcome of patients who were admitted to the pediatric intensive care unit (PICU) with bronchiolitis initially treated with high-flow oxygen through a nasal cannula (HFNC) at the emergency department and to determine predisposing factors for the need for non-invasive ventilation (NIV) or invasive endotracheal intubation (ETI). Methods: A retrospective descriptive study with a review of the clinical records was conducted. All patients less than 2 years of age with bronchiolitis without comorbidities that were admitted to the general PICU following treatment with HFNC at the emergency department between August 2017 and August 2019 were included in the study. Results: 145 patients were evaluated. Median age was 4.4 months (IQR 2-6 months). Median time from symptom onset to first consultation was 45.4 hours (IQR 24-72). Median modified TAL score on PICU admission was 8.4 (IQR 8-9). Overall 98,6% required mechanical ventilation (MV), 52.4% (75) in the NIV and 47.5% (68) in the ETI group. In 60% of the cases respiratory syncytial virus (RSV) was isolated. Co-infections were found in 5.5%. Of the patients with an RSV-positive isolate, 52.9% (46) required NIV and 47.1% (41) ETI. Patients had remained on HFNC for a median of 20 hours prior to PICU admission (IQR 6-24). Patients were on NIV for a median time of 23.3 hours (IQR 6-24) and on ETI for a median time of 17 hours (IQR 6-21). In the ETI group, mortality rate was 1.38%, corresponding to two patients, while the complication rate was 7.5%. Median length of PICU stay was 8.6 days (5-11) for the entire study population, 5.2 days (4-6) for patients on NIV, and 12 days (9-14) for those on ETI. Conclusions: Almost all patients treated with HFNC at the emergency department who required admission to the PICU needed MV. Neither TAL score nor time on HFNC allowed us to differentiate patients requiring NIV from those who needed ETI (AU)

Impacto de la cateterización venosa central percutánea bajo guía ecográfica en una terapia intensiva pediátrica polivalente / Impact of ultrasound-guided percutaneous central venous catheterization at a general pediatric intensive care unit

Introducción: La cateterización venosa central es un procedimiento usual en Unidades de Cuidados Intensivos (UCI). El ultrasonido (US) para guiar la cateterización, ofrece ventajas, permitiendo tener una imagen topográfica precisa del vaso, reduciendo las complicaciones, el tiempo y el número de punciones. Objetivo: determinar, si la US en la colocación de catéteres venosos centrales (CVC), podría disminuir el número de punciones y lograr la cateterización exitosa. Población y métodos: Estudio descriptivo, prospectivo de los CVC colocados mediante punción guiada por US, en una UCI polivalente del Hospital de Pediatría Juan P. Garrahan, entre el año 2018 al 2019. Población: pacientes de 1 mes a 18 años que requirieron colocación de un CVS por US. Se consideró significativo un valor de p< 0.05. Resultados: VYI en 66 pacientes (43,5%), VF fue en 86 pacientes (56,5%). 86 (56,5%) CVC, fueron insertados en el primer intento y 66 (43,5%), requirieron más de un intento. Las inserciones en VYI fueron exitosas en el primer intento en 46 pac. (53,5%) 20 pac. requirieron más de un intento (30,3%) p 0,004 OR 0,37 (IC 95% 0,18-0,78. En <6 meses los CVC colocados en VYI tuvieron menos riesgo de requerir más de un intento, con respecto a aquellos en los cuales se eligió la VF, p 0,0026 OR 0,31 (IC 95% 0,12 -0,75). 5,2% presentaron complicaciones, no hubo mortalidad relacionada al procedimiento. Conclusiones: La inserción de CVC guiados por US fue segura y significativamente exitosa en el primer intento cuando el vaso de elección fue la VYI, especialmente en < 6 meses (AU)

IIntroduction: Central venous catheterization is a common procedure in intensive care units (ICU). The use of ultrasound (US) to guide catheterization offers advantages, allowing for an accurate topographic image of the vessel, reducing complications as well as time and number of punctures. Objective: To determine whether the use of US for the placement of central venous catheters (CVCs) may decrease the number of punctures and achieve successful catheterization. Patients and methods: A descriptive, prospective study was conducted of CVCs placed by US-guided puncture at a general ICU of Hospital de Pediatría Juan P. Garrahan between 2018 and 2019. Patients from 1 month to 18 years of age who required US-guided placement of a CVC were included. A p< 0.05 was considered significant. Results: The internal jugular vein (IJV) was used in 66 (43.5%) and the femoral vein (FV) in 86 patients (56.5%). Overall, in 86 (56.5%) CVC were inserted on the first attempt and 66 (43.5%) required more than one attempt. Insertions into the VYI were successful on the first attempt in 46 (53.5%) patients and 20 (30.3%) patients required more than one attempt, p 0.004; OR 0.37 (95% CI 0.18-0.78). In patients <6 months CVCs placed in the IJV had a lower risk of requiring more than one attempt compared to those in which the FV was chosen, p 0.0026 OR 0.31 (95% CI 0.12 -0.75). Complications occurred in 5.2%; no procedure-related mortality was observed. Conclusions: US-guided insertion of CVC was safe and significantly successful on the first attempt when the vessel of choice was the IJV, especially in patients < 6 months (AU)

Modeling the Iatrogenic Pancreatic Cancer Risk After Islet Autotransplantation in Mouse.

Iatrogenic pancreatic cancer metastasis after islet infusion is a potential risk of islet autotransplantation performed after pancreatectomy. To model this risk, islets and/or pancreatic exocrine clusters obtained from a genetically engineered mouse model for pancreatic ductal adenocarcinoma (the LSL-KrasG12D/+ ;LSL-Trp53R172H/+ ;Pdx-1-Cre, termed KPC mouse) were transplanted via the portal vein in syngeneic wild type (WT) severely diabetic recipients in the following treatment groups: group A (n = 11) received KPC exocrine clusters in volume equal to 250 islet equivalents (IEQs); group B (n = 12) received 250 WT IEQs mixed with KPC exocrine clusters (1:1 volume ratio); group C (n = 5) received 250 KPC IEQs, and group D (n = 7) received 250 WT IEQs. The incidence of hepatic metastasis was assessed by magnetic resonance imaging and histology over the 13 months of follow-up. Overall survival was not different in the four groups. No mice developed liver metastases during the follow-up. Two mice developed spontaneous tumors: a liver hepatocellular tumor in group A and a malignant lymphoma in group D. Islets and/or exocrine clusters obtained by KPC mouse, a model that develops pancreatic cancer with 100% penetrance, do not retain the same risk of tumor development when transplanted via the portal vein in a syngeneic diabetic recipient.

Stem cells to restore insulin production and cure diabetes.

BACKGROUND: The advancement of knowledge in the field of regenerative medicine is increasing the therapeutic expectations of patients and clinicians on cell therapy approaches. Within these, stem cell therapies are often evoked as a possible therapeutic option for diabetes, already ongoing or possible in the near future. AIM: The purpose of this document is to make a point of the situation on existing knowledge and therapies with stem cells to treat patients with diabetes by focusing on some of the aspects that most frequently raise curiosity and discussion in clinical practice and in the interaction with the patient. In fact, at present there are no clinically approved treatments based on the use of stem cells for the treatment of diabetes, but several therapeutic approaches have already been evaluated or are being evaluated in clinical trials. DATA SYNTHESIS: It is possible to identify three large potential application fields: 1) the reconstruction of the ß cell mass; 2) the immunomodulation in type 1 diabetes (T1D); 3) the treatment of complications. In this study we will limit the discussion to approaches that have the potential for clinical translation, deliberately omitting aspects of basic biology and preclinical data. Also, we intentionally omit the treatment of the complications that will be the subject of a future document. Finally, an overview of the Italian situation regarding the storage of cord blood cells for the therapy of diabetes will be given.

Quantification of CatSper1 expression in human spermatozoa and relation to functional parameters.

STUDY QUESTION: Is CatSper1 expression in human spermatozoa related to semen parameter values and sperm functions? SUMMARY ANSWER: CatSper1 expression is positively related to progressive and hyperactivated (HA) motility, [Ca(2+)]i responsiveness to progesterone but not the acrosome reaction (AR). WHAT IS KNOWN ALREADY: The role of cationic channel of sperm (CatSper) in sperm functions is clear in animal models but less defined in human sperm cells. Current knowledge is mostly based on low specificity CatSper inhibitors showing agonistic and toxic effects on human spermatozoa and is thus of little help in clarifying the role of the CatSper channel in human sperm functions. STUDY DESIGN, SIZE, DURATION: CatSper1 protein expression was evaluated in 115 men undergoing semen analysis for couple infertility. CatSper1 expression was related to routine semen parameters, motility kinematic parameters and basal and progesterone-stimulated [Ca(2+)]i and the AR. PARTICIPANTS/MATERIALS, SETTING, METHODS: CatSper1 expression was evaluated (n = 85 normozoospermic, n = 30 asthenozoospermic patients) by immunofluorescence coupled to flow cytometry leading to quantitative measurement of the percentage of ejaculated sperm cells expressing the protein. Semen analysis was evaluated according to World Health Organization guidelines. Kinematic parameters were evaluated by a computer-aided sperm analysis system. [Ca(2+)]i was measured by a spectrofluorimetric method in fura-2-loaded spermatozoa. The AR was evaluated in live sperm cells by fluorescent-labeled lectin. MAIN RESULTS AND THE ROLE OF CHANCE: CatSper1 protein expression in spermatozoa was reduced in asthenozoospermic men (mean ± SD: 53.0 ± 15.5%, n = 30 versus 67.9 ± 17.1% in normozoospermic, n = 85, P < 0.01) and was significantly correlated with progressive (r = 0.36, P < 0.001), total (r = 0.35, P < 0.001) and HA (r = 0.41, P < 0.005) motility. In addition to a higher percentage of spermatozoa not expressing CatSper1, asthenozoospermic men showed a large number of spermatozoa with immunofluorescent signal localized outside the principal piece compared with those in normozoospermia. A significant positive correlation was found between CatSper1 protein expression and the increase of [Ca(2+)]i in response to progesterone (r = 0.36, P < 0.05, n = 40) but not with basal [Ca(2+)]i. No correlation was found with the AR, either basal or in response to progesterone. LIMITATIONS, REASONS FOR CAUTION: The study is partly descriptive. Furthermore, we cannot rule out the possibility that some round cells remain after a single round of 40% density gradient centrifugation or that this step may have removed some defective or slow swimming sperm, and therefore this preparation may not be representative of the entire sperm sample. Although our data suggest that CatSper1 may be a useful marker for infertility, and a possible contraceptive target, any clinical application is limited without further research. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate an association of CatSper1 expression with human sperm progressive and HA motility and provide preliminary evidence that lack of expression or mislocalization of CatSper1 in spermatozoa may be involved in the pathogenesis of asthenozoospermia. However, mechanistic studies are needed to confirm that the correlations between CatSper1 expression and sperm functions are causative. STUDY FUNDING/COMPETING INTERESTS: Supported by grants from Ministry of University and Scientific Research (PRIN project to E.B. and FIRB project to S.M.) and by Regione Toscana (to G.F.). L.T. was recipient of a grant from Accademia dei Lincei (Rome, Italy). The authors have no conflicts of interest to declare.

Long-term endoscopic follow-up in small bowel transplant recipients: single-center series.

BACKGROUND: The reliability of endoscopic findings after adult intestinal transplantation on short-term follow-up has been shown. The aim of this study was to evaluate in a long-term follow-up the diagnostic value of endoscopies compared with the biopsy value. METHODS: We evaluated 52 endoscopies over a period of 2 years (2 in each patient in 2010 and 1 in each patient in 2011, plus 1 endoscopy for suspected post-transplant lymphoproliferative disease [PTLD]) on 17 recipients transplanted between the years 2000 and 2006 (more than 5 years of follow-up). RESULTS: All the 52 endoscopic findings were comparable to biopsy definitive results: only 1 case of mild enteritis and 1 case of Epstein-Barr virus (EBV) chronic infection at biopsy were not diagnosed by endoscopy. One case of rectal PTLD and 1 of EBV-related enteritis were diagnosed by use of both procedures. Specificity was 98%: we did not calculate sensitivity because no episodes of rejection were diagnosed because recipients were stable in long-term follow-up. CONCLUSIONS: Endoscopy is a reliable procedure even on a long-term follow-up after intestinal transplantation, allowing a support to biopsy for diagnosis on adult recipients, especially for EBV infections and PTLD surveillance.

Induction therapy in adult intestinal transplantation: reduced incidence of rejection with "2-dose" alemtuzumab protocol.

The incidence of early rejection after intestinal transplantation correlates with heightened risk of graft loss and mortality. Many different induction or pre-conditioning protocols have been reported in the last 10 yr to improve outcomes; however, sepsis remains prevalent and diminishes long-term results. We recently began a "2-dose" alemtuzumab trial protocol - 15 mg at day 0 and 15 mg repeated on day 7 - with the hope of reducing our infection rate. We compared three different protocols used at our institution (daclizumab, conventional "4-dose" alemtuzumab, and "2-dose" alemtuzumab). There was a significantly lower rate of early rejection with the "2-dose" alemtuzumab protocol in our study group of mainly (88%) intestinal grafts without accompanying liver engraftment with its protective immunologic effect. Sepsis remained low. Longer follow-up will be required to evaluate the effects of this new protocol on longer-term outcomes.

Induction therapy with alemtuzumab (campath) in combined liver-kidney transplantation: University of Bologna experience.

BACKGROUND: Combined liver-kidney transplantation (LKT) is considered to be a safe procedure, but the appropriate immunosuppressive regimen is unclear. PATIENTS AND METHODS: Between January 1997 and October 2011, 55 patients were listed for LKT: 45 (82%) were effectively transplanted, 5 (9.2%) died whereon here the waiting list, 3 (5.5%) temporarily out of waiting list, 1 (1.8%) was on waiting list and 1 (1.8%) refused LKT. Five LKTs treated with cyclosporine (CyA) were excluded from the analysis. Mean recipient age was 50.32 ± 10.32 years (14-65), MELD score at time of LKT was 19.22 ± 4.69 (8-29), mean waiting list time was 8.14 ± 9.50 months (0.1-35.76), and follow-up, 4.09 ± 3.02 years (0.01-10.41). Main indications for LKT were policystic disease (n = 15; 37%), hepatitis virus C (HCV)-related cirrhosis (n = 9; 22%) metabolic disease (n = 5; 13%), hepatitis virus B (HBV) cirrhosis (n = 4; 10%), alcoholic cirrhosis (n = 4; 10%), and cholestatic disease (n = 3; 8%). Immunosuppressive regimen was based on tacrolimus and steroids in 40 cases with induction therapy with alemtuzumab (Campath; 0.3 mg/kg) in 13 of 40 instances cases administered on day 0 and day 7. RESULTS: Postoperative mortality was 2.5%. Acute cellular rejection episodes were biopsy-proven in 2 (5%) cases, post-LKT infections developed in 17 cases (42.5%), and de novo cancer developed in 3 (7.5%) cases. Similar 5-year overall survivals were obtained irrespective of the LKT indication: 100% in cholestatic and alcoholic cirrhosis patients, 86% in policystic disease, 75% in metabolic disease and HBV patients, and 66% in HCV cirrhosis. Overall survivals for the alemtuzumab vs without-induction therapy groups at 1, 3, and 5-years were 100%, 85.7%, and 85.7% vs 76%, 76%, and 70%, respectively (P = .04). CONCLUSION: An immunosuppressive regimen based on tacrolimus and steroids with induction therapy with alemtuzumab was safe, with excellent long-term results for combined LKT.

Mortality after steroid-resistant acute cellular rejection and chronic rejection episodes in adult intestinal transplants: report from a single center in induction/preconditioning era.

Steroid-resistant acute cellular rejection (ACR) and chronic rejection (CR) are still major concerns after intestinal transplantation. We report our experience from a single center on 48 adults recipients using 49 grafts from 2001 to 2011, immunosuppressing them initially with daclizumab initially and later Alemtuzumab. Overall patient survival was 41.9% at 10 years while graft survival was 38.5%. The steroid-resistant ACR population of 14 recipients (28.5%) experienced 50% mortality mainly due to sepsis, while the five (8%) CR recipients, included two survivors. All but 1 graft was placed without a liver. CR was often preceded by ACR episodes. Mortality related to steroid-resistant ACR and CR still affects the intestinal transplant population despite induction/preconditioning, especially in the absence of a protective liver effect of the liver. New immunosuppressive strategies are needed.

Negative regulation of type I interferon signaling: facts and mechanisms.

Initially described for their antiviral activities, type I Interferons are now recognized as central regulatory elements of the immune response, primarily for their effect on the differentiation of monocytes into dendritic cells and osteoclasts. They are routinely used in clinic for the treatment of several diseases, including viral hepatitis, multiple sclerosis and several forms of cancer. Interferons are however not devoid of toxic effects when high doses are administered to patients, indicating that interferon action must be timely and spatially down regulated. We review here the molecular mechanisms which have been described to shut off the interferon initiated signals.

Effect of resveratrol and catechin on PC12 tyrosine kinase activities and their synergistic protection from beta-amyloid toxicity.

beta-Amyloid peptide (beta-AP) is the main component of amyloid deposits around the cerebral vessel and in the brain parenchyma in Alzheimer's disease and Down's syndrome. In vitro studies in neuronal cells or in PC12 and Hela cell lines have shown that the aggregate form of beta-AP is toxic. Many genetic and environmental factors including metal ions, proteoglycans, plasma proteins and antioxidants modify beta-AP toxicity. We investigated the effect of two plant polyphenols--resveratrol and catechin--on soluble and particulate tyrosine kinase activity from PC12 cells and the protective action of these compounds against beta-AP (1-41) toxicity. beta-AP (1-41) decreased PC12 viability with an IC50 value of 1.1 +/- 0.14 x 10(-8) M. Resveratrol and catechin protected PC12 cells from beta-AP (1-41) toxicity. With 25 microM resveratrol the IC50 value increased to 2.2 +/- 0.19 x 10(-7) M. In the presence of beta-AP (1-41) resveratrol showed a concentration-dependent biphasic effect, and at a concentration of up to 40 microM it protected PC12 cells from beta-AP (1-41) toxicity. At concentrations higher than 40 microM, an inhibitory activity on cell proliferation appeared. This antiproliferative effect was also seen in the absence of beta-AP (1-41). With 100 microM catechin the IC50 value increased from 1.1 +/- 0.14 x 10(-8) M to 3.2 +/- 0.25 x 10(-7) M beta-AP (1-41). The protective effect was concentration dependent. Resveratrol and catechin had a synergistic protective action. In the presence of 40 microM catechin and 10 microM resveratrol or 20 microM resveratrol and 10 microM catechin, the toxicity determined by 10(-7) M beta-AP (1-41) was almost completely removed. Resveratrol and catechin had different effects on PC12 tyrosine kinase activity. With peptide 1-17 of gastrin as substrate, resveratrol inhibited particulate tyrosine kinases while it had no effect on soluble activity. With the same substrate, catechin increased the activity of soluble fraction while it inhibited particulate activity. When peptide 6-20 of cell division kinase p34cdc2 was utilized, catechin showed an opposite effect, inhibiting soluble tyrosine kinase activity and increasing particulate activity. With peptide 6-20, resveratrol inhibited both soluble and particulate activities. These results demonstrate that resveratrol and catechin have different activities on the signal transduction pathway involving protein phosphorylation. These differences may contribute not only to the different effects of these compounds on PC12 growth but also to the synergistic effect against beta-AP (1-41) toxicity. The different activity of resveratrol and catechin on signal transduction pathways, as well as the differences in metal chelation, partition coefficient between water and lipids, hydrogen donation redox potential and enzyme inhibition may be at least in part based on synergistic protection against beta-AP (1-41) toxicity.

Production of IL-4 and leukemia inhibitory factor by T cells of the cumulus oophorus: a favorable microenvironment for pre-implantation embryo development.

The nature and the functional activity of immunocytes present in the cumulus oophorus, a mass of cells surrounding the oocyte, were examined here for the first time. The cumuli oophorus were obtained from women who had taken part in an in vitro fertilization program and were suffering from blocked fallopian tubes. Both macrophages and CD4(+) T cells were detected in all cumuli. CD4(+) T cell clones, generated from T cells of these cumuli, showed higher potential to produce IL-4 and leukemia inhibitory factor (LIF) than CD4(+) T cell clones generated from peripheral blood or ovary specimens from the same women. More importantly, IL-4 and LIF, but not IFN-gamma mRNA was found to be constitutively expressed in vivo by cumulus oophorus cells. Progesterone is highly produced by the cumulus oophorus/oocyte complex. We recently showed that progesterone up-regulates the production of LIF by T cells and that the progesterone-induced LIF production is mediated by IL-4. Progesterone produced by cumulus granulosa cells may favor IL-4 production by T cells, which in turn can produce LIF. As the treatment with LIF enhances the in vitro growth and development of mammalian embryos, our data suggest that T cells present in the cumulus oophorus produce cytokines that may provide a microenvironment suitable for pre-implantation development of the mammalian embryo.

Reduced expression of INT-6/eIF3-p48 in human tumors.

The int-6 gene, originally identified as a common integration site for the mouse mammary tumor virus (MMTV) in mouse mammary tumors, encodes the p48 component of the eukaryotic translation initiation factor-3 (eIF3-p48). Int-6/eIF3-p48 is expressed in all adult tissues which have been tested and early in embryonic development. Int-6/eIF3-p48 has been highly conserved throughout evolution and the deduced amino acid sequence of the human gene product is identical to the mouse protein. Viral insertions at the Int-6/eIF3-p48 locus in mouse mammary tumors result in production of chimeric Int-6/eIF3-p48/MMTV products that may act as dominant negative oncoproteins. Int-6/eIF3-p48 has also been identified as a human protein that binds to the human T-cell leukemia virus type I Tax oncoprotein. The role of Int-6/eIF3-p48 in human carcinogenesis is unknown at the present time. In this study we have examined Int-6/eIF3-p48 gene status and expression in two of the most common forms of cancer in humans, breast and lung tumors. Sixty-two breast carcinomas and 78 non-small cell lung carcinomas (NSCLC) were investigated. LOH at the Int-6/eIF3-p48 locus was observed in 5 (21%) of 24 informative breast tumors and 10 (29%) of 34 informative lung tumors. A reduced expression of Int-6/eIF3-p48 was seen in 23 (37%) of breast cancer samples and 24 (31%) of NSCLC samples. An association between Int-6/eIF3-p48 expression and LOH at the Int-6/eIF3-p48 locus was observed. Int-6/eIF3-p48 expression was not related to commonly used pathological parameters in breast cancer patients, while in NSCLC patients int-6/eIF3-p48 expression was mainly seen in adenocarcinomas (P<0.0001). In conclusion, our data show for the first time a decreased expression of Int-6/eIF3-p48 in a consistent portion of human breast and lung carcinomas, frequently associated with LOH at the Int-6/eIF3-p48 locus. Additional studies on larger series of tumor specimens with long-term follow-up are needed to determine whether Int-6/eIF3-p48 expression may represent a new prognostic or predictive marker.

Visualization of interstitial cells of Cajal in living, intact tissues.

Interstitial cells of Cajal (ICC) appear to be a major element in pacing and signal transmission in the gastrointestinal tract. A prominent problem in the study of ICC has been the difficulty in observing them in intact tissues. We used several methods to visualize living ICC in freshly-dissected tissues: (1) Placing small crystals of the lipophilic dye DiI in the submucosal-circular muscle border in the mouse colon resulted in the labeling of living ICC-like cells. Two main morphological cell types, bipolar and multipolar, were noted. The DiI stain could be converted into a stable, electron-opaque product. Electron-microscopic observations showed that the labeled cells had the typical appearance of ICC reported in previous studies. (2) Living ICC in the region of the myenteric plexus (ICC-MP) in the small intestines of mice and guinea-pigs were observed with Nomarski optics. This enabled the visualization of ICC in living tissues, and the impalement of the cells with Lucifer yellow-filled microelectrodes. The dye-labeled cells had the morphological features of ICC-MP, and about 30% of them were found to be dye coupled to 1-21 other ICC. The identity of the cells as ICC was verified by electron-microscopy following photoconversion, and by c-kit immunohistochemistry. (3) Living ICC were labeled with a c-kit antibody that does not require tissue fixation. This resulted in the fluorescent staining of the entire ICC network. Single cells were labeled by dye injection, which provided a detailed picture of ICC morphology. This method was found to be suitable for a wide range of tissues. We expect that these three methods for identifying ICC in intact, living tissues will be useful for physiological and pharmacological investigations of ICC in a variety of gastrointestinal tissues.

The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT activation by interferon-alpha.

We have examined the effects of human papilloma virus (HPV) E6 proteins on interferon (IFN) signaling. Here we show that expression of the 'malignant' HPV-18 E6 in human HT1080 cells results in inhibition of Jak-STAT activation in response to IFN-alpha but not IFN-gamma. This inhibitory effect is not shared by the 'benign' HPV-11 E6. The DNA-binding and transactivation capacities of the transcription factor ISGF3 are diminished in cells expressing HPV-18 E6 after IFN-alpha treatment as a result of decreased tyrosine phosphorylation of Tyk2, STAT2 and STAT1. However, HPV-18 E6 does not affect the induction of tyrosine phosphorylation and DNA-binding of STAT1 by IFN-gamma. In addition, HPV E6 proteins physically interact with Tyk2. This interaction takes place preferably with HPV-18 E6 and to a lesser extent with HPV-11 E6. The E6/Tyk2 interaction requires the JH6-JH7 domains of Tyk2, which are important for Tyk2 binding to the cytoplasmic portion of IFN-alpha receptor 1 (IFNAR1). These findings demonstrate an inhibitory role of HPV-18 E6 in the IFN-alpha-induced Jak-STAT pathway, which may be explained, at least in part, by the ability of E6 to interact with and impair Tyk2 activation.

Lung tumours from non-smoking subjects: A p53-related genetic instability in a subset of cases.

Tumours developed in non-smoking patients represent about 10% of all lung neoplasms and show peculiar morphologic and genetic features. In a previous study, we found a constant association between p53 gene alterations and loss of heterozygosity (LOH) at the FHIT locus in a subset of non-smoking tumours (7 cases out of 35 analyzed), so we hypothesized that a genomic instability associated with p53 mutations could be the cause of FHIT LOH in these neoplasms. To test this hypothesis, in the same panel of tumours, we investigated the presence of LOH at 7 other microsatellite loci located on different chromosomes. Interestingly we found that all of the tumours with p53 alterations and LOH at the FHIT locus showed loss of heterozygosity at all of the informative tested loci. This association was statistically significant (p=0.0001). Our data indicate the presence of a generalized genomic instability in this subset of lung tumours. Since p53 alterations were mostly G:C --> A:T transitions and frameshift deletions, we are tempted to hypothesize that the genomic instability observed in non-smoking patients could be caused by particular p53 alterations. In fact, other kind of p53 mutations (G:C --> T:A transversions), frequently found in a series of 35 tumours of smokers used as control, were not associated with LOH at microsatellites loci. However, we cannot exclude that p53 alterations are a consequence and not the cause of the genomic instability. In this case, we have to admit that a gene(s), upstream of p53, is implicated in genome destabilisation in a subset of lung adenocarcinomas developed in non-smoking patients.

Induction of beta-R1/I-TAC by interferon-beta requires catalytically active TYK2.

The beta-R1/I-TAC (interferon-inducible T-cell alpha-chemoattractant) gene encodes an alpha-chemokine that is a potent chemoattractant for activated T-cells. We previously reported that beta-R1 was selectively induced by interferon (IFN)-beta compared with IFN-alpha and that the canonical type I IFN transcription factor interferon-stimulated gene factor 3 (ISGF3) was necessary but not sufficient for beta-R1 induction by IFN-beta. These findings suggested that beta-R1 induction by IFN-beta required an accessory component. To begin characterizing this signaling pathway, we examined the function of TYK2 protein in the IFN-beta-mediated induction of beta-R1. This study was motivated by the observation that beta-R1 could not be induced in TYK2-deficient U1 cells by IFN-beta (Rani, M. R. S., Foster, G. R., Leung, S., Leaman, D., Stark, G. R., and Ransohoff, R. M. (1996) J. Biol. Chem. 271, 22878-22884), an unexpected result because IFN-beta evokes substantial expression of IFN-stimulated genes (ISGs) in U1 cells through a TYK2-independent pathway. We now report beta-R1 expression patterns in U1 cells complemented with wild-type or mutant TYK2 proteins. Complementation with wild-type TYK2 rescued IFN-beta-inducible expression of beta-R1. Cells expressing kinase-deficient deletion or point mutants of TYK2 were refractory to induction of beta-R1 by IFN-beta despite robust expression of other ISGs. Transient transfection analysis of a beta-R1 promoter-reporter confirmed that transcriptional activation of beta-R1 by IFN-beta required competent TYK2 kinase. These studies indicate that the catalytic function of TYK2 is required for IFN-beta-mediated induction of beta-R1. Catalytic TYK2 is the first identified component in an accessory signaling pathway that supplements ISGF3/interferon-stimulated response element signaling for gene induction by type I IFNs.