A Copernican revolution of multigenic analysis: A retrospective study on clinical exome sequencing in unclear genetic disorders.

Despite the inevitable shift in medical practice towards a deeper understanding of disease etiology and progression through multigenic analysis, the profound historical impact of Mendelian diseases cannot be overlooked. These diseases, such as cystic fibrosis and thalassemia, are characterized by a single variant in a single gene leading to clinical conditions, and have significantly shaped our medical knowledge and treatments. In this respect, the monogenic approach inevitably results in the underutilization of Next-Generation Sequencing (NGS) data. Herein, a retrospective study was performed to assess the diagnostic value of the clinical exome in 32 probands with specific phenotypic characteristics (patients with autoinflammation and immunological dysregulation, N = 20; patients diagnosed with Hemolytic uremic syndrome N = 9; and patients with Waldenström macroglobulinemia, N = 3). A gene enrichment analysis was performed using the *. VCF file generated by SOPHiA-DDM-v4. This analysis selected a subset of genes containing pathogenic or likely pathogenic variants with autosomal dominant (AD) inheritance. In addition, all variants of uncertain significance (VUS) were included, filtered by AD inheritance mode, the presence of compound heterozygotes, and a minor allele frequency (MAF) cutoff of 0.05 %. The aim of the pipeline described here is based on a perspective shift that focuses on analyzing patients' gene assets, offering new light on the complex interplay between genetics and disease presentation. Integrating this approach into clinical practices could significantly enhance the management of patients with rare genetic disorders.

The treatment with Levonorgestrel Releasing Intrauterine System (LNG-IUS) in patients affected by menometrorrhagia, dysmenorrhea and adenomimyois: clinical and ultrasonographic reports.

OBJECTIVE: Adenomyosis is the consequence of the myometrial invasion by endometrial glands and stroma. Transvaginal ultrasonography plays a decisive role in the diagnosis and monitoring of this pathology. Our study aims to evaluate the efficacy of LNG-IUS (Levonorgestrel Releasing Intrauterine System) as medical therapy. We analyzed both clinical symptoms and ultrasonographic aspects of menometrorrhagia and dysmenorrhea in patients with adenomyosis and the control group. PATIENTS AND METHODS: A prospective cohort study was carried out on 28 patients suffering from symptomatic adenomyosis treated with LNG-IUS. Adenomyosis was diagnosed through transvaginal ultrasonography by an expert sonographer. A control group of 27 symptomatic patients (menorrhagia and dysmenorrhea) without a transvaginal ultrasonographic diagnosis of adenomyosis was treated in the same way. The two cohorts were compared to the efficacy of LNG-IUS on menorrhagia and dysmenorrhea.  Patients are evaluated at the time of LNG-IUS insertion and six months after for: increased uterine volume, globulous uterine morphology, uterine symmetry, alterations in the junctional zone, heterogeneous myometrial texture, presence of myometrial cysts, hyperechogenic lines crossing the myometrium, adenomyomas, menstrual blood loss and dysmenorrhea. RESULTS: After six months, the uterine volume decreased significantly in both cohorts (p=0.005; p=0.005). Furthermore, uterine symmetry, visibility of the junctional zone, heterogeneity of myometrial texture, presence of myometrial cysts, hyperechogenic lines and adenomyomas improved in patients affected by adenomyosis (p>0.001; p>0.001; p>0.001; p=0.014; p=0.025; p=0.014). The blood loss decreased significantly in both the cohorts (p<0.001) and particularly in adenomyotic patients. Pain relief was observed in all the patients (p<0.001). CONCLUSIONS: LNG-IUS can be considered an effective treatment for managing symptoms and improving uterine morphology.

Effects of dietary Tenebrio molitor meal inclusion in free-range chickens.

Insects are currently being considered as a novel protein source for animal feeds, because they contain a large amount of protein. The larvae of Tenebrio molitor (TM) have been shown to be an acceptable protein source for broiler chickens in terms of growth performance, but till now, no data on histological or intestinal morphometric features have been reported. This study has had the aim of evaluating the effects of dietary TM inclusion on the performance, welfare, intestinal morphology and histological features of free-range chickens. A total of 140 medium-growing hybrid female chickens were free-range reared and randomly allotted to two dietary treatments: (i) a control group and (ii) a TM group, in which TM meal was included at 75 g/kg. Each group consisted of five pens as replicates, with 14 chicks per pen. Growth performance, haematological and serum parameters and welfare indicators were evaluated, and the animals were slaughtered at the age of 97 days. Two birds per pen (10 birds/treatment) were submitted to histological (liver, spleen, thymus, bursa of Fabricius, kidney, heart, glandular stomach and gut) and morphometric (duodenum, jejunum and ileum) investigations. The inclusion of TM did not affect the growth performance, haematological or serum parameters. The morphometric and histological features were not significantly affected either, thus suggesting no influence on nutrient metabolization, performance or animal health. Glandular stomach alterations (chronic flogosis with epithelial squamous metaplasia) were considered paraphysiological in relation to free-range farming. The observed chronic intestinal flogosis, with concomitant activation of the lymphoid tissue, was probably due to previous parasitic infections, which are very frequently detected in free-range chickens. In conclusion, the findings of this study show that yellow mealworm inclusion does not affect the welfare, productive performances or morphological features of free-range chickens, thus confirming that TM can be used safely in poultry diets.

The Aquamantys® system as alternative for parenchymal division and hemostasis in liver resection for hepatocellular carcinoma: a preliminary study.

OBJECTIVE: To evaluate the clinical feasibility and safety of a new technique for liver resection using a new saline-coupled bipolar sealing device (Aquamantys®) that has shown high performance in the animal setting. PATIENTS AND METHODS: Twelve Child-Pugh A cirrhotic patients with hepatocellular carcinoma underwent partial hepatectomies using Aquamantys®. Our primary end-point was to observe occurrence of early specific surgical complications as bleeding, biliary leakage and abscess development. Our secondary end-point was to evaluate local recurrence along resection margin after a minimum follow-up of 1 year. RESULTS: One bisegmentectomy, five monosegmentectomies and six atypical resections were performed. Mean resection time was 45 minutes (range, 30-100 min). Mean blood loss was 20 mL (range 5-80 mL). Mean post-operative stay was 6 days (range 5-16 days). All specimens presented negative margins (R0) at pathological examination. No blood transfusion were required both intra-operatively and post-operatively. No mortality was observed within 30-days post-operatively. One fluid collection occurred after  6-7 bisegmentectomy and was successfully treated by ultrasound-guided percutaneous drainage. At 1 year follow-up two patients died: one because of new lesions into the liver and one because of distant metastases and multifocal new liver disease. Ten patient are alive disease free at 1 year follow-up. CONCLUSIONS: Liver resection using Aquamantys® is feasible and safe and allows to achieve almost bloodless parenchymal division with minimal necrosis and negative margins even in atipycal resection. Comparative trials are needed to confirm our preliminary results.

Solitary fibrous tumour of the pancreas: a case report.

The solitary fibrous tumour is an uncommon neoplasm of mesenchymal origin. It occasionally develops in extra pleural locations, and is very infrequent in the pancreas. A 40-year-old woman had an unsuspected solitary fibrous tumour detected by chance in the body of the pancreas. Detection of the mass occurred with imaging studies. CT and MRI images showed the well-circumscribed benign lesion, with 3 cm on its greatest diameter. Treatment consisted of partial pancreatectomy, and her postoperative recovery was uneventful.

Notch-1 stimulates survival of lung adenocarcinoma cells during hypoxia by activating the IGF-1R pathway.

Hypoxic microenvironment supports cancer stem cell survival, causes poor response to anticancer therapy and tumor recurrence. Inhibition of Notch-1 signaling in adenocarcinoma of the lung (ACL) cells causes apoptosis specifically under hypoxia. Here, we found that Akt-1 activation is a key mediator of Notch-1 pro-survival effects under hypoxia. Notch-1 activates Akt-1 through repression of phosphatase and tensin (PTEN) homolog expression and induction of the insulin-like growth factor 1 receptor (IGF-1R). The latter seems to be the major determinant of Akt-1 stimulation, as Notch-1 signaling affects Akt-1 activation in PTEN(-/-) ACL cells. Both downregulation of insulin receptor substrate 1 (IRS-1) and dominant-negative IGF-1R sensitized ACL cells to gamma-secretase inhibitor (GSI)-induced apoptosis. Conversely, overexpression of IGF-1R protected ACL cells from GSI toxicity. Inhibition of Notch-1 caused reduced IGF-1R expression, whereas forced Notch-1 expression yielded opposite effects. Chromatin immunoprecipitation experiments suggested Notch-1 direct regulation of the IGF-1R promoter. Experiments in which human ACL cells were injected in mice confirmed elevated and specific co-expression of Notch-1(IC), IGF-1R and pAkt-1 in hypoxic tumor areas. Our data provide a mechanistic explanation for Notch-1-mediated pro-survival function in hypoxic ACL tumor microenvironment. The results identify additional targets that may synergize with Notch-1 inhibition for ACL treatment.

Acute phase response in porcine reproductive and respiratory syndrome virus infection.

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1ß was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.

A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF.

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14(ARF). Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14(ARF) in a 'dose-dependent tug of war' fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14(ARF)) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14(ARF)) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.

Outcome analysis of breast cancer patients receiving breast-conserving surgery in Southern Italy.

The purpose of the current analysis was to evaluate the outcome of patients enrolled at the National Cancer Institute of Naples between 1997 and 2000, who underwent breast-conserving surgery. Between January 1997 and December 2000, 946 patients had been diagnosed with T1 or T2 (<3 cm) breast carcinoma. At the time of the present analysis (31-12-2005), all patients had been followed for >5 years. A Cox proportional hazards model was performed. Overall, 7-year Locoregional Relapse-free survival (LRFS) and Distant Relapse-free Survival (DRFS) rates were 95.9% and 88.4%, respectively. Seven-year DRFS was 91.2% and 79.3% in T1 and T2 stage, respectively (p<0.0001). Multivariate Cox analysis indicated that number of positive lymph-nodes and hormone receptor status were significantly associated with prognosis. Our findings confirm that early diagnosed breast cancer, treated with breast-conserving surgery, is associated with a very good prognosis in patients referred to an Institution which may be considered as representative of similar Cancer Institutes of Southern Italy. The risk of local relapse was found to be very low (4%), although a longer follow-up is needed to draw definitive conclusions.

Bochdaleck diaphragmatic hernia, complicated by an antenatal gastric perforation, presenting as a pneumothorax and a perforative peritonitis.

The presentation of congenital diaphragmatic hernia (CDH) at birth may fall outside the typical features (cyanosis, tachypnea and respiratory failure), manifesting, instead, also with others pictures that make the diagnosis difficult or even impossible. We report a case of CDH presenting as a pneumothorax and a perforative peritonitis due to an antenatal gastric perforation.

Born to be exported: COOH-terminal nuclear export signals of different strength ensure cytoplasmic accumulation of nucleophosmin leukemic mutants.

Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.

Imported malaria at Italy's National Institute for Infectious Diseases Lazzaro Spallanzani, 1984-2003.

Imported malaria is the most common cause of fatal infections in returning travellers. The increased amount of both tourist movement and migration has resulted in a growing number of people at risk of infection. In the present study, 507 malaria patients admitted to Italy's National Institute for Infectious Diseases in Rome between January 1984 and December 2003 were studied. Overall, 445 cases, or 87.7%, were acquired in Africa, of which 55% were acquired in five sub-Saharan countries. Plasmodium falciparum accounted for 393 (77.5%) of the imported cases. Patients consisted of short-term travellers (n = 213, 42%), long-term visitors (n = 134, 26.4%), and immigrants from endemic areas (n = 137, 27%). Malaria chemoprophylaxis was completed in less than one-quarter of all patients, with immigrants having the lowest rate of completion: only 3.6% of immigrants fully completed chemoprophylaxis compared to 31% of short-term travellers and 29.1% of long-term visitors (p < 0.001). Upon multivariate analysis, the lack of chemoprophylaxis was independently associated with the occurrence of severe malaria (p = 0.009). Severe malaria was reported in 59 (11.6%) individuals: all 11 deaths due to severe P. falciparum infection occurred in patients from sub-Saharan countries, two of whom were immigrants from countries where malaria is endemic. Malaria poses a serious health threat to individuals visiting endemic areas. Ensuring the correct chemoprophylaxis for all travellers, including immigrants from endemic areas, and providing prompt access to healthcare providers for unhealthy returning travellers are major points still to be addressed in Italy.

An immunohistochemical study of the tonsils in pigs with acute African swine fever virus infection.

An immunohistochemical study of the tonsils was carried out to gain further insight in the pathogenesis of acute African swine fever (ASF). Twenty-one pigs were inoculated by intramuscular route with a highly virulent isolate of ASF virus and painlessly killed at 1-7dpi. Viral antigen was highly distributed in the tonsil from 3 to 4dpi and an increase in the number of monocyte-macrophages was very evident at the same days post inoculation. This phenomenon was observed together with an increase of the expression of proinflammatory cytokines (Tumour necrosis factor alpha and Interleukin-1 alpha) and the apoptosis of lymphocytes studied by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) technique and haemorrhages. With these results, we can conclude that the tonsil is suffering similar lesions than those observed in other lymphoid organs in acute African swine fever, even when the route of inoculation is the intramuscular and not oral-nasal.

Palliative antecolic isoperistaltic gastrojejunostomy: a randomized controlled trial comparing open and laparoscopic approaches.

Gastric outlet obstruction is a common, often preterminal, event for patients with inoperable neoplasms of the distal stomach, duodenum, and biliopancreatic area. It can be surgically managed by open or laparoscopic gastrojejunostomy. This study aimed to compare the results of open and laparoscopic palliative gastrojejunostomy for patients with gastric outlet obstruction resulting from inoperable neoplasms. A total of 24 patients were randomized prospectively to undergo laparoscopic (12 patients) or open (12 patients) palliative laterolateral antecolic isoperistaltic gastrojejunostomy. All the procedures were completed as planned. The mean duration of surgery was not significantly different between the two groups (p = 0.75). The mean intraoperative blood loss was significantly less after laparoscopic gastrojejunostomy (LGJ) (p = 0.0001). Time to oral solid food intake was longer after open gastrojejunostomy (OGJ) (p = 0.04). Two patients in the OGJ group experienced postoperative delayed gastric empting, whereas no patients in the LGJ group experienced such a complication (p = 0.04). The mean postoperative stay was shorter in the LGJ group, but the difference did not reach statistical significance (p = 0.65). No readmissions were registered after a minimum follow-up period of 2 months. The findings show that LGJ is a safe, feasible, and effective alternative to OGJ. However, because the current data involved only a small number of patients, large studies still are required for further evaluation of the this operation's effectiveness.

Endogenous lipid pneumonia in a dog.

Endogenous lipid pneumonia has previously been diagnosed in dogs only once. This report describes a case in a dog with a persistent cough, in which the histological diagnosis was based on the presence of numerous foamy macrophages that filled the alveoli and contained small sudanophilic vacuoles. The appearance of endogenous lipid pneumonia in this animal was accompanied by Dirofilaria immitis infection and chronic bronchitis.

Evaluation of stray radiofrequency radiation emitted by electrosurgical devices.

Electrosurgery refers to the passage of a high-frequency, high-voltage electrical current through the body to achieve the desired surgical effects. At the same time, these procedures are accompanied by a general increase of the electromagnetic field in an operating room that may expose both patients and personnel to relatively high levels of radiofrequency radiation. In the first part of this study, we have taken into account the radiation emitted by different monopolar electrosurgical devices, evaluating the electromagnetic field strength delivered by an electrosurgical handle and straying from units and other electrosurgical accessories. As a summary, in the worst case a surgeon's hands are exposed to a continuous and pulsed RF wave whose magnetic field strength is 0.75 A m(-1) (E-field 400 V m(-1)). Occasionally stray radiation may exceed ICNIRP's occupational exposure guidelines, especially close to the patient return plate. In the second part of this paper, we have analysed areas of particular concern to prevent electromagnetic interference with some life-support devices (ventilators and electrocardiographic devices), which have failed to operate correctly. Most clinically relevant interference occurred when an electrosurgery device was used within 0.3 m of medical equipment. In the appendix, we suggest some practical recommendations intended to minimize the potential for electromagnetic hazards due to therapeutic application of RF energy.

Lymphocyte apoptosis and thrombocytopenia in spleen during classical swine fever: role of macrophages and cytokines.

Thirty-two Large White x Landrace pigs, 4 months old, were inoculated with the classical swine fever (CSF) or hog cholera virus strain "Alfort" in order to identify the mechanism responsible for the lymphopenia and thrombocytopenia observed in the spleen during the experimental induction of disease, by immunohistochemical and ultrastructural techniques. Results showed a progressive depletion of splenic lymphoid structures and evidence of platelet aggregation processes. Lymphoid depletion was due to lymphocyte apoptosis, which could not be ascribed to the direct action of the virus on these cells; direct virus action could play only a secondary role in the death of these cells. Absence of severe tissue and endothelial damage, together with moderate procoagulant cytokine levels in the serum, suggest that these pathologies can be ruled out as the cause of platelet aggregation and thrombocytopenia in CSF. Monocyte/macrophages were the main target cells for the CSF virus, and they exhibited phagocytic and secretory activation leading to the synthesis and release of tumor necrosis factor alpha, which proved to be the chief mediator, followed by IL-6, IL-1alpha, and C1q complement component. In view of their characteristics, TNF-alpha and, to a lesser extent, IL-1alpha and IL-6 appear to be the major cytokines involved in the pathogenesis of lymphocytopenia and thrombocytopenia; a clear spatial and temporal relationship was observed between these two phenomena.

Proinflammatory cytokines induce lymphocyte apoptosis in acute African swine fever infection.

Twenty-one pigs inoculated with a highly virulent isolate (E70) of African swine fever (ASF) virus were killed 1-7 days later; a further three animals served as uninfected controls. An early increase in TNF-alpha, IL-1alpha, IL-1beta and IL-6 expression was detected in lymphoid organs from infected animals, together with an increase in the serum concentrations of TNF-alpha and IL-1beta. These changes were accompanied by increased apoptosis of lymphocytes, and the presence of infected and uninfected macrophages showing changes indicative of secretory and phagocytic activation. The present study demonstrated an increase in the number of macrophages expressing TNF-alpha, IL-1 and IL-6 in proximity to lymphocytes undergoing apoptosis, supporting previous suggestions that in acute ASF proinflammatory cytokines induce lymphocyte apoptosis.