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Background: A new strain of WNV lineage 1 (WNV - 1) emerged in the Veneto Region, northern Italy, in 2021, eight years after the last outbreak of WNV - 1 in Italy. The virus, which co-circulates with WNV-2, has become endemic in the Region, where, in 2022, most human cases of neuroinvasive disease (WNND) reported in Europe have occurred., Methods: Comparative analysis of the epidemiology and clinical presentation of WNV-1 and WNV-2 infection in humans, as well as the temporal and geographic distribution of WNV-1 and WNV-2 among wild birds and Culex pipiens mosquitoes in Veneto, from May 16th to August 21st, 2022, to determine if the high number of WNND cases was associated with WNV-1., Results: As of August 21st, 2022, 222 human cases of WNV infection were confirmed by molecular testing, including 103 with fever (WNF) and 119 with WNND. WNV lineage was determined in 201 (90.5%) cases, comprising 138 WNV-1 and 63 WNV-2 infections. During the same period, 35 blood donors tested positive, including 30 in whom WNV lineage was determined (13 WNV-1 and 17 WNV-2). Comparative analysis of the distribution of WNV-1 and WNV-2 infections among WNND cases, WNF cases and WNV-positive blood donors showed that patients with WNND were more likely to have WNV-1 infection than blood donors (odds ratio 3.44; 95% CI 95% 1.54 to 8.24; p = 0.0043). As observed in humans, in wild birds WNV-1 had higher infectious rate (IR) and showed a more rapid expansion than WNV-2. At variance, the distribution of the two lineages was more even in mosquitoes, but with a trend of rapid increase of WNV-1 IR over WNV-2., Conclusions: Comparative analysis of WNV-1 vs WNV-2 infection in humans, wild birds, and mosquitos showed a rapid expansion of WNV-1 and suggested that WNV-1 infected patients might have an increased risk to develop severe disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of International Society of Travel Medicine.)

Background: In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy., Methods: Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood., Results: The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014-2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site., Conclusions: Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of International Society of Travel Medicine.)

USUTU virus (USUV) is an arbovirus maintained in the environment through a bird-mosquito enzootic cycle. Previous surveillance plans highlighted the endemicity of USUV in North-eastern Italy. In this work, we sequenced 138 new USUV full genomes from mosquito pools ( Culex pipiens ) and wild birds collected in North-eastern Italy and we investigated the evolutionary processes (phylogenetic analysis, selection pressure and evolutionary time-scale analysis) and spatial spread of USUV strains circulating in the European context and in Italy, with a particular focus on North-eastern Italy. Our results confirmed the circulation of viruses belonging to four different lineages in Italy (EU1, EU2, EU3 and EU4), with the newly sequenced viruses from the North-eastern regions, Veneto and Friuli Venezia Giulia, belonging to the EU2 lineage and clustering into two different sub-lineages, EU2-A and EU2-B. Specific mutations characterize each European lineage and geographic location seem to have shaped their phylogenetic structure. By investigating the spatial spread in Europe, we were able to show that Italy acted mainly as donor of USUV to neighbouring countries. At a national level, we identified two geographical clusters mainly circulating in Northern and North-western Italy, spreading both northward and southward. Our analyses provide important information on the spatial and evolutionary dynamics of USUTU virus that can help to improve surveillance plans and control strategies for this virus of increasing concern for human health., (© The Author(s) 2021. Published by Oxford University Press.)

Infection by human papillomavirus (HPV) represents one of the most common sexually transmitted diseases in both men and women worldwide. Recently, the detection of HPV virions in the semen of a large percentage of sexually active men has been associated with detrimental effects on both sperm parameters and on assisted reproductive technologies (ART) treatment outcomes. Conventional semen washing procedure used in ART have proved to be ineffective in removing HPV bound to sperm, requiring the identification of more effective and specific methods. In the present study, we assessed the possible use of hyaluronidase for the detachment of HPV from sperm cell surface. Semen samples from five normozoospermic control subjects (CTRL) were incubated with HPV virus-like particles (HPV-VLP) and treated with hyaluronidase by both a modified swim-up procedure (M-SU) and single-cell approach (SCA). The treatment with hyaluronidase was associated with the complete loss of HPV-VLP signal on sperms by both M-SU and SCA. In addition, semen samples from 12 HPV-positive infertile patients were treated with hyaluronidase 80 IU/mL by M-SU, resulting in the complete loss of HPV-DNA signal from sperm surface. Finally, the possible impact of hyaluronidase treatment on sperm parameters was assessed on both sperms from the five CTRL subjects and on further five oligo-astheno-terato-zoospermic (OAT) patients, both HPV negative. The treatment with hyaluronidase was equally associated with a slight reduction of sperm viability and progressive motility in both CTRL and OAT. In conclusion, the treatment with hyaluronidase removed efficiently and safely HPV virions bound to spermatozoa., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: To improve our understanding of the natural history of Zika virus (ZIKV) infection in humans, we described the dynamics of ZIKV RNA shedding in different body fluids and antibody responses in patients with acute infection., Methods: Twenty-nine adults with travel-associated infection and 1 case of sexual transmission were enrolled and followed up with weekly ZIKV RNA testing in blood, urine, saliva, and semen samples and antibody testing., Results: ZIKV RNA was detected in plasma, urine, and saliva of 57%, 93.1%, and 69.2% of participants, with estimated median times to clearance of 11.5 days (interquartile range [IQR] 6-24 days), 24 days (IQR, 17-34), and 14 days (IQR, 8-31), respectively. In 2 pregnant women, ZIKV RNA persisted in blood until delivery of apparently healthy infants. ZIKV RNA was detected in semen of 5 of 10 tested men; median time to clearance was 25 days (IQR 14-29), and the longest time of shedding in semen was 370 days. In flavivirus-naive patients, the median times to detection of ZIKV nonstructural protein 1 (NS1)-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies were estimated as 8 days (IQR, 5-15 days) and 17 days (IQR, 12-26 days), respectively. ZIKV NS1 IgM antibodies were undetectable in patients with previous dengue., Conclusions: Prolonged viremia and ZIKV RNA shedding in urine, saliva, and semen occur frequently in patients with acute ZIKV infection. At the time of diagnosis, about half of patients are ZIKV IgM negative. ZIKV NS1 IgM antibodies remain undetectable in patients with previous dengue. Estimates of the times to viral clearance and seroconversion are useful to optimize diagnostic algorithms.

Zika virus is a mosquito-borne flavivirus discovered in Uganda in 1947. The virus has emerged in recent years and spread in the Pacific Area and the Americas, where it has caused large human outbreaks. The factors involved in the virus's emergence are still unknown, but probably include its introduction in naïve environments characterised by the presence of high densities of competent Aedes spp. mosquitoes and susceptible human hosts in urban areas. Unique features of Zika virus infection are sexual and transplacental transmission and associated neurological morbidities, i.e. Guillain-Barré syndrome and fetal microcephaly. Diagnosis relies on the detection of viral nucleic acids in biological samples, while detection of a specific antibody response may be inconclusive because of the broad cross-reactivity of antibodies among flaviviruses. Experimental studies have clarified some mechanisms of Zika virus pathogenesis and have identified potential targets for antiviral drugs. In animal models, the virus can infect and efficiently replicate in the placenta and in the brain, and induce fetal demise or neural damage, recapitulating human diseases. These animal models have been used to evaluate candidate vaccines and promising results have been obtained., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: In hematopoietic stem cell transplantation (HSCT), late hemorrhagic cystitis (HC) has been associated with BK virus (BKV) infection. We assessed the value of plasma BKV load in predicting HC., Methods: Plasma and urine BKV-DNA load were assessed prospectively in 107 pediatric patients., Results: Twenty patients developed grade II and III HC, with 100-day cumulative incidence of 18.8%. At diagnosis of HC, the median load of BKV DNA was 2.3 × 10(3) copies/mL. A plasma BKV-DNA load of 10(3) copies/mL had a sensitivity of 100% and a specificity of 86% with a negative predictive value (NPV) of 100% and a positive predictive value (PPV) of 39% for HC. A urine BKV-DNA load of >10(7) copies/mL had a sensitivity of 86% and a specificity of 60% with a NPV of 98% and a PPV of 14% for HC. A BKV load of 10(3) copies/mL on plasma was significantly associated with HC in multivariate analysis (hazard ratio [HR], 6.1; P = .0006). Patients with HC had a significantly higher risk of mortality than patients who did not have HC (HR, 2.6; P = .018)., Conclusions: The above values were used to monitor plasma BKV-DNA load, and they provided a better prediction of patients at risk of HC than urine BKV-DNA load., (© The Author 2014. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: The search for short words that are absent in the genome of one or more organisms (neverwords, also known as nullomers) is attracting growing interest because of the impact they may have in recent molecular biology applications. keeSeek is able to find absent sequences with primer-like features, which can be used as unique labels for exogenously inserted DNA fragments to recover their exact position into the genome using PCR techniques. The main differences with respect to previously developed tools for neverwords generation are (i) calculation of the distance from the reference genome, in terms of number of mismatches, and selection of the most distant sequences that will have a low probability to anneal unspecifically; (ii) application of a series of filters to discard candidates not suitable to be used as PCR primers. KeeSeek has been implemented in C++ and CUDA (Compute Unified Device Architecture) to work in a General-Purpose Computing on Graphics Processing Units (GPGPU) environment., Availability and Implementation: Freely available under the Q Public License at http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=keeseek., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Human papillomavirus (HPV) infection is a very common sexually transmitted disease. A variable concordance and clearance time between partners of infected heterosexual couples have been shown., Aim: Aims of this study were evaluation of prevalent sites of HPV infection, frequency, genotype concordance, and course of viral infection in counseled and non-counseled infected heterosexual couples., Methods: Prospective, controlled study among 74 consecutive HPV-infected heterosexual couples seeking their first medical intervention for sexually transmitted infections., Main Outcome Measures: HPV detection and genotyping were performed at enrollment and during 24 months follow-up by polymerase chain reaction and INNO-LiPA assay in samples obtained from both partners. Samples were collected in males from coronal sulcus, urethra, semen, and oropharynx, and from cervix and oropharynx in females. Moreover, HPV fluorescence in situ hybridization was analyzed on semen samples., Results: The study was completed by 49 couples. The specific HPV-type concordance among infected couples was 69.4%. Among couples who declared performing oral sex, oropharyngeal infection was 22.7% of couples, and was more prevalent in females than males (18.1% and 4.5%, respectively). Thereafter, couples were randomly divided in two groups: a control group of 24 couples followed up for HPV-related lesions, and an experimental group counseled of 25 couples followed up for HPV-related lesions and counseled to avoid known risk factors for HPV infection. In patients who received counseling, the number of infected sites diminished faster during the follow-up and a higher number of couples had clearance. Only in the counseled group were there no infected couples at the end of the 24-month follow-up period., Conclusions: This study confirms that HPV infection affects many sites in both males and females and shows a significant prevalence of oropharyngeal infection in couples performing oral sex. Targeted counseling in heterosexual couples appears to reduce the number of infected sites and to speed up viral clearance., (© 2013 International Society for Sexual Medicine.)

Detection of West Nile virus (WNV) RNA in urine has been anecdotally described and proposed for the diagnosis of WNV infection. This study reports the routine use of real-time reverse-transcription polymerase chain reaction for the detection of WNV RNA in urine to support diagnosis of WNV infection during the large outbreak that occurred in northeastern Italy in 2012. Fourteen of 32 patients (43.8%) with symptomatic WNV infection, defined as neuroinvasive disease and fever, had detectable WNV RNA in urine at the time of diagnosis, at a higher rate and load and for a longer time than detection of WNV RNA in blood. Detection of WNV RNA in urine was less frequent (2 of 14 patients [14.2%]) in blood donors in whom WNV infection was identified by WNV nucleic acid amplification testing. Infectious virus was isolated from the urine of a patient with neuroinvasive disease and a high WNV RNA load in urine.

Next-generation sequencing technologies have fostered an unprecedented proliferation of high-throughput sequencing projects and a concomitant development of novel algorithms for the assembly of short reads. In this context, an important issue is the need of a careful assessment of the accuracy of the assembly process. Here, we review the efficiency of a panel of assemblers, specifically designed to handle data from GS FLX 454 platform, on three bacterial data sets with different characteristics in terms of reads coverage and repeats content. Our aim is to investigate their strengths and weaknesses in the reconstruction of the reference genomes. In our benchmarking, we assess assemblers' performance, quantifying and characterizing assembly gaps and errors, and evaluating their ability to solve complex genomic regions containing repeats. The final goal of this analysis is to highlight pros and cons of each method, in order to provide the final user with general criteria for the right choice of the appropriate assembly strategy, depending on the specific needs. A further aspect we have explored is the relationship between coverage of a sequencing project and quality of the obtained results. The final outcome suggests that, for a good tradeoff between costs and results, the planned genome coverage of an experiment should not exceed 20-30 ×.

Background: The ultimate goal of organ transplantation is the reestablishment of organ function and the restoration of a solid immunity to prevent the assault of potentially deadly pathogens. T cell immunity is crucial in controlling cytomegalovirus (CMV) infection. It is still unknown how preexisting antiviral T cell levels, prophylaxis, or preemptive antiviral strategies and pharmacological conditioning affect immune reconstitution., Methods: Seventy preemptively treated CMV-seropositive recipients, 13 prophylaxis-treated CMV-seronegative recipients of seropositive donor transplants, 2 seropositive recipients of seronegative donor kidneys, and 27 pretransplant subjects were enrolled in a cross-sectional study and analyzed for CMV viremia (DNAemia) and CMV-specific T cell response (interferon-gamma enzyme-linked immunospot assay) before transplantation and at 30, 60, 90, 180, and 360 days after transplantation., Results: CMV-seropositive transplant recipients displayed a progressive but heterogeneous pattern of immune reconstitution starting from day 60 after transplantation. CMV-seronegative recipients did not mount a detectable T cell response throughout the prophylaxis regimen. A single episode of CMV viremia (CMV copy number, 7000-170,000 copies/mL) was sufficient to prime a protective T cell immune response in CMV-seronegative recipients. Antithymocyte globulin treatment did not significantly affect CMV-specific T cell response., Conclusions: Baseline immunity, antiviral therapy but not antithymocyte globulin treatments profoundly influence T cell reconstitution in kidney transplant recipients.

The polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified mainly in respiratory samples from children and immunosuppressed patients. Investigation of 54 autopsy brain tissue samples from 22 subjects demonstrated that WUPyV DNA and KIPyV DNA could be detected in 1 of 4 human immunodeficiency virus (HIV)-positive individuals with progressive multifocal leukoencephalopathy (PML) and in 3 of 10 HIV-positive individuals without PML, but not in 8 HIV-negative individuals. Viruses were localized in all regions of the central nervous system that were analyzed, that is, the cerebral hemispheres, cerebellum, pons, and medulla oblongata. No specific histopathological findings were found to be associated with the presence of WUPyV and KIPyV.

Background: In pediatric kidney transplant recipients, viral infections occur soon after transplant and may be transmitted from the graft., Methods: This study of 75 pediatric kidney transplants investigated whether genome sequences of parvovirus B19, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and BK polyomavirus (BKV) could be detected in kidney graft samples (graft biopsy samples and preservation and washing solutions) collected before implantation and whether their presence was a risk factor for infections in the recipient., Results: B19 DNA was detected in approximately 30% of graft biopsy samples, preservation solutions, and washing solutions; EBV DNA was detected in approximately 20% of preservation and washing solutions but rarely in biopsy samples; and HCMV DNA and BKV DNA were rarely detected in graft biopsy samples. Seronegative recipients of B19 DNA-positive and EBV DNA-positive grafts had a significantly higher risk of infection during the early posttransplant period than did recipients of negative grafts. In particular, none of the B19-seronegative recipients of B19 DNA-negative grafts experienced infection soon after transplant, whereas most recipients of B19 DNA-positive grafts experienced infection within the first month after transplant., Conclusions: Molecular testing of donor grafts for viruses that infect circulating and resident cells in the graft-such as B19 in the kidney-could be useful (in association with donor/recipient serostatus) for identifying recipients at high risk for posttransplant infections.

Background: The relevance of viral infections in the development of allograft lesions is still unclear, although some viruses have been implicated. The present study investigated systemic and intrarenal viral infections in kidney transplant recipients and their association with the risk of acute rejection and chronic allograft injuries that are predictive of long-term dysfunction., Methods: The presence of DNA sequences of human herpesviruses, polyomaviruses, and parvovirus B19 was analyzed in renal allograft biopsy specimens obtained at baseline, after acute renal dysfunction, and during follow-up evaluation in 69 transplant recipients who were children or young adults. Results were correlated with clinical data, viral DNAemia, and results of renal function tests and allograft histology analyzed at the same time points., Results: Overall, viral DNA was detectable in 46% of baseline and 70% of follow-up biopsy specimens of kidney allografts, where it generally persisted. The most frequently detected viruses were B19 and human herpesvirus 6, already present in donor kidneys, and BK virus and Epstein-Barr virus, usually involving the allograft during follow-up. Among viruses, only the intrarenal persistence of B19 DNA and B19 DNAemia was associated with the development of chronic allograft injury, whereas human cytomegalovirus DNAemia was a risk factor for acute rejection., Conclusions: Parvovirus B19 seems to target the kidney electively. Its intrarenal persistence is associated with chronic kidney allograft injury.

Introduction: Activating mutations of the BRAF oncogene are frequently detected in papillary thyroid carcinoma (PTC) and have been associated with a worse prognosis. The amino acid substitution V600E accounts for 90% of all oncogenic BRAF mutations and is typically detected in classic PTCs, whereas other less frequent BRAF mutations seem to be associated with other PTC histotypes., Case: Screening for activating BRAF mutations in a series of 83 PTCs identified the most common V600E mutation in 39 cases (histologically, 38 classic PTCs and 1 sclerosing variant PTC) and a complex in-frame mutation involving amino acids V600-S605 in a stage III multicentric follicular variant PTC, occurring in a 50-year-old female patient, who was affected by hypothyroidism in autoimmune thyroiditis and had a family history of PTC and autoimmune thyroiditis. Since the identified BRAF mutation was novel in the literature, bioinformatic modeling was performed to predict its impact on BRAF activity. Although the mutation resulted in loss of a phosphorylation site in the activation loop of BRAF, it was predicted to increase BRAF kinase activity by mimicking an activating phosphorylation., Conclusions: This study, which reports a new BRAF mutation, highlights the usefulness of bioinformatic modeling in the prediction of functional effects of new mutations and indicates that mutation-specific screening tests might miss some rare BRAF mutations. These facts should be taken into consideration in the molecular diagnosis of thyroid cancer and in the design of therapeutic protocols based on inhibitors of the BRAF pathway.

Objective: Adrenocortical tumors may originate from the zona glomerulosa, zona fasciculata, or zona reticularis and be associated with syndromes due to overproduction of mineralocorticoids, glucocorticoids, or androgens respectively. We report an unusual case of recurrent adrenocortical carcinoma (ACC), which seems to contradict the paradigm of functional adrenal zonation., Case Report: A male patient presented with severe primary aldosteronism due to an ACC, which relapsed after adrenalectomy and adjuvant mitotane therapy. After removal of the tumor recurrence and eight cycles of chemotherapy with etoposide, doxorubicin and cisplatin, the patient presented again with ACC masses, but in association with overt Cushing's syndrome and normal aldosterone levels., Methods and Results: Extensive pathologic examination showed that this shift in steroid hormone production was paralleled by an attenuation of tumor cell atypia and polymorphism, whereas gene expression profile analysis demonstrated a change in expression of adrenal steroidogenic enzymes. Moreover, cancer progression was associated with overexpression of the inhibin-alpha subunit, which could have contributed to the phenotypic changes., Conclusions: This case of recurrent ACC demonstrates that adrenocortical cells can reverse their differentiation program during neoplastic progression and change their specific hormone synthesis, as a consequence of modifications in the expression profile of steroidogenic enzymes and cofactors. We hypothesize that this shift in steroid hormone secretion is a consequence of chromosome amplification induced by chemotherapy. These findings, besides opening new perspectives to study adrenocortical cell plasticity and potential, demonstrate how conventional clinical and pathologic evaluation can be combined with genomic analysis in order to dissect thoroughly the biology of cancer.

This study represents the first report of gene therapy for anaplastic thyroid carcinoma, one of the most aggressive solid tumors in humans. Two patients with end-stage anaplastic thyroid carcinoma were treated by direct intratumor injection of retroviral vector producer cells followed by ganciclovir. The retroviral vector carried the human IL-2 gene and the suicide gene thymidine kinase of herpes simplex virus type 1. Treatment was safe and associated with only mild adverse events. Transduction of tumor cells and production of T helper type 1 cytokines was demonstrated in tumor biopsies. Gene therapy led also to a marked increase in T helper type 1 cytokine expression in peripheral blood mononuclear cells. Radiological evaluation of injected tumor masses demonstrated local tumor necrosis.

The field of cancer gene therapy is in continuous expansion, and technology is quickly moving ahead as far as gene targeting and regulation of gene expression are concerned. This review focuses on the endocrine aspects of gene therapy, including the possibility to exploit hormone and hormone receptor functions for regulating therapeutic gene expression, the use of endocrine-specific genes as new therapeutic tools, the effects of viral vector delivery and transgene expression on the endocrine system, and the endocrine response to viral vector delivery. Present ethical concerns of gene therapy and the risk of germ cell transduction are also discussed, along with potential lines of innovation to improve cell and gene targeting.

Clinically silent adrenal masses discovered by imaging studies performed for unrelated reasons, i.e. adrenal incidentalomas, have become a rather common finding in clinical practice. However, only limited studies of incidence, prevalence, and natural history of adrenal incidentalomas are available. A comprehensive review of the literature shows the prevalence of adrenal incidentalomas to be 2.3% at autopsy and 0.5-2% at abdominal computed tomography scan. Most lesions are adrenocortical adenomas at histology, whereas the prevalence of adrenocortical carcinomas is relatively low. The risk of malignancy over time for masses defined as benign at diagnosis is estimated at about 1/1000, even though 5-25% of masses increase in size during follow-up. Hyperfunction develops in about 1.7% of cases and the risk is higher in patients with lesions larger than 3 cm. Cortisol hypersecretion is the most likely disorder that may ensue, and it remains subclinical in about two-thirds of cases. The lack of controlled studies precludes making specific management recommendations. Large perspective controlled studies to define the epidemiology, natural history, and possible associated morbidity of adrenal incidentalomas and their impact on the quality of life of patients are needed.

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome linked to mutations in the MEN1 gene, which encodes a 610-amino-acid nuclear protein termed menin. Because of the lack of a suitable detection protocol, the in situ expression pattern of menin in human tissues remains to be determined. In this study, we have developed an antimenin monoclonal antibody and an indirect immunofluorescence/laser-scanning microscopy protocol for analyzing menin expression in frozen tissue sections. Because neuroendocrine pancreatic tumors represent a key feature of MEN1, we focused this study on nontumoral pancreas and a small panel of neuroendocrine pancreatic tumors. Results showed that menin was readily detected in nontumoral exocrine cells. In contrast, most islet cells expressing insulin, glucagon, or somatostatin showed considerably weaker levels of menin expression; however, a subpopulation of pancreatic polypeptide-positive cells exhibited a signal comparable with that detected in adjacent exocrine cells. Sporadic endocrine tumors showed variable levels of menin expression, whereas a MEN1-/- gastrinoma scored negative. This report thus provides the first description of the expression pattern of menin in human pancreas in situ and lays the groundwork for further studies of other tissues and tumors.

Objective and Design: Based on our clinical experience with combined gene therapy of glioblastoma, we developed a retroviral vector expressing two therapeutic genes (i.e. thymidine kinase of herpes simplex virus, HSV-TK, and interleukin-2, IL-2) and evaluated its efficiency in vitro and in vivo., Methods: Expression of therapeutic genes in transduced thyroid carcinoma cells was analyzed by real-time RT-PCR. Ganciclovir sensitivity of infected cells was assessed in vitro in thyroid carcinoma cell lines and in vivo in nude mice bearing xenografted thyroid cancers. The combined effect of IL-2/HSV-TK was compared with the effect of IL-2 alone., Results: Expression of therapeutic genes was higher in differentiated than in anaplastic thyroid carcinoma cells. Ganciclovir treatment led to dose- and time-dependent killing of transduced cells in vitro. A bystander effect was demonstrated by using mixtures of infected and non-infected cells. In vivo studies showed a significant reduction of growth and the presence of an inflammatory infiltrate in transduced thyroid tumors expressing IL-2 alone, as compared with non-infected tumors. By using the retroviral vector expressing IL-2/HSV-TK, treatment with ganciclovir led to complete eradication of anaplastic tumors and a >80% reduction of the size of differentiated thyroid carcinomas. Histological analysis of tumor specimens showed extensive necrosis and inflammatory cell infiltrates. The combination of IL-2/HSV-TK plus ganciclovir was significantly more efficient than IL-2 alone in eradicating tumor masses. The bystander effect was also obtained in vivo., Conclusions: These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas.

Background: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial., Methods: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism., Results: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA., Conclusions: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

Gene therapy may be an effective approach to thyroid carcinoma refractory to conventional treatment. A transcriptionally targeted retroviral vector for gene therapy of thyroid carcinomas was generated replacing the viral enhancer with the enhancer sequence of the human thyroglobulin (TG) gene, yielding a chimeric long-terminal repeat. The TG enhancer was used to drive the expression of either a reporter gene (beta-galactosidase) or two therapeutic genes, i.e. the prodrug-activating enzyme thymidine kinase of herpes simplex virus (HSV-TK) and human IL-2, separated by an internal ribosome entry site. The corresponding vector having an unmodified long-terminal repeat was used as control. The targeted vector allowed selective transgene expression and cell killing in differentiated thyroid tumor cells but not in anaplastic thyroid carcinoma cells and nonthyroid cells, as demonstrated by quantitative RT-PCR and cytotoxicity assays. Nude mice injected with tumor cells underwent near complete or complete regression of tumors transduced with the control vector after ganciclovir treatment. On the other hand, infection with the thyroid-specific vector led to regression only of TG-expressing tumors. In addition, tumors expressing human IL-2 showed significant growth retardation, compared with nontransduced tumors while exhibiting signs of necrosis and presence of an inflammatory infiltrate. However, HSV-TK/IL-2 plus ganciclovir was significantly more efficient than HSV-TK/IL-2 alone in eradicating tumor masses. Our results indicate that replacement of viral enhancer with TG enhancer confers selectivity of transgene expression in thyroid cells. Thus, the combined thyroid-specific expression of two therapeutic genes (cytokine and suicide genes), although a safe tumor-targeted treatment, would allow an increased anticancer effect.

A hypercoagulable state and an increased incidence of thromboembolic complications are reported in Cushing's syndrome. The hypercoagulable state is related to an increase in plasma clotting factors, especially Factor VIII and von Willebrand factor complex, and to an impairment of fibrinolytic capacity. Retrospective analysis of postoperative thromboembolic events in a large group of patients with Cushing's syndrome, including 75 patients (group 1) evaluated in the period from 1972-1981 not receiving anticoagulants, and 232 patients (group 2), evaluated in the period from 1982-2000. Patients of group 1 underwent routine hemostatic function, i.e. prothrombin time and activated partial thromboplastine time. Patients of group 2 underwent a thorough investigation as to hemostatic parameters and received prophylactic treatment with heparin and/or warfarin. Patients with Cushing's syndrome showed various abnormalities of hemostatic parameters. A significant correlation between activated partial thromboplastine time and urinary free cortisol was observed. During follow-up, 15 patients (20%; mean follow-up, 9.4 +/- 6.4 yr) of group 1 and 14 (6.0%; mean follow-up, 6.6 +/- 4.2 yr) of group 2 showed thromboembolic complications. Of these patients, eight of group 1 and one of group 2 died. Survival analysis demonstrated a significantly higher morbidity and mortality due to thromboembolic events in group 1, not receiving anticoagulant prevention, than in group 2, treated with anticoagulants in the perioperative period until cure of the disease and normalization of clotting parameters. Cushing's syndrome is associated with a hypercoagulable state. An adequate anticoagulant prophylaxis can reverse this prothrombotic state and avoid postoperative thromboembolic events.

Objective: The natural course of adrenal incidentalomas, especially those with subclinical autonomous glucocorticoid production, i.e. subclinical Cushing's syndrome, and the risk that such conditions will evolve towards overt Cushing's syndrome are unknown., Design: Longitudinal follow-up evaluation of a series of 284 consecutive patients with adrenal incidentaloma., Methods and Results: Out of 284 consecutive patients with adrenal incidentaloma studied at our Institution in the last 15 years, 98 patients (23 with subclinical hypercortisolism) underwent surgery. Of 130 non-operated patients with a follow-up of at least 1 year, eight had subclinical hypercortisolism at diagnosis. We describe in detail four patients who developed overt Cushing's syndrome after 1-3 years of follow-up. Only one of these patients had subclinical hypercortisolism at first diagnosis. Estimated cumulative risk for a non-secreting adrenal incidentaloma to develop subclinical hyperfunction was 3.8% after 1 year and 6.6% after 5 years. For patients with masses with subclinical autonomous glucocorticoid overproduction, estimated cumulative risk to develop overt Cushing's syndrome was 12.5% after 1 year., Conclusions: In patients with adrenal incidentalomas the risk of progression towards overt Cushing's syndrome is not low, at variance with previous reports. A careful biochemical and hormonal follow-up is advisable in all patients who do not need surgery at first presentation.

Objective: Adrenocortical scintigraphy has demonstrated clinical utility in the morpho-functional characterization of adrenal tumors. The aim of this study was to identify possible relationships between the scintigraphic pattern and endocrine and/or morphological data in a series of adrenocortical carcinomas., Design and Methods: Twenty-one patients with adrenocortical carcinoma (11 nonfunctioning and 10 hormone-secreting) were investigated with 75Se-methyl-nor-cholesterol scintigraphy. Clinical, hormonal, radiological, and pathological data were analyzed., Results: The adrenal mass showed no radiocholesterol uptake in 18 cases (11 nonfunctioning and 7 functioning lesions). Contralateral normal adrenal gland was visualized in all patients with nonfunctioning tumors, whereas classic bilateral nonvisualization was observed in the 7 cases with hyperfunctioning masses. Three patients with cortisol-producing carcinomas showed radiotracer uptake by the mass, without visualization of the contralateral gland. At histology, the tumors were shown to be undifferentiated adrenocortical carcinomas; they had an aggressive clinical behavior., Conclusions: Radiocholesterol scintigraphy has an important role in diagnosing adrenocortical carcinomas, which typically are not visualized. However, 30% of hypersecreting adrenocortical carcinomas show an atypical increased tracer uptake, not predictive of the biochemical and histological features of the tumor.

Objective: To evaluate the roles of the CDKN1C (P57KIP2) gene, which encodes for the cyclin-dependent kinase inhibitor CDNC, and the TP53 tumor suppressor gene in adrenal tumorigenesis, as a means of investigating the molecular basis of sporadic adrenal tumors, which is unknown., Design: Screening for the presence CDKN1C and TP53 mutations and analyzing the expression pattern of CDNC, P53 and its downstream effector CDN1 (P21WAF1/CIP1) in a series of 79 sporadic adrenal tumors., Methods: Single-strand conformation polymorphism and sequencing were used for mutation analysis of CDKN1C and TP53 in blood and adrenal tissue samples. In a subgroup of 48 tissues, CDKN1C expression was evaluated by RT-PCR and immunohistochemistry. Immunohistochemical analysis of P53 and CDN1 was performed., Results: No somatic mutations of CDKN1C were found in the tumors analyzed, in spite of low/absent CDNC expression in adrenocortical adenomas and carcinomas. Mutations in the TP53 gene were present in 70% of adrenocortical carcinomas, associated with abnormal P53 and CDN1 expression, but not in benign neoplasms. In the normal adrenal cortex, CDNC expression was strictly nuclear and confined to the cortical zone (i.e. zona glomerulosa and reticularis), with no staining in the medulla., Conclusions: Mutations in the TP53 gene are frequent in adrenocortical carcinomas and might be used as a marker of malignancy. In the normal adrenal cortex, the zone-specific pattern of expression of CDNC suggests a role in adrenal differentiation.

Gene therapy for endocrine diseases represents an exciting new type of molecular intervention that may be a curative one. Endocrine disorders that might be treated by gene therapy include monogenic diseases, such as GH deficiency and hypothalamic diabetes insipidus, and multifactorial diseases, such as diabetes mellitus, obesity and cancer. Premises seem promising for endocrine tumours, but many combined approaches of cell and gene therapy are foreseeable also for other endocrine disorders. This review outlines the principles of gene therapy, describes the endocrine disorders that might take advantage of gene transfer approaches, as well as the gene therapy interventions that have already been attempted, their major limitations and the problems that remain to be solved.

In the human adrenal cortex, cortisol and aldosterone are synthesized by the isozymes 11beta-hydroxylase and aldosterone synthase, respectively, encoded by the 93% identical CYP11B1 and CYP11B2 genes. In vitro mutagenesis of CYP11B1 complementary DNA, resulting in the replacement of CYP11B1 codons by those encoding the corresponding amino acid residues of CYP11B2 enzyme (exon 5, Ser288Gly; exon 6, Val320Ala), yields a complementary DNA encoding a mutant enzyme with an efficient aldosterone synthase activity. Identical somatic mutations in the CYP11B1 gene in vivo would produce a gene encoding an enzyme with C18 activity and that would preserve ACTH responsiveness due to the retained 5'-promoter in the mutated CYP11B1 gene. An ACTH-responsive aldosterone synthase activity of this type is commonly seen in patients with aldosterone-producing adenomas (APA). We examined the occurrence of mutations in exons 5 and 6 of the CYP11B1 gene in APA from 10 patients with primary aldosteronism. Patients were selected on preoperative evidence of a 50% or greater plasma aldosterone decrease after short term dexamethasone trial and no aldosterone response to upright posture. DNA from adenomas was amplified by PCR using two pairs of primers spanning the regions of CYP11B1 gene, i.e. exons 3-5 and exons 6-9, where mutations could be located. Targeted regions were screened for mutations by automated sequencing of PCR products. No point mutations of the CYP11B1 gene over the two regions examined were found in APA. This argues against involvement of mutations in the pathogenesis of ACTH-responsive APA.

The natural course of adrenal incidentalomas and the risk that such lesions evolve toward hormonal hypersecretion or malignancy are still under evaluation. Of 246 consecutive patients with adrenal incidentaloma studied at our institution in the last 15 yr, 91 underwent surgery. Of the remaining patients, a group of 75 (52 females and 23 males; median age, 56 yr; range, 19-77 yr) with incidentally discovered asymptomatic adrenal masses (60 unilateral and 15 bilateral; median diameter, 2.5 cm; range, 1.0-5.6) was enrolled in an endocrine and morphological follow-up of at least 2 yr after diagnosis (median, 4 yr; range, 2-10). During follow-up, no patients developed malignancy; 9 showed mass enlargement, with appearance of a new mass in the contralateral gland in 2; 3 developed adrenal hyperfunction (overt Cushing's syndrome in 2, nonclinical hypercortisolism in 1); and 3 showed adrenal mass enlargement associated with adrenal hyperfunction (nonclinical hypercortisolism in 2, pheochromocytoma in 1). The estimated cumulative risks to develop mass enlargement and hyperfunction were 8% and 4%, respectively, after 1 yr, 18% and 9.5% after 5 yr, and 22.8% and 9.5% after 10 yr. Nine risk factors for adrenal mass enlargement or hyperfunction were arbitrarily selected and evaluated: sex, age, presence of obesity, hypertension, diabetes, abnormal endocrine tests, mass size, mass location, and scintigraphic uptake pattern. Three of them attained statistical significance: mass size of 3 cm or more at diagnosis and exclusive radiocholesterol uptake by the mass at scintigraphy had relevance for the occurrence of adrenal hyperfunction, whereas the presence of endocrine test abnormalities at diagnosis had predictive value for mass enlargement. It is concluded that subtle hormonal abnormalities are risk factors for mass size increase, which is not a sign of malignant transformation. Both mass size of 3 cm or more at diagnosis and exclusive radiocholesterol uptake, indicating higher risks of hyperfunction, should be considered to plan a more thorough endocrine follow-up.

The aim of the study was to investigate the effect of octreotide, a somatostatin analog drug potentially able to inhibit growth hormone (GH), on the circadian blood pressure profile in a group of patients with acromegaly. Ten patients with GH-secreting pituitary adenoma were studied before and 6 months after treatment with subcutaneous octreotide 0.2 to 0.6 mg/day. Twenty-four hour blood pressure and heart rate were measured every 15 min at daytime (07:00 to 22:59) and every 30 min at nighttime (23:00 to 06:59) using a TM-2420 recorder. No correlation was found between GH levels and 24-h blood pressure in baseline conditions. Untreated patients had a significant nocturnal decrease of both systolic and diastolic blood pressure (P < .01), and all showed a circadian systolic or diastolic blood pressure rhythm. During octreotide treatment, 24 h as well as nighttime systolic and diastolic blood pressures significantly increased (P < .05), whereas daytime systolic and diastolic blood pressures did not change. Treated patients did not have a nocturnal decline in both systolic and diastolic blood pressures (P = NS), and eight lost their systolic or diastolic blood pressure rhythm. In conclusion, blood pressure circadian rhythm seems to be maintained in acromegaly. Octreotide treatment is associated with an increase of 24-h and nighttime blood pressure, and with loss of circadian blood pressure rhythm. Splanchnic vasoconstriction by this drug, shifting blood to peripheral vessels, may explain this phenomenon.

A decrease in hypothalamic-pituitary-adrenal axis sensitivity to glucocorticoid feedback suppression seems to occur with aging. To investigate possible abnormalities in the inhibitory effect of glucocorticoids on ACTH secretion in the elderly, we evaluated the endogenous ACTH response to hydrocortisone (25 mg as an i.v. bolus) in 15 healthy aged (65-88 yr) and 15 healthy young (18-26 yr) men. Blood samples for ACTH and cortisol determinations were collected at -15, 0, 2, 5, 10, 15, 30, 45, 60, 90, 120, and 180 min. Hydrocortisone injection produced a rapid increase in plasma cortisol levels within the first 2 min in both groups. In old men, in concomitance with the cortisol increase, ACTH levels showed only a slight and nonsignificant decrease within the first 15 min, followed by a pronounced and significant decline thereafter. In young subjects, in concomitance with the plasma cortisol increase, a marked decrease in ACTH levels was observed within the first 15 min, followed by a less pronounced decline thereafter. The response curve of ACTH inhibition could be arbitrarily divided in two parts. The first part (from 0-60 min) showed a significant difference between old and young men, whereas the remaining part of the curve (from 60-180 min) showed no differences between the two groups. The slower response in glucocorticoid feedback inhibition of ACTH in old men supports the concept of some alteration in the central regulation by steroids in aging. Age-related vascular factors affecting cortisol penetration through the blood-brain barrier more than hippocampus-hypothalamus receptor abnormalities could be involved in the fast component of hypothalamic-pituitary-adrenal axis feedback regulation.

Hormonal and morphological data were investigated in 202 consecutive patients with adrenal incidentalomas (171 unilateral and 31 bilateral) in an attempt to assess subclinical hyperfunction or malignancy. In addition to the classical evaluation, scintiscan was carried out in a large number of these patients. In unilateral incidentalomas, 83% showed normal hormonal function, whereas 17% had biochemical signs of adrenal overactivity (hyperaldosteronism in 3, hypercortisolism in 17, medullary hyperfunction in 9). [75Se]Methylnorcholesterol scintigraphy depicted malignant, space-occupying lesions as decreased or absent radiotracer uptake by the mass, and cortical adenomas as increased or normal uptake. In cortical adenomas a relationship between radiocholesterol uptake and degree of functional autonomy was demonstrated. [123I]Metaiodobenzilguanidine scintiscan visualized 7 of 8 pheochromocytomas. In bilateral incidentalomas, abnormal adrenal function was more frequent, accounting for 29% of cases (hyperaldosteronism in 3, hypercortisolism in 3, adrenal insufficiency in 2, and congenital adrenal hyperplasia in 1). Malignant lesions were not scintigraphically visualized. [75Se]Methylnorcholesterol scan also provided functional information in the case of a cortisol-secreting adenoma and an aldosteronoma with a concomitant contralateral nonhypersecreting adenoma, showing the greatest uptake in the hyperfunctioning adenomas. In both unilateral and bilateral lesions, endocrine testing failed to differentiate benign from malignant tumors. Although hormonal assessment is mandatory to clarify the functional patterns, only morphofunctional examination by scintiscan seems to provide more data about the likelihood of malignancy.

A case of primary aldosteronism is presented in which computed tomography showed bilateral adrenal tumors. Adrenal scintigraphy during dexamethasone suppression revealed bilateral radiotracer uptake, with markedly predominant visualization of the left side. The left adrenal gland was removed, and an aldosterone-producing adenoma was confirmed by histologic examination. Fifteen months after surgery, serum potassium and plasma aldosterone were normal, and a marked decrease of blood pressure was observed, indicating that the right adrenal tumor was a benign nonfunctioning mass. Unilateral adrenalectomy on the side of the scintigraphically predominant lesion reversed the clinical syndrome of primary aldosteronism.

The effect of a surgical cure of hypercortisolism on hypertension in 54 patients with Cushing's syndrome was assessed. The correlation between preoperative duration of hypertension and posttreatment blood pressure was significant (P < .01). Restoration of normal cortisol was associated with blood pressure normalization in 39 out of 54 cases. Duration of hypertension of patients with normalized blood pressure was significantly shorter than that of patients with persistent hypertension postoperatively (P < .0001). Duration of hypertension, ie, long-lasting exposure to increased cortisol, appears to be the determinant of persistent hypertension following successful surgery in Cushing's syndrome.