Maria Rosaria Capobianchi

MYO1E Mutations and Childhood Familial Focal Segmental Glomerulosclerosis

BACKGROUND Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. METHODS We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. RESULTS We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. CONCLUSIONS MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

Use of Massively Parallel Ultradeep Pyrosequencing To Characterize the Genetic Diversity of Hepatitis B Virus in Drug-Resistant and Drug-Naive Patients and To Detect Minor Variants in Reverse Transcriptase and Hepatitis B S Antigen

ABSTRACT Direct population sequencing and reverse hybridization (line probe assay [LiPA])-based methods are the most common methods for detecting hepatitis B virus (HBV) drug resistance mutations, although only mutations present in viral quasispecies with a prevalence of ≥20% can be detected by sequencing, and only known mutations are detected by LiPA. Massively parallel ultradeep pyrosequencing (UDPS; GS FLX platform) was used to analyze HBV quasispecies in reverse transcriptase (RT) and hepatitis B S antigen (HBsAg) from five drug-naive patients and eight drug-resistant patients. Eight primer pairs were used to obtain partially overlapping amplicons, covering the RT gene from codons 1 to 288 and the complete overlapping HBsAg sequence. A 1% mutation frequency was selected as the cutoff based on an error rate estimated on plasmid DNA. This technology enabled simultaneous analysis of between 2,852 and 18,016 clonally amplified fragments from each patient. The results indicate that UDPS has a relative sensitivity much higher than both direct sequencing and LiPA. In addition, the UDPS results are quantitative, allowing establishment of the relative frequency of both known mutations and novel substitutions. Some of the detected RT substitutions led to changes also in HBsAg. On the whole, genotype D presented a higher heterogeneity than genotype A. Considering the high quantity of information that can be provided by a single test from one patient, the short turnaround time, the information on substitution frequency, and the detection of rare variants, there are strong advantages conferred by UDPS, and the new method could play a relevant role in the clinical management of HBV infection and therapy.

Spontaneous release of interferon gamma by intestinal lamina propria lymphocytes in Crohn's disease. Kinetics of in vitro response to interferon gamma inducers.

The spontaneous induced release of interferon gamma (IFN gamma) by cultured intestinal lamina propria lymphocytes was investigated in patients with Crohns disease. In contrast to normal lymphocytes, intestinal lymphocytes from these patients spontaneously released IFN gamma and seemed to contain IFN gamma in their cytoplasm. Autologous peripheral lymphocytes did not release IFN gamma. When stimulated with interferon inducers lamina propria lymphocytes from Crohns disease tissue showed an increase in IFN gamma release 24 hours after induction with no appreciable further increase over the next two days of culture, while in control cells, either peripheral or intestinal, IFN gamma release progressively increased, peaking 72 hours after induction. These findings indicate that in Crohns disease the intestinal lymphocytes are stimulated in vivo to produce IFN gamma and that the spontaneous IFN gamma production is compartmentalised to the gut lymphocytes. These data support the concept that locally released IFN gamma has a crucial role in cell interactions in the lamina propria and contribute to the locally occurring immune phenomena in Crohns disease, including the increased epithelial expression of major histocompatibility complex class II antigens.

Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveller returning from Haiti to Italy, February 2016

A man in his early 30s reported in January 2016 a history of fever, asthenia and erythematous rash during a stay in Haiti. On his return to Italy, ZIKV RNA was detected in his urine and saliva 91 days after symptom onset, and in his semen on day 188, six months after symptom onset. Our findings support the possibility of sexual transmission of ZIKV and highlight the importance of continuing to investigate non-vector-borne ZIKV infection.

Massively parallel pyrosequencing highlights minority variants in the HIV-1 env quasispecies deriving from lymphomonocyte sub-populations

BackgroundVirus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 amino acid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage.ResultsThe heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage. However, minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters with a predicted X4 phenotype were found.ConclusionThis study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present in archived proviral sequences and in actually replicating virions, which may have clinical and therapeutic relevance.

West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention

West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis antigenic group, has caused sporadic outbreaks in humans, horses and birds throughout many of the warmer regions of Europe for at least 20 years. Occasional cases of West Nile encephalitis have also been associated with infected blood transfusions and organ donations. Currently, WNV appears to be expanding its geographical range in Europe and causing increasing numbers of epidemics/outbreaks associated with human morbidity and mortality. This brief review reports on the current epidemic situation regarding WNV in Europe, highlighting the clinical, diagnostic and preventive measures available for controlling this apparently emerging human pathogen.

Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein

Objectives: To explore the possibility that HIV‐1 budding and cellular adhesion molecules co‐polarize at cell‐to‐cell contact sites. To investigate the incorporation of host‐cell‐derived adhesion molecules into HIV‐1. Methods: The cellular sites involved in HIV‐1 budding were examined by transmission electron microscopy. Single and double immunocytochemistry staining was used to evaluate the cellular distribution of the viral matrix protein and adhesion molecules. Quantitative flow cytometry was used to measure the cellular expression of adhesion molecules. An immunocapture technique was used to measure the presence of cell‐derived proteins on HIV‐1. The captured virus was measured by a p24 antigen assay. The infectivity of virus captured by monoclonal antibodies was tested by measuring the virus antigen yield in supernatants after the addition of sensitive cells. Results: Released and budding HIV‐1 was mainly localized at the cell‐to‐cell contact regions. This feature was consistent with a polarized staining for the virus matrix protein p18 at cell‐to‐cell contact regions. Intercellular adhesion molecules (ICAM)‐1 in HIV‐1‐infected cells were polarized on both isolated cells and syncytia, co‐localizing with HIV‐1 matrix protein. HIV‐1 incorporated all the adhesion molecules expressed by the host cells, although without quantitative correlation with their cellular expression. Conclusions: HIV‐1 is released at cell‐to‐cell membrane contact sites. Both ICAM‐1 and virus matrix protein co‐polarized on isolated cells and syncytia at the sites involved in the recruitment of uninfected cells. The impressive concentration of ICAM at cell sites where most virions are released may account for the acquisition of these membrane proteins by the HIV‐1 progeny, and may be important for the cell‐mediated spread. AIDS 1995, 9:329‐335

Cross-subtype Immunity against Avian Influenza in Persons Recently Vaccinated for Influenza

Seasonal influenza vaccination may induce heterosubtypic immunity against avian influenza virus (H5N1).

The Effect of Age on Response to Therapy With Peginterferon α Plus Ribavirin in a Cohort of Patients With Chronic HCV Hepatitis Including Subjects Older Than 65 Yr

OBJECTIVES:In many industrialized countries HCV infection is characterized by an increasing prevalence during ageing; however, data on the efficacy of treatment among older patients are scarce. This study was set up to evaluate the effect of age on the treatment of chronic HCV hepatitis with peginterferon α plus ribavirin.METHODS:We retrospectively reviewed medical records of 153 adult patients with chronic HCV hepatitis treated with combination therapy; 30 of them (19.6%) were 65 years of age or older.RESULTS:In multivariable analysis, age groups ≥40 years had similar odds of achieving sustained virologic response (P = 0.71) and significantly lower odds of sustained response compared with younger patients (odds ratio [OR] 0.16, 95% confidence interval [CI] 0.05–0.59, P = 0.006; OR 0.13, 95% CI 0.03–0.49, P = 0.002; OR 0.21, 95% CI 0.05–0.91, P = 0.037 for patients aged 40–49 years, 50–64 years, and older than 64 years, respectively). The effect of age was present in the 74 patients infected with genotype 1 or 4 (P = 0.04), while among the 79 patients with genotype 2 or 3 sustained virologic response rates were relatively uniform, with no statistically significant differences.CONCLUSIONS:The probability of good response to combination treatment with peginterferon α plus ribavirin is decreased for patients aged more than 40 years infected with genotype 1 or 4, but patients aged more than 65 had a similar rate of response to those aged 40–64 years. Combination treatment may be safely extended to elderly patients with no major contraindications.

Antiviral reactivities of γδ T cells

Abstract The complex antiviral immune mechanisms involve both adaptive and innate reactions mediated by γδ T lymphocytes, whose unique immunosurveillance contributions are analyzed here in different clinical and experimental settings. It is beyond any doubt that the fast, potent, cytotoxic as well as non-cytolytic antiviral activities of γδ T cells are critical in protecting the host against diverse viral pathogens.