Isabella Abbate

MYO1E Mutations and Childhood Familial Focal Segmental Glomerulosclerosis

BACKGROUNDnFocal 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.nnnMETHODSnWe 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnMYO1E 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.

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.

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

Combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing

BackgroundNext-generation sequencing (NGS) offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons). The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called in-silico recombinants.ResultsThe reconstruction algorithm was applied to error-free simulated data and reconstructed a high percentage of true variants, even at a low genetic diversity, where the chance to obtain in-silico recombinants is high. Results on empirical NGS data from patients infected with hepatitis B virus, confirmed its ability to characterise different viral variants from distinct patients.ConclusionsThe combinatorial analysis provided a description of the difficulty to reconstruct a quasispecies, given a determined amplicon partition and a measure of population diversity. The reconstruction algorithm showed good performance both considering simulated data and real data, even in presence of sequencing errors.

Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection.

Objectives:HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment. Methods:Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis. Results:Variants predicted to use CXCR4 were detected (frequency ≥0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3–56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants. Conclusion:Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.

Analysis of co‐receptor usage of circulating viral and proviral HIV genome quasispecies by ultra‐deep pyrosequencing in patients who are candidates for CCR5 antagonist treatment

UDPS combined with genotypic algorithms for prediction of HIV-1 co-receptor usage may provide quantitative data about the tropism of each variant present in the viral quasispecies. The aim of the present study was to assess co-receptor usage by ultra-deep pyrosequencing (UDPS), in comparison with the reference phenotypic test (Trofile), in patients who are candidates for CCR5 antagonist treatment, in both circulating and proviral HIV-1. Seventeen patients who were tested by Trofile were enrolled. UDPS of the V3 loop region was carried out on both plasma RNA and proviral DNA. Genotypic prediction of co-receptor usage was established by position-specific score matrices (PSSM) and confirmed, in discordant cases, with geno2pheno. Genetic heterogeneity of the RNA and DNA quasispecies was assessed as well. A total of 196,729 V3 sequences were considered (mean coverage per site, 6346). Concordance between phenotypic test and UDPS with PSSM was 0.82. Geno2pheno results were in line with those obtained with PSSM. Proviral quasispecies were more heterogeneous than those found in circulating HIV. In most patients eligible for CCR5 antagonist treatment, X4 variants were detected in proviral DNA, ranging from 1.0% to 52.7%. UDPS combined with genotypic algorithms for co-receptor usage prediction highlighted the presence of minority variants, with a discordant tropism with respect to the predominant population, in both circulating viral and proviral HIV. In most patients treated with Maraviroc the virological response was independent of the presence of X4 in proviral DNA. The clinical impact of minority X4 variants present in patients who are candidates for anti-CCR5 antagonists remains a crucial point to be addressed.

Activation of Vγ9Vδ2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication

Abstract Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vγ9Vδ2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified γδ T cells stimulated by non-peptidic antigens. Vγ9Vδ2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-γ revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate γδ T cells, were shown to induce the inhibition of HCV replication mediated by Vγ9Vδ2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vγ9Vδ2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.

Changes in host cell molecules acquired by circulating HIV-1 in patients treated with highly active antiretroviral therapy and interleukin-2.

ObjectiveTo analyse cell membrane proteins (CMP) acquired by HIV-1 present in the plasma of asymptomatic patients, and their modifications after a cycle of highly active antiretroviral therapy (HAART) and interleukin (IL)-2. Design and methodsPlasma samples from eight drug-naive asymptomatic subjects underwent immobilized antibody capture (IAC) to detect CMP on the surface of circulating HIV-1. The CMP considered were lymphocyte subset markers (CD45RA, CD45RO), activation markers (HLA-DR), adhesion molecules (LFA-3), costimulatory proteins (B7-2), lymph-node homing receptors (CD62L) and pro-apoptosis molecules (FasL). This analysis was repeated after one cycle of HAART + IL-2, after virus rebound. ResultsLFA-3, followed by CD45RO and HLA-DR, are the most represented CMP on the surface of circulating virions in naive asymptomatic patients; CD45RA, CD62L, B7-2 and FasL are detected only occasionally. After rebound, a significant reduction of CD45RO and HLA-DR, but not of LFA-3, is observed on virions, whereas CD45RA and CD62L, as well as other molecules, are not affected, remaining almost undetectable. ConclusionsAssuming that CMP on HIV-1 reflect the cellular origin of virions, activated T cells expressing CD45RO, HLA-DR, and LFA-3 may be the main source of HIV-1 in asymptomatic patients. After a cycle of HAART + IL-2, followed by therapy interruption, CD45RA and CD62L are detected on virions rarely, indicating that even during virus rebound, expanded naive T cells do not become a major target of virus replication. Furthermore, the presence of HLA-DR on rebound HIV-1 is decreased, consistent with decreased activation of the HIV-producing cells. More extensive investigation may clarify the significance of these findings with respect to pathogenesis.

underevaluation of Hiv-1 Plasma Viral Load by a Commercially Available Assay in a Cluster of Patients Infected With Hiv-1 A/g Circulating Recombinant Form (crf02)

The authors studied the correlation and agreement of commercially available assays in detection and quantification of the HIV-1 intersubtype A/G circulating recombinant form CRF02. The assays under comparison were Bayer Versant HIV-1 RNA, version 3.0; Roche Amplicor HIV-1 Monitor, version 1.5 (standard procedure); and Organon Teknika NucliSens HIV-1 RNA QT. Plasma samples from 114 patients infected with CRF02 were tested by the three assays under standard conditions. Although correlation among the assays was high and statistically significant for subtype B and CRF02, in the latter instance, NucliSens measured average viral load values (3.29 +/- 0.71 log(10) copies/mL) about 4 and >8 times lower than those obtained by Versant (3.90 +/- 0.90 log(10) copies/mL) and Amplicor (4.22 +/- 1.05 log(10) copies/mL), respectively. Furthermore, in a statistically significant percentage of CRF02-harboring samples, NucliSens produced viral load values undetectable or 1 log(10) lower than those obtained in Versant and Amplicor assays. Altogether, these data underline a low performance of NucliSens in detecting and quantifying viremia in plasma samples harboring the CRF02. These results are potentially important as global distribution of new HIV-1 subtypes is expanding, and recombinant strains, particularly CRF02, are emerging and becoming highly prevalent.

Monophyletic HIV type 1 CRF02-AG in a nosocomial outbreak in Benghazi, Libya.

A cluster of HIV-1 infection has been identified in Libya in 1999, involving 402 children admitted to El-Fath Childrens Hospital in Benghazi (BCH) during 1998 and 19 of their mothers. Nosocomial transmission has been indicated as responsible for the spread of infection. Out of this group, 104 children and 19 adult women have been followed at the National Institute for Infectious Diseases L. Spallanzani in Rome during 1 year. At BCH, all children had received intravenous infusions but not blood or blood products. A single child receiving a blood transfusion in 1997 and the 17 infected mothers were never hospitalized in Benghazi. In addition, two nurses were diagnosed as HIV-1 infected. In 40 subjects out of this group HIV-1 gag, env, and pol fragments were amplified and sequenced. The phylogenetic analyses showed that a monophyletic recombinant HIV-1 form CRF02-AG was infecting all of the HIV-1-seropositive patients admitted at BCH with no close similarities to the other CRF02-AG reported to GenBank. A different strain was found in the child infected by blood transfusion. The data thus suggest a highly contagious nosocomial spread of HIV-1 infection and possibly transmission of the virus from child to mother during breastfeeding in connection with primary HIV-1 infection.