Virologic Failures on Initial Boosted-PI Regimen Infrequently Possess Low-Level Variants with Major PI Resistance Mutations by Ultra-Deep Sequencing

It is unknown whether HIV-positive patients experiencing virologic failure (VF) on boosted-PI (PI/r) regimens without drug resistant mutations (DRM) by standard genotyping harbor low-level PI resistant variants. CASTLE compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with TVD in ARV-naïve subjects.To determine if VF on an initial PI/r-based regimen possess low-level resistant variants that may affect a subsequent PI-containing regimen.Patients experiencing VF on a Tenofovir/Emtricitabine+PI/r regimen were evaluated by ultra deep sequencing (UDS) for mutations classified/weighted by Stanford HIVdb. Samples were evaluated for variants to 0.4% levels. 36 VF subjects were evaluated by UDS; 24 had UDS for PI and RT DRMs. Of these 24, 19 (79.2%) had any DRM by UDS. The most common UDS-detected DRM were NRTI in 18 subjects: M184V/I (11), TAMs(7) & K65R(4); PI DRMs were detected in 9 subjects: M46I/V(5), F53L(2), I50V(1), D30N(1), and N88S(1). The remaining 12 subjects, all with VLs<10,000, had protease gene UDS, and 4 had low-level PI DRMs: F53L(2), L76V(1), I54S(1), G73S(1). Overall, 3/36(8.3%) subjects had DRMs identified with Stanford-HIVdb weights >12 for ATV or LPV: N88S (at 0.43% level-mutational load 1,828) in 1 subject on ATV; I50V (0.44%-mutational load 110) and L76V (0.52%-mutational load 20) in 1 subject each, both on LPV. All VF samples remained phenotypically susceptible to the treatment PI/r.Among persons experiencing VF without PI DRMs with standard genotyping on an initial PI/r regimen, low-level variants possessing major PI DRMs were present in a minority of cases, occurred in isolation, and did not result in phenotypic resistance. NRTI DRMs were detected in a high proportion of subjects. These data suggest that PIs may remain effective in subjects experiencing VF on a PI/r-based regimen when PI DRMs are not detected by standard or UDS genotyping

Prevalence and Clinical Significance of HIV Drug Resistance Mutations by Ultra-Deep Sequencing in Antiretroviral-Naïve Subjects in the CASTLE Study

CASTLE compared the efficacy of atazanavir/ritonavir with lopinavir/ritonavir, each in combination with tenofovir-emtricitabine in ARV-naïve subjects from 5 continents.Determine the baseline rate and clinical significance of TDR mutations using ultra-deep sequencing (UDS) in ARV-naïve subjects in CASTLE.A case control study was performed on baseline samples for all 53 subjects with virologic failures (VF) at Week 48 and 95 subjects with virologic successes (VS) randomly selected and matched by CD4 count and viral load. UDS was performed using 454 Life Sciences/Roche technology.Of 148 samples, 141 had successful UDS (86 subtype B, 55 non-B subtypes). Overall, 30.5% of subjects had a TDR mutation at baseline; 15.6% only had TDR(s) at <20% of the viral population. There was no difference in the rate of TDRs by B (30.2%) or non-B subtypes (30.9%). VF (51) and VS (90) had similar rates of any TDRs (25.5% vs. 33.3%), NNRTI TDRs (11.1% vs.11.8%) and NRTI TDRs (24.4% vs. 25.5%). Of 9 (6.4%) subjects with M184V/I (7 at <20% levels), 6 experienced VF. 16 (11.3%) subjects had multiple TAMs, and 7 experienced VF. 3 (2.1%) subjects had both multiple TAMs+M184V, and all experienced VF. Of 14 (9.9%) subjects with PI TDRs (11 at <20% levels): only 1 experienced virologic failure. The majority of PI TDRs were found in isolation (e.g. 46I) at <20% levels, and had low resistance algorithm scores.Among a representative sample of ARV-naïve subjects in CASTLE, TDR mutations were common (30.5%); B and non-B subtypes had similar rates of TDRs. Subjects with multiple PI TDRs were infrequent. Overall, TDRs did not affect virologic response for subjects on a boosted PI by week 48; however, a small subset of subjects with extensive NRTI backbone TDR patterns experienced virologic failure

Human Galectins Induce Conversion of Dermal Fibroblasts into Myofibroblasts and Production of Extracellular Matrix: Potential Application in Tissue Engineering and Wound Repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multi-functionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-beta 1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum. Copyright (C) 2011 S. Karger AG, Base

A pilot study to investigate respiratory ill health in people living with HIV

Background: Helping people living with HIV (PLWH) to maintain long‐term health as they age is central to HIV care and involves monitoring for chronic HIV‐associated co‐morbidities. Chronic lung disease (CLD) is recognised to be more common in PLWH as they age, but UK data on CLD prevalence and phenotype in PLWH are limited, particularly in female, non‐white, never smoking groups, and studies of progression over time are lacking. This pilot study aimed to obtain preliminary data on CLD in these subgroups and establish a simple scheme for monitoring lung health of PLWH for a future longitudinal study. Methods: Cross‐sectional pilot of CLD prevalence among PLWH on ART by age, gender, ethnicity and smoking status using convenience sampling of PLWH attending routine clinics. PLWH completed a web‐based questionnaire of self‐reported chronic cough, wheeze & phlegm production using the BOLD study questions; dyspnoea using the Medical Research Council (mMRC) scale; health related quality of life (EQ‐5D‐5L); respiratory, smoking and other lung exposure history. Forced Expiratory Volume (FEV)1 and Forced Vital Capacity (FVC) were measured by spirometry and z‐scores derived from Global Lung Initiative age, sex and ethnicity predicted values. We extracted HIV data from clinical records and evaluated the suitability and acceptability of the measurements. 50 HIV negative controls matched for smoking status were also assessed. Results: We recruited 150 PLWH, median age 46, 31% female, 47% white. 65% were never smokers, 45% reported childhood exposure to solid cooking fuel. 49% reported a history of PCP, TB or pneumonia and 26% a diagnosis of COPD, asthma or bronchiectasis. All groups had lung ill‐health (table) that correlated with EQ5D5L. Conclusion: CLD may affect all PLWH subgroups. These data and methods can inform a longitudinal study of CLD in UK PLWH

BF Integrase Genes of HIV-1 Circulating in São Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population

Acute radiation syndrome caused by accidental radiation exposure - therapeutic principles

Fortunately radiation accidents are infrequent occurrences, but since they have the potential of large scale events like the nuclear accidents of Chernobyl and Fukushima, preparatory planning of the medical management of radiation accident victims is very important. Radiation accidents can result in different types of radiation exposure for which the diagnostic and therapeutic measures, as well as the outcomes, differ. The clinical course of acute radiation syndrome depends on the absorbed radiation dose and its distribution. Multi-organ-involvement and multi-organ-failure need be taken into account. The most vulnerable organ system to radiation exposure is the hematopoietic system. In addition to hematopoietic syndrome, radiation induced damage to the skin plays an important role in diagnostics and the treatment of radiation accident victims. The most important therapeutic principles with special reference to hematopoietic syndrome and cutaneous radiation syndrome are reviewed

Dynamics of Hepatitis B Virus Quasispecies in Association with Nucleos(t)ide Analogue Treatment Determined by Ultra-Deep Sequencing

[Background and Aims]: Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. [Methods]: To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. [Results]: Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. [Conclusion]: Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection

Reappearance of Minority K103N HIV-1 Variants after Interruption of ART Initiated during Primary HIV-1 Infection

BACKGROUND: In the Zurich Primary HIV infection study (ZPHI), minority drug-resistant HIV-1 variants were detected in some acutely HIV-1-infected patients prior to initiation of early antiretroviral therapy (ART). Here, we investigated the reappearance of minority K103N and M184V HIV-1 variants in these patients who interrupted efficient early ART after 8-27 months according to the study protocol. These mutations are key mutations conferring drug resistance to reverse transcriptase inhibitors and they belong to the most commonly transmitted drug resistance mutations. METHODOLOGY/PRINCIPAL FINDINGS: Early ART was offered to acutely HIV-1-infected patients enrolled in the longitudinal prospective ZPHI study. Six patients harboring and eleven patients not harboring drug-resistant viruses at low frequencies prior to ART were included in this substudy. Minority K103N and M184V HIV-1 variants were quantified in longitudinal plasma samples after treatment interruption by allele-specific real-time PCR. All 17 patients were infected with HIV-1 subtype B between 04/2003 and 09/2005 and received LPV/r+AZT+3TC during primary HIV-1 infection (PHI). Minority K103N HIV-1 variants reappeared after cessation of ART in two of four patients harboring this variant during PHI and even persisted in one of those patients at frequencies similar to the frequency observed prior to ART (<1%). The K103N mutation did not appear during treatment interruption in any other patient. Minority M184V HIV-1 variants were detected in two patients after ART interruption, one harboring and one not harboring these variants prior to ART. CONCLUSION: Minority K103N HIV-1 variants, present in acutely HIV-1 infected patients prior to early ART, can reappear and persist after interruption of suppressive ART containing two nucleoside/nucleotide analogue reverse transcriptase inhibitors and a ritonavir-boosted protease inhibitor. TRIAL REGISTRATION: Clinicaltrials.gov NCT00537966

Identification of Hepatic Niche Harboring Human Acute Lymphoblastic Leukemic Cells via the SDF-1/CXCR4 Axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc null mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis