Quantitation of proviral load and the distribution of drug resistant mutations in human immunodeficiency virus infection.

Infection with Human Immunodeficiency virus type 1 (HIV-1) induces a chronic and progressive disease process with a broad spectrum of clinical manifestations from acute primary infection to AIDS which is often associated with opportunistic infections and AIDS. The course of the disease is also associated with increasing levels of viral replication and destruction of the immune system. Treatment of HIV-1 infection with single antiretroviral drugs provides a limited duration of benefit due to the emergence of drug resistant strains of the virus during monotherapy. The aim of this study was to examine levels of HIV-1 proviral DNA load and the distribution of HIV-1 zidovudine resistance mutations in a group of 79 patients who were receiving zidovudine monotherapy and also in multiple post-mortem tissues samples from patients who died of AIDS. A quantitative polymerase chain reaction (QPCR) assay was firstly developed to accurately measure HIV-1 proviral DNA load. The QPCR assay was based on coamplification of known copy number of a control sequence with patient HIV-1 DNA which allowed accurate quantitation. Measurement of proviral DNA load in post-mortem tissues from 11 patients who died of AIDS revealed high levels in lymph node and spleen samples, significant levels were also found in frontal lobe brain tissue. The sequence of the V3 loop of HIV-1 gp120 was obtained by direct sequencing of a selection of the tissues and 15 of the 20 sequences analysed showed a macrophage tropic, non syncytium inducing (NSI) phenotype. Mutations in HIV-1 reverse transcriptase which confer resistance to zidovudine were examined by a point mutation assay (PMA) which showed differences in levels of resistant genotypes between different tissues in individual patients. Separate evolution of drug resistant virus in different anatomical sites may reflect the efficiency of zidovudine in different tissues or the selective tissue tropism of HIV-1. The profiles of resistance at the five codons of HIV-1 RT examined were also different between patients and the presence of mutation at codon 41 and 215 was indicative of long term zidovudine monotherapy. Monitoring the emergence of drug resistant HIV-1 plays an important role in successful antiretroviral therapy and rapid PCR based assays for resistant genotypes have been demonstrated. In this thesis a Line Probe assay (LiPA) was evaluated for the detection of mutations at a number of codons of HIV-1 conferring Zidovudine resistance. Detection of resistant mutations by the LIPA test in PCR amplified DNA from a selection of PM tissues and whole blood samples showed 100% agreement with the sequence of HIV-1 RT obtained the samples. The LiPA test was both a rapid and sensitive assay for drug resistant mutations and wild type sequences. HIV-1 proviral DNA load and the presence of zidovudine resistant mutation at codons 41 and 215 was examined in 79 individual patients who had received zidovudine monotherapy. Expressing proviral DNA load per milliliter (ml) of whole blood or per unit of CD4 cells demonstrated how measurement of proviral load levels can be confounded by variation in cell numbers during the course of infection. Expression of proviral load per ml of blood showed a slight increase over time from baseline, whereas proviral load expressed per 103 CD4 cells showed a more significant change over time which mirrored the change in CD4 levels over time. Kaplan Meier analysis of proviral DNA load demonstrated that individuals with levels of > 4.0 log lo DNA copies had a shorter time for progression to a 50% CD4 cell decrease and also a shorter time to death. Further analysis of proviral DNA load as a marker for disease progression using Cox proportional hazard models showed a strong relationship between proviral load expressed per 10 CD4 cells and progression to death. The relationship of zidovudine resistance to proviral load, CD4+ T-cell count and disease progression was also examined. A significant correlation was found between presence of resistance mutation and a low CD4 cell count, resistance mutation was also strongly associated with AIDS and with a progression to death in a Cox proportional hazard model. The results presented in this thesis of measurements of proviral DNA load and drug resistance mutations combined with an in-depth statistical analysis provide an important insight into HIV-1 disease progression in a group of patients receiving zidovudine monotherapy

Natural products as a source of Coronavirus entry inhibitors

The COVID-19 pandemic has had a significant and lasting impact on the world. Four years on, despite the existence of effective vaccines, the continuous emergence of new SARS-CoV-2 variants remains a challenge for long-term immunity. Additionally, there remain few purpose-built antivirals to protect individuals at risk of severe disease in the event of future coronavirus outbreaks. A promising mechanism of action for novel coronavirus antivirals is the inhibition of viral entry. To facilitate entry, the coronavirus spike glycoprotein interacts with angiotensin converting enzyme 2 (ACE2) on respiratory epithelial cells. Blocking this interaction and consequently viral replication may be an effective strategy for treating infection, however further research is needed to better characterize candidate molecules with antiviral activity before progressing to animal studies and clinical trials. In general, antiviral drugs are developed from purely synthetic compounds or synthetic derivatives of natural products such as plant secondary metabolites. While the former is often favored due to the higher specificity afforded by rational drug design, natural products offer several unique advantages that make them worthy of further study including diverse bioactivity and the ability to work synergistically with other drugs. Accordingly, there has recently been a renewed interest in natural product-derived antivirals in the wake of the COVID-19 pandemic. This review provides a summary of recent research into coronavirus entry inhibitors, with a focus on natural compounds derived from plants, honey, and marine sponges

The Proangiogenic Potential of Mesenchymal Stem Cells and Their Therapeutic Applications

Mesenchymal stem cells (MSCs) can be isolated from many tissue types and following in vitro culture expansion, large numbers of patient-specific or allogenic cells can be produced for clinical applications. MSCs exhibit anti-inflammatory and immunomodulatory properties and are identified as lacking major histocompatibility complex (MHC) class II molecules. Cellular-based approaches using MSCs to enhance new blood vessel formation have shown promise in preclinical models and preliminary clinical trials. Transplantation of MSCs in vivo has significantly enhanced the formation of new blood vessels and promoted the healing of chronic wounds. The proangiogenic potential of MSCs can be further enhanced through gene delivery such as vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS) providing long-term therapeutic expression. In this chapter, we review recent advances on the isolation and characterization of MSCs and in vivo applications for promoting angiogenesis. Enhancement of angiogenesis is also required for improved healing in myocardial infarction and cerebral ischemia, and the use of MSCs in these areas will also be reviewed. Furthermore, the combination of MSCs with biomaterials has greatly improved their survival and potency with improved vascularization of tissue-engineered constructs and integration within the host. In summary, this chapter provides an overview of both the basic science supporting the proangiogenic properties of MSCs and their translational use

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

Introduction: A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia. Methods: Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-alpha-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated. Results: The second generation lentiviral vector rHIV-pWPT-EF1-alpha-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression. Conclusions: Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies

Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage.

BACKGROUND: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells. RESULTS: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors. CONCLUSION: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Gene Modification of Mesenchymal Stem Cells and Articular Chondrocytes to Enhance Chondrogenesis

Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role

Atomic force microscopy on chromosomes, chromatin and DNA: A review

The purpose of this review is to discuss the achievements and progress that has been made in the use of atomic force microscopy in DNA related research in the last 25 years. For this review DNA related research is split up in chromosomal-, chromatin- and DNA focused research to achieve a logical flow from large- to smaller structures. The focus of this review is not only on the AFM as imaging tool but also on the AFM as measuring tool using force spectroscopy, as therein lays its greatest advantage and future. The amazing technological and experimental progress that has been made during the last 25 years is too extensive to fully cover in this review but some key developments and experiments have been described to give an overview of the evolution of AFM use from 'imaging tool' to 'measurement tool' on chromosomes, chromatin and DNA. © 2012

Generation of diverse neural cell types through direct conversion

A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace, thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost. The process of neural direct conversion, in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency, shows great potential, with evidence of the generation of a range of functional neural cell types both in vitro and in vivo, through viral and non-viral delivery of exogenous factors, as well as chemical induction methods. Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells, with prospective roles in the investigation of neurological disorders, including neurodegenerative disease modelling, drug screening, and cellular replacement for regenerative medicine applications, however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option. In this review, we describe the generation of diverse neural cell types via direct conversion of somatic cells, with comparison against stem cell-based approaches, as well as discussion of their potential research and clinical applications

Generation of diverse neural cell types through direct conversion

A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace, thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost. The process of neural direct conversion, in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency, shows great potential, with evidence of the generation of a range of functional neural cell types both in vitro and in vivo, through viral and non-viral delivery of exogenous factors, as well as chemical induction methods. Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells, with prospective roles in the investigation of neurological disorders, including neurodegenerative disease modelling, drug screening, and cellular replacement for regenerative medicine applications, however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option. In this review, we describe the generation of diverse neural cell types via direct conversion of somatic cells, with comparison against stem cell-based approaches, as well as discussion of their potential research and clinical application