David J. Hooker

Genomic Structure of an Attenuated Quasi Species of HIV-1 from a Blood Transfusion Donor and Recipients

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation.

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH2-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.

A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA

Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late ‘point of no return’ step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR’s advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR’s utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.

Hepatitis C seropositivity is not a risk factor for sensory neuropathy among patients with HIV

Background: Sensory neuropathy (SN) is common in patients with HIV. Hepatitis C (HCV) coinfection is often cited as an HIV-SN risk factor, but data to support this are lacking. This collaboration aimed to examine the association between HCV serostatus and SN risk among ambulatory HIV-positive patients. Methods: Patients with HIV were assessed in cross-sectional studies in Baltimore, Jakarta, Johannesburg, Kuala Lumpur, Melbourne, and Sydney for SN (defined by both supportive symptoms and signs). HCV seropositivity was assessed as an SN risk using a χ2 test, followed by logistic regression modeling to correct for treatment exposures and demographics. Results: A total of 837 patients of African, Asian, and Caucasian descent were studied. HCV seroprevalence varied by site (Baltimore n = 104, 61% HCV+; Jakarta 96, 51%; Johannesburg 300, 1%; Kuala Lumpur 97, 10%; Melbourne 206, 16%; Sydney 34, 18%). HCV seropositivity was not associated with increased SN risk at any site, but was associated with reduced SN risk in Melbourne (p = 0.003). On multivariate analyses, the independent associations with SN were increasing age, height, and stavudine exposure. HCV seropositivity was not independently associated with an increased SN risk at any site, but associated independently with reduced SN risk in Baltimore (p = 0.04) and Melbourne (p = 0.06). Conclusions: Hepatitis C (HCV) seropositivity was not associated with increased sensory neuropathy risk among HIV-positive patients at any site. While we were unable to assess HCV RNA or liver damage, the data suggest that HCV coinfection is not a major contributor to HIV-SN. HCV = hepatitis C; SN = sensory neuropathy.

Syncytium-inducing phenotype and zidovudine susceptibility of HIV-1 isolated from post-mortem tissue.

ObjectiveTo establish the syncytium-inducing (SI) phenotype and zidovudine (ZDV) susceptibility of HIV-1 isolates obtained from autopsy specimens. MethodsIsolation of HIV was attempted from autopsy specimens obtained from 76 AIDS patients. Specimens of Iymph node, spleen, spinal cord, brain and cerebrospinal fluid (CSF) were processed and cultured with peripheral blood mononuclear cells (PBMC) from seronegative donors. Biological phenotype was determined in a T-Iymphocyte line (MT-2). ZDV susceptibility was evaluated in a PBMC-based assay. Sequencing of amino-acid codons in the reverse transcriptase gene previously shown to be associated with ZDV resistance was carried out on a subgroup of isolates. ResultsHIV was recovered from tissue specimens and CSF up to 5 days post-mortem, but recovery rate of infectious virus decreased as the time between autopsy and specimen processing increased. There was a lack of concordance between PBMC isolates and isolates from different tissue sites with respect to SI phenotype. ZDV-resistant virus was isolated from post-mortem specimens of patients who had received long-term ZDV therapy up until or shortly before their death. ZDV-sensitive virus re-emerged in the Iymph node of patients who ceased treatment several months prior to death. Phenotypically sensitive virus obtained from Iymph node tissue of three patients after a relatively short time off ZDV (4–6 months) retained some of the amino-acid substitutions known to be associated with resistance. ConclusionThe data suggests that ZDV resistance and re-emergence of sensitive virus does not originate in peripheral cells, and that these cells and tissues are seeded with virus present elsewhere, possibly in the germinal centres of the lymph node.

Measuring and monitoring apoptosis and drug toxicity in HIV patients by ligation‐mediated polymerase chain reaction

Apoptosis has a critical role in normal physiology while its dysregulation has causal links with certain pathologies. A biochemical hallmark of apoptosis, internucleosomal genomic DNA fragmentation, is detectable by ligation‐mediated polymerase chain reaction (LM‐PCR). Here we converted LM‐PCR into a new apoptosis quantifier by dividing trace quantities of 600 bp apoptotic amplicons into those of a single copy house‐keeping gene, generating the LM‐PCR ‘value’. Dynamic range was ∼17‐fold correlating with a ∼200‐fold difference in degree of apoptotic fragmentation. Inter‐ and intra‐gel reliability were both excellent, supporting LM‐PCRs utility with large sample sets. Validation experiments comprising cell exposure to staurosporine over time revealed LM‐PCR is as sensitive as caspase‐3/ELISA and more sensitive than terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling/flourescence‐activated cell sorting (TUNEL/FACS) for distinguishing low degrees of apoptosis (the spectrum most relevant in vivo). The LM‐PCR profile mirrored that of caspase‐3/ELISA but not TUNEL/FACS. We then applied this molecular tool to clinical investigation. Increased apoptosis is implicated in lipoatrophy (subcutaneous fat wasting), a serious, persistent toxicity of some nucleoside analogue reverse transcriptase inhibitors (NRTIs) used in anti‐HIV highly active antiretroviral therapy (HAART). We demonstrated in 105 peripheral blood mononuclear cell samples that elevated LM‐PCR values are seen during therapy with stavudine (d4T), a particularly toxic NRTI (P< 0.0001 versus no HAART, unpaired t‐test). Elevated values were also independently associated with clinical evidence of lipoatrophy (P= 0.007, multiple logistic regression modelling) but not with patient age, CD4 T‐cell count nor HIV viral load (P> 0.8 for each). Together these data demonstrate that LM‐PCR is a robust and reliable quantifier of apoptosis with potential for basic science and clinical investigation.

Ubisol-AquaTM: Coenzyme Q10 Prevents Antiretroviral Toxic Neuropathy in an In Vitro Model

BACKGROUND Peripheral neuropathy is the dose-limiting toxicity of stavudine and didanosine (nucleoside analogs used in HIV treatment) and is attributed to mitochondrial toxicity from these drugs. Acetyl L-carnitine (ALC) and co-enzyme Q(10) are proposed as neuropathy treatments, but evidence to support these is limited. METHODS We examined ALC and a water-soluble formulation of co-enzyme Q(10) (H(Q)O) for the prevention of d4T and ddI neurotoxicity using cultured fetal rat DRG as an in vitro model. RESULTS DdI (33microM) and d4T (50microM) caused clear toxicity (impaired neurite growth) by day 8 of DRG culture. H(Q)O at concentrations 1-100microM completely prevented the toxicity of 33microM ddI in vitro and ALC at concentrations 1-100 microM substantially (but incompletely) prevented ddI toxicity in this model. In contrast, ALC was ineffective at all concentrations tested for preventing the toxicity of 50microM d4T. H(Q)O showed dose-dependent efficacy for preventing d4T toxicity. H(Q)O (1microM) partially prevented d4T toxicity while 10 and 100microM H(Q)O completely prevented d4T toxicity in this model. CONCLUSIONS We find H(Q)O is superior to ALC for preventing the neurotoxicity of d4T (the HIV treatment most associated with neuropathy) and ddI in vitro. Further study is needed to clarify any clinical role for co-enzyme Q(10) co-administration with d4T and ddI and to assess whether this compound may have a role in treating established cases of neuropathy.

Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.

Apoptosis: a clinically useful measure of antiretroviral drug toxicity?

Antiretroviral therapy (ART) has improved life expectancy with HIV infection, but long-term toxicities associated with these medications are now a major global disease burden. There is a clear need to develop useful methods for monitoring patients on antiretroviral drugs for early signs of toxicity. Assays with predictive utility – allowing therapy to be changed before serious end organ damage occurs – would be ideal. Attempts to develop biochemical methods of monitoring ART toxicity have concentrated on the mitochondrial toxicity of nucleoside analogue reverse transcriptase inhibitors and have not generally lead to assays with widespread clinical applications. For example, plasma lactate and peripheral blood measurements of mitochondrial DNA associate with exposure to potentially toxic nucleoside analogue reverse transcriptase inhibitors but have not reliably predicted clinical toxicity. Better assays are needed, including markers of toxicity from additional drug classes. Apoptosis may be a potential marker of ART toxicity. Increased apoptosis has been demonstrated both in vitro and in vivo in association with various antiretroviral drug classes and a range of clinical toxicities. However, quantifying apoptosis on biopsy specimens of tissue (such as adipose tissue) is impractical for patient monitoring. Novel assays have been described that can quantify apoptosis using minute tissue samples and initial results from clinical samples suggest peripheral blood may have utility in predicting ART toxicities. The limitations and potential of such techniques for monitoring patients for drug side effects will be discussed.