Shirley Kwok

Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations

A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for latently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.

Detection of Human T-Cell Lymphoma/Leukemia Virus Type I DNA and Antigen in Spinal Fluid and Blood of Patients with Chronic Progressive Myelopathy

The presence of antibodies to human T-cell lymphoma/leukemia virus Type I (HTLV-I) has been associated with chronic progressive myelopathy. We attempted to isolate the virus from the blood and spinal fluid of patients with chronic progressive myelopathy and to define the clinical, radiologic, and electrophysiologic features of this disease. Ten of 13 patients from tropical countries and 2 of 8 from the United States had serum antibodies to HTLV-I. The virus was detected in cultures of peripheral-blood lymphocytes from three of seven patients by means of Southern blot hybridization. Using a sensitive in vitro enzymatic gene-amplification technique, we detected HTLV-I sequences in fresh peripheral-blood mononuclear cells of all of 11 patients tested who were positive for the antibody, and in cell cultures of the spinal fluid from 3 of the 11 tested. Magnetic resonance imaging of the cranium revealed periventricular lesions in the white matter of 3 of the 12 antibody-positive patients. Five of these patients had mild axonal sensorimotor polyneuropathy, and one had bilateral lumbar radiculopathy. Visual evoked potentials were abnormal in three seropositive patients, and brain-stem evoked responses were abnormal in two. The detection of the DNA and proteins of HTLV-I strengthens the proposition that this virus is involved in the pathogenesis of a subset of cases of chronic progressive myelopathy.

EVALUATION OF SCREENED BLOOD DONATIONS FOR HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION BY CULTURE AND DNA AMPLIFICATION OF POOLED CELLS

BACKGROUND Reports of transmission of the human immunodeficiency virus type 1 (HIV-1) from transfusions of screened blood and reports of silent, antibody-negative HIV-1 infections in persons at high risk continue to foster concern about the safety of the blood supply. Previous estimates of the risk of HIV-1 range from 1 in 38,000 to 1 in 300,000 per unit of blood but are based on either epidemiologic models or the demonstration of seroconversion in recipients. METHODS We isolated peripheral-blood mononuclear cells from blood that was fully screened and found to be seronegative, combined them into pools of cells from 50 donors, and tested them for HIV-1 by viral culture and the polymerase chain reaction, using protocols specifically adapted for this analysis. RESULTS The 1530 pools of mononuclear cells were prepared from 76,500 blood donations made in San Francisco between November 1987 and December 1989. Of these pools, 1436 (representing 71,800 donations) were cultured successfully; 873 (43,650 donations) were evaluated by the polymerase chain reaction. Only one pool was confirmed as HIV-1--infected by both methods. After adjustment for sample-based estimates of the sensitivity of the detection systems using culture and the polymerase chain reaction, the probability that a screened donor will be positive for HIV-1 was estimated as 1 in 61,171 (95 percent upper confidence bound, 1 in 10,695). CONCLUSIONS Silent HIV-1 infections are exceedingly rare among screened blood donors, so the current risk of HIV-1 transmission from blood transfusions, even in high-prevalence metropolitan areas, is extremely low.

Low incidence of HTLV infections in random blood donors with indeterminate Western blot patterns

Peripheral blood mononuclear cells (PBMCs) were recovered from platelet units of 61 blood donors who were HTLV‐I positive and 3 blood donors who were HTLV‐I negative on enzyme‐linked immunosorbent assay (ELISA). Western blot analyses were performed on the sera and DNA was prepared from the PBMCs and analyzed by the polymerase chain reaction (PCR). Of the 61 repeatably reactive samples, 2 were positive, 26 were negative, and 33 were interpreted as indeterminate on Western blot. HTLV‐II sequences were detected by PCR in one of the Western blot‐positive samples, as well as in one Western blot‐indeterminate sample that showed reactivity to p24 only. HTLV‐I sequences were detected in the second Western blot‐positive sample. HTLV sequences were not detected in the remaining samples, which suggested that the majority of individuals with indeterminate results on Western blots that used one set of commercially available reagents are not infected with HTLV. It is demonstrated in this study that PCR can be used not only to resolve the infection status of individuals with indeterminate Western blots but also to distinguish between HTLV‐I and HTLV‐II.

The application of quantitative polymerase chain reaction to therapeutic monitoring.

Use of polymerase chain reaction in HIV monitoring: The polymerase chain reaction can be used not only to detect the presence of viral sequences but also to provide a semiquantitative or a precise evaluation of the number of copies of genome present. Integral to the development of accurate and reproducible assays have been critical advances in polymerase chain reaction technology. In addition, standardization of protocols and reagents has proved invaluable. The HIV life cycle permits both DNA and RNA to be targeted. Although early cross-sectional studies provided little insight into disease progression, recent longitudinal studies have provided valuable information on HIV infection. ConclusionA rapid and simple quantitative assay for HIV RNA in plasma or sera has been developed that should prove valuable in determining the natural history of infection, dissecting viral pathogenesis and monitoring the efficacy of therapeutic intervention.

Significance of Human T-Lymphotropic Virus Type I Indeterminant Serological Findings among Healthy Individuals

Abstract. Follow‐up studies on 67 blood donors with indeterminant serological findings for human T‐lymphotropic virus (HTLV) type I by standard immunoassays showed no evidence of infection by polymerase chain reaction analysis for HTLV‐I or HTLV‐II nucleic acids or by antibody reactivity to a unique HTLV‐I recombinant envelope protein, MTA‐4. Among HTLV‐I‐ or ‐II‐infected individuals, a history of blood transfusion, past residence in established HTLV‐I endemic areas or some association with intravenous drug use were common. In contrast, 85% of indeterminant cases had none of these risk factors. These observations suggest that healthy individuals with indeterminant serology for HTLV‐I should not require additional studies.

A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis

Among several single‐nucleotide polymorphisms (SNPs) that correlate with fibrosis progression in chronic HCV, an SNP in the antizyme inhibitor (AzI) gene is most strongly associated with slow fibrosis progression. Our aim was to identify the mechanism(s) underlying this observation by exploring the impact of the AzI SNP on hepatic stellate cell (HSC) activity. Seven novel AZIN1 splice variants (“SV2‐8”) were cloned by polymerase chain reaction from the LX2 human HSC line. Expression of a minigene in LX2 containing the AZIN1 slow‐fibrosis SNP yielded a 1.67‐fold increase in AZIN1 splice variant 2 (AZIN1 SV2) messenger RNA (mRNA) (P = 0.05). In healthy human leukocytes, the SNP variant also correlated with significantly increased SV2 mRNA. Cells (293T) transfected with short hairpin RNA (shRNA) complementary to the exonic splicing chaperone SRp40 expressed 30% less SRp40 (P = 0.044) and 43% more AzI SV2 (P = 0.021) than control shRNA‐expressing cells, mimicking the effect of the sequence variant. LX2 cells transfected with AZIN1 full‐length complementary DNA expressed 35% less collagen I mRNA (P = 0.09) and 18% less α‐smooth muscle actin mRNA (P = 0.09). Transient transfection of AZIN1 SV2 complementary DNA into LX2 cells reduced collagen I gene expression by 64% (P = 0.001) and α‐smooth muscle actin by 43% (P = 0.005) compared to vector‐transfected controls, paralleling changes in protein expression. Both AZIN1 and AZIN‐SV2 mRNAs are detectable in normal human liver and reduced in HCV cirrhotic livers. The AZIN1‐SV2 acts via a polyamine‐independent pathway, as it neither interacts with antizyme nor affects the ability of AZIN1 lacking this variant to neutralize antizyme. Conclusion: An SNP variant in the AZIN1 gene leads to enhanced generation of a novel alternative splice form that modifies the fibrogenic potential of HSCs. (HEPATOLOGY 2011)

Evolutionary relationships between fungi, red algae, and other simple eucaryotes inferred from total DNA hybridizations to a cloned basidiomycete ribosomal DNA

Abstract DNA homologies between a cloned ribosomal DNA from Tyromyces unicolor , a basidiomycete, and whole DNA of representative fungi, algae, and other simple eucaryotes were determined by molecular hybridization experiments. The thermal stabilities of these hybrids indicate that basidiomycetes are more closely related to other fungi, including chytridiomycetes, than to red algae or protozoa, and that red algae are no more closely related to higher fungi than green algae, dictyostelids, and other lower eucaryotes.

An optimized five-gene multi-platform predictor of hormone receptor negative and triple negative breast cancer metastatic risk.

IntroductionOutcome predictors in use today are prognostic only for hormone receptor-positive (HRpos) breast cancer. Although microarray-derived multigene predictors of hormone receptor-negative (HRneg) and/or triple negative (Tneg) breast cancer recurrence risk are emerging, to date none have been transferred to clinically suitable assay platforms (for example, RT-PCR) or validated against formalin-fixed paraffin-embedded (FFPE) HRneg/Tneg samples.MethodsMultiplexed RT-PCR was used to assay two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4) in a pooled FFPE collection of 139 chemotherapy-naïve HRneg breast cancers. The prognostic value of the RT-PCR measured gene signatures were evaluated as continuous and dichotomous variables, and in conditional risk models incorporating clinical parameters. An optimized five-gene index was derived by evaluating gene combinations from both signatures.ResultsRT-PCR measured IR-7 and Buck-4 signatures proved prognostic as continuous variables; and conditional risk modeling chose nodal status, the IR-7 signature, and tumor grade as significant predictors of distant recurrence (DR). From the Buck-4 and IR-7 signatures, an optimized five-gene (TNFRSF17, CLIC5, HLA-F, CXCL13, XCL2) predictor was generated, referred to as the Integrated Cytokine Score (ICS) based on its functional pathway linkage through interferon-γ and IL-10. Across all FFPE cases, the ICS was prognostic as either a continuous or dichotomous variable, and conditional risk modeling selected nodal status and ICS as DR predictors. Further dichotomization of node-negative/ICS-low FFPE cases identified a subset of low-grade HRneg tumors with <10% 5-year DR risk. The prognostic value of ICS was reaffirmed in two previously studied microarray assayed cohorts containing 274 node-negative and chemotherapy naive HRneg breast cancers, including 95 Tneg cases where it proved prognostically independent of Tneg molecular subtyping. In additional HRneg/Tneg microarray assayed cohorts, the five-gene ICS also proved prognostic irrespective of primary tumor nodal status and adjuvant chemotherapy intervention.ConclusionWe advanced the measurement of two previously reported microarray-derived HRneg/Tneg breast cancer prognostic signatures for use in FFPE samples, and derived an optimized five-gene Integrated Cytokine Score (ICS) with multi-platform capability of predicting metastatic outcome from primary HRneg/Tneg tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype.

Multiple metabolic alterations exist in mutant PI3K cancers, but only glucose is essential as a nutrient source.

Targeting tumour metabolism is becoming a major new area of pharmaceutical endeavour. Consequently, a systematic search to define whether there are specific energy source dependencies in tumours, and how these might be dictated by upstream driving genetic mutations, is required. The PI3K-AKT-mTOR signalling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival, but has also been associated with metabolic dysregulation. In this study, we sought to define how mutations within PI3KCA may affect the metabolic dependency of a cancer cell, using precisely engineered isogenic cell lines. Studies revealed gene expression signatures in PIK3CA mutant cells indicative of a consistent up-regulation of glycolysis. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. Additional gene expression changes were also observed, suggesting that metabolic pathways other than glycolysis, such as glutaminolysis, were also affected. Nutrient dependency studies revealed that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients. This specific dependence on glucose for growth was further illustrated by studies evaluating the effects of targeted disruption of the glycolytic pathway using siRNA and was also found to be present across a wider panel of cancer cell lines harbouring endogenous PIK3CA mutations. In conclusion, we have found that PIK3CA mutations lead to a shift towards a highly glycolytic phenotype, and that despite suggestions that cancer cells are adept at utilising alternative nutrient sources, PIK3CA mutant cells are not able to compensate for glucose withdrawal. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies and tumour visualisation strategies.