Prognosis of patients treated with cART from 36 months after initiation, according to current and previous CD4 cell count and plasma HIV-1 RNA measurements

OBJECTIVES: CD4 cell count and plasma viral load are well known predictors of AIDS and mortality in HIV-1-infected patients treated with combination antiretroviral therapy (cART). This study investigated, in patients treated for at least 3 years, the respective prognostic importance of values measured at cART initiation, and 6 and 36 months later, for AIDS and death. METHODS: Patients from 15 HIV cohorts included in the ART Cohort Collaboration, aged at least 16 years, antiretroviral-naive when they started cART and followed for at least 36 months after start of cART were eligible. RESULTS: Among 14 208 patients, the median CD4 cell counts at 0, 6 and 36 months were 210, 320 and 450 cells/microl, respectively, and 78% of patients achieved viral load less than 500 copies/ml at 6 months. In models adjusted for characteristics at cART initiation and for values at all time points, values at 36 months were the strongest predictors of subsequent rates of AIDS and death. Although CD4 cell count and viral load at cART initiation were no longer prognostic of AIDS or of death after 36 months, viral load at 6 months and change in CD4 cell count from 6 to 36 months were prognostic for rates of AIDS from 36 months. CONCLUSIONS: Although current values of CD4 cell count and HIV-1 RNA are the most important prognostic factors for subsequent AIDS and death rates in HIV-1-infected patients treated with cART, changes in CD4 cell count from 6 to 36 months and the value of 6-month HIV-1 RNA are also prognostic for AIDS.

White blood cell count and mortality in patients with acute pulmonary embolism

Although associated with adverse outcomes in other cardiovascular diseases, the prognostic value of an elevated white blood cell (WBC) count, a marker of inflammation and hypercoagulability, is uncertain in patients with pulmonary embolism (PE). We therefore sought to assess the prognostic impact of the WBC in a large, state-wide retrospective cohort of patients with PE. We evaluated 14,228 patient discharges with a primary diagnosis of PE from 186 hospitals in Pennsylvania. We used random-intercept logistic regression to assess the independent association between WBC count levels at the time of presentation and mortality and hospital readmission within 30 days, adjusting for patient and hospital characteristics. Patients with an admission WBC count <5.0, 5.0-7.8, 7.9-9.8, 9.9-12.6, and >12.6 × 10(9) /L had a cumulative 30-day mortality of 10.9%, 6.2%, 5.4%, 8.3%, and 16.3% (P < 0.001), and a readmission rate of 17.6%, 11.9%, 10.9%, 11.5%, and 15.0%, respectively (P < 0.001). Compared with patients with a WBC count 7.9-9.8 × 10(9) /L, adjusted odds of 30-day mortality were significantly greater for patients with a WBC count <5.0 × 10(9) /L (odds ratio [OR] 1.52, 95% confidence interval [CI] 1.14-2.03), 9.9-12.6 × 10(9) /L (OR 1.55, 95% CI 1.26-1.91), or >12.6 × 10(9) /L (OR 2.22, 95% CI 1.83-2.69), respectively. The adjusted odds of readmission were also significantly increased for patients with a WBC count <5.0 × 10(9) /L (OR 1.34, 95% CI 1.07-1.68) or >12.6 × 10(9) /L (OR 1.29, 95% CI 1.10-1.51). In patients presenting with PE, WBC count is an independent predictor of short-term mortality and hospital readmission.

Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^

When to Monitor CD4 Cell Count and HIV RNA to Reduce Mortality and AIDS-Defining Illness in Virologically Suppressed HIV-Positive Persons on Antiretroviral Therapy in High-Income Countries: A Prospective Observational Study.

OBJECTIVE To illustrate an approach to compare CD4 cell count and HIV-RNA monitoring strategies in HIV-positive individuals on antiretroviral therapy (ART). DESIGN Prospective studies of HIV-positive individuals in Europe and the USA in the HIV-CAUSAL Collaboration and The Center for AIDS Research Network of Integrated Clinical Systems. METHODS Antiretroviral-naive individuals who initiated ART and became virologically suppressed within 12 months were followed from the date of suppression. We compared 3 CD4 cell count and HIV-RNA monitoring strategies: once every (1) 3 ± 1 months, (2) 6 ± 1 months, and (3) 9-12 ± 1 months. We used inverse-probability weighted models to compare these strategies with respect to clinical, immunologic, and virologic outcomes. RESULTS In 39,029 eligible individuals, there were 265 deaths and 690 AIDS-defining illnesses or deaths. Compared with the 3-month strategy, the mortality hazard ratios (95% CIs) were 0.86 (0.42 to 1.78) for the 6 months and 0.82 (0.46 to 1.47) for the 9-12 month strategy. The respective 18-month risk ratios (95% CIs) of virologic failure (RNA >200) were 0.74 (0.46 to 1.19) and 2.35 (1.56 to 3.54) and 18-month mean CD4 differences (95% CIs) were -5.3 (-18.6 to 7.9) and -31.7 (-52.0 to -11.3). The estimates for the 2-year risk of AIDS-defining illness or death were similar across strategies. CONCLUSIONS Our findings suggest that monitoring frequency of virologically suppressed individuals can be decreased from every 3 months to every 6, 9, or 12 months with respect to clinical outcomes. Because effects of different monitoring strategies could take years to materialize, longer follow-up is needed to fully evaluate this question.

Differential Cytoplast Requirement for Embryonic and Somatic Cell Nuclear Transfer in Cattle

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 microM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 micro g/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1-4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P < 0.05). In contrast, when skin fibroblasts were used as donor cells, the use of an MII cytoplast (vs. G1/S phase) was imperative for blastocyst development (30 vs. 6%, P < 0.05). Differential staining showed that parthenogenetic, embryonic, and somatic cloned BL contained 26, 29, and 33% presumptive inner cell mass (ICM) cells, respectively, which is similar to that of frozen-thawed in vivo embryos at a comparable developmental stage (23%). These data indicate that embryonic and somatic nuclei require different recipient cytoplast environment for remodeling/ reprogramming, and this is likely due to the different cell cycle stage and profiles of molecular differentiation of the transferred donor nuclei.

Cloning Animals by Somatic Cell Nuclear Transfer--Biological Factors

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.

Determinants of CD4 cell count decline after treatment interruption

Patients who had started HAART (Highly Active Anti-Retroviral Treatment) under previous aggressive DHHS guidelines (1997) underwent a life-long continuous HAART that was associated with many short term as well as long term complications. Many interventions attempted to reduce those complications including intermittent treatment also called pulse therapy. Many studies were done to study the determinants of rate of fall in CD4 count after interruption as this data would help guide treatment interruptions. The data set used here was a part of a cohort study taking place at the Johns Hopkins AIDS service since January 1984, in which the data were collected both prospectively and retrospectively. The patients in this data set consisted of 47 patients receiving via pulse therapy with the aim of reducing the long-term complications. ^ The aim of this project was to study the impact of virologic and immunologic factors on the rate of CD4 loss after treatment interruption. The exposure variables under investigation included CD4 cell count and viral load at treatment initiation. The rates of change of CD4 cell count after treatment interruption was estimated from observed data using advanced longitudinal data analysis methods (i.e., linear mixed model). Using random effects accounted for repeated measures of CD4 per person after treatment interruption. The regression coefficient estimates from the model was then used to produce subject specific rates of CD4 change accounting for group trends in change. The exposure variables of interest were age, race, and gender, CD4 cell counts and HIV RNA levels at HAART initiation. ^ The rate of fall of CD4 count did not depend on CD4 cell count or viral load at initiation of treatment. Thus these factors may not be used to determine who can have a chance of successful treatment interruption. CD4 and viral load were again studied by t-tests and ANOVA test after grouping based on medians and quartiles to see any difference in means of rate of CD4 fall after interruption. There was no significant difference between the groups suggesting that there was no association between rate of fall of CD4 after treatment interruption and above mentioned exposure variables. ^

Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

White blood cell count predicts reduction in coronary heart disease mortality with pravastatin

Background-Elevated serum inflammatory marker levels are associated with a greater long-term risk of cardiovascular events. Because 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins) may have an antiinflammatory action, it has been suggested that patients with elevated inflammatory marker levels may have a greater reduction in cardiovascular risk with statin treatment. Methods and Results-We evaluated the association between the white blood cell count (WBC) and coronary heart disease mortality during a mean follow-up of 6.0 years in the Long-Term Intervention With Pravastatin in Ischemic Disease (LIPID) Study, a clinical trial comparing pravastatin (40 mg/d) with a placebo in 9014 stable patients with previous myocardial infarction or unstable angina. An increase in baseline WBC was associated with greater coronary heart disease mortality in patients randomized to placebo (hazard ratio for 1 X 10(9)/L increase in WBC, 1.18; 95% CI, 1.12 to 1.25; P<0.001) but not pravastatin (hazard ratio, 1.02; 95% CI, 0.96 to 1.09; P=0.56; P for interaction=0.004). The numbers of coronary heart disease deaths prevented per 1000 patients treated with pravastatin were 0, 9, 30, and 38 for baseline WBC quartiles of <5.9, 6.0 to 6.9, 7.0 to 8.1, and >8.2X10(9)/L, respectively. WBC was a stronger predictor of this treatment benefit than the ratio of total to high-density lipoprotein cholesterol and a global measure of cardiac risk. There was also a greater reduction (P=0.052) in the combined incidence of cardiovascular mortality, nonfatal myocardial infarction, and stroke with pravastatin as baseline WBC increased ( by quartile: 3, 41, 61, and 60 events prevented per 1000 patients treated, respectively). Conclusions-These data support the hypothesis that individuals with evidence of inflammation may obtain a greater benefit from statin therapy.

Somatic cell gene therapy for diabetes mellitus: engineering a surrogate B-cell

Improved methods of insulin delivery are required for the treatment of insulin-dependent diabetes mellitus (IDDM) to achieve a more physiological profile of glucose homeostasis. Somatic cell gene therapy offers the prospect that insulin could be delivered by an autologous cell implant, engineered to secrete insulin in response to glucose. This study explores the feasibility of manipulating somatic cells to behave as a surrogate insulin-secreting β-cells. Initial studies were conducted using mouse pituitary AtT20 cells as a model, since these cells possess an endogenous complement of enzymes capable of processing proinsulin to mature insulin. Glucose sensitive insulin secretion was conferred to these cells by transfection with plasmids containing the human preproinsulin gene (hppI-1) and the GLUT2 gene for the glucose transporter isoform 2. Insulin secretion was responsive to changes in the glucose concentration up to about 50μM. Further studies to up-rate this glucose sensitivity into the mM range will require manipulation of the hexokinase and glucokinase enzymes. Intraperitoneal implantation of the manipulated AtT20 cells into athymic nude mice with streptozotocin-induced diabetes resulted in decreased plasma glucose concentrations. The cells formed vascularised tumours in vivo which were shown to contain insulin-secreting cells. To achieve proinsulin processing in non-endocrine cells, co-transfection with a suitable enzyme, or mutagenesis of the proinsulin itself are necessary. The mutation of the human preproinsulin gene to the consensus sequence for cleavage by the subtilisin-like serine protease, furin, was carried out. Co-transfection of human fibroblasts with wild-type proinsulin and furin resulted in 58% conversion to mature insulin by these cells. Intraperitoneal implantation of the mature-insulin secreting human fibroblasts into the diabetic nude mouse animal model gave less encouraging results than the AtT20 cells, apparently due to poor vascularisation. Cell aggregations removed from the mice at autopsy were shown to contain insulin secreting cells only at the periphery. This thesis provides evidence that it is possible to construct, by cellular engineering, a glucose-sensitive insulin-secreting surrogate β-cell. Therefore, somatic cell gene therapy offers a feasible alternative for insulin delivery in IDDM patients.