Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization,pharmacokinetic data and in vivo experiments

Summary The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.     

Ca2+-dependent actin remodeling in the contracting A7r5 cell

Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the -actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775–781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca²⁺]i by A₂₃₁₈₇ and thapsigargin on - and -actin remodeling. The effects of A₂₃₁₈₇ and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the - and -actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, -actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca²⁺]i resulted in partial or complete dissolution of -actin cables without further remodeling. In addition, PDBu-mediated -actin remodeling was blocked in the presence of A₂₃₁₈₇. Increased [Ca²⁺]i also caused dispersal of -actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for -actin cytoskeletal structure. The incubation of cells in calcium-free media prevented -actin dissolution by A₂₃₁₈₇/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of -actin cables by increased [Ca²⁺]i. The results suggest that the sustained elevation of [Ca²⁺]i beyond a threshold level initiates depolymerization of -actin but not -actin. It further appears that PDBu-induced -actin remodeling requires Ca²⁺ but increases of [Ca²⁺]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits -actin dissolution in the presence of A₂₃₁₈₇/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing -actin structure in the activated cell.     

A multicenter, randomized, double-blind study to evaluate the safety, tolerability, and efficacy of OptiMARK (gadoversetamide injection) compared with Magnevist (gadopentetate dimeglumine) in patients with liver pathology: results of a Phase III clinical trial

The purpose of this study was to evaluate the safety and efficacy of OptiMARK (gadoversetamide injection) compared with Magnevist (gadopentetate dimeglumine) in hepatic MRI of patients with suspected liver pathology. A Phase III, multicenter, randomized, double-blind, parallel group study was performed in adults with suspected liver pathology. All patients underwent contrast-enhanced computed tomography within 3 weeks prior to magnetic resonance scanning. Ninety-nine patients received OptiMARK, and 94 patients received Magnevist at a dose of 0.1 mmol/kg. Precontrast T1- and T2-weighted spin-echo imaging and T1-weighted gradient-echo imaging were performed, followed by T1-weighted gradient-echo imaging at 15-20 seconds, 1 minute, and 5 minutes after intravenous contrast injection. Three primary efficacy endpoints (confidence in lesion diagnosis, level of conspicuity, and lesion border delineation) were evaluated on the precontrast image set and compared with the pre plus postcontrast image set. Vital signs, physical examination, electrocardiograms (ECGs), and laboratory parameters (chemistry, hematology, and urinalysis) were measured at various time points. Adverse events were recorded. The study design and statistical analyses were chosen to demonstrate presumed equivalence of OptiMARK and Magnevist. There were no statistically significant differences in efficacy between OptiMARK and Magnevist as assessed by either blinded readers or the on-site principal investigators. No serious or unexpected adverse events were noted. Of the 193 patients receiving contrast media, 82 experienced a total of 154 adverse events. Thirty-three (21.4%) of these 154 adverse events were felt by the on-site investigators to be probably related to either study agent: 15 events in 9 patients in the OptiMARK group, and 18 events in 13 patients in the Magnevist group. Headache was the most common adverse event, occurring in 10.1% of the OptiMARK patients and 12.8% of the Magnevist patients. No clinically relevant trends were observed in any laboratory parameter or ECG findings in either treatment group. The results demonstrate the safety, efficacy, and equivalence of OptiMARK and Magnevist at a dose of 0.1 mmol/kg in hepatic magnetic resonance imaging of patients with suspected liver pathology.     

Measuring primary care patients' attitudes about dementia screening

OBJECTIVES: To develop a questionnaire that will capture patients' attitudes about dementia screening in primary care. METHODS: Cross-sectional study of 315 patients aged 65 and older attending urban and rural primary care clinics in Indianapolis and North Carolina. The Perceptions Regarding Investigational Screening for Memory in Primary Care (PRISM-PC) questionnaire was administered via face-to-face or telephone interview. RESULTS: The PRISM-PC questionnaire consists of two separate scales: the patient's acceptance of dementia screening scale and the patient's perceived harms and benefits of dementia screening scale. The face validity of the PRISM-PC questionnaire was based on a systematic literature review and the opinions of 16 clinician-investigators with experience in screening for dementia. Exploratory factor analyses for the acceptance scale revealed the presence of two dimensions: knowledge about dementia risk and testing for dementia. For the benefits and harms scale, exploratory factor analyses identified four dimensions: perceived benefits of screening, stigma of screening, suffering from screening, and impact of screening on patients' independence. The internal consistency of each of the above subscales was good with Cronbach's alpha ranging from 0.58-0.85. CONCLUSION: The PRISM-PC questionnaire captures primary care patients' acceptance, perceived harms, and perceived benefits of dementia screening.     

Aging and infectious diseases: do patterns of comorbidity vary by HIV status, age, and HIV severity?

Patterns of comorbidity among persons with human immunodeficiency virus (HIV) are not well described. We compared comorbidity among veterans with and without HIV infection. The sample consisted of 33,420 HIV-infected veterans and 66,840 HIV-uninfected veterans. We identified and clustered 11 comorbid conditions using validated International Classification of Diseases, 9th Revision, Clinical Modification codes. We defined multimorbidity as the presence of conditions in all clusters. Models restricted to HIV-infected veterans were adjusted for CD4 cell count and viral load. Comorbidity was common (prevalence, 60%-63%), and prevalence varied by HIV status. Differences remained when the veterans were stratified by age. In multivariable analyses, older HIV-infected veterans were more likely to have substance use disorder and multimorbidity. Renal, vascular, and pulmonary diseases were associated with CD4 cell count <200 cells/mm(3); hypertension was associated with CD4 cell count >200 cells/mm(3). Comorbidity is the rule, and multimorbidity is common among veterans with HIV infection. Patterns of comorbidity differ substantially by HIV status, age, and HIV severity. Primary care guidelines require adaptation for persons with HIV infection.     

The U3 promoter region of the acutely lethal simian immunodeficiency virus clone smmPBj1.9 confers related biological activity on the apathogenic clone agm3mc.

Infection with the acutely pathogenic molecular virus clone SIVsmmPBj1.9, cloned from isolate PBj14 of simian immunodeficiency virus (SIV) from sooty mangabey monkeys (Cercocebus atys), leads to acute viral and often lethal disease within days or weeks. SIVsmmPBj1.9 has the unique property of replicating in nonstimulated peripheral blood mononuclear cells from pig-tailed macaques. In contrast, molecular virus clone SIVagm3mc of SIV from African green monkeys (Cercopithecus aethiops), which is apathogenic in its natural host and in pig-tailed macaques, is unable to grow in nonstimulated peripheral blood cells. Chimeric proviruses were constructed by exchanging defined regions of SIVagm3mc against comparable regions of SIVsmmPBj1.9. Four of five hybrid viruses generated by transfection into the CD4-positive T-cell line C8166 replicated in T-cell lines permissive for SIVagm3mc replication and in stimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Three hybrid viruses displayed the distinct biological property of SIVsmmPBj14 to replicate in nonstimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Replication in nonstimulated peripheral blood cells was dependent on the presence of the U3 promoter region of SIVsmmPBj1.9 within the viral long terminal repeat.     

Cell-mediated immune proliferative responses to HIV-1 of chimpanzees vaccinated with different vaccinia recombinant viruses

The only animal that can be reproducibly infected with HIV, and that thus provides an experimental system for testing the effectiveness of prototype vaccines, is the chimpanzee. We compared proliferative responses to HIV and to vaccinia virus (VV) antigens of lymphocytes taken at various times from chimpanzees vaccinated with recombinant VV expressing different HIV genes. Animals were immunized with the original VV strain, as control, or with constructs expressing gp160 (VV160) given exclusively or in combination with one or two other constructs producing p25 (VV25), F/3'-orf (VVF), or the human interleukin-2 (IL-2) gene, which was included in an attempt to amplify immune responses. Irrespective of the HIV gene utilized, lymphocyte proliferation to HIV was usually weak and rapidly decreased after each inoculation, contrasting with strong and sustained responses to VV. Lack of adequate recall reactivity after challenge with fixed autologous lymphocytes expressing VV-produced HIV antigens indicated that vaccination resulted only in low levels of HIV-specific memory cell priming. The use of IL-2-producing VV did not lead to increased responsiveness. Reactivity to soluble purified gp160, but not to p25, could be detected in PBL from animals that had received both VV160 and VV25, while immunization with VVF resulted in a significant response to this protein in one of two animals. The transient nature of T cell reactivity to HIV might explain why, in similar studies, chimpanzees were not protected from infection with live HIV.