Ageing and chronic disease-related changes in the morphometric characteristics of ovarian follicles in cynomolgus monkeys (Macaca fascicularis).

STUDY QUESTION: How is the localisation of ovarian follicles affected by ageing and chronic diseases? SUMMARY ANSWER: Ovarian follicles shift deeper towards the medulla, due to thickening of the tunica albuginea (TA), with ageing and some major common chronic diseases. WHAT IS KNOWN ALREADY: The ovary undergoes morphological and functional changes with ageing. The follicular pool follows these changes with alterations in the amount and distribution of residual follicles. Diseases causing a chronic inflammatory process are associated with morphological changes and impaired ovarian function. STUDY DESIGN, SIZE, DURATION: We conducted a cross-sectional study, examining 90 ovaries from 90 female monkeys. The samples were collected from April 2018 to March 2019 at Tsukuba Primate Research Center in National Institutes of Biomedical Innovation, Health and Nutrition, Japan. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian samples were obtained from cynomolgus monkeys that died from natural causes or were euthanised. Ovarian sections were stained with haematoxylin and eosin (H&E) for histological analyses. In ovarian sections from 64 female macaques aged 0-25 years, a total of 13 743 follicles at different developmental stages (primordial, intermediary, primary, early secondary and late secondary) were assessed to determine the depth of each follicle from the outer surface of the ovarian cortex to the far end of the follicle, by using a digital imaging software. TA thickness was measured as sum of basal membrane and tunica collagen layer for each ovary under H&E staining. To explore the possibility of age-related trends in ovarian morphometric characteristics, samples were divided into four different age groups (0-3 years (pre-menarche), 4-9 years, 10-14 years and 15-20 years). To evaluate the effect of common chronic diseases on ovarian morphometric characteristics, macaques with diabetes mellitus (DM) (n = 10), endometriosis (n = 8) or inflammatory bowel disease (IBD) (n = 8) were compared to age-matched controls without chronic diseases. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian morphometric analysis revealed that the relative location of follicles became deeper in all age groups according to development of follicles (P < 0.05). Total follicle distance from the ovarian surface was increased with ageing (P < 0.05). In a sub-analysis according to developmental stage, only primordial and intermediary follicles were localised deeper with increasing age (P < 0.05). TA thickness was also increased with ageing (P < 0.05). The localisation of the total number of follicles became deeper in ovaries from monkeys with DM, endometriosis or IBD as compared to the control group (P < 0.05). With DM, analysis of follicles distance at almost each developmental stage was significantly deeper compared to controls (P < 0.05) with the exception of early secondary follicles. With endometriosis, follicles at primary and early and late secondary stages were significantly deeper compared to controls (P < 0.05). Also with IBD, follicles at primary and early and late secondary follicles were significantly deeper compared to controls (P < 0.001). The TA was thicker with DM and endometriosis compared to controls (P < 0.05), but not with IBD (P = 0.16). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Two-dimensional histology was used to assess follicle localisation. The possibility of minimal variations between the measured distance to the actual distance in a spherical structure cannot be excluded. Additionally, the severity of disease was not assessed. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first step towards enhancing our understanding of how ageing and chronic diseases affect the relative localisation of dormant and developing follicles. These observations, combined with possible future human studies, may have managerial implications in the field of fertility preservation and other conditions involving ovarian tissue cryopreservation. STUDY FUNDING/COMPETING INTEREST(S): The present work was supported by the Grant-in-Aid for Scientific Research B (19H03801) (to K.K.), Challenging Exploratory Research (18K19624), Japan Agency for Medical Research and Development, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Takeda Science Foundation and Naito Foundation (to K.K.). All authors have no conflicts of interest directly relevant to the content of this article.

Obstacles to using online health services among adults age 50 and up and the role of family support in overcoming them.

BACKGROUND: Using Online Health Services (OHS) could benefit older adults greatly and could also reduce the burden on the health system. Yet invisible obstacles or barriers appear to impede mass adoption of these services among this population group. The aim of the current research is to provide a qualitative picture of these invisible obstacles and to profile their main features, with special attention to the role of family members in supporting OHS use among this population group. METHODS: This qualitative study entailed a series of in-depth, semi-structured, open phone interviews conducted with 31 individuals age 50 and up in Israel, who constituted a sample of OHS users and non-users among older adults. RESULTS: Four major themes and primary observations emerge from our data: 1. While older adults are aware of OHS to some extent, they often do not fully understand the specific benefits of using these services; 2. Older adults need to acquire much more experience with OHS use. OHS user interfaces still have a long way to go for older adults to feel comfortable using them. People age 50 and up seem to be less concerned about privacy and security issues than about seemingly more trivial issues such as recovering forgotten passwords; 3. Family members can play key roles in helping older adults adopt OHS by providing technical support as well as encouragement; 4. Older adults have worthwhile recommendations for innovations and policy improvements that would facilitate wider adoption of OHS. CONCLUSIONS: The results of the current study reveal important nuances regarding the importance of awareness, user interface and experience for OHS use among older adults, as well as the critical role of family members in OHS adoption. Based on these findings, we recommend the following: expanding advertising on media channels to emphasize the benefits of OHS use; improving HMO websites to make them more user-friendly for older people; developing HMO-run community OHS guidance programs geared to older people to reduce the gap between required skills and user competencies, thus enabling older people to benefit from OHS use.

The effect of placenta previa on fetal growth and pregnancy outcome, in correlation with placental pathology.

OBJECTIVE: To compare the clinical characteristics and placental histopathology between pregnancies complicated by placenta previa and controls. STUDY DESIGN: Between 2009 and 2015, cesarean deliveries (CDs) of 119 pregnancies with placenta previa were identified from which maternal outcomes, neonatal outcomes and placental pathology were reviewed. Results were compared with CDs matched for maternal age and pregnancy complications (control group, n=119). Placental lesions were classified into maternal and fetal vascular supply lesions and inflammatory response. Composite neonatal outcome was defined as one or more of early neonatal complications. Small-for-gestational age (SGA) was defined as birth weight ⩽10th percentile. RESULTS: Placentas from the previa group had higher rates of weights <10th percentile (P<0.001) and of maternal and fetal vascular supply lesions (P<0.001, for both). Higher rate of SGA (P=0.003) and worse composite neonatal outcome (P<0.001) were also observed in the previa group as compared with controls. After controlling for potential confounding bias using multivariable logistic regression models, placenta previa remained statistically significantly associated with placental maternal (adjusted odds ratio (aOR) 2.48, 95% confidence interval (CI) 1.2-4.9, P=0.009) and fetal (aOR 7.05, 95% CI 2.4-20.2, P<0.001) vascular supply lesions, SGA (aOR 10, 95% CI 2.3-44.2, P=0.002) and adverse neonatal outcome (aOR 6.87, 95% CI 2.9-11.8, P<0.001). CONCLUSIONS: More placental vascular supply lesions, higher rate of SGA and worse neonatal outcome characterized pregnancies with placenta previa in the current study. These findings may suggest that abnormal placentation is accompanied by suboptimal implantation that interferes with fetal growth.

V3 variation in HIV-seropositive patients receiving a V3- targeted vaccine.

OBJECTIVE: To analyze V3 loop sequences of HIV-1 in three seropositive individuals who exhibited declines in viremia while receiving a V3-targeted vaccine. DESIGN: Retrospective analysis of case series at an HIV Clinic, University of Tel Aviv. PATIENTS: Three HIV-1-seropositive, PPD-DTHR-positive (PPD, Siebert purified protein derivative of tuberculin; DTHR, delayed type hypersensitivity reaction) individuals who had been inoculated with a mixture of PPD-cross-linked V3 peptides from five HIV strains and then exhibited declines in HIV-1 viremia during the course of vaccination in the absence of combination antiretroviral therapy and whose virus levels resurged once vaccine boosting was discontinued. RESULTS: Declines in viremia were observed even when the viral V3 sequences of the patients' HIV differed by at least one or two amino acid residues from those of the five peptides in the vaccine. Although viral mutants with amino acid substitutions within V3 appeared during vaccination, plasma virus loads remained at low levels for several months after these variants appeared. About a year after boosting was discontinued, anti-V3 peptide antibodies in the patients had declined and plasma virus returned to the prevaccination levels or higher. Compared with the isolates that predominated during the course of vaccination, the resurgent viruses contained zero to six amino acid residue differences in the V3 loop but few synonymous substitutions. CONCLUSIONS: Viruses with altered V3 sequences did emerge but did not result in increased viremia during the course of vaccination. In two individuals where V3 mutations were absent in the virus that re-emerged after vaccine boosting ceased, resurgence could not have been a consequence of V3 changes.

Need for an external proficiency testing program for cytokines, chemokines, and plasma markers of immune activation.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Ibeta, soluble TNF receptor II, soluble IL-2 receptor alpha, beta(2)-microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.

Progressive specific immune attrition after primary, secondary and tertiary immunizations with bacteriophage phi X174 in asymptomatic HIV-1 infected patients.

BACKGROUND: Antibody responses to immunization are often compromised in patients infected by HIV-1, and the use of childhood immunization in affected children is controversial. We investigated whether multiple immunizations with a T cell-dependent neoantigen, bacteriophage phiX174, induce selective immune attrition and post-vaccination viremia. METHODS: Seventeen asymptomatic, antiretroviral therapy-naïve HIV-1-infected patients with a CD4 cell count of 450 cells/microl or greater were immunized in 1990/1991 with three intravenous doses of bacteriophage phiX174. Group 1 received zidovudine (ZDV) during the primary and secondary immunization. Group 2 received ZDV exclusively during the tertiary immunization. Bacteriophage-specific antibodies of the IgM and IgG class, lymphocyte phenotypes (CD4+, CD8+, CD4+DR+, CD8+DR+, CD4+CD45RO+ and CD4+45RA+, CD4+CD45RO+DR+) and HIV-1 plasma viremia were measured sequentially. RESULTS: In both patient groups the primary, secondary and tertiary antibody responses, as expressed by geometric mean antibody titres and IgM to IgG switch, were impaired. Booster immunizations resulted in a progressive attrition of specific antibody responses to bacteriophage. Antibodies to tetanus toxoid remained stable. The HIV-1 viral loads, which were evaluated in archived specimens from eight patients, increased after immunization but returned to baseline approximately 4 weeks later. The humoral immune attrition and increases in plasma viremia were blunted by concomitant short courses of ZDV. DISCUSSION: Multiple boosters of immunizations in asymptomatic treatment-naive HIV-1-infected patients may result in a specific immune attrition and vaccine-induced viremia. Short-term monotherapy with ZDV may have blunted these adverse effects. Hyperimmunization of HIV-1-infected patients may be detrimental unless accompanied by antiretroviral therapy.

Immunologic responses of HIV-1-infected study subjects to immunization with a mixture of peptide protein derivative-V3 loop peptide conjugates.

V3 loop peptide sequences from several HIV-1 strains were covalently linked to purified protein derivative (PPD) of Mycobacterium tuberculosis. A mixture of PPD conjugates of V3 loop peptides from six different strains of HIV-1 induced a stronger antibody response than a single V3 peptide-conjugate administered to guinea pigs and humans. Sera from animals immunized with a PPD-six peptide-PPD conjugate neutralized multiple primary-isolate strains of HIV-1. Potent immune responses were noted only when animals were primed with bacillus Calmette-Guerin (BCG), PPD was covalently bound to the peptides, and PPD was used as the carrier protein. Based on these animal studies, an immunogen consisting of PPD-conjugated V3 loop peptides from five HIV-1 strains was tested in 7 HIV-1 seropositive PPD skin test positive study subjects. Vaccinees exhibited over time a uniform increase in neutralizing antibodies for both laboratory adapted and primary isolates of HIV-1, including strains from multiple clades. In 3 patients with baseline viral loads between 8000 and 12,000 RNA copies/ml, the viral load declined in 2 patients to <400 copies/ml and in 1 patient to 1200 copies/ml without concurrent administration of highly active antiretroviral therapy (HAART).

Receptor-mediated maternofetal transfer of immunoglobulins. Inhibition of transport of anti-HIV-1 immunoglobulin by generic immunoglobulins in the in vitro perfused placenta.

OBJECTIVES: The passage of immunoglobulin G (IgG) across the placenta is thought to involve Fc' receptors on the syncytiotrophoblast. To confirm the receptor dependency of this process we have studied the changes in the tissue content and transfer kinetics of immunoglobulins and hyperimmune serum to HIV (HIVIG) during in vitro dual placental perfusion. METHODS: Isolated lobules of term placentae from normal pregnancies were perfused in a model of maternal and fetal circulation. The perfused tissue was compared to fresh tissue samples from the same placenta for the content of IgG, IgG subclasses, IgM, cytokeratin, human placental lactogen and SP1 antigen by immunohistochemistry and by protein elution. RESULTS: The immunoglobulin staining faded by an average of 40% during the 1st hour of perfusion. In contrast, staining for cytokeratin, human placental lactogen and SP1 remained unchanged. During a 4-hour recycling of endogenous immunoglobulins in the maternal circulation, IgG and HIVIG crossed to the fetal side in a steady rate. The transport of HIVIG could be inhibited by preperfusion with an intravenous gammaglobulin preparation (IVIG). DISCUSSION: The transfer of IgG across the placenta occurs in a steady state rate consistent with a receptor-mediated mechanism. Furthermore, inhibition of HIVIG maternofetal transfer by IVIG further establishes the receptor-mediated transfer of immunoglobulins through the placenta.

Suppression of HIV-1 replication by propolis and its immunoregulatory effect.

In the current study we show that propolis, a non-toxic natural bee-hive product, suppresses HIV-1 replication and modulates in vitro immune responses. CEM cells were treated with propolis at nontoxic concentrations prior to or following infection with HIV-1. Propolis abolished syncytium formation at 4.5 micrograms/ml and inhibited it at lower doses in a concentration-dependent manner. Propolis decreased p24 antigen production by as much as 90-100% in a concentration-dependent manner. Furthermore, modulation of peripheral blood mononuclear cells (PBMCs) mitogenic responses upon the addition of propolis was noted, reducing the elevated responses to Concanavalin A (Con A) and enhancing suppressed mitogenic responses to pokeweed mitogen (PWM). In summary, propolis may constitute a non-toxic natural product with both anti HIV-1 and immunoregulatory effects.

CD4 confers susceptibility to human immunodeficiency virus type 1 infection in a rat fibroblast cell line.

A new model system is delineated that will enable study of CD4 cofactors and gp120 binding proteins other than CD4. We have previously described a nontransformed rat fibroblast cell line that can efficiently produce HIV-1 upon transfection with an HIV-1 infectious clone, in contrast to other nonhuman mammalian cell lines tested. In the present study we analyzed the susceptibility to HIV-1 infection of Rat2 cells expressing the human CD4 protein. We have used the mammalian expression vector pKS286, in which HIV-1 LTR drives the expression of the CD4 gene. We show that the Rat2 cell line, expressing the human CD4 (Rat2/CD4), is susceptible to fusion with and infection by HIV-1. The virus produced by the Rat2/CD4 cells was infectious. CD4 expression in the Rat2/CD4 was down-regulated over time, similarly to HIV-1 expression in HIV-1-transfected Rat2 cells. Transfection of the Rat2/CD4 cells with a tat expression vector reestablished the CD4 expression on the surface of those cells, as expected. We conclude that the expression of a gp120 binding protein on the Rat2 cells' surface suffices to render the Rat2 cells susceptible to HIV-1 infection.

L-cycloserine, an inhibitor of sphingolipid biosynthesis, inhibits HIV-1 cytopathic effects, replication, and infectivity.

Drugs that reduce viral production or prevent viral spread by interference with the host's cellular components are unlikely to induce resistance, in contrast to treatment modalities that interact with the HIV-1 life cycle. Two features make L-cycloserine (L-CS) a candidate drug of this kind: (a) L-CS is a potent inhibitor of the sphingolipid pathway (b) sphingolipids, galactocerebrosides, and sulfatides have been shown, by others, to bind gp120. In a feasibility and efficacy study, we have found that L-CS inhibits HIV-1 replication in a CD4+ lymphoid cell line (CEM) as documented by the reduction of syncytium formation, the number of HIV-1 infected cells, and p24 protein production. This observation may lead to a new strategy for the treatment of HIV-1 infection.

Safety and immunogenicity of a V3 loop synthetic peptide conjugated to purified protein derivative in HIV-seronegative volunteers.

OBJECTIVES: To develop a peptide-based model for a preventive vaccine for HIV-1 infection. DESIGN: Phase I trial in HIV-1-seronegative volunteers. PARTICIPANTS: Adult healthy subjects HIV-1-antibody-seronegative in an enzyme-linked immunosorbent assay, screened for tuberculin [purified protein derivative (PPD)] reactivity with 2 tuberculin units PPD-administered intradermally. INTERVENTIONS: Submicrogram doses of a PPD conjugate with a peptide of the primary neutralizing domain (PND) of HIV-1MN (PPD-MN-PND) were administered intradermally to tuberculin skin-test-positive and -negative volunteers. RESULTS: Antibodies to the MN-PND were measured after two immunizations in 10 out of 11 PPD skin-test-positive volunteers. After the fourth immunization high-affinity antibodies were detected, which persisted for over 1 year. High titers of MN-PND-specific immunoglobulin (Ig) G and IgA were detected in the serum and saliva of all volunteers tested. Serum antibodies were cross-reactive with PND peptide from some other HIV-1 strains but neutralized only the HIV-1MN prototype. Human leukocyte antigen (HLA)-B7-restricted MN-PND-specific cytotoxic T lymphocytes (CTL) were also detected. CONCLUSIONS: The PPD-MN-PND vaccine at submicrogram doses is safe and immunogenic in PPD skin-test-positive healthy adult volunteers. Long lasting humoral immune responses in the serum and saliva were possibly accompanied by HLA-B7-restricted CTL responses. This is a vaccine prototype that can be rapidly and inexpensively modified to include other peptide epitopes. It is especially suitable for use in a worldwide multibillion Bacillus Calmette-Guérin (BCG)-primed or tuberculosis-exposed population at risk for HIV-1 infection.

Improved brain delivery and in vitro activity of zidovudine through the use of a redox chemical delivery system.

OBJECTIVE: Improved therapy for AIDS dementia and related encephalopathies may be achieved through enhanced delivery of effective antiretroviral agents to the central nervous system (CNS). DESIGN: A novel chemical delivery system (CDS) was used, which utilized redox trapping of drugs in the brain. This study was aimed at defining the pharmacokinetics of a zidovudine (ZDV)-CDS as well as establishing its in vitro antiviral efficacy against HIV in both lymphocytes and in a neural cell line. RESULTS: ZDV-CDS administered parenterally to rats produced significantly higher brain levels of ZDV [area under the curve (AUC), 425 micrograms x min/g] than equimolar ZDV (AUC, 13.5 micrograms x min/g). Native ZDV uptake was minimal after 1 h when analyzed in CEM lymphocytes and in SKNMC neuroblastoma cell line. By contrast, marked uptake of ZDV-CDS was followed by biochemical conversion of ZDV-CDS to its main metabolites (ZDV-CDS quaternary salt, ZDV-Q+, and native ZDV). These improved uptake profiles were associated with greater in vitro virucidal effect. ZDV-CDS at 0.5 microM was 80% more effective than ZDV in suppressing p24 production in a lymphocyte culture infected with 6000 median tissue culture infective doses (TCID50) of the HIV N1T strain and 50% more effective at 0.05 microM. Furthermore, syncytia formation was completely suppressed at a ZDV-CDS dose of 0.5 microM (600 TCID50) but native ZDV at the same dose was ineffective. Finally, while ZDV (at 0.5 microM) is not active in reducing viral replication in an SKNMC neural cell line, the ZDV-CDS complex significantly suppressed p24 synthesis. CONCLUSION: The ZDV-CDS complex is capable of delivering higher ZDV doses to lymphocytes and neural cells, with improved antiretroviral activity.

HIV type 1 infection of human cortical neuronal cells: enhancement by select neuronal growth factors.

Neuronal cells in culture respond to neuronal growth factors by secondary cellular pathways similar to those described for activation of lymphocytes and macrophages. In HIV-1-infected T lymphocytes and in macrophages, these pathways were shown to converge on nuclear factors that bind and stimulate the HIV-1 LTR and lead to enhancement of HIV-1 expression. In the current study we have investigated whether the same mechanisms also enhance HIV-1 production by neural cells. We have demonstrated that HIV/N1T replication in HCN-1A cells, a human cortical neuronal cell line, is enhanced threefold by nerve growth factor (NGF) and by fibroblast growth factor (FGF), but not by epidermal growth (EGF) factor, or phorbol ester. HCN-1A cells also responded to HIV/N1T infection with pronounced morphological changes, indicative of a differentiation-like process. The cells diminished in size and small neurites were observed.

Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease.

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, > 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.

The establishment of rodent cell lines persistently producing HIV-1.

Animal cells differ in susceptibility to HIV-1 infection. To identify rodent cells which are permissive to HIV-1 replication, we transfected murine and rat cells with an infectious clone of HIV-1 and a vector containing the chloramphenicol acetyl transferase gene under the control of HIV-1 LTR. Three groups of transfectants were distinguished: (i) Cells which permit neither HIV-1 LTR activation nor viral protein expression; (ii) Cells which permit activation of the HIV-1 LTR but not HIV-1 protein expression; and (iii) Cells which are fully permissive to both HIV-1 LTR activation and virus production. The latter included rat embryonal fibroblastoid (Rat2) cells, which, in short-term transfection assays, produced titers of HIV-1 proteins similar to transfected T lymphoid cells. To establish persistently infected cells, Rat2 cells were stably transfected with a plasmid containing an infectious clone of HIV-1/N1T-A and a neo gene, yielding several G-418-resistant, HIV-1-producing cell cultures. Of these, Rat2/A1 and Rat2/A2 cell cultures expressed up to 60 ng HIV-1 p24 core antigen per 1 x 10(6) cells 3 days after cell subculture over a period of 3 months. Southern blot hybridization revealed that Rat2/A1 and Rat2/A2 carried one to two HIV-1 DNA copies per cell; no rearrangements or deletions in viral DNA were present. Restriction endonuclease analysis of HIV-1 DNA in Rat2/A2 cells suggested clonal expansion of cells containing integrated HIV-1 genome. Virus produced by the Rat2/A1 cells was infectious in human T cells. These data demonstrate that some rodent cells have no inherent restriction to persistent and efficient production of infectious HIV-1.

PC12 cells differentiate into chromaffin cell-like phenotype in coculture with adrenal medullary endothelial cells.

Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

Neurotrophic activity of monomeric glucophosphoisomerase was blocked by human immunodeficiency virus (HIV-1) and peptides from HIV-1 envelope glycoprotein.

Glucophosphoisomerase (GPI), a glycolytic enzyme, was recently described to share 90% sequence homology with neuroleukin, a recently discovered growth factor which promotes motor neuron regeneration in vivo, survival of peripheral and central neurons in vitro, and affects B cell immunoglobulin synthesis. Interestingly, neuroleukin activity was described to be antagonized by the human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120), with which neuroleukin was found to share partial sequence homology. In this study, reduced GPI demonstrated similar activity to neuroleukin in a novel bioassay using human and rat neuroblastoma cell lines. In the presence of reduced GPI, these cells were found to differentiate, in terms of enhanced neurite extension at a reduced proliferation rate. These results demonstrate the existence of a novel growth factor activity of an evolutionary ancient enzyme. The nonreduced commercial form of GPI, probably the dimer, was found to be inactive in this bioassay. Using the neuroblastoma cells model system, we further investigated the significance of the region of homology to HIV-1 envelope glycoprotein (gp120) as the putative binding site of GPI to its receptor on neuronal cells.