Improved retention associated with community-based accompaniment for antiretroviral therapy delivery in rural Rwanda.

BACKGROUND: Minimizing death and ensuring high retention and good adherence remain ongoing challenges for human immunodeficiency virus (HIV) treatment programs. We examined whether the addition of community-based accompaniment (characterized by daily home visits from a community health worker, directly observed treatment, nutritional support, transportation stipends, and other support as needed) to the Rwanda national model for antiretroviral therapy (ART) delivery would improve retention in care, viral load suppression, and change in CD4 count, relative to the national model alone. METHODS: We conducted a prospective observational cohort study among 610 HIV-infected adults initiating ART in 1 of 2 programs in rural Rwanda. Psychosocial and clinical characteristics were recorded at ART initiation. Death, treatment retention, and plasma viral load were assessed at 1 year. CD4 count was evaluated at 6-month intervals. Multivariable regression models were used to adjust for baseline differences between the 2 populations. RESULTS: Eighty-five percent and 79% of participants in the community-based and clinic-based programs, respectively, were retained with viral load suppression at 1 year. After adjusting for CD4 count, depression, physical health quality of life, and food insecurity, community-based accompaniment was protective against death or loss to follow-up during the first year of ART (hazard ratio, 0.17; 95% confidence interval [CI], .09-.35; P < .0001). In a second multivariable analysis, individuals receiving accompaniment were more likely to be retained with a suppressed viral load at 1 year (risk ratio: 1.15; 95% CI, 1.03-1.27; P = .01). CONCLUSIONS: These findings indicate that community-based accompaniment is effective in improving retention, when added to a clinic-based program with fewer patient support mechanisms.

Cat allergen-induced blood basophil reactivity in vitro predicts acute human nasal allergen challenge responses in vivo.

BACKGROUND: Basophil histamine release (BHR) to allergen has been used as a confirmatory test to support the clinical diagnosis of allergic disease. OBJECTIVE: Among subjects reporting respiratory cat allergy, we hypothesized that cat-induced BHR in vitro would predict nasal allergen challenge (NAC) response in that same individual. We therefore compared the magnitude of cat allergen-induced BHR to NAC outcome and serological measures of cat-specific IgE and the ratio of cat-specific IgE to total IgE. METHODS: Forty-two subjects with a history of cat allergy, positive cat puncture skin test (PST) and detectable cat-specific IgE (> 0.1 kAU/L, ImmunoCap) participated with consent. Subjects were grouped as positive or negative cat allergen-induced BHR, with a positive result defined as the release of ≥ 20% of the total cellular histamine content. The majority of subjects also underwent a NAC with a positive result defined as ≥ 5 total sneezes. RESULTS: Subjects with a positive compared with a negative cat allergen BHR had higher cat-specific IgE levels at 5.40 ± 1.24 kAU/L (n=25) vs. 1.55 ± 0.73 kAU/L (n=17, P=0.01) as well as a higher cat-specific IgE/total IgE ratio [6.1 ± 1.4% (n=25) vs. 1.6 ± 0.9% (n=17, P=0.01)]. Of the 31 subjects who underwent a NAC, a positive NAC was observed in 78% (18/23) with a positive cat allergen BHR compared with 37% (3/8) with a negative cat allergen BHR, giving a positive predictive value of 78% and a negative predictive value of 63%. The diagnostic sensitivity and specificity of a positive BHR to predict a positive NAC was 86% and 50%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: A positive cat allergen-induced BHR is associated with higher cat-specific IgE levels, a higher cat-specific to total IgE ratio and is predictive of a positive cat-induced NAC [ClinicalTrials.gov NCT00604786].

Purification, crystallization and preliminary X-ray diffraction studies of alpha-toxin of Clostridium perfringens.

Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C. The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

Discrimination between twenty isolates of herpesvirus simiae (B virus) by restriction enzyme analysis of the viral genome.

Twenty isolates, obtained from adult breeding monkeys, were all identified as herpesvirus simiae (B virus) by neutralisation with polyclonal B virus antiserum. Subsequent analysis of restriction enzyme profiles produced by digestion of DNA from the isolates enabled discrimination to be made between them. In particular Cynomolgus monkey isolates could be distinguished from those of Rhesus animals. One isolate (isolate 9) could not be typed either as B virus or as the antigenically related herpesvirus SA8, despite neutralisation by B virus antiserum. Unlike herpes simplex virus, B virus isolates could not be divided into oral and genital types on the basis of restriction enzyme profiles.

Nitric oxide modulates articular sensory discharge and responsiveness to bradykinin in normal and arthritic rats in vivo.

Nitric oxide is implicated in peripheral nociceptive processing. This study determined the effects of the nitric oxide synthase inhibitor, L-NAME, on neural discharge from articular C-fibre afferents innervating normal and arthritic ankle joints in anaesthetized rats. Intra-arterial injection of L-NAME (10-20 mg kg(-1)) increased neural discharge in normal and arthritic ankle joints, whereas D-NAME (30 mg kg(-1)) had no effect. The excitation induced by L-NAME (20 mg kg(-1)) was reduced by co-injecting the nitric oxide precursor, L-arginine (50 mg kg(-1)). L-NAME (20 mg kg(-1)) also enhanced responsiveness to bradykinin (10 microg) but only in arthritic rats, whereas L-arginine (50 mg kg(-1)) reduced the excitation by bradykinin (30 microg) in both groups. These results provide evidence that nitric oxide modulates articular C-fibre activity and reduces responsiveness to bradykinin.

"Oryctes" virus replication: Electron microscopic observations on infected moth and mosquito cells.

"Oryctes" virus replicates efficiently in continuous moth (Spodoptera frugiperda) and mosquito (Aedes albopictus) cell cultures. Progeny virus was detected by bioassay and electron microscopy 15 hr after infection in both cell cultures. S. frugiperda cells permitted replication morphologically similar to that observed in vivo occurring in the cell nucleus. A. albopictus cells permitted replication of the virus solely in the cytoplasm.

Frog virus 3 replication: electron microscope observations on the sequence of infection in chick embryo fibroblasts.

The replication of frog virus 3 in primary chick embryo fibroblasts has been studied by examination of thin sections with the electron microscope and the assay of infectious viurs. Uptake of frog virus 3 by the cells was observed to occur by pinocytosis and this may be the method of entry. Early in infection (i h p.i.) marked margination of the nuclear chromatin occurred and the chromatin remained in this condition throughtout the infection. Foci of infection were first detected in the cytoplasm of cells 24 h p.i. when production of infectious virus commenced. These foci appeared as electron translucent areas containing fine grains, surrounded by degenerate mitochondria. The foci usually contained virus particles. At this time budding of virus particles at the plasma membrane occurred. Later in infection at 36 and 48 h p.i. large numbers of virus particles were detected in the cytoplasm of cells either scattered loosely throughtout the cell, arranged as clusters or in paracrystalline arrays. Extensive budding at the plasma membrane then took place. Virus particles were detected in the nucleus of the cells at these late stages and it is possible that the virus may infect and replicate at this site. Throughout the productive stages of infection aberrant forms of the virus, namely particles devoid of cores, incompletely assembled particles and elongated bacilliform particles were noticed.

Baculovirus replication: effects of inhibitors of macromolecular synthesis.

A number of inhibitors of DNA, RNA, protein and polyamine synthesis have been used to elucidate the mode of replication of baculoviruses. An overview of the viruses, the antimicrobial inhibitors used, and the effects of the drugs are presented. Although certain inhibitors of protein synthesis and DNA synthesis are useful in determining the program of expression of the viral genome into immediate early, delayed early, late and very late phases of synthesis, few drugs have proved useful as antimicrobial agents. Bromovinyldeoxyuridine (BVDU) is a potent inhibitor of baculovirus replication inhibiting viral DNA synthesis by blocking the virus induced DNA polymerase. Preliminary experiments suggest that BVDU suppresses baculovirus disease in insect larvae.

Biochemical and biophysical properties of a Mamestra brassicae multiple enveloped nuclear polyhedrosis virus.

A multiply enveloped nuclear polyhedrosis virus from Mamestra brassicae has been shown to be morphologically similar to other baculoviruses. The virus particles contain 13 polypeptides of which 4 are associated with the nucleocapsid. The polyhedron comprises of one major polypeptide of molecular weight 28,000. The DNA has a mol. wt. of about 1.15 X 10(8), which is larger than that reported for other baculoviruses. The DNA is a circular, supercoiled molecule of CG mol. fraction 0.448. DNA fragments produced by a range of restriction enzymes are presented as an aid to identification.

Interrelationships of the proteins of two parvoviruses (densonucleosis virus types 1 and 2).

Densonucleosis viruses (types 1 and 2) contain four major structural polypeptides with a total molecular weight in excess of the coding capacity of the DNA. Peptide maps obtained by limited proteolysis of isolated (125)I-labeled proteins of both virus types indicate a common origin of the virus proteins and homology between the different viruses. The structure of densonucleosis virus type 2 and its homologous top component (naturally occurring empty particles) was compared by proteolysis using several proteases and the bifunctional cross-linking reagents dimethylsuberimidate (DMS) and dimethylmalonimidate. Similar susceptibilities of both components with proteases were obtained. The top components alone were accessible to the action of the cross-linking reagent DMS. The lowest molecular weight major structural polypeptide was most resistant to the action of the proteases and DMS.

Frog virus 3 replication: induction and intracellular distribution of polypeptides in infected cells.

The synthesis of the polypeptides induced in frog virus 3-infected cells was analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled cell extracts. Purified frog virus 3 contained 22 polypeptides, with molecular weights in the range 9 x 10(3) to 114 x 10(3). All of the structural and an additional seven nonstructural polypeptides were detected in infected cell lysates. The following three classes of induced polypeptides (under temporal control) were observed in BHK cells: at 2 h, four alpha polypeptides; at 4 h, 13 beta polypeptides; and at 6 h, the remaining 12 gamma polypeptides. The total molecular weight of the infected cell-specific polypeptides (ICPs) was approximately 1.5 x 10(6), which accounts for about 30% of the coding capacity of the viral genome. At least 10 of the induced polypeptides were phosphorylated, but none was glycosylated or sulfated. No evidence for posttranslation cleavage of polypeptides in pulse-chase and inhibition experiments was obtained. The synthesis of gamma polypeptides was not detected in the presence of the viral DNA replication inhibitors cytosine arabinoside and hydroxyurea, but halogenated nucleotides apparently had no effect. These results suggest that alpha and beta polypeptides are "early" events and that detectable gamma polypeptide synthesis is dependent on the production of progeny viral DNA. The regulation of frog virus 3-induced polypeptide synthesis in infected BHK cells was examined by using inhibitors of protein and RNA synthesis and amino acid analogs. These experiments confirmed the existence of three sequentially synthesized, coordinately regulated classes of polypeptides, designated alpha, beta, and gamma. The requirements for the synthesis of each class were as follows: (i) alpha polypeptides did not require previous cell protein synthesis; (ii) beta polypeptides required a prescribed period of alpha polypeptide synthesis and new mRNA synthesis; and (iii) gamma polypeptides required prior synthesis of functional beta polypeptides and new mRNA synthesis. alpha polypeptide synthesis was controlled by beta and gamma polypeptides, and alpha and beta polypeptides were involved in the suppression of host cell polypeptide synthesis. Indirect evidence was obtained for the temporal regulation of frog virus 3 transcription. The intracellular distribution of virus-induced polypeptides in cells infected with frog virus 3 was investigated by using standard cell fractionation techniques. Most of the 29 induced polypeptides were bound to structures within the nucleus, and only two ICPs were not associated with purified nuclei. When isolated nuclei were incubated in an infected cell cytoplasm preparation, all of the nuclear ICPs were incorporated in vitro. All of the ICPs were associated with ribosomal and rough endoplasmic reticulum fractions of infected cells, and a number of ICPs were found on smooth intracellular membranes. Most of the ICPs were also associated with purified plasma membranes of infected cells, and one polypeptide (ICP 58) was highly enriched in the plasma membrane compared with whole cell extracts or purified frog virus 3.

Iridescent virus type 22 DNA.

Double stranded DNA extracted from iridescent virus type (IV 22) was characterized by its buoyant density in CsCl, thermal denaturation profile and guanine plus cytosine content. The DNA was linear with a molecular weight of 130--142 x 10(6) determined by reassociation kinetics, contour length measurements and restriction endonuclease analysis.

Neutralisation of Trichoplusia ni nuclear polyhedrosis virus with antisera to two forms of the virus.

Two forms of a baculovirus, Trichoplusia ni nuclear polyhedrosis virus, one released at the plasma membrane of cells (cell released virus: CRV) and the other derived from polyhedra (polyhedron derived virus: PDV) occur naturally. Antisera raised against the two forms specifically neutralised the homologous form. The envelope proteins of the two forms were shown to be the major proteins involved in serum neutralisation and to be responsible for the antigenic diversity of the virus forms.

Polyamines contained by two densonucleosis viruses.

Densonucleosis viruses 1 and 2 both contain the three polyamines putrescine, spermidine, and spermine in complete virus particles but not in their respective top components. The polyamines, with spermidine predominant, comprise 1.41% of the virus particle by weight, which is sufficient to neutralize 26% of the single-stranded DNA contained within the particles.

The DNA contained by nuclear polyhedrosis viruses isolated from four Spodoptera spp. (Lepidoptera, Noctuidae); genome size and configuration assessed by electron microscopy.

The mol. wt. of the DNA from four nuclear polyhedrosis viruses isolated from Spodoptera littoralis, S. exempta, S. exigua and S. frugiperda were determined to be 84, 80, 68 and 74 X 10(6), respectively, by electron microscopy. The molecules were demonstrated to exist as double-stranded relaxed circular or supercoiled DNA, though linear forms of DNA were also observed.

The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus in cultured Spodoptera frugiperda cells.

The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus have been determined. Two inhibitors of protein synthesis-cycloheximide and puromycin-were irreversible inhibitors of virus multiplication. Three inhibitors of nucleic acid synthesis-actinomycin D, cytosine arabinoside and camptothecin- prevented virus multiplication; only camptothecin was reversible. Rifampicin had no effect on virus multiplication.

Frog virus 3 replication: electron microscope observations on the terminal stages of infection in chronically infected cell cultures.

An examination of BHK, CEF, and FHM cells chronically infected with frog virus 3 has been made by scanning and transmission (thin section, freeze fracture, and surface replica) electron microscopy. With minor differences the pattern of virus development is similar in all three cell line. Virus particles were detected in cell nuclei which subsequently became degenerate very late in infection. Three inclusions were associated with frog virus 3 cytoplasmic foci of infection; lamella structures, extensive microtubule formation (in BHK and FHM cells), and linear crystalline structures. The last two structures may play a role in creating or maintaining the cell rounding c.p.e. revealed by scanning electron microscopy. Very late in infection most BHK and FHM, but not CEF, cells are stripped of the plasma membrane. Replicas of frozen fractured BHK cells featured cytoplasmic foci of infection, budding at the plasma membrane, and showed that at early times when virus is detected in the nucleus, the nuclear membranes are intact and morphologically unaltered. Budding at the plasma membrane was better resolved by scanning and as surface replicas. This demonstrated that sparse to profuse localized budding occurred. Frequently virus particles were located singly, or as multiples, at the end of, or along, cytoplasmic protrusions which occur both on the body of the cells and at the cytoplasmic/coverslip 'interface'.

Baculovirus replication: phosphorylation of polypeptides synthesized in Trichoplusia ni nuclear polyhedrosis virus-infected cells.

A number of polypeptides synthesized specifically in Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus (T. ni MNPV)-infected Spodoptera frugiperda cells are phosphorylated both early and late in infection. Certain non-structural proteins and the major basic internal protein are the main phosphoproteins detected in infected cells. The polyhedron protein was not phosphorylated. Many cell proteins continue to be phosphorylated throughout infection. Pulse-chase experiments have shown that some polypeptides are stably phosphorylated whereas other polypeptides (including the major basic protein) have phosphates which cycle on and off. One polypeptide was substantially labelled only after a chase with unlabelled orthophosphate. Fractionation of cells into nucleus and cytoplasm showed that polypeptides located in both the cytoplasm and nucleus were phosphorylated.

The detection of intracellular retrovirus-like entities in Drosophila melanogaster cell cultures.

A Drosophila melanogaster cell line has been examined for the presence of retrovirus particles. When these cells were disrupted and analysed on sucrose density gradients a subcellular fraction with a density of 1.22 g/ml was found to possess endogenous DNA polymerase activity and could catalyse polymerization of deoxynucleotide triphosphates in response to added template primers. The latter activity had the cation and template primer responses expected for reverse transcriptase. A high mol. wt. polyadenylic acid-containing RNA was also purified from this fraction and could be dissociated by heat treatment into 30 to 35S and smaller species. Electron microscopy revealed the presence of torroidal forms reminiscent of intracytoplasmic A-type retrovirus particles within the Drosophila cells. Similar forms were found associated with the subcellular fraction of 1.22g/ml. We concluded that our D. melanogaster cell line contains retroviruses similar, but not identical, to the A-type particles previously described in mammalian and avian cells.