Microbiological aspects of peritonitis in patients on continuous ambulatory peritoneal dialysis.

The objective of the study was to identify the microbiological spectrum and drug-sensitivity pattern of peritonitis in patients on continuous ambulatory peritoneal dialysis. This was a prospective study done over a period of a year-and-a-half at a tertiary-care hospital in a hilly state of India. The effluent dialysate bags from 36 consecutive patients with peritonitis were studied. One hunderd ml dialysate fluid was processed under aseptic conditions by lysis centrifugation method. Microscopy and culture was done from the deposits for bacteriological, fungal, and mycobacterial isolates. They were identified by colony morphology and their biochemical reactions. Drug susceptibility testing was done by Kirby-Bauer disc diffusion method. In 36 dialysates, 33 (91.6%) dialysates were culture-positive and in 3 (8.4%), the culture was negative. A total of 36 microorganisms were isolated in 33 cultures. Among the 36 microorganisms, 19 (52.8%) isolates were gram-positive, 10 (27.8%) were gram-negative, 5 (13.9%) were fungi, and 2 (5.6%) were mycobacterial isolates. All gram-positive organisms were sensitive to ampicillin, amoxi-clavulanic acid, cefazolin, clindamycin, and vancomycin. Neither a methicillin-resistant Staphylococci aureus nor a vancomycin-resistant Enterococcus was isolated in gram-positive isolates. Gram-negative organisms were sensitive to ciprofloxacin, ceftriaxone, ceftazidime, cefepime, gentamicin, piperacillin-tazobactam and imipenem. One of the gram-negative isolate was an extended spectrum beta-lactamase producer. Gram-positive peritonitis was more frequent than gram-negative peritonitis in our continuous ambulatory peritoneal dialysis patients. Mycobacterial causes were responsible for peritonitis in patients with culture-negative peritonitis which was not responding to the conventional antimicrobial therapy.

Replication and immunogenicity of Ad7-, Ad4-, and Ad5-hepatitis B virus surface antigen recombinants, with or without a portion of E3 region, in chimpanzees.

Human adenovirus vectors containing intact or largely deleted E3 region were used to construct adenovirus-hepatitis B recombinant viruses (Ad-HepB) and shown to produce substantial amount of recombinant protein, hepatitis B surface antigen (HBsAg), in tissue culture. Previously we showed that these viruses were able to elicit good anti-HBs antibodies in a dog model. In the present study, the Ad-HepB viruses were evaluated for replication and immunogenicity in chimpanzees which sustain permissive infection by human adenoviruses. Recombinants containing entire E3 region showed better replication pattern than their E3 deleted counterparts as evidenced by longer duration and high titers of virus shedding. The effect of E3 region was also seen in the antibody titers against HBsAg in that the E3 containing viruses showed better response than the E3 deleted viruses. The importance of E3 region for the development of adenovirus vectored vaccines is further discussed.

Adenovirus vectored vaccines.

Human recombinant adenoviruses (Ad) have been employed to develop experimental vaccines against a number of infectious agents. Ad-vectored vaccines express recombinant proteins, including any post-translational modifications, into functioning replicas of the native proteins capable of eliciting neutralizing antibodies in both abortive and permissive animal models. Human Ad types 4, 5, and 7 were used to construct recombinant viruses that express the respiratory syncytial virus F or G glycoproteins, the hepatitis B surface antigen, and the HIV env or gag genes. The recombinant Ad-HIV viruses are of particular interest and have been examined for their immunogenicity in dogs and chimpanzees. Dogs were immunized intratracheally with Ad-env recombinants (10(9) pfu/dog). Excellent humoral anti-HIV responses, including neutralizing antibodies, were detected in the sera following booster immunization (12-18 weeks after primary immunization) with a second Ad-env recombinant made in a different Ad serotype (heterotypic booster). Chimpanzees were immunized in two ways, orally with lyophilized virus (10(9) to 10(10) pfu/virus) in enteric-coated capsules or intranasally (10(7) pfu/virus). Intranasal immunization was superior to oral immunization with respect to replication of recombinant viruses as well as induction of anti-Ad and anti-HIV antibodies. Administration by both routes resulted in stimulation of cellular immune responses, as measured by antigen proliferation assays. Anti-HIV antibodies were detected in chimpanzee secretions (salivary, nasal, rectal, vaginal) taken from animals following intranasal immunization with a heterotypic recombinant. Intranasal administration effectively primed chimpanzees to produce high-titred (320-640) serum neutralizing antibodies to HIV following boosting with a baculovirus-derived env (gp160) subunit vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees.

Recombinant human adenovirus (Ad) type 4-, 5-, and 7-vectored vaccines expressing either the HIV env or gag-protease genes were tested for immunogenicity in three chimpanzees. The first phase of the vaccination protocol consisted of a primary and two booster immunizations with Ad-HIVs by the oral route of administration, followed by a single booster immunization with Gag and/or Env subunit vaccines. The second phase of the vaccination protocol consisted of intranasal administration of Ad-HIVs previously administered by the oral route. Following the first phase adenovirus was shed into stools for only 1-7 days and modest type-specific anti-adenovirus neutralizing antibody titers were induced. Strong anti-Env binding antibody responses were detected in all three animals following the second oral booster immunization. One chimpanzee responded with a low-titered type-specific neutralizing antibody response to HIV. Cell-mediated immune responses to Env were not detected after the primary vaccination, but were detected following all booster immunizations. Administration of the Gag subunit vaccine boosted both humoral and cell-mediated immune responses to Gag antigens. In contrast, the Env subunit vaccine boosted cellular but not humoral immune responses. In the second phase of the vaccination protocol, both virus shedding and anti-adenovirus responses were enhanced. All three chimpanzees responded to the intranasal administration of Ad7-HIVs with boosted anti-HIV serum responses, including low-titered type-specific neutralizing antibodies, elicited anti-HIV antibodies at secretory sites, and stimulated cell-mediated immune responses to both Gag and Env antigens.

Conservation of essential sequences in the major late promoter and tripartite leader of the simian adenovirus type 30.

We report here the cloning and sequencing of the major late promoter (MLP) and the tripartite leader (TPL) from simian adenovirus type 30 (sAd30) and the comparison of the sAd30 nucleotide (nt) sequence with that of human adenoviruses (hAd). The nt sequence homology between sAd30 and hAd2 is 75% from -66 to +190 relative to the cap site. This sAd30 MLP segment contains the upstream regulatory sequence element, TATA box, and downstream regulatory sequence elements that are homologous to hAd MLP. The sAd30 upstream regulatory sequence has a small palindromic DNA sequence GTCACGTGAC, and the TATA box contains the sequence of ATAAA instead of TATAAA. The sAd30 TPL was located on the sAd30 genome as identified by sequence homology with the hAd counterpart. The splice sites of TPL introns were confirmed by sequence analysis of cDNAs synthesized from sAd30-infected cells. There is a 74.2% nt sequence homology between the TPL of sAd30 and hAd2. The conservation of these sequence elements during evolution of Ad suggests that they are essential for the transcription and translation of Ad ML transcripts.

High level expression of the envelope glycoproteins of the human immunodeficiency virus type I in presence of rev gene using helper-independent adenovirus type 7 recombinants.

The effect of rev (art/trs) gene on the level of HIV-1 envelope (env) expression using recombinant adenovirus was investigated. Recombinant adenoviruses expressing either the envelope or the rev gene of the human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the gene into an expression cassette. The expression cassette contained the adenovirus type 7 major late promoter, followed by leader 1 of the adenovirus tripartite leader and a portion of intron between leaders 1 and 2, leaders 2 and 3, and a hexon polyadenylation signal. The cassette was then inserted at the terminal region between the E4 and ITR regions of the adenovirus 7 genome with a concomitant E3 region deletion (80-87 m.u.). A549 cells infected with the recombinant virus containing the env gene produced the envelope glycoproteins gp160, gp120, and gp41. HIV-1 envelope gene expression was greatly enhanced (20- to 50-fold) in the cells that were simultaneously infected with the recombinant adenovirus containing the rev gene as measured by ELISA and Western blotting. Interestingly, this effect was observed despite the lack of the 5' down splice site for rev and seems to be post-transcriptional. Another recombinant adenovirus which contains both the rev and the env genes was constructed by inserting the rev gene in the deleted E3 region and the env gene in the terminal cassette. This double recombinant virus expressed high levels of env antigen in A549 cells similar to those attained upon co-infection with two separate recombinant viruses containing the rev or env gene. Furthermore, the rev gene nucleotide sequence could be altered without altering the amino acid sequence and its sequences truncated by 17 amino acids from the C-terminus had no effect of rev function.

Adenovirus vaccine strains genetically engineered to express HIV-1 or HBV antigens for use as live recombinant vaccines.

Types 4 and 7 adenovirus are currently used as live, oral vaccines for the prevention of adenovirus respiratory disease in military recruits. These vaccine strains have been genetically engineered in order to express HIV-1 or HBV antigens in infected cells. A dog model was developed to evaluate the immunogenicity of these recombinant vaccines. Dogs inoculated with live adenovirus-HBV recombinant vaccine produced antibody against hepatitis B surface antigen.

Distinctive properties of the hepatitis B virus envelope proteins.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.

Expression of HBV surface antigen or HIV envelope protein using recombinant adenovirus vectors.

Recombinant adenoviruses were constructed that contained either the HBsAg coding sequence or the HIV envelope protein coding sequence. The recombinant adenoviruses can replicate normally in cultured human cells. Cells infected with the adenovirus-HBV recombinant secreted HBsAg into the tissue culture medium. This HBsAg had immunological and physical properties similar to those of the 22-nm particles found in human serum. Expression of HIV envelope protein in cells infected with the adenovirus-HIV recombinant was demonstrated using cytoimmunofluorescence and immunoprecipitation. A hamster model was developed to evaluate the immunogenic properties of adenovirus-HBV recombinants. Hamsters inoculated intranasally with live adenovirus-HBV recombinant produced antibody against both adenovirus and hepatitis B virus surface antigen.

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence.

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.

Expression of hepatitis B surface antigen with a recombinant adenovirus.

Early region 1 of the adenovirus type 5 genome was replaced with a DNA sequence containing the gene coding for the hepatitis B surface antigen (HBsAg) flanked by the major late promoter from adenovirus 2 and processing and polyadenylylation signals from simian virus 40. In one type of hybrid virus only the adenovirus 2 major late promoter, including just 33 base pairs of the adenovirus type 2 tripartite leader, preceded the coding region of the HBsAg gene. In another, this region was preceded by both the adenovirus major late promoter and almost the entire tripartite leader. The structure of the substituted sequence in each of the recombinant viral DNAs was identical to that in the plasmids used to construct the viruses. Approximately equivalent amounts of HBsAg-specific mRNA were produced late in infection with each recombinant virus. Although HBsAg production was detected late in infection of the hybrid virus not containing the full tripartite leader sequence, its level was 1/70th of that obtained with the hybrid virus containing this sequence. One likely interpretation is that the presence of the tripartite leader at the 5' end of this mRNA is critical for the synthesis of HBsAg polypeptide in the late stage of infection. HBsAg produced upon infection with the hybrid adenoviruses was glycosylated and secreted into the culture medium as particles that were essentially indistinguishable from the 22-nm particles found in human serum.

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes.

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.

Immunomodulating activity of 3-(p-chlorophenyl)thiazolo-[3,2-a] benzimidazole-2-acetic acid (Wy-18,251, NSC 310633) in mice implanted with Lewis lung tumor. Effect of Wy-18,251 on lymphocyte proliferation and phagocytosis.

Proliferation of B lymphocytes is depressed in Lewis lung tumor bearing mice. Treatment of these mice with Wy-18,251 (5 mg/kg) significantly increased the responsiveness of their splenic T cells to concanavalin A in cell culture. Levamisole (5 mg/kg) acted more weakly than Wy-18,251. Neither Wy-18,251 nor levamisole elevated the depressed B cell mitogenesis. Wy-18,251 significantly increased macrophage phagocytosis against 51chromium labeled opsonized chicken red blood cells. Levamisole behaved differently: it either had no effect on phagocytosis, or depressed it.