Development of Neonatal Airway Management Simulator for Evaluation of Tracheal Intubation.

The long-term goal of this study is a training system that can simulate medical cases and advise physicians based on quantitative evaluation of neonatal resuscitation. In this paper, we designed and manufactured a neonatal airway management simulator for quantitative evaluation of tracheal intubation. This robotic simulator is equipped with 25 sensors of 6 types, which detect motions that lead to complications, inside the manikin replicated a neonate. A performance experiment of the developed sensor and an evaluation experiment with physicians were conducted. We observed that an erroneous operation in the laryngoscopy can be detected by the sensors in our simulator.

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payer's patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework.

To establish simian/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among subtype B envelope, four subtype E V3 sequences were substituted into SHIV(MD14), a SHIV clone bearing an envelope derived from a CXCR4 (X4)/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an only V3-chimera clone capable of replicating in human and macaque peripheral blood mononuclear cells (PBMCs), was propagated in pig-tailed macaque PBMCs and in cynomolgus macaque splenic mononuclear cells. The propagated virus stocks were intravenously inoculated into respective macaque species. SHIV-TH09V3 infected both macaque species as shown by plasma RNA viremia, isolated viruses from PBMCs and plasma, and antibody production against viral proteins. To assess how the substituted V3 sequence affected coreceptor usage, SHIV-TH09V3 stocks propagated in vitro and after isolation from macaques were verified for their corecepor usage by GHOST cells assay. SHIV-TH09V3 maintained R5-tropic phenotype both in vitro and after isolation from macaques, in contrast to the X4/R5-dual tropic SHIV(MD14). This indicates the substituted V3 sequence among the backbone of SHIV(MD14) governs coreceptor usage. Future study of infecting macaques with SHIV-TH09V3 and SHIV(MD14) will focus on differences of the outcome caused by the different V3 sequences in connection with coreceptor usage.

Induction of effective antitumor immune responses in a mouse bladder tumor model by using DNA of an alpha antigen from mycobacteria.

One of the main objectives of cancer immunotherapy is the activation and increase in number of antitumor effector cells. Recently, genetically modified tumor cell vaccines have been proposed for elicitation of antitumor effector cells. Native alpha antigen (alpha Ag) (also known as MPT59 and antigen 85B) of mycobacteria, which cross-reacts among mycobacteria species, may play an important biological role in host-pathogen interaction because it elicits various helper T-cell type 1 immune responses. To assess the induction of antitumor immune responses by alpha Ag, mouse tumor cell lines transfected with cDNA of alpha Ag from Mycobacterium kansasii were established, and the possibility of producing a tumor cell vaccine for induction of antitumor effects was explored. Transfection of tumor cell lines with an alpha Ag gene lead to primary tumor rejection and the establishment of protective immunity to nontransfected original tumor cell lines in Mycobacterium bovis bacillus Calmette-Gurin (BCG)-primed and unprimed mice. Mice immunized with tumor cell lines transfected with the alpha Ag gene showed delayed-type hypersensitivity responses in vivo and proliferative responses together with induction of interferon-gamma of spleen cells against nontransfected wild-type tumor cell lines in in vitro experiments. Moreover, immunization of mice with alpha Ag-expressing tumor cells elicited tumor-specific and cytotoxic T lymphocyte (CTL) epitope peptide-specific CD8+ CTLs. The results of this study provided evidence of the potential usefulness of alpha Ag in tumor cell vaccines.

Isolation and characterization of a full-length molecular DNA clone of Ghanaian HIV type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range.

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.

Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides.

Generally, major histocompatibility complex (MHC) class I presentation of peptide antigens only occur for proteins' which are actively synthesized and processed intracellularly, so that immunization with a cytotoxic T lymphocyte (CTL) target peptide does not usually elicit effective CTL responses. In the present study, we explored the use of epitope peptides by in vivo electroporation to introduce directly into the cytoplasm for the vaccine elicitation of virus-specific CTLs in a mouse system. BALB/c mice were immunized with human immunodeficiency virus (HIV) env (P18, residues 311-320) or hepatitis C virus (HCV) NS5 (P17, residues 2423-2434) with or without electric pulses. Effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides. The results of the present study suggest that a peptide vaccine against a virus using electric administration is effective in eliciting virus specific CTLs.

Closely related HIV-1 CRF01_AE variant among injecting drug users in northern Vietnam: evidence of HIV spread across the Vietnam-China border.

To investigate the nature of recent HIV outbreaks among injecting drug users (IDUs) near the Vietnam-China border, we genetically analyzed 24 HIV-positive blood specimens from 2 northern provinces of Vietnam (Lang Son and quang Ninh) adjacent to the China border, where HIV outbreaks among IDUs were first detected in late 1996. Genetic subtyping based on gag (p17) and env (C2/V3) sequences revealed that CRF01_AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01_AE sequences among IDUs in Quang Ninh and Lang Son showed significant clustering with those found in nearby Pingxiang City of China's Guangxi Province, sharing a unique valine substitution 12 amino acids downstream of the V3 loop. This particular subtype E variant, uniquely found among IDUs in northern Vietnam and southeastern China, is designated E(v). The genetic diversity of CRF01_AE distributed in Quang Ninh (1.5 +/- 0.6%) and Pingxiang City (1.9 +/- 1.2%) was remarkably low, indicating the emerging nature of HIV spread in these areas. It is also noted that the genetic diversity of CRF01_AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01_AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01_AE had been introduced via the sexual route. The data in the present study provide additional evidence that HIV outbreaks among IDUs in northern Vietnam were caused by the recent introduction of a highly homogeneous CRF01_AE variant (E(v)) closely related to that prevailing in nearby southern China.

Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar.

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.

Lipase-catalyzed asymmetric desymmetrization of prochiral 2,2-disubstituted 1,3-propanediols using 1-ethoxyvinyl benzoate.

The lipase-catalyzed asymmetric desymmetrization of the prochiral 2,2-disubstituted 1,3-propanediols was studied using various types of 1-ethoxyvinyl esters (1a-i). Although 1a-e with aliphatic acyl groups were not sufficient, use of the benzoate (1f) in combination with Candida rugosa lipases converted acyclic diols (2, 6) and cyclic diols (11-14) to the optically active compounds (3f, 7f, 15f-18f), bearing a quaternary carbon center, with moderate-to-high optical yields. These products were fairly stable against racemization under acidic conditions.

Polymorphism in the interleukin-4 promoter affects acquisition of human immunodeficiency virus type 1 syncytium-inducing phenotype.

The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4(+) cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 -589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.

Convenient enzymatic resolution of alcohols using highly reactive, nonharmful acyl donors, 1-ethoxyvinyl esters

1-Ethoxyvinyl esters 3, a new type of acyl donors for enzymatic resolution of racemic alcohols, were disclosed to be superior to the contemporary major reagents, vinyl esters 1 and isopropenyl esters 2. Three features of 3 are noticeable: (1) 3 generates ethyl acetate as a single coproduct, which does not affect any enzymes, while acetaldehyde liberated from 1 deactivates some kinds of lipases. (2) The reactivity of 3 was not less than that of 1 and much higher than that of 2, and the optical purity of the products was as high as that of 1 and 2. Especially, it was generally observed that 3 showed higher reactivity than 1 for reactions using Candida rugosa lipases, one of the most commonly employed lipases, having liberal applicability to substrates but sensitive to acetaldehyde. Twelve examples of the kinetic resolution of racemic secondary alcohols (5 and 10) and one desymmetrization of meso-alcohol 7 were presented employing the acetate 3a or the octanoate 3b and four types of lipases. (3) A one-pot procedure for the preparation of 3 from the corresponding carboxylic acid and the subsequent enzymatic resolution of alcohols, which has not been reported using 1 or 2, was elucidated. The chemical and optical yields of the products by this procedure were similar to those obtained using isolated 3.

Convergent evolution of reverse transcriptase (RT) genes of human immunodeficiency virus type 1 subtypes E and B following nucleoside analogue RT inhibitor therapies.

Changes in the drug susceptibility, gene lineage, and deduced amino acid sequences of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) subtype E following 3'-azido-3'-deoxythymidine (AZT) monotherapy or AZT-2', 3'-dideoxyinosine combination therapy were examined with sequential virus isolates from a single family. The changes were compared to those reported for HIV-1 subtype B, revealing striking similarities in selected phenotype and amino acids independent of differences in the RT backbone sequences that constantly distinguish the two subtypes. Particularly, identical amino acid substitutions were present simultaneously at four different positions (D67N, K70R, T215F, and K219Q) for high-level AZT resistance. These data suggest that HIV-1 subtypes E and B evolve convergently at the phenotypic and amino acid levels when the nucleoside analogue RT inhibitors act as selective forces.

Inhibition of immunoglobulin E response to Japanese cedar pollen allergen (Cry j 1) in mice by DNA immunization: different outcomes dependent on the plasmid DNA inoculation method.

To develop a new immunotherapy for Japanese cedar (Cryptomeria japonica; CJ) pollinosis, we evaluated the use of DNA immunization by inoculating mice with plasmid DNA encoding Cry j 1 as a CJ pollen major allergen (pCACJ1). Repeated intramuscular (i.m.) inoculation of BALB/c mice with pCACJ1 produced anti-Cry j 1 antibody responses, which were predominately of the immunoglobulin G2a (IgG2a) type. Furthermore, this inoculation suppressed immunoglobulin E (IgE) and IgG1 antibody responses to subsequent alum-precipitated Cry j 1 injections. Splenic T cells isolated from mice inoculated with pCACJ1 i.m. secreted interferon-gamma (IFN-gamma), but not interleukin (IL)-4, in vitro upon stimulation with Cry j 1 as well as with p277-288, a peptide corresponding to the T-cell epitope of Cry j 1. In contrast, inoculation of BALB/c mice with pCACJ1 by gene gun injection caused response predominantly of the IgG1 type, and enhanced production of anti-Cry j 1 IgE antibodies to subsequent alum-precipitated Cry j 1 injections. Splenic T cells isolated from pCACJ1-innoculated mice by gene gun injection secreted both IFN-gamma and IL-4 in vitro, upon stimulation with Cry j 1 as well as with p277-288. These findings suggest that i.m. inoculation with pCACJ1 effectively elicits Cry j 1-specific T helper 1 (Th1)-type immune responses, resulting in inhibition of the IgE response to Cry j 1.

A group of V3 sequences from human immunodeficiency virus type 1 subtype E non-syncytium-inducing, CCR5-using variants are resistant to positive selection pressure.

In a human immunodeficiency virus type 1 (HIV-1)-infected individual, immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the gp120 third variable (V3) loop. Recently, we suggested on the basis of sequencing C2/V3 segments from an HIV-1 subtype E-infected family that a V3 sequence lineage group of the non-syncytium-inducing (NSI) variants (group 1) was relatively resistant to positive selection pressure (35). To better understand the relationship between the intensity of positive selection pressure and cell tropism of the virus, we determined the linkage between each V3 genotype and its function of directing coreceptor preference and MT2 cell tropism. The biological characterization of a panel of V3 recombinant viruses showed that all of the group 1 V3 sequences could confer an NSI/CCR5-using (NSI/R5) phenotype on HIV-1(LAI), whereas the group 2 V3 sequence, which was more positively charged than the group 1 sequence, dictated mainly a syncytium-inducing, CXCR4-using (SI/X4) phenotype. Phylogenetic analysis of C2/V3 sequences encoding group 1 or 2 V3 suggested that the variants carrying group 1 V3 are the ancestors of the intrafamilial infection and persisted in the family, while the variants carrying group 2 V3 evolved convergently from the group 1 V3 variants during disease progression in the individuals. Finally, a statistical test showed that the V3 sequence that could dictate an NSI/R5 phenotype had a synonymous substitution rate significantly higher than the nonsynonymous substitution rate. These data suggest that V3 sequences of the subtype E NSI/R5 variants are more resistant to positive selection pressure than those of the SI/X4 variants.

An infectious DNA clone of HIV type 1 subtype C.

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.

Genetic similarity of HIV type 1 subtype E in a recent outbreak among injecting drug users in northern Vietnam to strains in Guangxi Province of southern China.

To investigate the molecular epidemiology of a recent HIV-1 outbreak in northern Vietnam and its relation to the epidemic in surrounding areas, we analyzed 17 HIV-positive blood specimens from 3 heterosexuals, 2 sexually transmitted disease patients, and 12 injecting drug users (IDUs), collected in 4 provinces near Hanoi in 1998. These were compared with the specimens from Ho Chi Minh City (n = 10) and An Giang Province (n = 10) in southern Vietnam and with published sequences from neighboring countries. Genetic subtyping based on the env C2/V3 sequences revealed that HIV-1 subtype E predominated throughout Vietnam in all risk populations; the exception was one typical United States-European-type HIV-1 subtype B detected in a patient in Ho Chi Minh City, the first case of HIV infection identified in Vietnam in 1990. The HIV-1 subtype E sequences identified in 9 of the 12 IDUs from northern provinces were closely related phylogenetically to those in IDUs in nearby Guangxi Province of China, and also shared a common amino acid signature downstream of the env V3 loop region. The low interperson nucleotide diversity among IDUs in northern Vietnam supports the view that HIV-1 subtype E was introduced recently among IDUs in northern Vietnam. These data indicate a linkage between HIV-1 circulating among IDUs in northern Vietnam and southern China, and suggest recent transborder introductions as the likely source of HIV-1 subtype E in northern Vietnam.

Sendai virus-based production of HIV type 1 subtype B and subtype E envelope glycoprotein 120 antigens and their use for highly sensitive detection of subtype-specific serum antibodies.

We previously described a Sendai virus (SeV)-based expression system for the recombinant gp120 of HIV-1 subtype B (rgp120-B), which has permitted the production of antigenetically and functionally authentic gp120 at a concentration as high as 6 microg/ml of culture supernatant (Yu D et al.: Genes Cells 1997;2:457-466). Here the same procedure was successfully applied to the production of HIV-1 subtype E gp120 (rgp120-E). The remarkable production of the proteins by the SeV expression system enabled us to use crude culture supernatants for serological and functional studies of gp120s. The immunological authenticity of rgp120-E was verified by patient sera and anti-V3 loop monoclonal antibodies specific for HIV-1 subtypes B and E. CD4-binding properties were corroborated by FACS analyses. The rgp120s were then used in an enzyme immunoassay (rgp120-EIA) to detect antibodies in the sera of HIV-1-infected individuals, and the performance was assessed in comparison with a conventional V3 loop peptide EIA (V3-EIA). The initial evaluation of a serum panel (n = 164) consisting of 76 subtype E and 88 subtype B sera revealed that the rgp120-EIA was nearly 1000-fold more sensitive than the V3-EIA and was able to detect subtype-specific antibody with 100% sensitivity and with a complete correlation with the genotypes, whereas the V3-EIA failed to detect 9 and 24% of the same subtype E and B sera, respectively. Furthermore, a study employing a panel of 28 international sera with known genotypes (HIV-1 subtypes A through F) confirmed the remarkable specificity of this method. An EIA reactivity higher than 1.0 was an unambiguous predictor of HIV-1 subtype E and B infections. The data imply the presence of strong subtype-specific epitopes for antibody bindings to these rgp120s.