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The accumulation of full-length HIV genome sequences in the different regions of the world led to a new view to the HIV pandemic. It is recognized that subtypes and intersubtype recombinants are equally important in the pandemic. In this article, we will discuss the latest findings on molecular epidemiology of HIV and the related subjects.

Objective: To identify and characterize cytotoxic T-cell (CTL) epitopes for HIV-1 clade E using eight known HLA-A*1101-restricted HIV-1 clade B epitopes., Methods: Induction of clade E-specific CTL was examined by stimulating peripheral blood mononuclear cells (PBMC) from clade E-infected Thai individuals with the clade E-specific peptide corresponding to the clade B epitopes. Cross-clade and clade-specific CTL recognition for these epitopes was analysed using CTL clones and bulk CTL specific for these epitopes. To clarify the presentation of these epitopes in HIV-1-infected T cells, CTL recognition for the clade E-specific and cross-clade epitopes was investigated using CD4CXCR4 cells infected with an HIV-1 clade E clone., Results: Three epitopes, which are identical among clades A-E, were recognized as cross-clade CTL epitopes in both individuals. Clade B and E sequences corresponding to three epitopes were recognized as clade-specific epitopes in clade B-infected and clade E-infected individuals, respectively. In contrast, clade E-specific peptides corresponding to two other clade B epitopes failed to elicit clade E-specific CTL. CTL specific for the three cross-clade and three clade E-specific epitopes effectively lysed target cells infected with HIV-1 clade E virus., Conclusions: These six epitopes are found to be processed naturally in HIV-1 clade E-infected cells. We show here that a strategy utilizing HIV-1 clade B epitopes is very useful for identifying clade E CTL epitopes.

The southeastern region of Yunnan province is a key site for drug trafficking and HIV-1 infection spread from the west of Yunnan and Laos to southeastern China. To investigate the prevalence of HIV-1 infection and hepatitis C virus (HCV) coinfection among injection drug users (IDUs) in southeastern Yunnan, three cohorts of 285 addicts, including 242 IDUs and 43 oral drug users, living in the cities of Gejiu and Kaiyuan and the county of Yanshan were studied. HIV-1 and HCV infections were detected by enzyme-linked immunosorbent assay and/or polymerase chain reaction. Data on the age, sex, risk behavior, drug use history, employment, ethnic background, and marriage status were obtained by interview. The overall prevalence of HIV-1 infection was 71.9%. The rate of HCV coinfection among 138 HIV-1-infected IDUs was 99.3%. Most HIV-infected IDUs were 20 to 35 years old (86.7%) and were ethnic Han (75.9%), suggesting that the epidemic in Yunnan is no longer confined to non-Han ethnic minorities. HIV prevalence in female IDUs (81.2%) was significantly higher than in male IDUs (68.2%) ( p <.05). The prevalence of HIV infection reached 68.4% after 1 year of injection drug use. Needle/syringe sharing is the major high risk factor for the spread of HIV-1 and HCV infections. Large-scale educational campaigns are urgently needed to reduce the spread of HIV and HCV infection in these regions.

An efficient lipase-catalyzed desymmetrization of prochiral 2,2-disubstituted 1,3-propanediols was developed using 1-ethoxyvinyl 2-furoate 1b, for which the well-known method using vinyl or isopropenyl acetate has had limited success due to low reactivity and easy racemization of the products through acyl group migration. The reagent 1b is highly reactive and converts various prochiral 1,3-diols to the monoesters having a chiral quaternary carbon center with 82-99% ee. These products were stable against racemization under acidic conditions, and their furoyl groups were compatible with oxidative conditions. Prolonging the reaction time led to the kinetic resolution of the monoesters resulting in an increase of their optical purity. The similar desymmetrization of meso cis-1,2-cycloalkanediols gave the monoesters with 82-97% ee without racemization.

We have isolated replication-competent molecular clones of HIV-1 circulating recombinant form CRF01&#95;AE with different coreceptor usages. After lambda phage cloning of unintegrated circular proviral DNAs derived from a CRF01&#95;AE strain (HIV-1NH1), isolated in Japan, the infectious molecular clone, designated p93JP-NH1, was reconstituted. 93JP-NH1 showed an X4 and R5 phenotype in NP2 cell-based coreceptor utilization assays and exerted robust replication in human T cell lines, including MT2, M8166, and PM1 cells, whereas it propagated modestly in peripheral blood mononuclear cells. The CRF01&#95;AE molecular clone with R5 phenotype (p93JP-NH2env) was then constructed by replacing the env gene of p93JP-NH1 with that of a nearly isogenic CRF01&#95;AE R5 strain isolated from an epidemiologically linked case. The phylogeny and recombination break-point analysis confirmed that these clones shared an A/E recombinant structure similar to that of the prototype CRF01&#95;AE strain, CM240. These replication-competent CRF01&#95;AE molecular clones with different coreceptor usages would be useful tools for the study of CRF01&#95;AE, one of the most prevalent strains in Asia.

In plastic surgery, 3D models of the affected part are often used for the purpose of visualizations and surgical simulations. The optimal models for web-based surgical simulations keep high accuracy in affected parts and keep low accuracy in other parts. Consequently, the data size becomes small. In this research, we propose a method to generate free-form surfaces based on Lattice Structure from polygonal meshes. The polygonal meshes are generated automatically from CT and MRI data using Marching Cubes. By changing the resolution of input images, the accuracy of output meshes is controlled. Free-form surfaces based on Lattice Structure are fitted to polygonal meshes. Lattice Structure is a method to manage a free-form surface with a simple base polygonal mesh. The data size is quite small because surface shape is converted and saved as a simple polygon. A free-form surface is quickly generated and high accuracy is maintained. Moreover, users can input character lines and they are reflected as boundaries of patches. The models generated with this method are partly accurate and compact. These data make it possible to simulate surgery on the WWW, because they can be quickly transferred

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.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01&#95;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.

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02&#95;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&#95;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.

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.

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&#95;AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01&#95;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&#95;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&#95;AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01&#95;AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01&#95;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&#95;AE variant (E(v)) closely related to that prevailing in nearby southern China.

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01&#95;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&#95;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&#95;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.

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.

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.

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.

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.

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.

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.

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.

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.

By PCR screening, we found an extremely high prevalence of TT virus (TTV) in the general populations from different geographic regions. This suggests that TTV may be a common DNA virus with no clear disease association in humans. TTV genotyping by phylogenetic analysis was also performed.

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1LAI. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E., (Copyright 1999 Academic Press.)

It has been suggested that immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the third variable (V3) loop of the gp120 of human immunodeficiency virus type 1 (HIV-1). Here we present evidence, on the basis of sequencing 147 independently cloned env C2/V3 segments from a single family (father, mother, and their child), that the intensity of positive selection is related to the V3 lineage. Phylogenetic analysis and amino acid comparison of env C2/V3 and gag p17/24 regions indicated that a single HIV-1 subtype E source had infected the family. The analyses of unique env C2/V3 clones revealed that two V3 lineage groups had evolved in the parents. Group 1 was maintained with low variation in all three family members regardless of the clinical state or the length of infection, whereas group 2 was only present in symptomatic individuals and was more positively charged and diverse than group 1. Only virus isolates carrying the group 2 V3 sequences infected and induced syncytia in MT2 cells, a transformed CD4(+)-T-cell line. A statistically significant excess of nonsynonymous substitutions versus synonymous substitutions was demonstrated only for the group 2 V3 region. The data suggest that HIV-1 variants, possessing the more homogeneous group 1 V3 element and exhibiting the non-syncytium-inducing phenotype, persist in infected individuals independent of clinical status and appear to be more resistant to positive selection pressure.

RANTES (regulated on activation normal T cell expressed and secreted) is one of the natural ligands for the chemokine receptor CCR5 and potently suppresses in vitro replication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. Previous studies showed that peripheral blood mononuclear cells or CD4(+) lymphocytes obtained from different individuals had wide variations in their ability to secrete RANTES. These findings prompted us to analyze the upstream noncoding region of the RANTES gene, which contains cis-acting elements involved in RANTES promoter activity, in 272 HIV-1-infected and 193 non-HIV-1-infected individuals in Japan. Our results showed that there were two polymorphic positions, one of which was associated with reduced CD4(+) lymphocyte depletion rates during untreated periods in HIV-1-infected individuals. This mutation, RANTES-28G, occurred at an allele frequency of approximately 17% in the non-HIV-1-infected Japanese population and exerted no influence on the incidence of HIV-1 infection. Functional analyses of RANTES promoter activity indicated that the RANTES-28G mutation increases transcription of the RANTES gene. Taken together, these data suggest that the RANTES-28G mutation increases RANTES expression in HIV-1-infected individuals and thus delays the progression of the HIV-1 disease.

To study the molecular epidemiology of HIV-1 spread in Myanmar and the interplay with the epidemic in surrounding Southeast Asian countries, we determined the HIV-1 subtypes prevailing in Myanmar. Thirty HIV-positive blood specimens were sampled in the capital city, Yangon, and an additional 459 sera were collected nationwide in 1995. Genetic subtyping based on the env C2/V3 sequence and serologic data, using a V3 peptide enzyme immunoassay (PEIA), revealed three patterns of HIV spread in different geographic regions in Myanmar: (1) in the capital city, Yangon, HIV-1 subtype B' ("Thai-B" cluster within subtype B) predominated both in IDUs and heterosexuals; (2) in the cities near the border with Thailand, including Tachelaik and Kawthaung, where heterosexual transmission is a major pathway of HIV-1 spread, HIV-1 subtype E was predominantly distributed among the commercial sex workers and heterosexuals; (3) in central and northeast Myanmar, both HIV-1 subtypes B' and E occurred in a mixed distribution, without showing any significant segregation by risk group. In addition, the PEIA data implied the occurrence of other subtype(s) in these areas. The interperson nucleotide sequence variations in env C2/V3 regions of B' and E, prevailing in Yangon, were 6.7 +/- 2.1 and 7.1 +/- 0.7%, respectively. They were similar to those levels observed in Thailand. These findings are consistent with the view that HIV spread in Myanmar might have taken place at about the same time as that in Thailand, and that multiple entries and exchanges of HIV-1 with neighboring countries are important factors contributing to the current distribution of subtypes in Myanmar.

Previous serological studies have demonstrated that some 60% of intravenous drug abusers (IVDAs) in urban areas of the former South Vietnam are infected with HTLV-II. In the present report we have attempted to characterize the viruses using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of the provirus long terminal repeat (LTR) region. RFLP analysis of nine samples demonstrated that all were infected with the HTLV-IIb subtype. The HTLV-IIa subtype was not detected. Phylogenetic analysis of the nucleotide sequences demonstrated that the viruses clustered closely with HTLV-IIb isolates present in IVDAs from the New York City area. The present molecular analysis together with the previously reported absence of HTLV-II infection in North Vietnam supports the view that HTLV-II may have been introduced from the United States to this part of Asia by military personnel during the Vietnam conflict.

Although the efficacy of extracranial/intracranial bypass for reduction of risk of ischemic stroke has been denied, we have encountered patients in whom bypass operation seems to have improved his or her clinical course. The efficacy of bypass should be evaluated not by the patency of the bypass but by the extent of collateral circulation brought about by the bypass. We retrospectively analyzed our patients to determine whether the extent of bypass flow could be predicted from the results of preoperative studies. In 18 hemispheres of 18 consecutive patients who underwent extracranial/intracranial bypass surgery, correlation between the extent of bypass flow and the multiple preoperative factors including the angiographic findings were investigated. The bypass function was highly predictable in the light of preoperative studies. In all of 10 hemispheres in patients under 70 years of age and with occlusive lesions in the proximal portion of the middle cerebral artery, collateral circulation through the bypass developed to an extensive or a moderate degree. In 9 of 10 hemispheres in which an interval between the latest attack and the diagnosis of hemodynamic failure was 4 weeks or longer, collateral circulation through the bypass was shown to have developed to an extensive or a moderate degree. Our results indicate that extensive or moderate collateral circulation through the bypass can be expected only in patients under 70 years of age, with lesions in the proximal portion of the middle cerebral artery, and in whom an interval between the latest attack and diagnosis of hemodynamic failure was 4 weeks or more.

We describe a robust expression of human stromal cell-derived factor-1alpha (SDF-1alpha) and SDF-1beta, the members of CXC-chemokine family, with a novel vector system based upon Sendai virus, a non-segmented negative strand RNA virus. Recombinant SDF-1alpha and SDF-1beta were detected as a major protein species in culture supernatants, reached as high as 10 microg/ ml. This remarkable enrichment of the products allowed us to use even the crude supernatants as the source for biological and antiviral assays without further concentration nor purification and will thus greatly facilitate to screen their genetically engineered derivatives.

A 49-year-old male with tuberculous meningitis was reported. When admitted to our hospital with mild right hemiparesis, he was alert but he developed disorientation 7 days later. A diagnosis of tuberculous meningitis was reached because of elevated levels of adenosine-deaminase at 19.6U/liter in the cerebrospinal fluid. MRI showed a marked enhancement in the basal cisterns and an enhanced intraparenchymal lesion in the brainstem. Chronological changes of MRI findings did not closely correlate with the clinical course. Slight meningeal enhancement on MRI seems to remain for a long time without active tuberculous meningitis, and absence of the meningeal enhancement on MRI is not necessarily appropriate as a marker of cure of tuberculous meningitis.

To better understand the pyramidal tract of the internal capsule, we evaluated the relationship between the localization of thalamic hemorrhage and motor weakness. On an axial CT scan at the level of the pineal body, two lines were drawn as follows: line-a between the lateral edge of the anterior horn and the lateral edge of the trigone, line-b vertical to the sagittal line and passing the midpoint of the third ventricle. The location of the hematoma was classified into three types according to localization of the center of the hematoma and lateral extension beyond line-a as follows: type A (anterior), type P (posterior) and type PL (postero-lateral). Discrepancy of motor weakness between upper extremities and lower extremities was higher in patients with hematoma of type P and type PL (p < 0.05) than in those with hematoma of type A. Improvement of motor weakness on discharge was higher in patients with type P (p < 0.01) than in those with type A. We concluded that most of the pyramidal tract fibers were located in the third quarter of the posterior limb of the internal capsule but a small number of pyramidal tract fibers were located in the anterior two-thirds of it. A greater number of cortico-spinal fibers to the upper extremities than to the lower extremities occupy the third quarter of the posterior limb of the internal capsule.

A high rate of human T-lymphotropic virus type II (HTLV-II) infection has been documented in intravenous drug abusers (IVDAs) in South Vietnam. We have investigated the molecular characteristics of the virus and have shown that one HTLV-II subtype is predominant in Ho Chi Minh City. This molecular subtype, HTLV-IIb, was identified in a number of South Vietnamese by nucleotide sequence analysis of the long terminal repeat (LTR) region. HTLV-IIa was not found. These findings suggest that HTLV-IIb is endemic in IVDAs in South Vietnam, although IVDAs in urban areas in North America are predominantly infected with HTLV-IIa.

We analyzed the clade distribution of B and E in HIV-1 isolates in Japan by a nested PCR method using 5'-CCCACAAGATTTAAATATG-3' of the gag gene as clade B primer and 5'-CCCACAAGATTTAAACTCC-3' of the gag gene as clade E primer. Seventy-two anti-HIV-1 confirmatory positive serum samples were collected during a period of 1991-1996 in two hospitals in Yokohama City. Peripheral blood mononuclear cells were obtained from the buffy coat of these samples and extracted DNA were used for nested PCR. The 72 cases comprised of 11 Japanese hemophiliacs, 14 Japanese male homosexuals, 19 Japanese male heterosexuals, 5 Japanese female heterosexuals and 23 Thai female heterosexuals. Of these 36 were clade B and 35 were clade E and one case showed positive PCR results for both B and E primers. Almost all male Japanese hemophiliacs and homosexuals in our sample have clade B, while the female Thai heterosexuals have clade E, irrespective of the year of isolation. As for Japanese male heterosexuals, through 1993, clade B was predominant but since 1994, the predominate clade switched to clade E. Although the number of Japanese female heterosexuals in our sample is small, clade B was isolated in 2 cases even after 1994.

The adsorption isotherms of water, the differential heat of adsorption, and the entropy of adsorbed water were investigated to elucidate the structure of stability of cyclodextrins (CyD) and branched CyD's. The amount adsorbed on branched CyD's increased with increasing relative humidity. The steep rise in the amount adsorbed at higher relative humidities indicated that the structure of branched CyD's was affected by the water molecules at these higher relative humidities. The bonding force between alpha-CyD's and water molecules was stronger than that between their branched CyD's and water, while that between beta-CyD's and water was weaker than that between their branched CyD's and water. We have used activated carbons for the recovery of organic solvents. There are polar groups on the activated carbon surface. Therefore, the recovered organic solvents were degraded by these groups. However, such polar groups are not expected for the hydroxyl groups of the CyD's. The amounts of 1,1,1-trichloroethane, benzene, and toluene included in natural CyD's and their branched CyD's were measured. The amounts of organic solvents included in the CyD's depended upon the cavity radii and the glucosyl and maltosyl functional groups of the CyD's and the radii of the organic solvent molecules.

Previous work has shown the usefulness of severe combined immunodeficient (SCID) mice as in vivo models for the growth of normal human haemopoietic cells and leukaemic cells. Many approaches have been made to improve the engraftment of human haemopoietic cells in SCID mice. We established transgenic mice producing human granulocyte-macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. Endogenous serum hGM-CSF levels were detected by ELISA [mean 9585 pg/ml (line A, n = 4); mean 1610 pg/ml (line B, n = 4)]. Expression of hGM-CSF was observed in all organs tested including the heart, lung, liver, kidney, spleen, thymus, bone marrow and brain of hGM-CSF transgenic (hGMTg) mice. Morphological analysis of organs and peripheral blood cell counts showed no differences between hGMTg mice and their littermates. Murine Ba/F3 cells expressing functional hGM-CSF alpha beta receptor (BAF/alpha beta cells) could be successfully engrafted in hGMTg SCID mice. The cells invaded multiple organs and caused death within a few weeks of transplantation, although they infiltrated only the spleen of their littermates. These results showed that these hGM-CSF-producing SCID mice are useful as an in vivo assay system for investigating leukaemogenesis.

A 34-day-old male presented with a rare neonatal ruptured aneurysm of the distal posterior inferior cerebellar artery manifesting as a 10-day history of enlargement of head circumference. Magnetic resonance imaging revealed hydrocephalus and a round infratentorial enhanced lesion which compressed the medulla. Left vertebral angiography demonstrated an aneurysm on the telovelotonsillar segment of the left posterior inferior cerebellar artery. Ventriculoperitoneal shunt emplacement and proximal artery clipping were performed. The cerebrospinal fluid was bloody, suggesting aneurysm rupture had caused hydrocephalus. His postoperative course was uneventful, and neurological and developmental findings were normal 7 months later. Present neuroimaging, surgical, and neuro-anesthesiology techniques allow successful surgical intervention in cases of neonatal ruptured aneurysm.

Susceptibility to HIV infection was examined in macrophages differentiated from human monocytes by macrophage colony-stimulating factor (M-CSF) or granulocyte/macrophage colony-stimulating factor (GM-CSF). The replication of macrophage-tropic human immunodeficiency virus type-1 (HIV-1), which was determined by reverse transcriptase (RT) activity, was significantly suppressed in macrophages induced by GM-CSF (GM-type macrophages) but not in those induced by M-CSF (M-type macrophages). Multinucleated giant cells were formed only in M-type macrophages after HIV infection. However, the expression of CD4 molecules on the surface of both types of macrophages was similar and the proviral DNA was detectable in cell lysates of both macrophages, although the amount of proviral DNA in M-type macrophages was higher than that in GM-type macrophages. Many steps have been defined in HIV infection and replication, such as adsorption of HIV to the cell surface, internalization of the viral core into the cytoplasm, uncoating of viral RNA, reverse transcription and integration of proviral DNA into cellular DNA, transcription and translation of proviral DNA, assembly of viral components, and budding of virus particles. Our findings suggested that the suppression of HIV-1 replication in macrophages induced by GM-CSF is mainly due to a disturbance at certain steps of replication after synthesis of the proviral DNA. Thus, the suppression of HIV replication in GM-type macrophages may provide a model of the latency of HIV infection in vivo.

We experienced a case of successful acute revascularization using a long vein graft. A 68-year-old man was admitted to our department due to transient ischemic attack of the left hemiparesis. CT scan showed no infarction, but PAO-SPECT revealed moderate hypoperfusion in the right ACA and MCA area. Cerebral angiography demonstrated right IC occlusion at its origin and moderate collateral circulation via leptomeningeal anastomosis from the PCA area, and via the external carotid system, especially directly from STA. But the STA was very narrow. Three days after admission, left hemiparesis appeared again and deteriorated severely. This time the hemiparesis persisted. Although MRI demonstrated little infarction in the right frontal lobe, we decided to carry out revascularization on the same day. Right saphenous vein was harvested for a graft because of the narrow STA. The facial artery and angular artery was selected as a donor and a recipient respectively, to avoid a clamp of the EC and a craniotomy of the STA running area. Finally we performed a facial artery-vein graft-angular artery (M4) bypass. The patient showed no complication and the left hemiparesis improved enough to allow the patient to walk by himself. Revascularization using vein graft is dangerous for acute ischemia due to the possibility of a complication such as brain edema and hemorrhagic infarction. The usual style of vein graft bypass is an EC-vein graft-M2 or M3 bypass. Using this style, high pressure inside the EC is carried intracranially.(ABSTRACT TRUNCATED AT 250 WORDS)

A case of unusual early myoclonic encephalopathy was reported. He showed massive and/or fragmentary myoclonic seizures soon after birth until 20 months before death. These seizures were resistant to various kinds of anti-epileptic drugs. The EEG pattern of suppression-burst was continuously seen in both waking and sleep states from 2 months to 19 months of age. He was supported with mechanical ventilator because of the central respiratory dysfunction since 4 days after birth until death. This case was electroclinically considered as that of early myoclonic encephalopathy. The central respiratory dysfunction has not been reported in early myoclonic encephalopathy.

Heterosexual transmission of the human immunodeficiency virus (HIV) appears to occur readily all over the world. Recent reports have suggested an association between HIV and sexually transmitted disease (STD). We conducted a case study among foreign female prostitutes who were seropositive to HIV to determine the prevalence of STD. In this study, we checked the prevalence of STD infection in 19 HIV seropositive female prostitutes. Overall, 84 percent were seropositive for Chlamydia trachomatis, 37% for HBs, 32% for Treponema pallidum (Tp) and 6% for hepatitis C virus (HCV). The high frequency of a history of STD may be associated with an increased risk of HIV infection acquired through heterosexual contact. Prevention of heterosexually transmitted HIV infection will require an extensive educational program aimed specifically at the risk associated with the number and selection of sexual partners and at promoting safer sexual practices.

A 61-year-old male presented with recurrent malignant histiocytosis of the brain manifesting as nausea and headache. Malignant histiocytosis is a disorder of proliferating histiocytes characterized by a rapidly progressive and fatal course, but central nervous system involvement is relatively rare. Magnetic resonance (MR) imaging demonstrated cerebrospinal fluid (CSF) dissemination of histiocytes as a low-intensity area on the T1-weighted image with marked gadolinium-diethylenetriaminepenta-acetic acid enhancement and a high-intensity area on the T2-weighted image. CSF cytological examination revealed an increased level of atypical histiocytes. Brain and spine irradiation, and intrathecal methotrexate and prednisolone administration induced remission. MR imaging is particularly useful for the diagnosis of meningeal dissemination of malignant histiocytosis.

We encountered a case of HIV-1 infection in a previously healthy man, which was characterized by rapid progression to AIDS and death within 7 months in association with high levels of antigenemia throughout the clinical course and no humoral immune response for at least 6 months. Genetic changes of the third variable domain (V3) of the envelope gene of HIV-1 in serum samples were analyzed at four time points during his rapid clinical course. The nucleotide changes were confined to a maximum of three substitutions among 105 nucleotides of the V3 region. A major population of the viral clones in this patient showed one amino acid substitution from aspartic acid (a negatively charged amino acid) to lysine (a positively charged amino acid) at position 30 from the first cysteine of the V3 loop. This substitution was thought to be associated with phenotypic changes, and viruses with this sequence in the V3 region had a strong syncytium-inducing ability in MT-4 cells. It appears that the lack of a humoral immune response accelerated disease progression in our patient and a genetic change that appeared to produce a phenotypic change occurred at an early stage of the disease.