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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.

Amino Acid Sequence↗

Cytogenetic subtyping of 220 salivary gland pleomorphic adenomas: correlation to occurrence, histological subtype, and in vitro cellular behavior.

Based on the hypothesis that three main cytogenetic subtypes of salivary gland pleomorphic adenomas can be distinguished which may also represent different etiologic entities, we investigated whether these subtypes correspond to clinical, histologic, or biologic features of 220 tumors karyotyped (including 117 tumors with detailed clinical history and histologic subtyping). The following results were obtained. As compared with the group of patients showing salivary gland pleomorphic adenomas with an apparently normal karyotype, the patients in the "8q12-group" were significantly younger (51.1 years versus 39.3 years, p < 0.001). The distribution of histologic subtypes also showed highly significant differences between the groups. Whereas the breakpoint in the 8q12 group was always mapped to a single band, no exact localization of the breakpoint in the group of tumors showing chromosome number 12 abnormalities was possible. In most cases, however, the breakpoints were clustered to 12q15. Finally, all tumors with 8q12 breakpoints showed a characteristic in vitro cellular morphology which was also observed in a few tumors with an apparently normal karyotype but in none of the tumors with the 12q13-15 breakpoint.

Adenoma, Pleomorphic↗

Multiplicity of muscarinic autoreceptor subtypes? Comparison of the distribution of cholinergic cells and cells containing mRNA for five subtypes of muscarinic receptors in the rat brain.

In situ hybridization was used to compare the microscopic distribution in the rat brain of cells containing mRNA for choline acetyltransferase (ChAT) (i.e. cholinergic cells) with that of cells containing mRNA for the five subtypes of muscarinic receptors, in an attempt to establish the potential role as autoreceptors (i.e. muscarinic cholinoceptors present in cholinergic cells) of the different muscarinic receptor subtypes. [32P]alpha-dATP-labelled synthetic oligonucleotides were used as hybridization probes in serial sections. Transcripts for all five subtypes of muscarinic receptors were detected in cells co-distributing with ChAT mRNA-containing cells in one or more regions of the brain. Cells containing m2, m3, m4 or m5 mRNAs were observed in the regions of the basal forebrain where cholinergic cells are located (medial septum/diagonal band nuclei, ventral pallidum, basal nucleus of Meynert). m2, m3 and m5 mRNAs were abundant in the parabigeminal nucleus. m2, m3 and m4 transcripts were detected in the pedunculopontine and laterodorsal tegmental nuclei. m1, m2 and m3 mRNAs were present in several cranial nerve nuclei. The present results suggest that muscarinic autoreceptors belonging to the five subtypes cloned to date may exist.

Animals↗

Pharmacologic characterization of cloned alpha 1-adrenoceptor subtypes: selective antagonists suggest the existence of a fourth subtype.

In membranes prepared from Cos-7 or HeLa cells expressing one of three individual cloned alpha 1-adrenoceptor subtypes, competition with 2-[(beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-tetralone ([125I]HEAT) by the selective compounds [+]-niguldipine, 5-methyl-urapidil, and benoxathian reveals high affinity for the cloned alpha 1C-adrenoceptor subtype and low affinity for both the cloned alpha 1A-adrenoceptor and alpha 1B-adrenoceptor. Competition with [125I]HEAT by spiperone revealed high affinity for the cloned alpha 1C-adrenoceptor, intermediate affinity for the cloned alpha 1B-adrenoceptor, and low affinity for the cloned alpha 1A-adrenoceptor. Combining pharmacological properties previously described for alpha 1-adrenoceptor subtypes in rat membranes and here described from cloned receptors, these data suggest the existence of a fourth distinct alpha 1-adrenoceptor subtype.

Adrenergic alpha-Agonists↗

Distribution of alpha1-adrenoceptor subtype mRNA and identification of subtype responsible for renovascular contraction in human renal artery.

This study was intended to quantify the amounts of the alpha1-adrenoceptor subtype mRNAs in human renal artery and to demonstrate the distribution of receptor subtypes responsible for the contraction of the renal artery. RNase protection assay showed that the mean amount of alpha1a mRNA was much greater than that of alpha1b or alpha1d mRNAs in both the main and branch renal arteries. However, the abundance of alpha1a mRNA in human renal artery was much less than in our previous data in the prostate. In situ hybridization showed that all alpha1 subtype mRNAs were localized in the smooth muscle cells of the tunica media of the artery, and the distribution pattern of these three mRNAs in the main artery was the same as in the branch artery. However, the intensity of signals for alpha1d and alpha1b antisense RNAs probes was lower than that for the alpha1a antisense RNA probe. In the functional study, concentration-response curves to noradrenaline pretreated with KMD-3213, an alpha1A/L-adrenoceptor selective antagonist, seemed to be biphasic in nature. Chloroethyclonidine (CEC) failed to inactivate the noradrenaline-induced contraction, and prazosin showed relatively low affinity with a pA2 value of 8.8. These data suggest that the alpha1A/L-adrenoceptor mediates primarily those responses to noradrenaline in this artery. The other alpha1-adrenoceptor subtypes could also mediate the secondary contractile response to noradrenaline in this artery.

Adrenergic alpha-Antagonists↗

Somatostatin receptor subtype 1 (sst(1)) regulates intracellular 3',5'-cyclic adenosine monophosphate accumulation in rat embryonic cortical neurons: evidence with L-797,591, an sst(1)-subtype-selective nonpeptidyl agonist.

Somatostatin (SRIF) initiates its biological activities by interacting with five homologous G-protein-coupled receptor subtypes (sst(1--5)). In the mammalian nervous system, sst(1--5) receptor mRNA expression patterns have been localized by in situ hybridization studies, or at the protein level with receptor-specific antibodies. Cortical responses to SRIF have been demonstrated, although a functional relationship between an SRIF effect and an individual receptor subtype is lacking. The recent development of novel, subtype-selective SRIF receptor ligands now provides a means to correlate receptor subtype expression patterns with the corresponding biological function. In cultured monolayers of E17-18 rat embryonic cortical neurons, 10(-7) M SRIF-28 inhibited 10(-6) M forskolin-stimulated cAMP accumulation by 37%, a level of inhibition that was mimicked by L-797,591, a potent sst(1)-selective agonist. SRIF-14 or L-797,591 inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent fashion, with EC(50)s (effective concentration for 50% maximal response) of 8.0 x 10(-10) M and 7.0 x 10(-10) M, respectively. No similar concentration-dependent effect on forskolin-stimulated cAMP levels was observed with sst(2)-, sst(3)- or sst(4)-selective agonists. Furthermore, both SRIF-14 and L-797,591 inhibited 10(-7) M CRH-induced cAMP in the embryonic neurons. These results are the first evidence demonstrating that sst(1) regulates intracellular cAMP levels in embryonic neurons and may inhibit CRH-mediated effects in the embryonic cortex.

Amides↗

Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT1 receptor subtypes to adenylyl cyclase.

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10-12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 microM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

Adenylyl Cyclases↗

Distinct distribution of four Ca2+-dependent subtypes of protein kinase C in rat olfactory bulb; definite expression of betaII-subtype in the accessory olfactory bulb.

The localization of four subtypes of Ca2+-dependent protein kinase C (PKC) in the main and accessory olfactory bulb was examined by immunocytochemistry by using specific antibodies against each PKC subtype. In the main olfactory bulb, alpha-PKC was densely localized in a large number of granule cells and in a few tufted cells, and faint immunoreactivity was seen in some periglomerular cells. betaI-PKC was intensely found in periglomerular cells and tufted cells. gamma-PKC immunoreactivity was present in the external plexiform layer, the internal plexiform layer, and the granular layer, but the immunoreactivity was found only in the neuropils. Little, if any, betaII-PKC was seen in the main olfactory bulb. On the other hand, the intense immunoreactivity for betaII-PKC was seen in periglomerular cells of the accessory olfactory bulb. The betaI-PKC and gamma-PKC were also present in periglomerular cells of the accessory olfactory bulb, while alpha-PKC was localized only in granule cells. Double staining study in the accessory olfactory bulb showed that betaII-PKC was present in the GABAergic periglomerular cells, while betaI-PKC localized to the non-GABAergic periglomerular cells; gamma-PKC was expressed in both GABAergic and non-GABAergic cells. These findings suggest that four calcium-dependent subtypes of PKC play different roles in the olfactory bulb and definite expression of betaII-PKC strongly suggested the involvement of this subtype in a specific function in the accessory olfactory bulb.

Animals↗

Effect of systemic insulin and angiotensin II receptor subtype-1 antagonist on endothelin-1 receptor subtype(s) regulation and binding in diabetic rat heart.

This study reports on the regulation and remodeling role of endothelin-1 (ET-1) and its receptor subtypes, ET(A)-Rs/ET(B)-Rs, at the coronary endothelium (CE) and cardiomyocyte (CM) sites. It is carried out in normal and normotensive rats with streptozotocin-induced diabetes mellitus receiving different treatment modalities. Normal rats were divided into two groups, namely a placebo (N) and a losartan-treated (NL), and diabetic rats into four groups receiving placebo (D), insulin-treated (DI), losartan-treated (DL), and insulin/losartan-treated (DIL) respectively. Binding kinetics of ET-1 to ET(A)-Rs/ET(B)-Rs on CE and CMs were assessed in the above groups to try to explain the effect of therapeutic doses of an angiotensin II receptor subtype-1 blocker on the dynamics of this ligand and its receptor in insulin supplemented diabetic animals. Each group was divided into two subgroups: CHAPS-untreated and CHAPS-treated rat hearts perfused with [125I]ET-1 to respectively estimate ET-1 binding affinity (tau = 1/k-n) to its receptor subtype(s) on CE and CMs using mathematical modeling describing a 1:1 reversible binding stoichiometry. Heart perfusion results revealed that insulin treatment significantly decreased tau on CE but not on CMs in diabetic rats. In diabetics treated with losartan, an increase in tau value on CE but not on CMs was noted. Cotreatment of diabetic rats with insulin and losartan normalized tau on CE but decreased it on CMs. Western blot, using snap-frozen heart tissues, revealed increase in ET(A)-R density in all diabetic groups. However, significant decrease in ET(B)-R density was observed in all groups compared to the normal, and was reconfirmed by immunohistochemical analysis. In conclusion, coadministration of insulin and losartan in nonhypertensive animals suffering from diabetes type 1 may offer new cardiac protection benefits by improving coronary blood flow and cardiomyocyte contractility through modulating ET-1 receptor subtypes density and affinity at CE and CM sites.

Angiotensin II Type 1 Receptor Blockers↗

Near full-length genome analysis of HIV type 1 CRF02.AG subtype C and CRF02.AG subtype G recombinants.

HIV-1 CRF02.AG strains are prevalent in west and west-central Africa, suggesting a longstanding presence of these subtype A/G recombinants in the global epidemic. Cocirculation of CRF02.AG strains with other group M subtypes may give rise to HIV-1 recombinants constituting a mosaic genome comprising fragments of three different subtypes. We report on the genetic analysis of the near-full-length genomes of such recombinants (VI1035 and VI1197) as well as CRF02.AG strains in Belgian individuals. VI1035 and VI1197 may be the result of successful "second-generation" recombinations of HIV-1 strains CRF02.AG with, respectively, subtype C (VI1035) and G (VI1197) strains in a dually infected individual.

Africa, Central↗

Reanalysis of full-length HIV type 1 group M subtype K and sub-subtype F2 with an MS-DOS bootscanning program.

Five new complete HIV-1 group M genome sequences have been published (Triques et al., AIDS Res Hum Retroviruses 2000;16:139-151). One of these clustered consistently with subtype F sequences, while two others were identified as representatives of a subcluster within the subtype F clade, called F2, and the two remaining sequences were described as a new subtype K. We reanalyzed these sequences by means of bootscanning and phylogeny, using a newly developed MS-DOS bootscanning program. Although our analysis does not contradict the existence of the new subtype K, it also indicates that in some regions the F2 sequences do not cluster with the F1 clade. This suggests that some fragments in the F2 sequences have an uncertain origin, and care should be taken when F2 sequences are used in analyses.

Genetic Techniques↗

Comparative study on subtypes of hepatitis B surface antigen (HBs Ag) in two regions of southern Japan, Okinawa and Kumamoto. Prevalence and unusual subtypes.

A total of 209 hepatitis B surface antigen (HBs Ag)-positive sera from two possibly ethnologically different regions of southern Japan, Okinawa and Kumamoto, were subtyped by counterelectrophoresis using monospecific antisera to a, d, y, w, and r antigenic determinants. There was a marked difference in the prevalence of the w and r determinants in the two regions; the frequency of the HBs Ag/adr subtype was 95% in Kumamoto and 47% in Okinawa, suggesting that HBs Ag subtypes may be one of the markers reflecting an ethnological difference between the peoples of the two regions. Seven and three serum specimens in Okinawa and Kumamoto respectively showed unusual HBs Ag subtypes: four carried adwr (two specimens), aywr, or adyw determinants, five had only a, and the remaining one carried ar.

Animals↗

Frequent detection of hepatitis C virus subtype 3a (HCV-3a) isolates in Thailand by PCR using subtype-specific primers.

By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or -2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and -1b) and 2 (HCV-2a and -2b).

Adult↗

Detection, typing, and subtyping of enteric adenoviruses 40 and 41 from fecal samples and observation of changing incidences of infections with these types and subtypes.

Monoclonal antibody (MAb) preparations specific for the enteric adenoviruses of subgenus F (AdF) were generated and evaluated as typing reagents in virus neutralization tests and enzyme-linked immunosorbent assays (ELISAs). A panel of 11 genome types of adenovirus 40 (Ad40), 24 genome types of Ad41, and 47 adenovirus prototype strains was used to determine the specificities of the MAbs in the two assays. In this way two MAbs, MAb 40-1 (anti-Ad40) and MAb 41-1 (anti-Ad41) were selected. These two MAbs showed strict type specificity in both assays. A third MAb reacted in an ELISA with all 47 human adenovirus types. With two other MAbs, three antigenic subtypes of Ad41 could be distinguished by their reactivities in virus neutralization tests and ELISAs. On the basis of the five selected MAbs, a sensitive ELISA system was developed for the direct detection and simultaneous typing and subtyping of Ad40 and Ad41 present in stool specimens. The five MAbs were also used to study the epidemiology of infections with Ad40 and Ad41 in The Netherlands in the period 1981 through 1989. It was shown that there were no significant fluctuations in the annual incidence of the cluster of enteric adenoviruses as a whole. This cluster should therefore be considered to belong to the "endemic" rather than the "epidemic" adenoviruses. The relative incidence of Ad40 infections compared with that of Ad41 infections changed considerably during the period studied; the proportion of Ad41 infections rose from about 30% in 1981 to about 95% in 1986, after which it stabilized at 90 to 95%. The proportion of one of the subtypes of Ad41 (Ad41 subtype M3) increased from about 40 to 80% in the same period.

Adenovirus Infections, Human↗

Hepatitis C virus (HCV) subtype prevalence in Chiang Mai, Thailand, and identification of novel subtypes of HCV major type 6.

Subtype analysis of hepatitis C viruses (HCVs) obtained from patients with chronic liver disease in Chiang Mai, Thailand, was performed. Of 46 HCV isolates, 13 (28%) were shown to belong to HCV subtype 3a (HCV-3a), 10 (22%) to belong to HCV-1a, 7 (15%) to belong to HCV-1b, 1 (2%) to belong to HCV-3b, and 1 (2%) to belong to a variant group, as determined from partial nucleotide sequences of the NS5B region of the viral genome. Analysis of 5' untranslated region sequences identified five other isolates (11%) of HCV type 1 and two other isolates (4%) of type 3. Detailed phylogenetic positions for the variant described above and those previously obtained from blood donors and drug addicts in Chiang Mai were determined by a six-parameter neighbor-joining method on the basis of core, E1, and NS5B region sequences. The results revealed that those sequence variants represent novel subtypes of HCV type 6. The HCV type 6 isolates appear to be antigenically different from isolates of HCV types 1 and 2, as determined by a serotyping method that utilizes recombinant peptides corresponding to a portion of the NS4 protein. The significance of subtype analysis around this area is discussed.

Adult↗

Quantitative analysis of serum neutralization of human immunodeficiency virus type 1 from subtypes A, B, C, D, E, F, and I: lack of direct correlation between neutralization serotypes and genetic subtypes and evidence for prevalent serum-dependent infectivity enhancement.

Human immunodeficiency virus type 1 (HIV-1) M group strains have been assigned to date to nine distinct genetic subtypes, designated A through I, according to phylogenetic analyses of nucleotide sequences of their env or gag genes. Whether there is any relationship between phylogenetic subtypes and the neutralization serotypes is not clear, yet defining the nature of any such relationship by mathematical means would be of major importance for the development of globally effective HIV-1 vaccines. We have therefore developed a quantitative method to analyze serum neutralization of HIV-1 isolates and to identify HIV-1 neutralization serotypes. This method involves calculations of the neutralization index, N(i), a newly defined parameter derived from plots generated from in vitro neutralization assays, calculations of pairwise serum-virus vector distances, and cluster analyses. We have applied this approach to analyze three independent neutralization matrices involving primary HIV-1 strains and sera from genetic subtypes A, B, C, D, E, F, and I. Detailed serum and HIV-1 isolate cluster analyses have shown that in general, the identified neutralization serotypes do not directly correlate with HIV-1 genetic subtypes. These results suggest that neutralization serotypes do not during natural HIV-1 infection are not governed by antibodies directed against simple epitopes within gp120 monomers. A significant proportion (28%) of 1,213 combinations of sera and HIV-1 isolates caused serum-dependent infectivity enhancement [negative N(i) values] rather than neutralization. We also noted that negative N(i) values tended to correlate better with certain HIV-1 isolates rather than with HIV-1-positive sera. Syncytium-inducing variants of HIV-1 were slightly more likely than non-syncytium-inducing variants to undergo serum-dependent infectivity enhancement, although the latter variants could clearly be susceptible to enhancement.

Amino Acids↗

Failure of a human immunodeficiency virus type 1 (HIV-1) subtype B-derived vaccine to prevent infection of chimpanzees by an HIV-1 subtype E strain.

Generation of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) must overcome problems associated with extensive genetic diversity. Although we previously reported vaccine-induced protection of chimpanzees against infection with an HIV-1 strain different from the one used to make the immunogens, both the HIV-1 vaccine and challenge strains were classified within subtype B. To determine whether the HIV-1-specific immunity elicited might also prevent infection by a strain of HIV-1 from a different clade, the same chimpanzees were given booster inoculations with the rgp160-MN/LAI (recombinant hybrid gp160 molecule) and V3-MN immunogens and then were challenged by intravenous inoculation of a comparable dose of a subtype E HIV-1 from the Central African Republic. Both animals became infected with the subtype E virus, indicating that intraclade vaccine-mediated protection does not predict interclade protection, at least in the context of intravenous challenge and the HIV-1 strains used. This study has important implications for planned phase III efficacy trials of similar vaccine preparations in Thailand where HIV-1 subtype B and E strains cocirculate.

AIDS Vaccines↗

Epidemiology of ischemic stroke subtypes according to TOAST criteria: incidence, recurrence, and long-term survival in ischemic stroke subtypes: a population-based study.

BACKGROUND AND PURPOSE: The purpose of this study was to determine the incidence, recurrence, and long-term survival rates of ischemic stroke subtypes by a mechanism-based classification scheme (Trial of ORG 10172 in Acute Stroke Treatment, or TOAST). METHODS: We identified all 583 residents of the city of Erlangen, Bavaria, Germany, with a first ischemic stroke between 1994 and 1998. Multiple overlapping sources of information were used to ensure completeness of case ascertainment. The cause of ischemic stroke was classified according to the TOAST criteria. Patients were followed up at 3 months and 1 and 2 years after stroke onset. RESULTS: The age-standardized incidence rates for the European population (per 100 000) regarding ischemic stroke subtypes were as follows: cardioembolism, 30.2 (95% CI 25.6 to 35.7); small-artery occlusion, 25.8 (95% CI 21.5 to 30.9); and large-artery atherosclerosis, 15.3 (95% CI 12 to 19.3). When age-adjusted to the European population, the incidence rate for large-artery atherosclerosis was more than twice as high for men than for women (23.6/100 000 versus 9.2/100 000). Two years after onset, patients in the small-artery occlusion subgroup were 3 times more likely to be alive than those with cardioembolism. Ischemic stroke subtype according to the TOAST criteria was a significant predictor for long-term survival, whereas subtype was not a significant predictor of long-term recurrence up to 2 years, both before and after adjustment for age and sex. CONCLUSIONS: Epidemiological observational studies that possess wide access to appropriate diagnostic technologies and apply standardized etiologic classifications provide a much better understanding of underlying risk factors for initial stroke, recurrence, and mortality.

Age Distribution↗