Improved amplification of genital human papillomaviruses

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.     

Cytogenetic and biological characterization of two new human plasma cell lines

Two new human plasma cell lines designated as ACB-885 and ACB-1085 have been established from a 39-year-old patient with multiple myeloma. These cell lines have definitive plasma cell features by morphologic examination, and essentially all of the cells are positive for cytoplasmic IgG kappa immunoglobulin. These cells are negative for standard T-cell surface markers and mature B-cell markers, such as B1, B2, and HLA-DR, but are strongly positive for the antigen defined by OKT-10. The cells are negative for Epstein-Barr virus. The cell lines have a doubling time of 30-35 hours and a growth fraction approaching 100%. Cytogenetic analysis showed a 2n chromosome number of 45-46 with very similar karyotypic abnormalities in both the plasma cell lines and the original tumor material. One of the chromosomes in each of the pairs of chromosomes number #1, #2, #6, #7, #8, #10, #12, #13, and #22 were consistently missing. These were replaced by eight marker chromosomes that resulted from chromosomal rearrangements involving mainly these missing chromosomes. Almost all of the breakpoints occurring in the marker chromosomes were identified, and eight of these breakpoints have been reported in other studies of myeloma plasma cells. Homogeneously staining regions were observed in two marker chromosomes suggesting gene amplification in these chromosomal regions.     

Typing and molecular characterization of human papillomaviruses in genital warts from South African women.

Condyloma acuminata from 12 women were examined for the presence of human papillomavirus DNA. Six of the women had HPV 6a, two had HPV 11a, one had a new HPV 6 subtype, and one a new HPV 11 subtype. The new HPV 6 subtype could be distinguished from other HPV 6 subtypes on the basis of Pst I fragments as well as Hind II fragments. The new HPV 11 subtype had a different Hpa II restriction endonuclease pattern. Restriction maps of both new subtypes were constructed. Two of the biopsies did not contain detectable HPV DNA when probed with HPV 6, HPV 11, and HPV 16. Biopsies were taken from normal tissue 1 cm away from the condyloma in 11 of the patients. Only one of these normal tissue biopsies was positive for HPV by Southern blot hybridization.     

Infection of the oral mucosa with defined types of human papillomaviruses

Biopsies from 9 different oral papillomatous proliferations were analysed for human papilloma viral (HPV) sequences of types 1 to 19 and 21 to 26 by Southern blot analysis with 32p-labelled cellular DNA. HPV sequences were detected in 7 out of 9 biopsies obtained from individual patients. Of three cases with the clinical diagnosis focal hyperplasia Heck, two contained HPV-6 related sequences and one HPV 13. In addition, one tongue base papilloma contained HPV 11. A papilloma of the palate revealed HPV 11 sequences. HPV 6 could be demonstrated twice in the ramaining 4 oral papillomatous proliferations. Two biopsies remained negative. The data shows that HPV DNA can be regularly demonstrated in oral papillomas.     

The biological properties of E6 and E7 oncoproteins from human papillomaviruses

More than 100 different human papillomavirus (HPV) types have been isolated so far, and they can be sub-grouped in cutaneous or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts. A sub-group of human mucosal HPVs, referred to as high-risk HPV types, is responsible for approximately 5% of all human cancers, which represents one-third of all the tumours induced by viruses. Epidemiological and biological studies have shown that HPV16 is the most oncogenic type within the high-risk group. Emerging lines of evidence suggest that, in addition to the high-risk mucosal HPV types, certain cutaneous HPVs are involved in skin cancer. HPV-associated cancers are intimately linked to HPV persistence and the accumulation of chromosomal rearrangements. The products of the early genes, E6 and E7, of the high-risk mucosal HPV types play a key role in both events. Indeed, these proteins have developed a number of strategies to evade host immuno-surveillance allowing viral persistence, and to alter cell cycle and apoptosis control, facilitating the accumulation of DNA damage/mutations. Often, the two oncoproteins target the same cellular pathways with different mechanisms, showing a strong synergism in promoting cellular transformation and neutralizing the immune response. Here, we review most of the findings on the biological properties and molecular mechanisms of the oncoproteins E6 and E7 from mucosal and cutaneous HPV types.     

Physicochemical properties of two distinct types of virus-like particles from Helminthosporium victoriae

Two serologically distinct types of virus-like particles (VLPs) were isolated from Helminthosporium victoriae. The first, which was isolated from three normal and two diseased isolates, sedimented at a rate of 190 S when centrifuged in linear-log sucrose density gradients. The second, which was found only in the two diseased isolates, had a sedimentation coefficient of 145 S. No VLPs were detected in two other normal isolates of the fungus. The 190 and 145 S VLPs both were polyhedral and 35 to 40 nm in diameter and had typical nucleoprotein absorbancy spectra. The 190 S type of VLP was electrophoretically distinct from the 145 S VLP when electrophoresed on either polyacrylamide gels or cellulose acetate strips. Equilibrium density gradient centrifugation in cesium chloride revealed a single density component for the 190 S VLPs with a buoyant density of 1.4325 g/cm³ The 145 S VLPs could be resolved into two to four components with buoyant densities of 1.3813 to 1.4138 g/cm³. SDS polyacrylamide gel electrophoresis of the 190 S VLPs revealed two major proteins of molecular weight 83,000 and 88,000 present in equimolar amounts. The 145 S type of VLP had three major proteins with molecular weights of 92,000, 97,000, and 106,000; these were also present in equimolar amounts. Both the 145 and 190 S VLPs contained double-stranded RNA (dsRNA). The 190 S type of VLP had a single species of dsRNA with a molecular weight of 3.0 × 10⁶. There were four classes of dsRNA associated with the 145 S VLPs, and their molecular weights were 2.4, 2.2, 2.1, and 2.0 × 10⁶.     

Prevalence of high- and low-risk oncogenic human papillomaviruses in patients with external genital pathology

The paper presents the results of examining 49 patients with genital papillomas, vulvar and vaginal leukoplakia, dysplasia, and cancer. The findings may suggest that human papillomavirus plays an important role in the development of vulvar and vaginal lesions and reconsider the importance of high- and low-risk oncogenic genotypes in the development of benign neoplasms, precancerous conditions, and malignant tumors of the vulva and vagina.     

Minor capsid protein of human genital papillomaviruses contains subdominant, cross-neutralizing epitopes

Vaccination with virus-like particles (VLP), comprising both L1 and L2 of human papillomavirus (HPV) genital types 6, 16, and 18, induces predominantly type-specific neutralizing antibodies. L2 polypeptide vaccines protect animals against experimental challenge with homologous papillomavirus and cross-reactive epitopes are present in HPV L2. To assess L2-specific cross-neutralization of HPV genotypes, sheep were immunized with purified, bacterially expressed HPV6, 16, or 18 L2. In addition to neutralizing the homologous HPV type in vitro, antisera to each HPV L2 also cross-neutralized both heterologous HPV types. This suggests that unlike VLP-based prophylactic HPV vaccines, an L2 polypeptide vaccine may provide broad-spectrum protection.     

Isolation and characterization of two distinct types of HVJ (Sendai virus) spikes

Solubilization of the envelope of HVJ (Sendai virus) with alkali-Tween 20 and subsequent fractionation by sucrose density electrofocusing in the presence of Tween 20 resulted in the separation of two biologically distinct glycoproteins: one protein banded at pH 4.9 without hemagglutinin and neuraminidase activities (HANA^?) and the other at pH 6.5 with both hemagglutinin and neuraminidase activities (HANA⁺). Morphologically, both fractions consisted of dumbbell-shaped spikes, with HANA^? spikes exhibiting more definite contour in or shape of knob and shaft than HANA⁺ spikes. Serologically, HANA^? and HANA⁺ spikes were distinct from each other in micro-Ouchterlony test. Moreover, SDS-polyacrylamide gel electrophoresis revealed that HANA^? spikes were composed of two species of glycoproteins, VP4, (MW, 51,000) and SGP (MW, 15,000), while HANA⁺ spikes contained single glycoprotein, VP2 (MW, 67,000).Radioactivity of HANA^? spike fraction did not adsorb onto chicken erythrocyte ghosts, whereas radioactivity, neuraminidase, and hemagglutinin activity of HANA⁺ spike fraction adsorbed onto the ghosts. No hemolytic activity was detected in either HANA^? or HANA⁺ spike fractions.     

Isolation and characterization of two distinct human rotavirus strains with G6 specificity.

Two new human rotavirus (HRV) strains, PA151 and PA169, with subgroup I specificity and a long RNA pattern, yet with a serotype G (VP7) specificity different from those of any of the six well-established HRV serotypes (G1 to G4, G8, and G9), were isolated 3 months apart from two children with acute gastroenteritis in Sicily, southern Italy, in the winter season of 1987 and 1988. The HRV isolates were adapted to growth in cell cultures and were then characterized by neutralization and RNA-RNA (Northern blot) hybridization. Cross-neutralization studies with type-specific immune sera to RV serotypes 1 to 10 showed the antigenic relatedness of the two strains with serotype 6 bovine strains UK and NCDV. Monoclonal antibodies to VP7 of UK were able to recognize UK and NCDV strains as well as both HRV isolates. Cross-hybridization studies showed a genetic relatedness of PA151 and PA169 to bovine strains for all genes except gene 4. Gene 4 of PA151 appeared to be genetically related to that of AU228 (a human strain of subgroup I and with serotype G3 specificity that belongs to a feline genogroup), whereas gene 4 of PA169 appeared to be unique, yet it was related to gene 4 of two recently reported subgroup I HRV strains, one (PA710) with serotype G3 specificity and the other (HAL1271) with serotype G8 specificity. The new HRV strains must be taken into consideration when deciding strategies for the development of an effective RV vaccine.     

Structural characterization of CD6: properties of two distinct epitopes involved in T cell activation

Studies from our laboratory have shown that anti-T12, a mAb which recognizes CD6, is a macrophage-dependent mitogen for human T cells and can augment T cell autoreactivity in vitro. To obtain additional information regarding the potential biological role of CD6 we sought to further characterize its biochemical properties. The CD6 molecule on 125I-surface-labeled T cells and by Western blot analysis was a monomer of mol. wt 130,000 under reducing conditions and mol. wt 117,000 under non-reducing conditions, suggesting the presence of intrachain disulfide bonds. The polypeptide contains a protease sensitive site. In activated T cells, the protein was serine phosphorylated. Analysis of biosynthetically labeled CD6 in the presence of tunicamycin revealed a reduction in mol. wt from 130,000 to 100,000, indicating that the polypeptide is extensively N-glycosylated. The mAb, anti-2H1, had been shown to activate T cells in combination with PMA or the anti-T11(3) mAb but, unlike anti-T12, not in the presence of macrophages alone. The present studies demonstrate by sequential immunoprecipitation that these two mAbs recognize the same polypeptide. However, Western blot analysis and indirect immunofluorescence cross-blocking studies demonstrate that the two mAbs recognize different determinants on CD6. Anti-T12 recognizes an epitope that is present only under non-reducing/non-denaturing conditions, while anti-2H1 recognizes an epitope that is also preserved under reducing/denaturing conditions. A direct comparison of activation properties of the mAbs confirmed that anti-T12 was optimally mitogenic in the presence of macrophages but not PMA, while, conversely, anti-2H1 was optimally mitogenic in combination with PMA but not macrophages, suggesting that the differences in epitope specificity may account for the distinct activation properties of each mAb. Taken together, the structural and functional data strongly suggest that the CD6 membrane glycoprotein may function as a physiologically important receptor structure on human T lymphocytes.     

Molecular cloning and characterization of the DNA of two papillomaviruses from monkeys

Benign and malignant lesions from monkeys were analyzed for the presence of papillomavirus (PV) DNA. By hybridization with different PV DNA probes under conditions of lowered stringency, two tumors were found to contain PV-specific DNA sequences: (1) a cutaneous papilloma from a Colobus monkey; and, (2) a lymph node metastasis of a squamous cell carcinoma of the penis from a Rhesus monkey. Analysis of the DNA of the papilloma from the Colobus monkey indicated the presence of extrachromosomal DNA whereas analysis of DNA from the Rhesus tumor suggested the presence of integrated viral DNA. The physical size (7.8 and 8.1 kb), colinear alignment to HPV-5, and cross-hybridization with other PV types under low stringency indicate that the two genomic DNA clones represent new PV types that have been tentatively designated as Rhesus papillomavirus type 1 (RhPV 1) and Colobus guereza papillomavirus type 2 (CgPV 2). A putative viral-host DNA junction fragment was also isolated from the Rhesus genomic library. Nucleotide sequences very closely related to RhPV 1 were observed by in situ hybridization in a laryngeal carcinoma from the Colobus guereza monkey. This report communicates the finding of novel papillomaviruses associated with a benign cutaneous tumor and genital and laryngeal malignancies in non-human primates which may have significance as a putative system for the study of papillomavirus-induced genital and laryngeal malignancies in humans.     

O-phenylenediamine oxidation by phorbol myristate acetate-stimulated human polymorphonuclear leukocytes: characterization of two distinct oxidative mechanisms

O-Phenylenediamine (OPD) oxidation has been extensively utilized for the measurement of peroxidase-mediated catabolism of hydrogen peroxide. However, until now this system has not been evaluated for the measurement of hydrogen peroxide produced upon activation of the hexose monophosphate shunt (HMPS) in polymorphonuclear leukocytes (PMNs). OPD oxidation by phorbol myristate acetate (PMA)-stimulated PMNs was mediated by both hydrogen peroxide and superoxide produced by the activation of the HMPS. Furthermore, OPD oxidation by an oxidative mechanism independent of the HMPS was observed by the PMA stimulation of PMNs obtained from patients with chronic granulomatous disease (CGD). This HMPS-independent OPD oxidation was inhibited by superoxide dismutase or 1 mM potassium cyanide (KCN). Superoxide dismutase, catalase, or 1 mM potassium cyanide inhibited 50% OPD oxidation obtained with PMA-stimulated normal PMNs. PMA treatment of purified human myeloperoxidase (MPO) produced OPD oxidation which is inhibited by superoxide dismutase or 1 mM KCN. These data indicate that OPD oxidation observed with CGD PMNs is mediated by a PMA-induced oxidase activity of myeloperoxidase. OPD oxidation in the presence of 1 mM KCN is a method comparable in sensitivity with ferricytochrome c reduction for the evaluation of HMPS activity. Furthermore, the OPD assay can measure myeloperoxidase oxidase activity in PMA-stimulated PMNs.     

人乳头瘤病毒基因分型液态芯片的构建

目的:通过构建人乳头瘤病毒基因分型的液态芯片,建立高通量、灵敏和特异的HPV型别检测系统.方法:在LuminexTM平台上,采用国际公认的标准HPV质粒建立13种基因分型HPV芯片,选择引物及探针,将探针有效的偶联到微球上.结果:用LuminexTM平台建立了13型HPV芯片,对单一或两种标准质粒混合的样品均可准确分型.对分型检测的阳性标本进行基因测序,经美国NCBI数据库BLAST比对符合其检测型别.结论:利用LuminexTM平台构建了13种HPV 型别液相芯片的诊断体系,具有高通量、快速准确等特点,适用于大规模临床样本的HPV基因分型的诊断.     

Isolation and characterization of six new genome types of human adenovirus types 1 and 2.

Several of the 41 types of human adenovirus have been divided into genome types based on aberrant restriction endonuclease digestion patterns of viral DNA. In the process of screening a large number of clinical adenovirus isolates by restriction endonuclease digestion of viral DNA, we have identified nine isolates from eight patients which were identified by neutralization as adenovirus type 1 or 2 but had aberrant cleavage patterns. Cleavage sites for the enzymes SmaI, EcoRI, HindIII, KpnI, and HpaI were mapped. The variants could be placed in six distinct groups based on cleavage patterns. The designation genome types 1a, 1b, 1c, 2b, 2c, and 2d are proposed for these isolates.     

Low immunoglobulin E flags two distinct types of immune dysregulation

During the last two decades, hyper‐immunoglobulin (Ig)E syndromes have been characterized clinically and molecularly in patients with genetically determined primary immunodeficiencies. However, the detection of low IgE levels, defined here as below detection limit in the routine clinical immunology laboratory, has received little attention. We analysed the association of serum IgA, IgM and IgG levels (including IgG subclasses) with low, normal or high serum IgE levels in patients evaluated in a single‐centre out‐patient immunodeficiency and allergy clinic. The correlation of serum IgE levels with IgG subclasses depended on the clinical phenotype. In patients with immunodeficiencies, IgE correlated with IgG2 and IgG4 but not with IgG3. In contrast, in patients referred for signs of allergy, IgE correlated with IgG3 but not with IgG2. A low IgE result was associated with low IgG3 and IgG4 in allergy referrals, while immunodeficiency referrals with a low IgE result had significantly lower IgG1, IgG2 and IgG4 levels. Hierarchical clustering of non‐IgE immunoglobulin profiles (IgM, IgA, IgG, IgG1–4) validated that non‐IgE immunoglobulin levels predict the clinic referral, i.e. phenotype, of low‐IgE patients. These results suggesto guide the clinical management of patients with low serum IgE levels.     

Blood monocytes consist of two principal subsets with distinct migratory properties

Peripheral blood monocytes are a heterogeneous population of circulating leukocytes. Using a murine adoptive transfer system to probe monocyte homing and differentiation in vivo, we identified two functional subsets among murine blood monocytes: a short-lived CX(3)CR1(lo)CCR2(+)Gr1(+) subset that is actively recruited to inflamed tissues and a CX(3)CR1(hi)CCR2(-)Gr1(-) subset characterized by CX(3)CR1-dependent recruitment to noninflamed tissues. Both subsets have the potential to differentiate into dendritic cells in vivo. The level of CX(3)CR1 expression also defines the two major human monocyte subsets, the CD14(+)CD16(-) and CD14(lo)CD16(+) monocytes, which share phenotype and homing potential with the mouse subsets. These findings raise the potential for novel therapeutic strategies in inflammatory diseases.     

A scanning electron microscopic study of the human ovarian surface epithelium: characterization of two cell types

The surface of the ovary has two types of epithelial cells. We have called these A and B cells and they are found in their own respective zones (A and B). To assess the scanning electron microscopic features of these cell types, 65 ovarian samples were collected from biopsies taken from 35 women with normal ovaries. Biopsies included developing follicles, corpora lutea and ovarian capsules. Type A cells were cuboidal and sometimes tall, with a mean diameter of 6.49 microns, and a mean density of microvilli of 6.48/microns 2. Type B cells, on the other hand, were flat squamous cells with broader and flat apices with mean diameters and microvillus density of 11.71 microns and 3.88/microns 2 respectively. The A and B zones were common to all surfaces including the distending follicle. Type A cells overlying the distended surface of a follicle had a mean diameter of 7.03 microns compared to a mean of 6.05 microns for the capsular surface. Type B cell diameters and the microvillus density of both types were more variable and did not differ significantly over any of the surfaces. We suggest that previous human studies which identified flattening of cells over the distending follicle were probably observing B cells. The relationship of the B zones to papillae and surface bridges on the ovarian surface, and the association of these with ovulation sites, suggests that B cells are probably metaplastic cells derived in response to chronic surface injury with ovulation.     

Conservation of two distinct types of 100S ribosome in bacteria

In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the ‘hibernating ribosome’. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF.