Integrin expression in oral hairy leukoplakia and normal tongue epithelium.

OBJECTIVE: To describe the expression of integrins in the epithelium of oral hairy leukoplakia (HL) and compare to that of normal lateral tongue epithelium. MATERIALS AND METHODS: Immunohistochemistry to identify integrins (alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1) was performed, using a standard biotin-streptavidin-peroxidase technique on five clinically and histologically confirmed frozen biopsy specimens of HL and five normal lateral tongue control tissues. RESULTS: Expression of integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 was seen both in HL epithelium and in normal control tissue. alpha 5 expression was not seen in HL or in control tissue epithelium. alpha 2 and alpha 3 were expressed mainly in the basal and suprabasal layers; alpha 6 expression was most intense on the basal surface of the basal cells, alpha v was expressed in the basal and suprabasal layers with more expression seen in the higher differentiated cell layers than the other integrins. beta 1 expression was seen in the basal and suprabasal layers only. No apparent difference between HL and normal oral mucosa was noted in the staining pattern of the various integrins. CONCLUSION: Integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 are expressed in HL and the expression pattern is not different from that of normal oral mucosa. alpha 5 is not expressed in HL or in normal oral epithelium.

Expression of Epstein-Barr virus BMRF-2 and BDLF-3 genes in hairy leukoplakia.

The high level of Epstein-Barr virus (EBV) replication found in hairy leukoplakia (HL) provides a unique opportunity to study EBV expression in the oral epithelium. Screening of a cDNA library from an HL biopsy revealed expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs demonstrated several nucleotide changes from the B95-8 sequence. In all six different HL strains studied, only one amino acid change was found in BMRF-2 relative to B95-8 and two amino acid changes were found in the BDLF-3 ORF. mRNA expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer; immunoelectron microscopy revealed that BMRF-2 was associated with the nuclear chromatin. BDLF-3 protein expression was observed in the perinuclear space and cytoplasm of the prickle cells. BDLF-3 has recently been identified as a virion-associated protein, but the functions of BMRF-2 and BDLF-3 have not been elucidated.

Epstein-Barr virus BMRF-2 and BDLF-3 expression in hairy leukoplakia.

Hairy leukoplakia (HL) is a lesion found on the side of the tongue of immunocompromised individuals, including those with human immunodeficiency virus (HIV) infection. The lesion has unique histopathologic features and is characterised by high-level Epstein-Barr virus (EBV) replication, multiple EBV strains, and extensive inter- and intra-strain recombination. Expression of EBV genes spanning the entire viral life cycle from latency-associated genes to late, replicative genes has been detected in the lesion. HL thus provides a unique opportunity to study EBV expression in oral epithelium, and to study expression of novel EBV genes. We therefore constructed a cDNA library from an HL biopsy and detected expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs revealed few amino acid changes from the B95-8 sequence. Expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer. BDLF-3 protein expression was observed in the peri-nuclear space and Golgi compartment. The function of these proteins is currently under investigation.

Sequence variation of latent membrane protein-1 of Epstein-Barr virus strains associated with hairy leukoplakia.

The Epstein-Barr virus (EBV) latent membrane protein (LMP)-1 is expressed in hairy leukoplakia (HL), but data on LMP-1 sequence variation of HL isolates are limited. Variation in the LMP-1 repeat region and presence of a 30-nt deletion were studied using DNA scrapings from 28 HL lesions. cDNAs from 3 different HL isolates were sequenced, 2 from lymphocyte cell lines (LCLs) generated using HL biopsy material. The deletion was found in 16 (57%) of 28 HL scraping, and multiple repeat region variants were seen in 13 scrapings (46%). HL LMP sequence were similar to those described in nasopharyngeal cancer and lymphoma tissues, including two motifs of four amino acid changes relative to B95-8 upstream and downstream of the repeat region, respectively. Generation of LCLs using HL biopsy material confirmed the ability of HL EBV strains to infect and transform lymphocytes.

Investigation of an outbreak of adult diarrhea rotavirus in China.

In 1987 an epidemic of diarrhea associated with adult diarrhea rotavirus (ADRV) occurred in Qinhuangdao City, China, affecting more than 200 persons and causing 2 deaths. The outbreak was introduced by a person returning from an epidemic area and was spread initially to his family members and subsequently to the entire community. Adults were at greater risk of diarrhea than children 0-4 y of age and, the duration of illness increased significantly with increasing age. ADRV was identified by ELISA and electron microscopy. The electropherotypes of all positive specimens were identical, consistent with the single point-source introduction of the virus. Seroconversion was detected in 6 of 7 ill persons with a blocking ELISA. Both asymptomatic infection and person-to-person spread identified in this epidemic suggest that current emphasis on preventing waterborne transmission may not control the introduction of ADRV into new areas. The predisposition of adults for more severe disease with ADRV is similar to the pattern observed with other enteric viruses such as the Norwalk agent and hepatitis A.

Simultaneous administration of rhesus rotavirus vaccine and oral poliovirus vaccine: immunogenicity and reactogenicity.

Rotavirus vaccine could be administered most efficiently if it were incorporated into routine childhood immunizations and did not interfere with the immune response to the other vaccines, principally oral poliovirus vaccine (OPV). We conducted a placebo-controlled randomized trial giving oral rhesus rotavirus vaccine (RRV) (strain MMU 18006) alone and together with a child's first dose of OPV and diphtheria-tetanus toxoids-pertussis to examine the possible interaction of these vaccines. A total of 102 infants 2 to 3 months of age were randomized into 3 groups to receive (1) RRV with OPV, (2) placebo with OPV and (3) RRV 2 weeks after OPV. All infants were given diphtheria-tetanus toxoids-pertussis. Serum samples were collected at the time of OPV immunization and 3 to 5 weeks later. Three to 5 weeks after OPV immunization 60% of infants had a 4-fold rise in neutralization titer to at least one of the three poliovirus serotypes. The rate of antibody response to poliovirus did not differ by RRV groups but a lower rate was correlated with a shorter interval (3 vs. 5 weeks) between OPV vaccination and antibody measurement. Fifty-six percent of infants had a 4-fold rise of IgA and 62% had a 4-fold rise of neutralizing antibody to RRV; this rise did not differ according to time of OPV immunization. RRV was not associated with side effects and may be safely given with OPV to infants 2 to 3 months of age.

Seroepidemiology of adult diarrhea rotavirus in China, 1977 to 1987.

In 1982, large outbreaks of diarrhea that were caused by group B adult diarrhea rotavirus (ADRV) occurred throughout the People's Republic of China. Until 1982, group B rotavirus had never been associated with disease in humans. To determine whether ADRV was a new virus introduced in 1982 or had been present before that time, we examined antibody titers of ADRV in gamma globulin (pooled immunoglobulin) pools that were prepared during 1977 to 1987 in four cities in the People's Republic of China (Shanghai, Lanzhou, Wuhan, and Chandu). ADRV antibodies were assayed by using a blocking enzyme-linked immunosorbent assay. Antibodies were present in most Chinese gamma globulins tested, including those collected in Shanghai before the 1982 epidemic, and absent from American reference pools. The highest titers of antibody to ADRV (3,200) were found in gamma globulins collected in 1983 in Shanghai just after the epidemic, and these were fourfold higher than titers present in the preceding years. The quality of the gamma globulins stored for up to 12 years was tested by measuring levels of immunoglobulin G to group A rotavirus; these were equally high in gamma globulin pools prepared in the United States and in all samples from the People's Republic of China. Serum samples from patients from an outbreak of ADRV had elevated titers to ADRV 3 and 16 months after the onset of symptoms. These findings, as well as other epidemiologic findings on ADRV, suggest that the organism is an important and continuing cause of diarrhea in the People's Republic of China, was present before the first epidemic in 1982, and represents a risk to surrounding populations in Asia.

Group C rotavirus infections in patients with diarrhea in Thailand, Nepal, and England.

Atypical rotavirus obtained from fecal specimens of six patients with diarrhea from Thailand, Nepal, and England were characterized by using polyacrylamide gel electrophoresis and immune electron microscopy. The electropherotypes were characteristic of the porcine reference group C rotavirus strain but demonstrated considerable strain-to-strain variation. Human convalescent group C sera had a high titer (1:320) when tested against the human isolates and a low titer (1:40) when tested against a porcine reference strain (Cowden). When porcine antiserum (Cowden) was tested against the human isolates, the titers ranged from 1:40 to 1:320, indicating significant antigenic diversity between strains. Group C rotavirus appears to have a worldwide distribution as an agent associated with diarrhea in children and adults.

Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system.

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SA11 gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 micrograms/10(6) cells) when Spodoptera frugiperda cells were infected with baculovirus recombinants containing SA11 gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intracellularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.

Two glycoproteins are produced from the rotavirus neutralization gene.

The major neutralization antigen of rotaviruses is an outer capsid glycoprotein, VP7, with an apparent molecular weight of 38,000 (38K). The simian rotavirus SA11 genome segment 9, which codes for VP7, contains two in-phase initiation codons, each of which is followed by a sequence that codes for a region of hydrophobic amino acids. We have determined that this gene is functionally bicistronic by analyzing the synthesis of VP7 in SA11-infected cells and in cell-free translation systems programmed with hybrid-selected, segment 9 specific mRNA and dog pancreatic microsomes. The translation of hybrid-selected gene 9 mRNA in wheat germ extracts yielded two distinct polypeptides of molecular weights 37K and 35.3K. In vitro translation in the presence of microsomes yielded one diffuse band of 38K that was converted into the 37K and 35.3K precursor bands by digestion with endoglycosidase H. Studies with a variant of SA11 that lacks the glycosylation site in VP7 confirmed these precursor-product relationships and extended them by indicating that the glycoprotein produced by translation from the first AUG contained a cleaved signal sequence whereas the glycoprotein produced by translation from the second AUG contained an uncleaved signal sequence. Immunoprecipitation with monospecific anti-VP7 serum and improved gel electrophoresis conditions allowed us to show that both VP7s were expressed at similar times in infected cells and both were found in purified virus particles of several different rotavirus strains. Whether these two VP7 glycoproteins are functionally distinct remains to be determined.

ST:LT:CFA/II plasmids in enterotoxigenic Escherichia coli belonging to serogroups O6, O8, O80, O85, and O139.

Colonization factor antigen II-producing enterotoxigenic Escherichia coli serotypes possessed at least one large plasmid. Loss of colonization factor antigen II correlated with either complete or partial loss of the large plasmid. Complete loss of the plasmid always correlated with complete loss of enterotoxin production. Three of five deletion events also correlated with the loss of toxin production.

CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate.

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.

[Diarrhea outbreak and bacterial resistance to ampicillin in newborns]. / Brote de diarrea y resistencia bacteriana a la ampicilina en neonatos.

Twenty-eight healthy neonates from the San Juan de Dios Hospital were studied to determine the pattern of antibiotic resistance of indigenous intestinal bacteria. Sixty-eight per cent of infants had enterobacteriaceae resistant to several wide-spectrum antibiotics, including ampicillin; 28 per cent of the cultures had plasmid-mediated ampicillin resistance. In the course of the study, an outbreak of 10 acute cases of diarrhea occurred, not associated to any of the commonly known agents, including the rotaviruses. Enterobacteriaceae multiple resistant were isolated from all cases; ampicillin-resistant strains were found in all; those resistances were mediated by transmissible plasmids. Several sites of the Neonatology Ward were sampled and two yielded E. coli with resistance to 8 drugs transmissible in vitro. The outbreak was controlled after strict hygienic measures were established in the ward. A following sample showed a decrease in indigenous antibiotic-resistant strains, especially E. coli; however, plasmid mediated resistant Klebsiella was still isolated several months later. The need to regulate the use of antibiotics; to educate the medical personnel and the public in general and to modify the hospital norms and regulations are discussed in the paper.

[Infectious agents in diarrhea of hospitalized children in Costa Rica]. / Agentes infecciosos en la diarrea del niño hospitalizado en Costa Rica.

Shigella, toxigenic Escherichia coli (stable toxin) and rotaviruses were frequently found among 50 children hospitalized with diarrhea studied during one year. These agents were less common among 45 controls without diarrhea, of comparable age and from the same wards as the cases reported. There was a greater frequency of respiratory symptoms in the diarrhea associated with rotaviruses. These were characterized by frequent bowel movements and vomiting and often fever. The bacterial diarrheas showed, in general, a more severe clinical picture than the viral ones. The rotaviruses had a low endemic level during April-October, but their prevalence increased in December and January; in such months these viruses were found in more than 50 per cent of the diarrheal cases.

Characterization of an R-plasmid associated with ampicillin resistance in Shigella dysenteriae type 1 isolated from epidemics.

Ampicillin-resistant strains of Shigella dysenteriae type 1 isolated in epidemics in Mexico, Central America, and Bangla Desh were examined for the presence of plasmid deoxyribonucleic acid (DNA) by gel electrophoresis. All strains contained a heterogeneous population of plasmids. Transfer experiments to Escherichia coli K-12 indicated that the ampicillin resistance determinant (Ap(r)) was located on a 5.5-megadalton (Mdal) plasmid identical in all Shiga strains examined, as judged by DNA hybridization and by its molecular properties. This 5.5-Mdal plasmid contained the ampicillin transposon (TnA) sequences. There was not a high degree of homology between the Shiga Ap(r) plasmid DNA and DNA obtained from Ap(r)Salmonella typhi strains isolated from typhoid epidemics in Mexico, previous to the dysentery outbreaks. Although low, the degree of reassociation observed indicated that probably part of the TnA sequence was present in S. typhi DNA. The DNA hybridization experiments showed, in addition, that there was a high degree of homology among Ap(r) plasmids isolated from different enterobacteria, and this identity was confirmed by restriction endonuclease activity. These results together with their similarities in molecular and replicative properties indicate that the Ap(r) plasmids, as was suggested for the Sm(r) Su(r) plasmids, possibly evolved once and then epidemiologically spread in the Enterobacteriaceae.