Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus.

Most human immunodeficiency virus (HIV) transmissions in women occur through the cervicovaginal mucosa, which is coated by a bacterial biofilm including Lactobacillus. This commensal bacterium has a role in maintaining a healthy mucosa and can be genetically engineered to produce antiviral peptides. Here, we report a 63% reduction in transmission of a chimeric simian/HIV (SHIV(SF162P3)) after repeated vaginal challenges of macaques treated with Lactobacillus jensenii expressing the HIV-1 entry inhibitor cyanovirin-N. Furthermore, peak viral loads in colonized macaques with breakthrough infection were reduced sixfold. Colonization and prolonged antiviral protein secretion by the genetically engineered lactobacilli did not cause any increase in proinflammatory markers. These findings lay the foundation for an accessible and durable approach to reduce heterosexual transmission of HIV in women, which is coitally independent, inexpensive, and enhances the natural protective effects of the vaginal microflora.

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.

Regulation of Epstein-Barr virus promoters in oral epithelial cells and lymphocytes.

Hairy leukoplakia (HL) is a proliferative lesion of the tongue that supports abundant Epstein-Barr virus (EBV) replication. Previous work showed high-level expression of the EBV BMRF2 gene in HL. To characterize the regulation of BMRF2 expression in HL, we mapped the 5' ends of the BMRF1 and BMRF2 transcripts and showed that BMRF2 is expressed from a novel internal promoter within the BMRF1 coding region. Mechanisms of BMRF2 regulation were compared in oral epithelial cells and B lymphocytes, as were those of BMRF1 and BDLF3, early and late EBV transcripts, respectively, that are also known to be expressed in HL. Basal activity of the putative BMRF2 promoter was 10-fold higher in HSC-3 epithelial cells than in B lymphocytes. The BMRF2 and the BDLF3 promoters were responsive to induction by phorbol ester, but unlike the BMRF1 promoter, they were not responsive to BZLF1 transactivation. By mutational analysis, the major activity of the BMRF2 promoter mapped to a 50-bp region, which includes a TATA-like element and a GC box. The BMRF2 promoter may be regulated differentially from the BMRF1 promoter and more closely resembles that of BDLF3. This novel BMRF2 promoter likely belongs to a class of viral promoters that is more responsive to mechanisms known to induce epithelial cell differentiation, consistent with its high level of expression in HL.

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.

Apoptosis-associated markers in oral lichen planus.

Hypothesizing that loss of basal cells in oral lichen planus is due to apoptosis, we evaluated LP specimens for apoptosis-regulating proteins [positive regulators Bcl-xS, Bax, Fas/Fas-ligand, p53, and negative regulators (anti-apoptotic) Bcl-2, Bcl-xL and compared results with reactions in normal mucosa and chronically inflamed gingiva. Also, sections were evaluated with an in situ TUNEL assay that identifies apoptotic DNA fragments. Basal keratinocytes in normal buccal mucosa, nonspecific gingivitis, and LP were negative for Bcl-2 protein, but melanocytes and lymphoid cells were positive. Keratinocyte staining for Bcl-x was negative to weak in normal buccal mucosa and gingivitis, and moderate in LP. Keratinocytes (especially upper prickle cells) in all tissues stained similarly for Bax at weak to moderate levels. Also, no differences in Fas and Fas-ligand staining were evident. Prominent p53-positive staining was seen in all LP biopsies (10-100% of basal keratinocytes) but not in normal buccal mucosa and gingivitis. Few basal keratinocytes in 5/10 LP cases exhibited a positive in situ signal for DNA fragment-associated apoptosis. That the Bcl-2 family of proteins and Fas/Fas-ligand were detected in normal and diseased tissues, and were occasionally expressed differently in oral LP, supports the notion that apoptosis is a potential mechanism of keratinocyte loss, especially in LP. The pattern of p53 staining in oral LP suggests over-expression of wild-type protein; a phenomenon that would arrest the cell cycle to allow repair of damaged DNA, or trigger apoptosis. While immunohistochemical evidence for apoptosis-associated basal keratinocyte death in LP was slight, it appeared that it may be p53 protein, and possibly Bcl-x associated.

Structure and function of the murine cytomegalovirus sgg1 gene: a determinant of viral growth in salivary gland acinar cells.

The salivary gland has long been recognized as an important target organ for cytomegalovirus replication in the infected host. A viral gene, denoted sgg1, plays an important role for replication in the salivary gland even though it is dispensable for growth in other organs or in cultured cells. The nucleotide sequence of this gene and of cDNA clones representing two spliced transcripts (1.5 and 1.8 kb in size) has been determined. The more abundant 1.5-kb transcript contains a 312-amino-acid (aa) open reading frame (ORF) and encodes the corresponding 37-kDa protein (Sgg1) when expressed in transfected COS-7 cells. The 1.8-kb transcript initiates upstream of the 1.5-kb transcript and contains a 108-aa ORF in addition to the 312-aa ORF. This longer cDNA also encodes the 37-kDa protein Sgg1, although at lower abundance than the 1.5-kb cDNA. Sgg1 localizes to the cytoplasm of COS-7 cells, which is consistent with the predicted structural characteristics of the 312-aa ORF: a type 1 integral membrane protein. During viral infection, expression of both sgg1 transcripts is highest at early times (8 to 12 h) after infection; only the 1.5-kb transcript is present, at low levels, late in infection. A recombinant virus, RM868, carrying a lacZ-gpt insertion within sgg1, fails to express Sgg1 protein and exhibits reduced growth in the salivary gland. RM868 retains the capacity to disseminate in the infected mouse and to enter serous acinar cells, although it fails to replicate efficiently in this cell type. These results suggest that sgg1 is critical for high levels of viral replication in the salivary gland.

Cytomegalovirus determinant of replication in salivary glands.

Murine cytomegalovirus carrying a deletion mutation disrupting the expression of a gene dispensable for growth in cultured cells was found to disseminate poorly in the mouse. The mutation resulted in a dramatic decrease in the expression of a 1.5-kb major and a 1.8-kb minor beta transcript from a region adjacent to the ie2 gene in the viral genome. Nucleotide sequence determination indicated that 323 bp, including a predicted polyadenylation signal, was deleted from this beta gene. In cultured cells, the plaque morphology and growth characteristics of the mutant were similar to those of parental or rescued wild-type viruses. Following intraperitoneal inoculation of BALB/c mice, growth of the mutant in the salivary gland was dramatically reduced 10,000-fold, while growth in the liver and spleen was not dramatically affected. The beta gene was thus denoted sgg1 (salivary gland growth gene 1). Neither intranasal infection nor direct inoculation into the salivary glands completely overcame the restriction of growth in this organ, suggesting that the sgg1 gene encoded a determinant of tissue tropism. To investigate the impact of the sgg1 mutation on virus dissemination via the blood, the virus titer in peripheral blood leukocytes was determined. No difference was found between the sgg1 mutant and rescued wild-type virus. Thus, murine cytomegalovirus sgg1 gene products appear to be involved in entry or replication of virus in salivary gland cells.

Septicemia and septic arthritis caused by Streptococcus pneumoniae in a cat: possible transmission from a child.

Streptococcus pneumoniae serotype 23F was isolated from the blood and synovial fluid of an acutely ill, 15-year-old castrated male cat and from the nasopharynx of that regularly played with it, an infant child. Information presented supports the hypothesis that the infection was transmitted from child to cat.