The gut and oral microbiome in HIV disease: a workshop report.

Recent years have seen a massive expansion in our understanding of how we interact with our microbial colonists. The development of new, rapid sequencing techniques such as pyrosequencing and other next-generation sequencing systems have enabled us to begin to characterise the constituents of our diverse microbial communities, revealing the astonishing genetic richness that is our microbiome. Despite this, our ignorance of how these communities change over the course of an HIV infection is profound. Whilst some steps have been made to characterise the HIV microbiome at selected sites, these reports are still limited and much remains to be done. It has become apparent, however, that host-microbiota interactions are perturbed during HIV infections, with microbial translocation of potential pathogens linked to a variety of different HIV complications, including more rapid progression of disease. The use of probiotics and prebiotics has been investigated as treatments to alleviate symptoms for a variety of conditions, and is now being proposed for the treatment of symptoms associated with HIV. However, this is a new area of investigations and many questions remain unanswered. What we know about both of these topics is a drop in the ocean compared with what we need to know. In this article, we report on a workshop where these two major under-investigated research areas were presented, and future directions explored and discussed.

Innate immunity in HIV-1 infection: epithelial and non-specific host factors of mucosal immunity- a workshop report.

The interplay between HIV-1 and epithelial cells represents a critical aspect in mucosal HIV-1 transmission. Epithelial cells lining the oral cavity cover subepithelial tissues, which contain virus-susceptible host cells including CD4(+) T lymphocytes, monocytes/macrophages, and dendritic cells. Oral epithelia are among the sites of first exposure to both cell-free and cell-associated virus HIV-1 through breast-feeding and oral-genital contact. However, oral mucosa is considered to be naturally resistant to HIV-1 transmission. Oral epithelial cells have been shown to play a crucial role in innate host defense. Nevertheless, it is not clear to what degree these local innate immune factors contribute to HIV-1 resistance of the oral mucosa. This review paper addressed the following issues that were discussed at the 7th World Workshop on Oral Health and Disease in AIDS held in Hyderabad, India, during November 6-9, 2014: (i) What is the fate of HIV-1 after interactions with oral epithelial cells?; (ii) What are the keratinocyte and other anti-HIV effector oral factors, and how do they contribute to mucosal protection?; (iii) How can HIV-1 interactions with oral epithelium affect activation and populations of local immune cells?; (iv) How can HIV-1 interactions alter functions of oral epithelial cells?

Modulation of the orodigestive tract microbiome in HIV-infected patients.

More than 37 million people are living with human immunodeficiency virus 1 (HIV), and more people than ever received lifesaving antiretroviral therapy worldwide. HIV-1 infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the GI microbiome and its association with host immune activation. The data indicated that the microbiome was different in HIV-positive and HIV-negative individuals. The initial sequence analysis of saliva indicated that there were major differences in the phyla of Bacteroidetes, Firmicutes, Proteobacteria, and TM7. Phylum Tenericutes was only seen in HIV-positive saliva. At the family level, we identified differences in Streptococcacea, Prevotellaceae, Porphyromonadaceae, and Neisseriaceae, whereas data from various sites in GI tract indicated that Prevotella melaninigencia, Fusobacterium necrophorum, Burkholderia, Bradyrhizobium, Ralstonia, and Eubacterium biforme were predominant but differentially present at various sites. Furthermore, there was a decrease in seven proteins associated with the alternative complement pathway and an increase in 6 proteins associated with the lectin and classical complement pathways. The correlation with a shift in complement pathways suggests that compromised immunity could be responsible for the observed dysbiosis in the GI microbiome.

Point-of-care oral-based diagnostics.

Many of the target molecules that reside in blood are also present in oral fluids, albeit at lower concentrations. Oral fluids are, however, relatively easy and safe to collect without the need for specialized equipment and training. Thus, oral fluids provide convenient samples for medical diagnostics. Recent advances in lab-on-a-chip technologies have made minute, fully integrated diagnostic systems practical for an assortment of point-of-care tests. Such systems can perform either immunoassays or molecular diagnostics outside centralized laboratories within time periods ranging from minutes to an hour. The article briefly reviews recent advances in devices for point-of-care testing with a focus on work that has been carried out by the authors as part of a NIH program.

Translational and clinical applications of salivary diagnostics.

There have been significant advances in techniques for the detection of biomarker signals in the oral cavity (e.g., ELISAs for proteins, PCR for RNA and DNA) as well as the engineering and development of microfluidic approaches to make oral-based point-of-care (POC) methods for the diagnosis for both local and systemic conditions a reality. In this section, we focus on three such approaches, namely, periodontal disease management, early markers for systemic diseases, and salivary markers useful for pharmacogenomic studies. Novel approaches using non-invasive, salivary samples and user-friendly devices offer results that are as sensitive and specific as laboratory-based analyses using blood or urine.

Antiviral activities in human saliva.

In this review, the authors survey the large number of antibacterial and antiviral proteins present in human saliva. Of interest, most of these antibacterial proteins display antiviral activity, typically against specific viral pathogens. The review focuses on one protein that interacts with both bacteria and viruses-gp340, originally referred to as salivary agglutinin. In the oral cavity, soluble gp340 binds to and aggregates a variety of bacteria, and this is thought to increase bacterial clearance from the mouth. However, when bound to the tooth surface, gp340 promotes bacterial adherence. In the oral cavity, most gp340 is found soluble in saliva and can function as a specific inhibitor of infectivity of HIV-1 and influenza A. In contrast, in the female reproductive track, most gp340 is bound to the cell surface, where it can promote HIV-1 infection.

Mucosal fluids and biomarkers of clinical disease: workshop 3B.

Diagnostic tests for a range of oral and systemic diseases using fluids sampled from the mouth are under intense investigation and are increasingly being used. Methods exist for identification of HIV antibody and nucleic acid and for other viral infections of the mouth, such as Kaposi sarcoma herpes virus or human herpesvirus-8, which may coexist with HIV. A number of commercial test kits are available, with variable evidence of sensitivity, specificity, and utility. There is intense research on sophisticated but potentially facile handheld in-office devices for many disease markers. Challenges to their uptake require well-designed studies on their practical reliability and utility, with appropriate controls. A range of ethical, social, and political issues need to be addressed in such studies.

Uridylate kinase activity: effect of isoproterenol.

Isoproterenol stimulates cell proliferation in mouse salivary glands. Prior to the stimulation of DNA synthesis, (3)H-uridine incorporation into RNA is decreased. This decreased incorporation results from a depression of uridylate kinase activity.

In-ovo monochromatic green light photostimulation enhances embryonic somatotropic axis activity.

Previous studies demonstrated that in ovo photostimulation with monochromatic green light increases body weight and accelerates muscle development in broilers. The mechanism in which in ovo photostimulation accelerates growth and muscle development is not clearly understood. The objective of the current study was to define development of the somatotropic axis in the broiler embryo associated with in ovo green light photostimulation. Two-hundred-forty fertile broiler eggs were divided into 2 groups. The first group was incubated under intermittent monochromatic green light using light-emitting diode (LED) lamps with an intensity of 0.1 W\m2 at shell level, and the second group was incubated under dark conditions and served as control. In ovo green light photostimulation increased plasma growth hormone (GH) and prolactin (PRL) levels, as well as hypothalamic growth hormone releasing hormone (GHRH), liver growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-1) mRNA levels. The in ovo photostimulation did not, however, increase embryo's body weight, breast muscle weight, or liver weight. The results of this study suggest that stimulation with monochromatic green light during incubation increases somatotropic axis expression, as well as plasma prolactin levels, during embryonic development.

Interaction of DPPC monolayers with soluble surfactants: electrostatic effects of membrane perturbants.

We studied the effects of four soluble surfactants on DPPC monolayers to elucidate the action of these membrane perturbants. The presence of nonionic N-9 and amphoteric C31G strongly affected the pure DPPC isotherm, while anionic SDS and cationic DTAB had little effect. The impact of surfactant on DPPC domain shape in the liquid condensed-liquid expanded coexistence region showed the opposite result. Neutral surfactants had minimal effect on the shape of DPPC domains; charged surfactants, on the other hand, induced a new shape transition at high surface pressures previously unreported for DPPC domains. All of these results are discussed with particular attention given to electrostatic effects at the interface.

Multiple forms of brain adenylate cyclase: stimulation by Mn2+.

Mn2+-stimulated adenylate cyclase (ATP pyrophosphate-lyase-(cyclizing), EC 4.6.1.1) activity in detergent solubilized preparations from mouse brain. While NaF-stimulated activity was decreased by both solubilization and storage at 0--4 degrees C, the ability of the enzyme to be stimulated by Mn2+ was maintained for up to one week. By including Mn2+ in the assay of adenylate cyclase in gel fractions after isoelectric focusing, two distinct peaks of enzyme activity (pI1 - 5.8, pI2 = 6.4) were detected, suggesting the existence of more than one type of catalytic subunit in mouse brain cell membranes.

Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase.

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.

Isolation of matrix vesicles by isoelectric focusing in Pevikon-Sephadex.

We have investigated the use of an isoelectric focusing (IEF) technique for isolating and characterizing matrix vesicles. Focusing was performed on crude preparations of matrix vesicles isolated from collagenase digests of chick epiphyseal cartilage and purified by discontinuous sucrose gradient centrifugation. Crude and partially purified vesicle preparations were subjected to flat bed IEF in a slurry of Pevikon-Sephadex. Partially purified matrix vesicles focused as a narrow band (pI congruent to to 6.5). Alkaline phosphatase, solubilized from matrix vesicles, focused with a pl of 4.0-4.5. The IEF profile of matrix vesicles also differed from that of chondrocyte membranes. Thus, the membrane pls were congruent to to 5.4 and 6.6-7.8, respectively. The latter peak probably corresponded to the pl of the matrix vesicle preparation. This observation lends support to the view that vesicles originate from distinct regions of the chondrocyte membrane.

Oral diagnostic testing for detecting human immunodeficiency virus-1 antibodies: a technology whose time has come.

An oral fluid-based test for antibodies to human immunodeficiency virus (HIV), equivalent to serum in its accuracy but safer and easier to use, is now available in the United States. The development of the oral test involved overcoming technical obstacles to the use of oral fluid as a testing medium, including low immunoglobulin G (IgG) titers, suboptimal assay performance, protease degradation of IgG, high viscosity, and lack of a standardized method of specimen collection, all of which contribute to suboptimal assay performance. The currently available oral HIV test utilizes a collection device to isolate a mucosal transudate component of oral fluid rich in IgG. A vial containing a preservative solution facilitates the transport of stable, low-viscosity specimens to the laboratory for testing with an ELISA and confirmatory Western blot assay, specifically designed for use with oral fluid. Non-HIV medical conditions and oral pathologies do not appear to affect oral test results. Hopefully, the availability of a more patient-friendly, portable diagnostic test for antibodies to HIV will facilitate identification of greater numbers of infected individuals with the ultimate goals of early identification, early treatment, and prevention of disease transmission.

Human submandibular saliva aggregates HIV.

Incubation of HIV with human whole, parotid, or submandibular saliva leads to a decrease in viral infectivity in Sup-T1 cells. The effect is most pronounced with submandibular saliva. Inhibition is seen within 2 min, and increases with time. There is little inhibition seen after incubation of saliva with HSV, and no effect with adenovirus, suggested that there is some viral specificity. Electron microscopic studies revealed that HIV-saliva aggregates are trapped in 0.45-microns pore size nitrocellulose filters. If these inhibitory effects are manifest in vivo, this could account for the low level of virus detected in oral secretions.

Human submandibular saliva specifically inhibits HIV type 1.

Studies from a number of laboratories have shown the presence of factor(s) in whole, parotid, and submandibular human saliva capable of inhibiting HIV-1 infectivity in vitro. Data from our laboratory suggested that the level of anti-HIV-1 activity is higher in submandibular than parotid or whole saliva. Previous results obtained with pooled submandibular saliva from seronegative individuals included a filtration step following saliva-virus interaction. In this article, we present data on the HIV-1 inhibitory activity of individual submandibular saliva samples collected from 15 donors. We show that although anti-HIV activity is quantitatively similar in most individuals (9 of 15), some (4 of 15) are much less active than others and some (2 of 15) lack inhibitory activity. We also show that for most individuals the level of anti-HIV inhibitor is similar with or without a filtration step. However, 2 of the 15 samples demonstrated activity only after filtration. The quantitative and qualitative anti-HIV activity of individual saliva samples appeared to reflect differences in the individual donors. We further show that the anti-HIV activity of submandibular saliva is demonstrated not only against laboratory strains of HIV-1 but is similarly active against three clinical HIV-1 isolates. In contrast, submandibular saliva had little effect on the infectivity of HIV-2 or SIV.

Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate.

A highly desirable approach to prevention of human immunodeficiency virus type 1 (HIV-1) transmission during sexual intercourse is the development of nontoxic, topical, broad spectrum microbicides effective against transmission of cell-associated and cell-free virus. Toward this end, the HIV-1 inactivation potential of surface active agents C31G and an alkyl sulfate, sodium dodecyl sulfate (SDS) was assessed. Because of its extensive use as a microbicidal agent, nonoxynol-9 (N-9) was used as a reference against which C31G and SDS were compared. Viral inactivation was measured using HIV-1 LTR-beta-galactosidase indicator cells (expressing CD4 or CD4/CCR5) derived from HeLa cells, a cell line of human cervical adenocarcinoma origin. In experiments which examined inactivation of cell-free HIV-1, C31G was generally more effective than N-9. Viral inactivation by SDS occurred at twice the concentration necessary to achieve similar levels of inactivation using either N-9 or C31G. Using HeLa and HeLa-derived cells in cytotoxicity studies, it was demonstrated that SDS is as much as 11 and five times less cytotoxic than N-9 or C31G, respectively, during 48 h of continuous exposure. SDS (unlike C31G and N-9) can inactivate non-enveloped viruses such as human papillomavirus (HPV) [Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D., Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R., Kreider, J.W., 1999. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob. Agents Chemother. 43(2), 314-321]. Since addition of SDS to C31G or N-9 may make the resulting microbicidal mixtures broadly effective against both enveloped and non-enveloped viruses, several surface active agent combinations were evaluated for their abilities to inactivate HIV-1. Addition of SDS to either C31G or N-9 resulted in mixtures that were only slightly less effective than equivalent concentrations of C31G or N-9 alone. To investigate inactivation of cell-associated infectivity, HIV-1 IIIB-infected SupT1 cells were treated with N-9, C31G, or SDS. Inactivation of cell-associated infectivity required higher microbicide concentrations than were needed for inactivation of cell-free virus. However, the relative activities of N-9, C31G, or SDS were similar to those seen in assays of inactivation using cell-free virus. These studies suggest that C31G and SDS may be attractive candidates for human trials as topical microbicides effective against HIV-1 transmission since both function at concentrations that provide effective viral inactivation with low levels of cytotoxicity. SDS microbicides (used alone or with other microbicides) may provide the added advantage of protection from HPV infection.

Rapid reversal of compensatory renal hypertrophy after withdrawal of the stimulus.

The permanence of the early phases of compensatory renal hypertrophy was tested by interrupting vascular parabiosis between an anephric rat and a normal rat after 48 hours. At the time of interruption, the weights and the ratio of ribonucleic acid (RNA) content to deoxyribonucleic acid (DNA) content of the kidneys were the same as those of the remaining kidney in a single rat subjected to unilateral nephrectomy, previously reported. Within 12 hours after parabiosis was stopped, renal mass and nucleic acid concentrations returned to normal. Compensatory hypertrophy could be produced again by unilateral nephrectomy. Regression of the early phase of compensatory hypertrophy appears to be faster than muscular atrophy produced by disuse or denervation. Compensatory hypertrophy can be activated at least twice.

Onset of cell proliferation in the shortened gut: growth after subtotal colectomy.

Cell proliferation and remodeling of the small intestine were studied after subtotal colectomy in the rat. After an initial fall in nucleic acid contents, there was a trend to increasing amounts of RNA throughout the intestine on the fourteenth postoperative day; by the thirtieth day RNA and DNA contents were increased about 20 percent. DNA synthesis increased after 5 days. Although initially villi became shorter and crypts shallower, higher villi, deeper crypts, and an increased rate of cell migration were present after 14 days. Subtotal colectomy stimulates nucleic acid synthesis and remodeling of the small bowel within 2 weeks. Changes are more marked in ileum.

Distribution of topical medication in the human vagina as imaged by magnetic resonance imaging.

OBJECTIVE: To assess the use of magnetic resonance imaging (MRI) to determine the varying distribution of a vaginally placed gel over time and with different levels of patient activity. DESIGN: Prospectives interventional trial. SETTING: University medical center. PATIENT(S): One nulliparous volunteer with normal menstrual cycles and no gynecologic disease who underwent seven MRI scans of the pelvis. INTERVENTION(S): Five mL of a commercially available topical spermicide was mixed with gadolinium-chelate magnetic resonance contrast material and introduced with a standard applicator. T1-weighted three-dimensional MRI was done to assess the distribution of the gel. MAIN OUTCOME MEASURE(S): Gel thickness and distribution. RESULT(S): The initial bolus of gel was delivered into the upper portion of the vagina, above the urogenital diaphragm. Thereafter, it spread into the vaginal fornices and "flattened" to cover the lateral aspects of the vagina. Without ambulation, the majority of spread was confined to the upper vagina. With ambulation and longer elapsed time, the gel spread further in the upper vagina and into the lower vagina, and significant vaginal surface coverage increased significantly. CONCLUSION(S): Magnetic resonance imaging can be used to monitor the spread of vaginally placed products and to evaluate coverage of topical drugs used for prevention and treatment, including those used for HIV prophylaxis.