Bacteriorhodopsin induces a light-scattering change in Halobacterium halobium.

When suspensions of Halobacterium halobium are exposed to bright light, the light-scattering properties of the bacteria change. This light-scattering response can produce a transmission decrease of about 1% throughout the red and near-infrared region. The action spectrum for the light-scattering response appropriately matches the absorption spectrum of bacteriorhodopsin. The response is eliminated by cyanide p-trifluoro-methoxyphenylhydrazone, a proton ionophore, and by triphenylmethylphosphonium, a membrane permanent cation. A mild hypertonic shock induces a similar light-scattering change, suggesting that bright light causes the bacteria to shrink about 1% in volume, thereby producing the light-scattering response.

Membrane characteristics and osmotic behavior of isolated rod outer segments.

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na(+) influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na(+) influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na(+) permeability constant of about 2.8 x 10(-6) cm/s and an osmotic permeability coefficient of greater than 2 x 10(-3) cm/s.

Early receptor potential: photoreversible charge displacement in rhodopsin.

When the eye is illuminated by an intense flash, the visual pigment rhodopsin begins to pass rapidly through a series of intermediate states, eventually becoming bleached. If a second flash is delivered during the lifetimes of these intermediates the rhodopsin can be photoregenerated. A fast electrical response of the visual receptors, the early receptor potential, is elicited by the first flash. A similar response is elicited by the second flash, but the polarity of this response is reversed. Moreover, this response can be separated into three components, each arising from the action of light on a different intermediate. It is likely that all these fast responses, including the early receptor potential, arise from charge displacements in the visual-pigment molecule.

Limulus rhodopsin: rapid return of transient intermediates to the thermally stable state.

Spectral transitions of rhodopsin in single cells of the Limulus ventral eye were observed both with flash photometry and by measuring the early receptor potential. Even with repetitive stimulus flashes the rhodopsin did not bleach; after each flash the spectral intermediates decayed rapidly to the initial thermally stable state. The pigment returns to the stable state in a time comparable with the duration of the late receptor potential.

Light-stimulated electrical responses from skin.

When skin is exposed to an intense flash of light, an early electrical response can be detected from its surface. The signals that occur during the first milliseconds after the flash are similar to electrical signals recently observed in the eye from the cell layers containing melanin. Possibly the melanin in skin augments, but does not directly generate, this early electrical response. In addition, a late response, which arises hundreds of milliseconds after the flash, also occurs in skin. Unlike the early response, the late response is sensitive only to violet and shorter wavelengths of light and hence is probably mediated by a pigment other than melanin.

Controlled release of antibodies for long-term topical passive immunoprotection of female mice against genital herpes.

Current methods for sexually transmitted diseases (STD) prophylaxis, which can be disruptive and inconvenient, must be used before each act of sexual intercourse, so a method that provides protection over the course of many acts is desirable. We used a mouse model of vaginally-transmitted herpes simplex virus 2 (HSV-2) infection to test polymeric controlled-release devices for sustained passive immunoprotection. Vaginal disks were prepared by dispersing a monoclonal antibody to HSV-2 (III-174) within a matrix of poly(ethylene-co-vinyl acetate); these disks released 2 to 40 micrograms/day of antibody into buffered water. When disks were placed in the vagina, large amounts of III-174 (5 to 3,000 ng) were recovered from the vaginal fluid over the next 8 days. Mice were vaginally challenged with 10 ID50 of HSV-2 either 3 or 7 days after disk placement; no mice receiving III-174 disks became infected, while 65% of control mice receiving identical disks with nonspecific IgG did. Controlled-release disks with III-174 provided significant protection against HSV-2 infection (p < 0.005). This new technology for long-term STD prophylaxis should increase user compliance, a factor limiting the efficacy of current methods.

A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes.

The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosal immunoprotection against sexually transmitted diseases. To investigate the suitability of plant-expressed Mabs for vaginal preventive applications, we compared a humanized anti-herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soybean. We found these Mabs to be similar in their stability in human semen and cervical mucus over 24 h, their ability to diffuse in human cervical mucus, and their efficacy for prevention of vaginal HSV-2 infection in the mouse.

Vaginal lactic acid elicits an anti-inflammatory response from human cervicovaginal epithelial cells and inhibits production of pro-inflammatory mediators associated with HIV acquisition.

Inflammation in the female reproductive tract (FRT) is associated with increased HIV transmission. Lactobacillus spp. dominate the vaginal microbiota of many women and their presence is associated with reduced HIV acquisition. Here we demonstrate that lactic acid (LA), a major organic acid metabolite produced by lactobacilli, mediates anti-inflammatory effects on human cervicovaginal epithelial cells. Treatment of human vaginal and cervical epithelial cell lines with LA (pH 3.9) elicited significant increases in the production of the anti-inflammatory cytokine IL-1RA. When added simultaneously or prior to stimulation, LA inhibited the Toll-like receptor agonist-elicited production of inflammatory mediators IL-6, IL-8, TNFα, RANTES, and MIP3α from epithelial cell lines and prevented IL-6 and IL-8 production by seminal plasma. The anti-inflammatory effect of LA was mediated by the protonated form present at pH≤3.86 and was observed with both L- and D-isomers. A similar anti-inflammatory effect of LA was observed in primary cervicovaginal cells and in an organotypic epithelial tissue model. These findings identify a novel property of LA that acts directly on epithelial cells to inhibit FRT inflammation and highlights the potential use of LA-containing agents in the lower FRT as adjuncts to female-initiated strategies to reduce HIV acquisition.

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

We have investigated, with osmotic techniques, the light-regulated Na+ channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na+ permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na+ channels are highly sensitivive to membrane oxidation. Using the continuous flow technique, we investigated the H+ and Ca++ dependence of the Na+ channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H+ activity decreases PNa (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca++ across membranes, the Na+ channels are blocked when the external Ca++ activity is increased from 10(-7) to 10(-5) M. This high sensitivity to both H+ and Ca++ ions suggests that high field strength anionic sites may exist in or near the Na+ channels and that the channels are blocked when these sites bind H+ or Ca++ ions.

Dark ionic flux and the effects of light in isolated rod outer segments.

We have determined the permeability properties of freshly isolated frog rod outer segments by observing their osmotic behavior in a simple continuous flow apparatus. Outer segments obtained by gently shaking a retina are sensitive but nonideal osmometers; a small restoring force prevents them from shrinking or swelling quite as much as expected for ideal behavior. We find that Na(+), Cl(-), No(3) (-), glycerol, acetate, and ammonium rapidly enter the outer segment, but K(+), SO(4) (=), and melezitose appear impermeable. The Na flux is rectified; for concentration gradients in the physiological range, 2 x 10(9) Na(+) ions/sec enter the outer segment, but we detect no efflux of Na(+), under our conditions, when the gradient is reversed. Illumination of the outer segment produces a specific increase in the resistance to Na(+) influx, but has no effect on the flux of other solutes. This light-dependent Na(+) resistance increases linearly with the number of rhodopsin molecules bleached. We find that excitation of a single rhodopsin molecule produces a transient ( approximately 1 sec) "photoresistance" which reduces the Na(+) influx by about 1%, thus preventing the entry of about 10(7) Na(+) ions. At considerably higher light levels, a stable afterimage resistance appears which reduces the Na influx by one-half when 10(6) rhodopsin molecules are bleached per rod. We have incorporated these findings into a model for the electrophysiological characteristics of the receptor.

The cat/feline immunodeficiency virus model for transmucosal transmission of AIDS: nonoxynol-9 contraceptive jelly blocks transmission by an infected cell inoculum.

OBJECTIVES: To develop an animal model to study transmucosal lentivirus transmission, and to determine whether topical application of contraceptive jelly can block transmission by an infected cell incoulum. DESIGN: Feline immunodeficiency virus (FIV), a lentivirus similar to HIV, causes an AIDS-like disease in domestic cats. HIV is transmitted primarily across mucosal surfaces, and infected cells may be important in this transmission. We tested the ability of FIV-infected cells to transmit infection across the vaginal, rectal and oral mucosa of the cat, and whether a vaginal contraceptive jelly could prevent such transmission. METHODS: An inoculum consisting of 2 million FIV-infected primary cat T cells was administered vaginally, rectally or orally to female cats that had received either no pretreatment or pretreatment with a contraceptive jelly containing the detergent nonoxynol-9 as spermicide. Transmission was detected by monitoring recipient animals for viral antibodies and by viral cultures of blood leukocytes. RESULTS: A single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina or rectum (10 out of 11 animals became infected). Pretreatment of the vagina (five animals) or rectum (four animals) with contraceptive jelly protected all animals from transmission by the highly infectious inoculum. CONCLUSIONS: The cat/FIV model provides an efficient means to study transmucosal transmission of lentivirus infections, and for assessing vaginal barrier methods that could block transmission. One such method, nonoxynol-9 contraceptive jelly, effectively prevents transmucosal transmission by an FIV-infected cell inoculum.

Preventing infectious disease with passive immunization.

Antibodies can prevent infectious diseases by providing passive immune protection. Here we review successful clinical trials of passive immunization and consider some of the unique qualities monoclonal antibodies are now beginning to offer for developing methods for passive immunization against a wide range of infectious diseases.

Macromolecules released from polymers: diffusion into unstirred fluids.

Polymers that release macromolecules may be useful for preventing and treating human disease. In certain applications of polymeric controlled release, like drug therapy of brain disease and immunoprotection of mucus epithelia, effectiveness may be limited by diffusion through an unstirred fluid near the polymer. Using computer-assisted epifluorescence microscopy, we have examined the local distribution of fluorescently labelled macromolecules released from an ethylene-vinyl acetate copolymer matrix into unstirred layers of phosphate-buffered water and mid-cycle human cervical mucus. Diffusion coefficients in the fluid were determined by observing the concentration profiles as a function of time. Diffusion coefficients determined for fluorescein, bovine serum albumin, and three classes of human immunoglobulins (IgG, sIgA and IgM) in phosphate-buffered water were in good agreement with literature values. For fluorescein, albumin and IgG, diffusion in mucus was comparable with diffusion in water: the largest molecule tested was slowed by only a factor of 3.

Comparison of an anti-HSV-2 monoclonal IgG and its IgA switch variant for topical immunoprotection of the mouse vagina.

An IgG2a monoclonal antibody (Mab) directed against glycoprotein D of herpes simplex virus 2 (HSV-2) was compared with an IgA heavy chain Mab switch variant to investigate the effect of isotype for topical immunoprotection of the murine vagina. The IgA Mab, a mixture of monomeric and polymeric IgA, was indistinguishable from its IgG parent in an in vitro HSV-2 neutralization assay. When these class switched Mabs were delivered to the mouse vagina, we also found no significant difference between the IgG and IgA for preventing vaginal transmission of HSV-2 infection. The implications of these results for active and passive immunization strategies against vaginal transmission of genital herpes infections are discussed.

Antigen-releasing polymer rings and microspheres stimulate mucosal immunity in the vagina.

Several recent studies suggest that direct application of antigen to the vaginal surface may enhance local IgA secretion, but the most effective methods for stimulating immunity at the vaginal surface have not been identified. We used antigen-loaded, biocompatible, vaginal rings to provide controlled and sustained antigen delivery directly to the vaginal mucosal surface. Mice were primed with ferritin, either subcutaneously or orally by ferritin-loaded polymer microspheres, and vaginally boosted by insertion of a ferritin-loaded polymer ring. We found that the vaginal rings were a convenient method for providing controlled antigen delivery to the vagina. Subcutaneously primed mice receiving ferritin-loaded vaginal rings had ferritin-specific IgA in their mucus secretions, while mice receiving blank rings did not. Oral priming with ferritin-loaded poly(lactic acid) microspheres also produced significant levels of ferritin-specific IgA in the vaginal secretions, but required the presence of cholera toxin. Controlled ferritin delivery to mucosal surfaces, either by oral, biodegradable microspheres or vaginal rings, provides a convenient and reliable method for enhancing vaginal IgA production in mice.

The rate at which human sperm are immobilized and killed by mild acidity.

OBJECTIVE: To determine the rate at which mild acidity immobilizes and kills human sperm and to evaluate an acidic microbicide, BufferGel, for sperm immobilization. DESIGN: Controlled in vitro study. SETTING: An academic research university and hospital andrology lab. PATIENT(S): Eight volunteer male sperm donors. INTERVENTION(S): Semen samples were treated with hydrochloric acid (HCl) or BufferGel. MAIN OUTCOME MEASURE(S): Sperm motility was measured by using a computerized automated semen analyzer and video microscopy. Sperm membrane permeability and intracellular pH were measured by using fluorescent techniques. RESULT(S): In semen acidified with HCl to pH 4.0, sperm were rapidly immobilized (within 1 min) and were irreversibly immobilized (killed) within 10 minutes. The speed of immobilization and of killing were both linearly proportional to hydrogen ion activity over a pH range of 7.5-4.0. Across the same range, the intracellular pH of human sperm equilibrated to within 0.5 pH units of extracellular pH within 1-2 minutes. BufferGel immobilized sperm significantly faster than HCl from pH 4.0-6.0. CONCLUSION(S): Exposure to mild acidity rapidly acidifies the intracellular pH of human sperm and is rapidly spermicidal. BufferGel accelerates acid immobilization of sperm.

Rat cauda epididymal fluid is a mucus.

Immobilin, the highly viscoelastic glycoprotein isolated from rat cauda epididymal fluid, exhibits all of the key biochemical characteristics of a mucin: 1) it has a very high molecular weight (will not pass through a 10(6) dalton cut-off filter; 2) it contains 56% carbohydrate, with low or undetectable levels of mannose, xylose and uronic acid; 3) the carbohydrates (primarily galactose, N-acetylglucosamine and N-acetylgalactosamine) are arranged in short, oligosaccharide chains (4-20 monosaccharides per chain); 4) these oligosaccharide chains can be cleaved by NaOH in the presence of NaBH4, suggesting O-glycosidic linkages; and 5) the protein core is pronase-resistant. Immobilin, however, contains no detectable sialic acid, and 67% of the oligosaccharides are uncharged, indicating that immobilin is less acidic than most other mucins.

Tests of vaginal microbicides in the mouse genital herpes model.

Microbicide candidates were selected that have demonstrated activity against sperm or sexually transmitted disease pathogens in vitro, and the efficacy of these agents for preventing vaginal transmission of genital herpes infection was evaluated in the progestin-treated mouse. Each agent was delivered to the vaginas of mice approximately 20 sec prior to delivering a highly infectious herpes simplex virus-2 inoculum. The following agents provided significant protection: anti-HSV monoclonal antibodies III-174 and HSV8, modified bovine beta-lactoglobulin (beta-69), carrageenan, concanavalin A, chlorhexidine, dextran sulfate (average molecular weight 8,000 and 500,000), fucoidan, neem, nonoxynol-9, polystyrene sulfonate, and povidone-iodine. Two agents, gramicidin and heparan sulfate, though highly effective in vitro, were not protective in vivo at the doses tested.

Contraceptive testing of vaginal agents in rabbits.

Development of new vaginal products, such as microbiocides and contraceptives, requires in vivo testing of their effect on fertility. Rabbits, unlike smaller laboratory animals such as rats and mice, which inseminate in the uterus, inseminate vaginally and thus are valuable as animal models for testing vaginal agents for contraceptive effects. Rabbits are inexpensive and easy to handle compared to nonhuman primates, and have frequently been used for testing the effects of vaginal agents on fertility. We review the pertinent literature and report findings that provide guidance for effectively using and improving the rabbit contraceptive model in testing new vaginal products.