The interaction of bee melittin with lipid bilayer membranes.

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.

The effect of synthetic polymers on the electrical and permeability properties of lipid membranes.

1. The effect of two series of hydrophilic and hydrophobic polymers on the stability, conductivity and permeability towards water and leucine of black lipid membranes and liposomes is reported. 2. The changes in properties of these membrane preparations is related to bulk phase viscosity and dielectric measurements together with monolayer studies. 3. The hydrophobic polymers dramatically increase membrane stability, had no effect on conductivity, but increased the permeability coefficient of leucine. 4. The hydrophilic polymers produced minor, but significant changes to membrane properties. 5. It is concluded that not only basic polymers but also neutral and acidic macromolecules can interact strongly with lipid membranes.

The induction of immune tolerance in delayed contact sensitivity by the use of chemically related substances of low immunogenicity.

Immune tolerance in delayed contact sensitivity to pentadecylcatechol can be induced by a series of derivatives substituted in the 6 position of the ring. Some of these derivatives have the property of being very poor sensitizers and having very low dermal toxicity. Thus, sensitization and tolerance have different biologic mechanisms and are associated with different properties of these chemicals.

Purulent ocular discharge in neonates: significance of Chlamydia trachomatis.

We obtained cultures for bacteria and chlamydiae from 100 infants with conjunctivitis that began during the first month of life. Sixty-nine infants were evaluated during well-child visits (group A); 31 were seen specifically for the ocular discharge (group B). Potentially pathogenic bacteria, predominantly Staphylococcus aureus, were cultured from one third of the infants in each group. Chlamydia trachomatis was recovered from three infants (4%) in group A and from ten (32%) in group B. Three infants with chlamydial conjunctivitis (two in group A, one in group B) had only mild inflammation. Initial treatment with topical antibiotics was unsuccessful in eliminating the organism from seven of 11 infants.

Surface features of the conjunctiva and cornea.

Guinea pig corneal and conjunctival surfaces were examined by transmission electron microscopy and cytochemistry. Some specimens of cornea and conjunctiva were examined morphologically; others were stained with ruthenium red or tannic acid before examination to enhance the detection of glycoproteins at cell surfaces. The epithelia were covered by microvilli and on the cornea also by microplicae. These surface projections were the shortest (150 nm) over the central cornea, and became progressively longer (approximately 300 nm) over the tarsal and fornical conjunctiva. There was a filamentous cell coat (glycocalyx) on the microvilli and microplicae that was best demonstrated in specimens stained with tannic acid. The glycocalyx extended approximately 300 nm from the tips and lateral surfaces of the microvilli and microplicae. Although there were slight local variations in its thickness, the maximum thickness of the cell coat was uniform over the cornea and conjunctiva. Heavy deposits at the cell surface after ruthenium red staining indicated that the cell coat contained many highly charged polyanions. The density of the ruthenium red stain obscured the fine structure of the filaments in the cell coat. The glycocalyx forms a scaffolding that is believed to bind mucus, with its content of immunoglobulins, by weak chemical interactions to the epithelial surface. Therefore, the microvilli, microplicae, and glycocalyx that were demonstrated in this study provide the structural framework that supports and binds a complex of related factors, including tears, mucus, and immunoglobulins, that have the common function of protecting the eye.

Unusual structure of rat conjunctival epithelium. Light and electron microscopy.

The conjunctiva of the adult Sprague-Dawley rat was studied by light microscopy of 3 micron glycol methacrylate sections of whole eyes with lids and by electron microscopy of conjunctiva from the lower fornix. Rat conjunctiva is unique among species studied. All the superficial epithelial cells are squamous cells rather than polyhedral or columnar cells. Furthermore, the goblet cells are aggregated into clusters rather than distributed randomly throughout the epithelium. These clusters are not found at the lid margin or limbus, but are present in the palpebral and bulbar conjunctivae and achieve maximal size and number near the fornix. The stratified squamous epithelium is typical, composed of a layer of basal cells, an intermediate zone of wing cells, and an upper zone of several layers of squamous cells. Dividing cells are seen only in the basal layer. Occasional mononuclear leukocytes are found in the basal and intermediate layers. The goblet cell clusters are largely composed of columnar cells. Goblet cells predominate, but there are also occasional tuft cells, characterized by thick microvilli at their apices. Basal cells form only an incomplete layer beneath the columnar cells, which in places span the entire epithelium. The conjunctiva of the adult rat has few cells with potential for immunological activity. It does not contain appreciable numbers of plasma cells, and lymphoid follicles are absent.

Herpes simplex viral infection of the mouse trigeminal ganglion. Immunohistochemical analysis of cell populations.

Several distinct populations of sensory neurons in the ophthalmic region of the mouse trigeminal ganglion have been identified by their reactivity to antibodies raised against substance P (SP), calcitonin gene-related peptide (CGRP), cell-surface glycoconjugates SSEA3 and LD2, and the plant lectin, Bandeiraea simplicifolia lectin 1, isolectin 4 (BSIL4). Thirty-six percent of the neurons in the ophthalmic portion of the mouse trigeminal ganglion express CGRP and 17%, SP. All neurons that express SP also express CGRP. Forty percent of the neurons in the ophthalmic region of the ganglion are recognized by monoclonal antisera to SSEA3, and 66% of this population also express the neuropeptides SP or CGRP. The neuronal population recognized by BSIL4 is identical to the population with the LD2 epitope. This population of cells (BSIL4/LD2) does not express the SSEA3 glycoconjugate and is largely nonpeptidergic. All four populations of sensory neurons (SP, CGRP, SSEA3, and LD2/BSIL4) can be infected by herpes simplex virus (HSV). However, the relative proportion of SSEA3- and LD2/BSIL4-labeled cells that were infected productively with HSV was much less than expected based on the relative size of the populations of these neurons in the ophthalmic region of the ganglion.

Resistance to reinfection with a chlamydial agent (guinea pig inclusion conjunctivitis agent).

Although most chlamydial infections are chronic or recurrent, infection of the guinea pig's eye with guinea pig inclusion conjunctivitis (GPIC) agent induces a marked resistance to reinfection. To characterize this resistance to GPIC agent, we compared the disease and infection in previously infected guinea pigs with that in animals infected for the first time. In animals experiencing primary infection, even the lowest dose (10 egg-lethal doses [ELD50]) produced the disease and chlamydial inclusions in conjunctival smears, but the incubation period became progressively shorter with the highest inocula (10(4) and 10(5) ELD50). In animals with previous infection only these two highest inocula produced disease and infection, but the disease was short-lived, and replication of the agent was severely limited. The mechanism of this resistance may be due to secretory antibody in the tears, cellular immunity, or other local factors.

Demonstration of the mucous layer of the tear film by electron microscopy.

The mucous layer on the ocular surface maintains the stability, spread, and coherence of the tear film and is essential for normal vision. In spite of its importance, the precise thickness and localization of mucus on the surface of the eye are not known because it is not preserved in conventional electron-microscopic preparations. The authors used two different methods to show mucus on the guinea pig cornea and conjunctiva. First, the authors precipitated mucous glycoproteins by adding a quaternary ammonium compound, either cetylpyridinium chloride or hexadecyltrimethylammonium bromide, to aldehyde fixatives. This procedure stabilized the mucus over the goblet cells and adjacent epithelium, although the mucous layer was not preserved uniformly in other areas. Tannic acid intensely stained mucus precipitated by these methods and showed it to be 0.8 micron thick on the cornea and 1.4 micron thick on the conjunctiva. To confirm these results, the authors also prepared specimens of cornea and conjunctiva by freeze substitution. This technique preserved the mucus in a smooth, uninterrupted layer. The thickness of the mucus was somewhat variable; it measured 1.0 micron over the cornea and varied from 2.0 to 7.0 micron over the conjunctiva because of the greater irregularity of the tissue. The authors' results show that mucus constitutes a considerable part of the precorneal tear film and is thicker than was recognized formerly.

Serum and tear antibodies to Chlamydia after reinfection with guinea pig inclusion conjunctivitis agent.

Repeated inoculation of th eyes of guinea pigs with the naturally occurring Chlamydia psittaci agent, guinea pig inclusion conjunctivitis (GPIC), showed that animals gradually become susceptible to reinfection with the passage of time after primary infection. Higher levels of serum IgG antibody had a significant association with resistance to challenge inoculation only with a high dose (250 ELD50) but not with a low dose (25 ELD50) inoculum. With each inoculum, however, some animals with high serum antibody were susceptible. the presence of antibodies in tears did not correlate with resistance to the first low-dose challenge inoculation, but both tear IgG and secretory antibody did have a significant association with resistance on the second rechallenge with a high-dose inoculum. Topical treatment of the eye with immune serum or tears during primary infection reduced the amount of agent in the conjunctiva only during the period of application. Local treatment of the eye with heat-killed vaccine prior to primary infection did not produce detectable antibody or protect animals against challenge inoculation; this local immunization did "prime" the animals, however, so that they had an accelerated antibody response after infection. Although there is abundant evidence that local immunity has an important role in resistance to challenge inoculation with GPIC, serum and tear antibody levels correlate equally well with resistance to repeated ocular challenge inoculation. Effective immunization procedures for this chlamydial infection then would involve stimulation of both local and systemic immune responses.

Chlamydial infection of subcutaneous conjunctival transplants in guinea pigs.

The development and testing of candidate vaccines for trachoma are constrained because only humans and nonhuman primates are susceptible to conjunctival infection with Chlamydia trachomatis. Guinea pig inclusion conjunctivitis (GPIC), an analogous disease of guinea pigs, provides a useful, less expensive model to study ocular chlamydial infections. GPIC is caused by a Chlamydia psittaci strain whose external constituents are very similar to those of C. trachomatis. To develop a better model for studying GPIC immunity, conjunctival pockets were established under the abdominal skin of guinea pigs by subcutaneous implantation. Up to six implants could be produced in each animal. The success rate of implantation was 79.0% (n = 148). These pockets were then infected with GPIC. The organism was recovered from the autografts indicating local replication, and tests for serum antibody by microimmunofluorescence showed production of GPIC-specific antibody of IgG and IgM classes after infection. There was minimal antibody response after moderate inoculating doses to the implants, and the titers increased more slowly than after eye infection with GPIC; with higher concentration of the inoculum, however, the antibody response increased to the same levels as with the ocular challenge but more slowly. Inoculation of pockets with GPIC also produced acute inflammatory changes in infected autografts (n = 101). Histologic examination of infected grafts showed chlamydial inclusions in epithelial cells and significant infiltration with lymphocytes and polymorphonuclear cells. Subcutaneous autografts may provide a useful model for chronologic studies of chlamydial infection. The delayed immunologic response, however, suggests that these pockets of implanted epithelium do not have full access to the immune system.

A microscopic study of herpes simplex virus retinopathy in mice.

ICR white mice were inoculated with herpes simplex virus (HSV) type I in the anterior chamber of one eye. Animals were killed at intervals of 2, 4, 6, 8, and 10 days and both eyes were obtained for light and electron microscopic study of retinal changes. HSV retinopathy developed in 42 (91%) of 46 inoculated eyes. Fourteen (88%) of sixteen noninoculated eyes examined after the sixth postinoculation day developed HSV retinopathy. The earliest signs of retinopathy in the inoculated eye were peripheral retinal vasculitis and inflammatory cells throughout the nerve fiber layer on day 2. No virus was found in retinal tissue until day 4, at which time disruption of outer retinal layers (outer nuclear layer and layer of rods and cones) was observed in the peripheral retina. The earliest signs of retinopathy in the noninoculated eye were isolated foci of outer retinal disruption in the posterior retina on day 6. The inflammation accompanying early retinal changes of HSV retinopathy were more severe in the inoculated eye. Electron microscopy of both eyes revealed viral particles in the inner nuclear and ganglion cell layers at the time of outer retinal disruption, but viral particles were seen only rarely in the outer retinal layers at this stage. Early disruption of normal retinal architecture may be due to infection and destruction of Muller cells. The retinopathy progressed in both eyes to total destruction of the retina by day 10. Viral infection of the retinal pigment epithelium occurred, but viral particles were seen only rarely in the underlying choroid. This model may be useful for the study of HSV retinopathy in humans.

An animal model for cicatrizing trachoma.

An animal model of cicatrizing trachoma was developed in cynomolgus monkeys. This model is consistent with our hypothesis that repeated ocular inoculation of Chlamydia trachomatis, BOUR strain, mimics the repeated reinfection that occurs naturally in endemic human trachoma. A chronic follicular conjunctivitis developed, and scarring later appeared in the superior tarsal conjunctiva. The organism was reisolated after the infection and was also demonstrated cytologically. Specific antichlamydial antibodies of both the IgM and IgG types appeared in the sera of the monkeys. Histopathologic examination of conjunctiva showed a marked lymphocytic response and the presence of germinal centers; areas of conjunctival scar tissue were also examined. Efforts to produce a similar model in rhesus monkeys were less successful.

An animal model of trachoma II. The importance of repeated reinfection.

An animal model of chronic cicatrizing trachoma has been produced by repeated ocular inoculation with Chlamydia trachomatis serotype E, a genitally transmitted strain. We have now produced a chronic follicular conjunctivitis on cynomolgus monkeys by repeated inoculation with C. trachomatis serotype A, which has been isolated from an area of endemic trachoma. This disease was similar in all respects to that which followed infection with the serotype E strain. Cynomolgus monkeys inoculated with a single dose of serotype E of C. trachomatis strain developed an acute, self-limited follicular conjunctivitis, which was intense for 4 weeks and then slowly subsided. The organism could be reisolated only during the first 4 weeks after inoculation. On reinoculation at 15 and 30 weeks after the initial infections, these animals demonstrated only a mild and transitory clinical response, and the agent could be recovered for only up to 14 days after inoculation. In contrast, repeated weekly reinoculation with either serotype led to a chronic progressive clinical response in these animals, although after the first 6 weeks the agent was isolated only occasionally. This chronic disease was shown not to be due to hypersensitivity to the egg yolk components in which the organism was grown. These data suggest that the serotype of the chlamydial organism may not be as important in determining the clinical course of disease as is the frequency or persistence of exposure to the chlamydial agent. Although a single inoculum produced an acute follicular conjunctivitis, repeated inoculation is needed to produce the chronic disease characteristic of trachoma in this animal model.

Herpetic eye disease in rabbits after inoculation of autonomic ganglia.

Since herpes simplex virus (HSV) can cause persistent infection of autonomic ganglia of both humans and experimentally infected animals, we followed the pattern of eye disease and viral growth after HSV inoculation of one superior cervical ganglion in rabbits. Of 27 inoculated animals, eye disease or detectable virus developed in 18. Anterior uveitis was the most common clinical manifestation (94%), but conjunctivitis and dendritic keratitis were also frequent (60%). All 12 uveal-retinal specimens tested and five of seven ipsilateral superior cervical ganglia had detectable virus. If recurrent herpetic iritis in humans is associated with persistent infection of the superior cervical ganglion, autonomic mediators might trigger episodes of virus shedding. In patients with herpetic iritis, then, the use of epinephrine and other adrenergic agonists or antagonists should be avoided.

Vitamin A in experimental herpetic keratitis.

Systemic vitamin A palmitate at three dosage levels was evaluated for its effect on experimental herpes simplex virus (HSV) keratitis in corticosteroid-treated rabbits. High-dose vitamin A palmitate reduced the severity of herpetic keratitis, but the low or moderate doses did not, and none of the vitamin A treatments affected virus recovery. Vitamin A treatment, however, produced substantial weight loss in uninfected rabbits and in corticosteroid-treated, HSV-infected rabbits. Steroid treatment alone significantly increased the antibody response to HSV, but this effect was not enhanced by vitamin A. The slight beneficial effect on HSV keratitis could be attributed to the known effect of vitamin A on promoting epithelial healing rather than an enhancement of immunity. For this purpose, local application of vitamin A may be just as effective and much less toxic than administration for systemic effect.

Leprosy in a trachomatous population.

In an Egyptian leprosy hospital, 17% of 133 patients had a visual acuity of less than 3/60. Corneal opacity, phthisis bulbi, and cataract accounted for 85% of blindness. Leprosy and trachoma together produce blinding corneal opacity by exposure, leprous keratitis, and trichiasis and entropion. Inturned lids, a late result of conjunctival scarring due to childhood trachoma, were less frequent in patients with lepromatous leprosy than in patients with tuberculoid leprosy; because conjunctival scarring from trachoma depends on cell-mediated immunity, patients with lepromatous leprosy may not have had severe trachomatous scarring develop due to their lifelong abnormality in cellular immunity. In patients with leprosy, even when complicated by trachoma, simple measures to prevent or restore vision include medical treatment of leprosy, surgical correction of lid deformities, sector iridectomy for constricted pupils or central corneal opacities, and cataract extraction.

Herpesvirus in sensory and autonomic ganglia after eye infection.

In herpesvirus hominis (HVH) infections, virus harbored in the sensory ganglia is now thought to be the main source of recurrent infection at peripheral sites. Experimental HVH infection of the external eye in rabbits produces an acute infection and then latent infection of the trigeminal ganglion. In this study, acute infection of the automatic ganglia serving the eye (superior cervical and ciliary) as well as trigeminal ganglia occurred after HVH inoculation of rabbits' corneas with a herpes type 1 strain (RE). Latent virus infection was detected in the trigeminal ganglion of one of five animals tested six months after initial infection. Since the superior cervical and other autonomic ganglia serving the eye become infected during acute herpes simplex virus infection of the external eye in rabbits, it is possible that these ganglia are also sources of reinfection in recurrent herpetic disease of the eye. Following the initial eye disease with this virus strain, HVH shedding could not be demonstrated even after induction attempts by topically applied epinephrine or systemic use of cyclophosphamide. Thus, establishment of latent HVH infection in the ganglia and chronic shedding of virus into the external eye is not a constant feature of this animal model, but may depend on the specific strain of herpesvirus used.

Incidence of cataract extraction in Pima Indians. Diabetes as a risk factor.

The incidence of visually disabling cataract was estimated by the rate of first cataract extraction in a population of Pima Indians in Arizona. The annual age-specific rates of cataract surgery (first and second eyes) were 3.7 to 5.9 times as high as the estimated US rates. Diabetes was a strong risk factor for first cataract surgery in all age and sex groups except in men aged 75 to 84 years. Overall, with age and sex controlled, the rate of first cataract surgery was 2.2 times as high (95% confidence interval, 1.3 to 3.9) in diabetic as in nondiabetic subjects. Cataract surgery was related to the duration and type of treatment of diabetes. Insulin-treated diabetics had about five times the rate of those with normal glucose tolerance. The rate of cataract surgery was lowest in subjects with normal glucose tolerance, somewhat higher in those with impaired glucose tolerance, and even higher with increasing duration of diabetes.

Cataract extraction. Risk factors in a health maintenance organization population under 60 years of age.

Risk factors for cataract extraction in a young (less than 60 years of age) urban health maintenance organization population were evaluated in a case-control study. The subjects (72 case-control pairs) subscribed to the Kaiser Permanente Medical Care Program in the San Francisco Bay area and had cataract extraction between 1976 and 1980. All patients had visual acuity of at least 20/40 OU, documented before development of cataracts. Thirty-six (50%) of the 72 cataract extraction patients had at least one known risk factor for cataract formation, including trauma, intraocular inflammation, diabetes mellitus, syphilis, oral or topical steroid use, or previous eye surgery. Male patients were found to be a mean of 4.3 years younger than female patients, and diabetics were found to be a mean of 3.5 years older than nondiabetics. Variables found to be related to cataract extraction in univariate analysis included diagnosis of diabetes mellitus, a family history of cataracts, pulse rate, white blood cell count, and syphilis.