The coincidence of HSV-1 ocular cultures with HSV-1 corneal epithelial defects in rabbits after experimental penetrating keratoplasty.

Penetrating keratoplasty (PKP) in conjunction with postoperative corticosteroids may reactivate latent herpes simplex virus type 1 (HSV-1) to cause persistent postoperative epithelial defects. The clinical diagnosis of HSV keratitis after penetrating keratoplasty is difficult because the postoperative appearance may be nondendritic and, therefore, not characteristic of HSV-1 infection. Presently, the most reliable method to diagnose HSV-1 under these conditions is to culture eyes for the presence of HSV-1. To determine the coincidence of positive HSV-1 ocular cultures with HSV-1 epithelial defects, 15 rabbits (20 eyes) latently infected with HSV-1 underwent autograft PKP with postoperative corticosteroids. Daily ocular cultures and slit-lamp examinations were performed on postoperative days 1-8 and 10. Viral shedding occurred in 15 of 19 (79.0%) of the eyes postoperatively. Superficial punctate keratopathy (SPK) was observed in 19 of 19 (100%) of the eyes and coincided with positive HSV-1 cultures 24% of the time. Dendritic lesions were observed in three of 19 (15.8%) of the eyes; the dendrites coincided with positive HSV-1 cultures 60% of the time. Finally, epithelial ulcers were seen in eleven of 19 (57.9%) of the eyes, thus coinciding with HSV-1 positive cultures 29% of the time. The greatest coincidence of positive HSV-1 cultures with nondendritic epithelial lesions occurred on postoperative day number 4. The results suggest that an epithelial lesion following PKP and postoperative corticosteroids could represent HSV infection, even if a single HSV ocular culture is negative.

Corneal nerve disruption reactivates virus in rabbits latently infected with HSV-1.

Trauma, inflammation, and neuronal stimulation or damage can reactive latent herpes simplex virus type 1 (HSV-1). The innervation density of the corneal epithelium is 300-600 times that of skin and, therefore, corneal nerve disruption could provide a strong stimulus for HSV-1 reactivation. This study has documented HSV-1 ocular reactivation following three methods of corneal nerve disruption in rabbits. Twenty HSV-1 latently infected rabbits (26 eyes) were divided into three groups: 7 rabbits received uniocular cryogenic injury, 7 rabbits underwent uniocular anterior superficial keratectomy, and 6 rabbits had binocular transection of the corneal nerves at the corneoscleral limbus which, in contrast to the other treatments, produced minimal epithelial change. Opposite eyes in the first two groups of rabbits were left undisturbed to serve as HSV-1 infected controls. Three additional rabbits, not infected with HSV-1, underwent gold chloride impregnation of the corneal nerves for light microscopic documentation of corneal nerve damage induced by each procedure. On all HSV-1 infected eyes, daily HSV-1 ocular cultures were obtained for 7 consecutive days. All three procedures resulted in marked corneal nerve destruction and degeneration. HSV-1 shedding occurred in 5/7 (71%) of the eyes that underwent cryogenic lesioning; in 5/7 (71%) of the eyes that underwent anterior keratectomy; and in 8/12 (67%) of the eyes that had the corneal nerves transected at the corneoscleral limbus. Only 4 (29%) of the 14 control eyes had positive HSV-1 ocular cultures. This investigation provides strong evidence that corneal nerve disruption is correlated with ocular HSV-1 reactivation.

An improved technique for experimental penetrating keratoplasty in rabbits.

Penetrating keratoplasty is difficult to perform, especially for the beginning ophthalmologist or corneal surgeon. This investigation describes, step by step, a simplified technique of performing experimental keratoplasty on rabbit eyes. Use of this method resulted in 20 of 24 (83%) rabbit eyes maintaining technically successful clear grafts without anterior synechiae or angle-closure glaucoma for up to 3 months postoperatively.

Cryogenic induced ocular HSV-1 reactivation is enhanced by an inhibitor of the lipoxygenase pathway.

Reactivation of latent herpes simplex virus type 1 (HSV-1) has been shown to occur in response to localized inflammation. Prostaglandins and lipoxygenase products [eg. hydroxyeicosatetraenoic acids, (HETEs)] are associated with inflammation and, therefore, may play a role in HSV-1 infection and reactivation. In the rabbit cornea, alkali injury, cryogenic injury, and acute HSV-1 infection promote the synthesis of HETEs. Recently, a platelet activating factor antagonist, ginkgolide B (BN 52021) has been found to specifically inhibit the corneal synthesis of HETEs after alkali injury. If the induction of HETEs after injury is related to HSV reactivation and severity of infection, BN 52021 may alter HSV reactivation and the severity of infection by reducing the production of HETEs. To study the effect of BN 52021 on HSV-1 reactivation, cryogenic corneal lesions were produced in ten HSV-1 latently infected rabbits. Five rabbits were treated with topical and intravenous BN 52021 while the remaining five rabbits received topical artificial tears and intravenous saline. In the BN 52021 treated group, 90% (9/10) of the eyes and 53% (35/66) of the total ocular cultures were positive for HSV-1. In the control group, 60% (6/10) of the eyes, and 27% (18/66) of the ocular swabs were positive for HSV-1. The total number of positive cultures was significantly greater (p less than .05) in the BN 52021 treated rabbits. By increasing the number of positive HSV ocular cultures, BN 52021 appeared to act similarly to other inhibitors of arachidonic acid metabolism such as steroidal and nonsteroidal anti-inflammatory agents.

Penetrating keratoplasty in rabbits induces latent HSV-1 reactivation when corticosteroids are used.

The increased incidence of corneal graft failure in patients with herpes simplex virus (HSV) keratitis may be due in part to reactivation of latent HSV following surgical corneal trauma and postoperative corticosteroid therapy. To determine the onset, frequency, and nature of HSV recurrences following penetrating keratoplasty (PKP), 21 HSV type 1 (HSV-1) latently infected rabbits underwent unilateral autograft PKP. Opposite unoperated eyes served as HSV-1 latently infected controls. Corneal autografts were performed so that immunologic graft rejection would not be confused with recurrent HSV-1 stromal disease. After PKP, 11 of the 21 eyes were treated with dexamethasone. Ocular cultures and slit-lamp examinations were performed daily for the first postoperative 8 days and every other day thereafter for 82 days. Nine (82%) of the 11 dexamethasone-treated PKP eyes, 2 (20%) of the PKP eyes not treated with dexamethasone, and 3 (17%) of the 18 unoperated eyes had positive HSV-1 ocular cultures. Geographic ulcers appeared only in the PKP eyes treated with dexamethasone; 9 (82%) of the 11 PKP eyes treated with dexamethasone developed geographic ulcers. Between the 24th and 90th postoperative days, stromal keratitis appeared in 5 (56%) of the 9 PKP eyes treated with dexamethasone and in 2 (25%) of the 8 PKP eyes not treated with dexamethasone. Autograft PKP with postoperative corticosteroids significantly increased HSV-1 ocular shedding, epithelial ulceration, and stromal keratitis. This experimental model provides a useful tool to further investigate the development and treatment of HSV-1 epithelial and stromal recurrences after PKP.

Expulsive choroidal hemorrhage in rabbits. A histopathologic study.

Expulsive hemorrhage is a catastrophic complication of intraocular surgery that can result in total loss of vision. Suprachoroidal effusion and hemorrhage may precede the development of expulsive hemorrhage; however, the relationship remains unclear. After sedation with intravenous pentobarbital sodium, 11 rabbits were given lactated Ringer's solution and heparin sodium intravenously. The right eyes were proptosed, and the central cornea, lens, and anterior vitreous were removed. After surgery, all 11 eyes (100%) developed choroidal effusion, choroidal hemorrhage, or expulsive hemorrhage. The rabbits were killed at various intervals after surgery so that the eyes could be enucleated and processed for light microscopy. Histologic examination revealed four sequential stages of expulsive hemorrhage as follows: (1) There was engorgement of the choriocapillaris. (2) Suprachoroidal effusion occurred mainly near the posterior pole. (3) As the effusion enlarged, stretching and tearing of choroidal vessels as well as tearing of the vessels and attachments at the base of the ciliary body occurred. (4) Massive extravasation of blood, primarily from the torn vessels at the ciliary body base, resulted in suprachoroidal hemorrhage and expulsion of blood through the surgical wound. This experimental model may provide new information relating to the cause and prevention of expulsive choroidal hemorrhage.

Oral acyclovir reduces the incidence of recurrent herpes simplex keratitis in rabbits after penetrating keratoplasty.

To determine if acyclovir sodium prevents postoperative herpes simplex virus type 1 (HSV-1) recurrences, 21 rabbits harboring latent HSV-1 underwent uniocular autograft penetrating keratoplasty. All operated-on eyes were treated with topical and subconjunctival dexamethasone sodium phosphate. Ten of the 21 rabbits also received oral acyclovir (intravenous acyclovir was given at the time of surgery). Postoperatively, 9 (82%) of 11 operated-on eyes in rabbits not treated with acyclovir had positive HSV-1 ocular cultures. In acyclovir-treated rabbits, however, none of the 10 operated-on eyes had positive ocular cultures. In addition, 9 (82%) of 11 of the operated-on eyes had geographic ulcers develop in the non-acyclovir-treated rabbits, compared with 1 (10%) of 10 in the acyclovir-treated rabbits. Finally, stromal keratitis appeared in 5 (56%) of 9 of the operated-on eyes in non-acyclovir-treated rabbits and 1 (12%) of 8 of the operated-on eyes in acyclovir-treated rabbits. The results of this study indicate that acyclovir significantly lowered the incidence of HSV-1 ocular shedding, geographic ulceration, and stromal keratitis in a rabbit autograft penetrating keratoplasty model.

Characterization of ligand binding to mitochondrial benzodiazepine receptors.

We have evaluated the affinity and density of binding sites for [3H]Ro5-4864 and [3H]PK11195 in intact and fragmented rat kidney mitochondria. These sites are known as peripheral-type or mitochondrial benzodiazepine receptors (MBR) and the preceding paper provided evidence that they function in vitro as modulators of mitochondrial respiratory control (1). In this report, MBR density, localization, and ligand specificity were investigated. In intact mitochondria, there were approximately the same number of binding sites for [3H]PK11195 as for [3H]Ro5-4864, and their apparent Kd values were identical. However, in mitochondrial fragments, there were 80% more binding sites for [3H]Ro5-4864 than for [3H]PK01195. Rat kidney mitochondria were fractionated by decompression and digitonin-based methods into outer and inner membrane-containing fractions before and after incorporation of the MBR-specific photoaffinity ligand [3H]PK14105. Assays of selective mitochondrial membrane markers and [3H]Ro5-4864 binding or specifically bound [3H]PK14105 revealed that the receptors were found in the mitochondrial outer membrane. We also evaluated the binding of a large number of structurally and pharmacologically diverse compounds to MBR by studying their ability to inhibit the binding of both 3H-ligands. These compounds had affinities ranging from 0.015 to 100 microM and, with a few exceptions, were similar in their abilities to bind to MBR in intact and fragmented mitochondria. However, there was considerable variation in the ratios between drug potencies at displacing [3H]Ro5-4864 and [3H]PK11195. This represents a new form of evidence that these two radioligands do not label identical sites on the receptor. Thirteen of the drugs, including [3H]Ro5-4864 and [3H]PK11195, were analyzed as to the nature of the inhibition and, with only two exceptions, were competitive inhibitors. One drug, Konig's polyanion, was uncompetitive whereas the other, cyclosporin A, was a noncompetitive inhibitor. These studies revealed several new classes of MBR ligands and suggest that the relationship between ligand structure and binding affinity is highly complex.

Mitochondrial benzodiazepine receptors mediate inhibition of mitochondrial respiratory control.

Drugs that bound to the peripheral-type or mitochondrial benzodiazepine receptors in rat kidney mitochondria produced several effects on mitochondrial respiration with succinate and malate/pyruvate as substrates. These drugs increased state IV and decreased state III respiration rates, which resulted in a significant decrease in the respiratory control ratio. ADP: O ratios were not affected. The receptor binding affinities of a set of 10 compounds (Ro5-4864, PK11195, diazepam, mesoporphyrin IX, flunitrazepam, deuteroporphyrin IX, dipyridamole, dibutylphthalate, cyclosporin A, and CL259,763) correlated over a concentration range of almost 4 orders of magnitude with their potencies at inhibiting respiratory control (r = 0.95). The anxiolytic benzodiazepine clonazepam had no effect on mitochondrial respiratory control and bound with negligible affinity to the receptor. The magnitude of the effect of Ro5-4865 on respiration increased in parallel with the density of mitochondrial benzodiazepine receptors in mitochondria from liver, kidney, and adrenal. These results suggest that ligand binding to mitochondrial benzodiazepine receptors results in inhibition of mitochondrial respiratory control. This effect may help to explain the pleiotropic effects of receptor ligands on intact cells.

A 'dot-immunobinding assay' on nitrocellulose membrane for the determination of the immunoglobulin class of mouse monoclonal antibodies.

A rapid and convenient procedure for the determination of the immunoglobulin class of mouse monoclonal antibodies is described. Hybridoma supernatants or other dilute solutions of monoclonal antibodies are spotted onto nitrocellulose membrane strips, and each strip is incubated with a different rabbit antiserum specific for a mouse Ig class. The strips are then incubated with protein A conjugated to peroxidase, and finally with the peroxidase substrate 4-chloro-1-naphthol. A positive reaction produces a dark blue spot. The procedure is sensitive, economical, fast, and safe. Many monoclonal antibodies can be tested at one time, and most of the reagents can be re-used. The procedure has been applied to a new series of monoclonal antibodies recognizing plasma membrane antigens of rat thymocytes.

Rat dendritic cells function as accessory cells and control the production of a soluble factor required for mitogenic responses of T lymphocytes.

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred at a ratio of only one dendritic cell per 200 periodate-treated lymphocytes. This evidence thus indicates strongly that in rats, dendritic cells--not macrophages--function as accessory cells. Further, the number of dendritic cells in a preparation governed the magnitude of the mitogenic response and was limiting in the case of unfractionated lymph node cells. In addition, when oxidized with periodate or with neuraminidase plus galactose oxidase, the dendritic cell served as a very potent indirect stimulator of untreated responder lymphocytes. Both functions of the dendritic cell appeared to lack species specificity, since mouse dendritic cells were very active when tested with rat responder lymphocytes. A soluble factor (accessory cell-replacing factor), produced by cultures of lymph node or spleen cells subjected to oxidative mitogenesis, enabled otherwise unresponsive mitogen-treated lymphocytes to respond. Dendritic cells were required for the production of this factor but may not be solely responsible for its production.

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.

A deoxyribonucleic acid-replication intermediate in the growing mosquito.

In previous experiments on growth and aging in the yellow-fever mosquito, Aedes aegypti, a low mol. wt. (500000) DNA species was found in the supernatant fraction after ultracentrifugation of homogenates of rapidly-growing larvae. This DNA species, "sDNA", constituted 30-40% of total DNA in 2-4-day-old larvae, but was less than 5% in older larvae, pupae and adults. We have now isolated and characterized sDNA and initiated experiments to determine its metabolic role. Isolated sDNA has the same physical and chemical characteristics as bulk DNA, "pDNA", and differs only in size. In CsCl isopycnic centrifugation the buoyant densities of sDNA and pDNA were 1.700 and 1.697 g/cm3 respectively. The "melting" temperature of both DNA species was 84 degrees C. Base compositions calculated from these data and other methods were 38.9 mol% of guanine-plus-cytosine for sDNA, and 38.5 for pDNA. Also, the size of newly-synthesized DNA was investigated by pulse-labelling and pulse-chase experiments. In neutral sucrose gradients the labelled DNA component after a 2h pulse had a sedimentation coefficient of about 8S, but after a 4h pulse sedimented in a broad band from 10-19S. In alkaline sucrose gradients a single peak around 7S was observed for pulse times up to 4h. After a 6h pulse and a 1 day "chase", labelled DNA species had sedimentation coefficients ranging from 10-15S in alkaline sucrose, and after a 2-day chase the values were 17-31S, similar to those of pDNA under alkaline conditions. These results suggest that sDNA represents an intermediate form in the replication of DNA in mosquito larvae.

Periodate and concanavalin A induce blast transformation of rat lymphocytes by an indirect mechanism.

When rat lymph node cells, rendered incapable of division by treatment with mitomycin C, were then reacted with sodium metaperiodate (NaIO4), they stimulated the transformation of untreated, syngeneic lymph node cells in vitro. Unequivocal evidence that untreated lymph node cells responded in this situation was obtained by means of sex chromosome karyotype analysis. In experiments in which the ratio of responder to stimulator cells was varied between the limits of pure responder and pure stimulator cells at a constant cell density, [3H]thymidine incorporation increased linearly to a maximum at a ratio of 1:1, and then decreased linearly. These results suggest a predominantly bicellular reaction in which one periodate-treated cell stimulates only one responder cell. In experiments in which stimulator cells were reacted with periodate, but not treated with mitomycin C, and then mixed at a 1:1 ratio with untreated, syngeneic lymph node cells, karyotype analysis showed that both periodate-treated and untreated lymph node cells responded in significant numbers. Lymph node cells were cultured at various cell densities under three different conditions: one group was made up entirely of cells treated with periodate; a second group consisted of a 1:1 mixture of cells treated with periodate and untreated cells; the third group contained normal cells cultured in the presence of soluble concanavalin A. Expressed as [3H]thymidine incorporated per 10(6) cells, a progressive increase was found at the lower cell densities for all three groups, and identical slopes (about 2.5) were obtained when the results were plotted on a full logarithmic scale. Mitomycin C-treated lymph node cells incubated with concanavalin A and then thoroughly washed were found to be capable of transforming untreated, syngeneic lymph node cells. Maximal [3H]thymidine incorporation also occurred at a ratio of 1:1. These results show that, similarly to periodate stimulation, mitogenic stimulation by concanavalin A probably operates through an indirect mechanism, analogous to a mixed lymphocyte reaction. It is possible that other mitogenic agents also operate indirectly.

Studies of individual amino acid residues of the decapeptide tyrocidine A by proton double-resonance difference spectroscopy in the correlation mode.

The cyclic decapeptide antibiotic tyrocidine A was studied by two relatively new methods, viz., correlation proton magnetic resonance (pmr) spectroscopy and double-resonance difference pmr spectroscopy. The correlation method of spectral accumulation provided pmr spectra of good resolution, and in addition the signal-to-noise ratio achieved per unit time of accumulation was much higher than that achieved by use of the conventional continuous wave (cw) method. Furthermore, when protonated solvents are used, the correlation mode of accumulation has a distinct advantage over pulse and fast Fourier transform (fft) methods currently in use. Double-resonance difference (drd) spectra of individual amino acid residues in tyrocidine A were obtained by the correlation method when the decoupling frequency was maintained at the center frequency of the appropriate C-alpha proton multiplet and at a level of power that totally decoupled vicinal C-alpha and C-beta protons; the resolution of these spectra was good, and the signal-to-noise ratio was high. The distinct patterns and spectral positions of the drd spectra were characteristic of the particular type of amino acid residue and, therefore, could be used as the basis for making assignments. Furthermore, the drd spectra revealed the spectral positions of individual C-alpha and C-beta proton transitions and therefore, upon spectral analysis, could provide the chemical shifts and coupling constants of these protons. Positions of transitions were revealed even though they were hidden by overlap in the corresponding conventional single- or double-resonance spectra.