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Biomedical subjects

D Pavan-Langston

Publications and source records attributed to D Pavan-Langston.

At least 37 records · Page 2Linked to original sources

Detection of herpes simplex virus thymidine kinase and latency-associated transcript gene sequences in human herpetic corneas by polymerase chain reaction amplification.

Herpes simplex virus (HSV) latency in sensory ganglion neurons is well documented, but the existence of extraneuronal corneal latency is less well defined. To investigate the possibility of extraneuronal latency during ocular HSV infection, corneal specimens from 18 patients with quiescent herpes simplex keratitis (HSK) were obtained at the time of keratoplasty. Polymerase chain reaction (PCR) amplification followed by southern blot hybridization with a radiolabeled oligonucleotide probe was done to detect the presence of HSV-1 genome in these human corneal samples. Two pairs of oligonucleotides from the region of the HSV thymidine kinase (TK) gene and the latency-associated transcript (LAT) gene were used as primers in the PCR amplification. The DNA sequences from either the TK or the LAT gene were identified in 15 of 18 HSK corneas (83%). These results demonstrate that the HSV genome was retained, at least in part, in human corneas during quiescent HSV infection, giving further support to the concept of corneal extraneuronal latency.

Adult↗

Multicenter clinical evaluation of the Du Pont Herpchek HSV ELISA, a new rapid diagnostic test for the direct detection of herpes simplex virus.

A new 4h rapid enzyme-immunoassay for direct detection of herpes simplex virus (HSV) antigen (Du Pont Herpchek) was evaluated with 743 clinical samples collected at obstetrics and gynecology (OB/GYN), sexually transmitted diseases (STD), and ophthalmology clinics. The sensitivity and specificity of Herpchek was 98.0% and 98.4% respectively compared to virus isolation in cell culture. Confirmatory blocking ELISA tests, clinical history and follow up indicate that the true specificity of the test is 100%.

Adult↗

Ocular varicella-zoster virus infection in the guinea pig. A new in vivo model.

Corneal intrastromal inoculation of guinea pigs with approximately 10(4) plaque-forming units of live, adapted varicella-zoster virus (VZV) resulted in reproducible, acute, superficial corneal disease in all animals. The culture-positive VZV ocular infection progressed to involve 30% to 40% of the corneal surface in a diffuse punctate keratitis and 10% to 15% of this surface with microdendrites, characteristic of VZV-induced ocular disease. Retrograde dissemination of VZV to the trigeminal ganglia, midbrain, cerebellum, and superior cervical ganglia was demonstrated by whole-cell coculture VZV recovery. Central nervous system VZV dissemination, manifested by transient neurologic symptoms and pneumonitis, was evident in 60% of the animals. Varicella-zoster virus spread to the trigeminal ganglion during acute and early-latent infection was evident by electron microscopy.

Animals↗

Extraneuronal herpetic latency: animal and human corneal studies.

HSV DNA has been previously detected by both in situ and dot blot hybridization in neuronal tissues latently infected with herpes simplex virus (HSV), but not in extraneuronal tissues. The present study, using dot blot hybridization with a cloned full-length HSV DNA probe and subtractive hybridization assays for detecting HSV RNA, reveals both the presence and activity of the HSV genome in 100% of HSV latently infected rabbit corneas tested. Studies on human herpetic corneas taken at keratoplasty using slot blot hybridization with a cloned full-length HSV DNA probe demonstrated positive binding (hybridization) to the probe in 50% of samples tested but no binding to normal human control DNA. These studies confirm earlier, less sensitive virus recovery assays and implicate the cornea as an extraneuronal site of HSV latency and reactivation.

Adult↗

Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease.

This study evaluated the continued presence of herpes simplex virus (HSV) nucleic acid sequences after resolution of acute herpetic stromal keratitis in the rabbit ocular model. Forty-four rabbits were inoculated bilaterally with 10(5) plaque-forming units of RE strain HSV-1 by intrastromal injection. All eyes were cultured for the presence of HSV during acute disease and immediately before the animals were killed. Full-thickness corneal buttons were then removed and processed for in situ hybridization with a 3H-labelled HSV DNA probe representing the full-length HSV genome. HSV nucleic acid sequences were detected autoradiographically at all time intervals examined. HSV nucleic acid sequences were localized in the epithelium and the anterior stromal keratocytes during acute disease and in all corneal layers during latent infection. Retention of HSV nucleic acid sequences, either HSV DNA or HSV RNA, or both, in corneal tissues (epithelium, stroma, and endothelium) may be a contributing factor in the development of HSV-induced stromal keratitis.

Animals↗

An in vitro study of ophthalmic antiviral agent toxicity on rabbit corneal epithelium.

Using an in vitro system we measured the corneal epithelial cytotoxicity and the antiviral activity of the antiviral agents idoxuridine (IDU), trifluridine (TFT), ethyldeoxyuridine (EDU), and (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU). Confluent rabbit corneal epithelial cell cultures were established, and the antiviral agents were added for 5, 30, or 60 min at a range of concentrations including that used clinically (IDU 0.1%, TFT 1.0%, BVDU 0.1%, EDU 2.0%). Twelve hour [3H]thymidine incorporation then was measured and expressed as % inhibition of control cultures. In separate experiments confluent corneal epithelial cell monolayers were inoculated with 10(4) plaque forming units (PFU) of HSV type 1 (McKrae strain) for 1 h, and IDU 0.1%, TFT 1.0%, and BVDU 0.1% were added to the culture for determination of PFU inhibition. Significant dose-, but not time-dependent, toxicity was observed at the clinical concentrations of IDU, TFT, and EDU. Toxicity was absent for BVDU. TFT and IDU were the most toxic, and EDU was of intermediate toxicity. IDU, TFT, and BVDU showed significant antiviral activity in this corneal epithelial cell culture system (TFT greater than BVDU greater than IDU). The results of this in vitro study paralleled the findings of previous in vivo corneal epithelial toxicity studies of IDU, TFT, and BVDU. Our data, however, suggest that EDU has a potential for clinical toxicity and further studies are recommended. Our model may be useful in the future toxicologic study of new antiviral agents.

Animals↗

Rapid detection of herpes simplex virus (HSV) antigen in human ocular infections.

The new HERPCHEK (Dupont, No. Billerica, MA) enzyme immunosorbent assay (EIA) was used in a double-blind clinical study for rapid and specific detection of ocular herpes simplex virus (HSV) infection. This 4-hour assay can be used to demonstrate conclusively the presence of HSV antigen without culture and thereby rapidly differentiate between HSV and other clinically similar ocular infectious diseases. Ocular samples were collected from 180 individuals including 30 patients with acute HSV, 90 with latent HSV (ie, currently asymptomatic but with a positive history), 11 with acute or latent varicella zoster virus, 30 with nonherpetic infections (due to adenovirus, Acanthamoeba or bacteria), and 19 normal controls. A clinical diagnosis was made by one of us (DPL) and duplicate tear-film samples obtained by swabbing the conjunctival cul-de-sac and cornea. Coded samples were tested by routine viral culture on Vero cell monolayers and also were run independently in the HERPCHEK test. During active HSV infection, the HERPCHEK correlated 100% with clinical diagnosis, and virus culture correlated 90% with clinical diagnosis. In all latent HSV ocular infections, other nonherpetic ocular infections and normal samples, both the HERPCHEK and culture assays were negative.

Antigens, Viral↗

Molecular analysis of acyclovir-induced suppression of HSV-1 replication in rabbit corneal cells.

An in vitro HSV drug-suppression model has been established in rabbit corneal cell (SIRC) monolayers. Confluent SIRC monolayers were inoculated with McKrae strain HSV-1 (0.014 PFU/cell) and subsequently suppressed with acyclovir (ACV) (40 micrograms/ml) after adsorption for 1 hr at 37 degrees C. On days 0-5 postinoculation (PI), infectious HSV-1 was detected in approximately 50% of the SIRC cell cultures by standard supernatant and cell-free homogenate co-culture assays. On days 6-10 postsuppression, infectious HSV was detected in only 7-19% of the cultures. After removal of ACV suppression in the cultures (day 11 PI), HSV cytopathology developed to a 3-4+ level within 2-5 days. Dot blot hybridization of DNA extracted from the cultures indicated that the HSV genome was retained consistently in SIRC cells at a level of 0.0015-0.015 copies per cell during active ACV-suppression. During ACV-suppressed infection, approximately 0.9% of the SIRC cells contained the HSV genome as demonstrated by blot hybridization. After removal of ACV, a 1000-fold increase in HSV DNA concentration in SIRC cell cultures was detected.

Acyclovir↗

Effect of immunization and immunosuppression on induced ocular shedding and recovery of herpes simplex virus in infected rabbits.

Immunization and immunosuppression were evaluated during latent ocular herpes simplex virus, type 1 (HSV-1) infection in the rabbit, using the following parameters: (1) ability to recover virus from preocular tearfilm cultures; (2) reactivation of latent infection by direct electrical stimulation; and (3) recovery of virus from latently infected ganglia by whole-cell co-cultivation. Immunization prior to ocular inoculation of virus significantly reduced both the titer of virus shed into the tearfilm and the duration of virus shedding during primary ocular infection. Half of the non-immunized control rabbits died secondary to virus encephalitis, whereas none of the immunized rabbits died. The immunized rabbits could not be induced to shed virus by electrically stimulating the trigeminal ganglion directly. Immunosuppression of latently infected rabbits with high-dose cyclophosphamide (300 mg kg-1) enhanced virus shedding in the tearfilm and increased mortality due to viral encephalitis. Low-dose cyclophosphamide immunosuppression (40 mg kg-1) did not increase mortality because of viral encephalitis. Tearfilm virus shedding secondary to electrical induction in high-dose and low-dose cyclophosphamide animals was higher than that of control, non-immunosuppressed animals.

Animals↗

HSV-induced reactivation: contribution of epinephrine after corneal iontophoresis.

A modified, bilateral iontophoresis technique was used to evaluate herpes simplex virus (HSV) reactivation and epinephrine distribution and concentration in full-thickness corneas and in trigeminal ganglia of rabbits. Infectious virus was recovered from 58% of epinephrine-induced eyes 24-96 hours after iontophoresis. A histochemical adrenochrome oxidation method localized epinephrine exclusively in the corneal epithelium and anterior stromal lamellae after iontophoresis. Epinephrine introduced iontophoretically into the corneal epithelium and stroma did not reach the ganglion level as detected by the assay. Radiometric-enzymatic assay of corneal and ganglionic tissues demonstrated a transient increase in total corneal catecholamine levels from an average of 417 micrograms to an average of 778 micrograms ; concentrations returned to approximate control values 24 hours after iontophoresis. Ganglion total catecholamine levels increased from an average of 606 micrograms to an average of 1211 micrograms for a 12-hour period and remained elevated at 24 hours after iontophoresis. Epinephrine acting directly on the corneal epithelium and epinephrine metabolites at the ganglion level could act synergistically to induce ocular and ganglionic HSV reactivation.

Animals↗

Prophylactic topical cyclosporine in experimental herpetic stromal keratitis.

We determined the anti-inflammatory prophylactic effect of topical cyclosporine in a rabbit model of herpetic stromal keratitis. In a first study, we established that presensitization of rabbits prior to corneal intrastromal injection of herpes simplex virus produced a severe stromal keratitis beginning on day 7 after the intrastromal challenge. Twenty-four presensitized rabbits were thereafter divided into three groups for topical treatment five times a day: (1) 1% cyclosporine in a vehicle solution, started on day 4 after intrastromal challenge, plus 1% trifluridine started on day 7 after challenge, (2) the vehicle solution for cyclosporine, started on day 4, plus 1% trifluridine started on day 7, and (3) the vehicle solution for cyclosporine alone, started on day 4. The severity of stromal disease was significantly decreased in eyes treated with combined cyclosporine-trifluridine, whereas trifluridine alone had no effect on the stromal disease. Epithelial disease did not seem to be worse in eyes treated with combined cyclosporine-trifluridine; the incidence and duration of positive cultures, however, were increased in this group.

Animals↗

Antiviral medications and corneal wound healing.

Masked controlled rabbit studies were done to determine the toxic effects on corneal wound healing of the antiviral ointments 0.5% idoxuridine, 3% Ara A, and 3% acyclovir, and the antiviral drops 0.1% idoxuridine, 3% Ara AMP, and 1% trifluridine. Ara A, acyclovir, trifluridine and idoxuridine drops had no significant effects on the rate of closure of epithelial wounds. Idoxuridine ointment given 5 times a day significantly retarded the rate of epithelial wound closure, but not when given 4 times a day. Only Ara AMP caused a retardation of epithelial healing and an actual increase in the defect after 4 days of treatment. Histopathologically all drugs, except acyclovir, showed a toxic effect on the regenerating epithelium. All drugs, except acyclovir, showed retarded stromal wound healing with reduced bursting strength and collagen content. Ara AMP had increased bursting strength and collagen content possibly because of greater inflammation. Acyclovir, in comparison to all the other medications studied, appeared to have minimal to no toxic effects on experimental epithelial and stromal wound healing, and on this basis is the agent of choice for use in herpes simplex stromal keratitis with ulceration and as a prophylactic agent for long-term use after penetrating keratoplasty.

Animals↗

In vitro effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5'-amino-5-iodo-2',5'-dideoxyuridine and 2-deoxy-D-glucose on latent ganglionic herpes simplex virus infection.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and 5'-amino-5-iodo-2',5'-dideoxy-uridine AIdUrd, blocked the reactivation of latent ganglionic herpes simplex virus in vitro. Furthermore, BVDU, but not AIdUrd, blocked the multiplication of reactivated latent virus and transiently suppressed emergence of reactivated virus from the sensory ganglia after removal of drug from the medium. 2-Deoxy-D-glucose (2-DG) neither prevented the in vitro reactivation of latent virus nor blocked the further multiplication of reactivated latent virus.

Animals↗

Effect of combined acyclovir and vidarabine on infection with herpes simplex virus in vitro and in vivo.

Acyclovir and vidarabine exerted a less-than-additive effect on the replication of herpes simplex virus type 1 (HSV-1) in green monkey kidney cells. The synthesis of viral DNA was reduced much more (97%) than that of cellular DNA (45%) by combined acyclovir and vidarabine in cell culture. Furthermore, the combined drugs, given topically or systemically, were more effective than the individual drugs in diminishing the development of clinical signs of HSV-1 infection in hairless mice. However, mortality among animals given topical acyclovir plus vidarabine was not significantly lower than that among animals given topical acyclovir only. Systemic acyclovir (50 mg/kg per day) plus vidarabine (100 mg/kg per day), given for five consecutive days starting 24 hr after viral inoculation, significantly reduced the frequency of development of latent HSV-1 infection in the trigeminal ganglia of mice, whereas systemic acyclovir or vidarabine alone did not.

Acyclovir↗

Herpes simplex ocular disease.

The present epidemic of genital herpes has led to broad concern among both physicians and lay persons. For those who specialize in cornea and external disease, this new awareness provides a much needed opportunity to discuss ocular herpetic disease. Through ongoing medical education, great strides are being made toward early diagnosis and appropriate therapy. The primary care physician occupies a pivotal position in these efforts.

Antiviral Agents↗

Herpes simplex acyclovir-resistant mutant in experimental keratouveitis.

Acyclovir is a potent and selective antiviral agent. Unfortunately, drug-resistant (acyclovir-resistant) mutants have already been reported in herpes simplex virus type 1 (HSV-1) orofacial infections. We have developed a laboratory acyclovir-resistant HSV-1 mutant. The natural course of acyclovir-resistant HSV-1 keratitis was found to be less virulent than that observed in wild type HSV-1 keratitis, but the rate of ganglionic latency was as high as that induced by the parental strain. In vitro studies and in vivo observation of rabbit corneas infected with acyclovir-resistant HSV-1 both demonstrated a significant sensitivity to vidarabine and bromovinyldeoxyuridine ([E]-5-[2-bromovinyl]-2'-deoxyuridine). The thymidine kinase activity of the acyclovir-resistant mutant was 69% of that of the wild type HSV-1.

Acyclovir↗