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Biomedical subjects

B M Gebhardt

Publications and source records attributed to B M Gebhardt.

At least 37 records · Page 2Linked to original sources
PubMed1997

Nerve growth factor antibody stimulates reactivation of ocular herpes simplex virus type 1 in latently infected rabbits.

Anti-nerve growth factor (anti-NGF) antibody has been shown to induce reactivation of latent herpes simplex virus type 1 (HSV-1) in vitro. We found that systemically administered anti-NGF induces ocular shedding of HSV-1 in vivo in rabbits harboring latent virus. Rabbits in which HSV-1 latency had been established were given intravenous injections of goat anti-NGF serum daily for 10 days beginning 42 days after primary viral infection. Tears were assayed for virus for 12 days beginning on the day of the first injection. All eight rabbits given high titer anti-NGF had infectious virus in their tears at least once during the 12-day period. Fifteen of 16 eyes were positive and the average duration of viral shedding for these eyes was 4.0 days. Latently infected rabbits receiving daily injections of nonimmune goat serum or saline for 10 consecutive days were controls. Only six of the 16 (38%) eyes from rabbits receiving nonimmune goat serum shed virus. Only one of 12 eyes from untreated rabbits shed virus. Sera from control rabbits had no detectable anti-NGF activity; titers in anti-NGF-treated rabbits ranged between 1:1000 and 1:10,000. NGF deprivation may act as a neuronal stressor and may share a common second messenger pathway with heat- or cold-stress induced reactivation of latent HSV-1.

Animals↗
PubMed1996

Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1.

Following ocular infection, herpes simplex virus type 1 (HSV-1) establishes latency in trigeminal ganglion (TG) neurons. Using reverse transcription-PCR, cytokine gene expression was analyzed in the TGs of mice infected with HSV-1. IL-2, TNF-alpha, IFN-gamma, IL-10, and RANTES mRNAs were readily detected in TGs taken from mice 7 days postinoculation (PI). Likewise, IL-2, IL-6, IL-10, and IFN-gamma protein were detected by ELISA of TG homogenates. Between 5 and 45 days PI, IL-10, IFN-gamma, TNF-alpha, and RANTES mRNAs were detected in nearly 100% of latently infected TGs (latent infection was confirmed by reverse transcription-PCR detection of HSV-1 latency-associated transcripts). T cell-associated cytokine and chemokine mRNAs (IL-2, IL-10, IFN-gamma, and RANTES) were still detected in the majority of latently infected TG samples taken between 60 and 135 days PI. In contrast, these cytokine mRNA species were rarely detected in uninfected TGs. Measurement of serum Abs to HSV-1 at different times revealed that anti-HSV-1 Ab concentrations approached a plateau in mice by 30 days PI but remained at high levels 67 and 125 days PI. Although there was molecular evidence of an ongoing immune response to HSV-1 in latently infected TG, histologic analysis indicated that very few mononuclear cells remained in the ganglion 60 days PI. Collectively, the results suggest that residual lymphocytes encounter viral Ag during HSV-1 latency with sufficient frequency to remain activated. The paradox of a persistent immune response against a latent infection is discussed.

Animals↗
PubMed1996

Quantitative analysis of polymerase chain reaction products by dot blot.

Quantitative analysis of polymerase chain reaction (PCR) products is usually accomplished by gel electrophoresis and Southern blotting. We have developed an alternative technique that allows PCR products to be directly quantitated from unfractionated samples. The PCR was used to amplify genomic (endogenous) DNA sequences (actin) and exogenous DNA (herpes simplex virus-1 (HSV-1) ribonucleotide reductase) isolated from the trigeminal ganglia of rabbits to demonstrate the dot blot method of PCR product analysis. Two primer pairs (actin and ribonucleotide reductase) were coamplified, resulting in two different PCR products. Duplicate aliquots of the PCR products were applied to separate nylon membranes and hybridized with 32P-labeled oligonucleotide probes. Each radioactive probe was specific for target (HSV-1 DNA) or control (actin DNA) products. Quantitation using a laser scanning PhosphorImager and ImageQuant software demonstrated that the dot blot method can be used to rapidly analyze a large number of PCR samples.

Animals↗
PubMed1996

Synthetic scleral reinforcement materials. II. Collagen types in the fibrous capsule.

Attainment of a steady state in fibrous-capsule formation around a polymeric implant is indicative of minimization of the foreign-body response, and is characterized by thin capsule walls, few macrophages, and the replacement of type III collagen with type I collagen within the capsular matrix. We implanted four general types of extraocular band materials (porous, solid, composite, and patched) in rabbit eyes, and examined the proportions of types III and I collagen in the surrounding fibrous-capsule walls at intervals after surgery. Immunohistochemical analysis showed that 18 months after implantation of the band materials, the capsules adjacent to the porous-material surfaces had virtually no type III collagen remaining, while the solid-surface-material capsules still contained more than trace amounts (greater than 5%). In that the bands were implanted around the outside of the globe, which provides a dynamic environment in constant motion, it may be that the cellular ingrowth permitted by the porous materials increased the stability of the implants, thereby lessening the foreign body response and allowing the collagen replacement characteristic of the steady state to be completed. In contrast the solid-surface implants, which had little cellular ingrowth, may have continued to undergo sufficient micromovement within the capsule walls to sustain a low-level activation of the wound-healing response, resulting in thicker capsule walls and residual type III collagen a year and a half after implantation surgery.

Animals↗
PubMed1996

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG), a thymidine kinase inhibitor, suppresses herpes virus reactivation in mice.

In cells of the nervous system, which have little or no cellular thymidine kinase, the pharmacologic inhibition of viral thymidine kinase may prevent the reactivation of herpes virus, which requires phosphorylated thymidine for replication. We tested a newly synthesized inhibitor of viral thymidine kinase, 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG) for its capacity to suppress the reactivation of herpes simplex virus type 1 (HSV-1) in vivo. Mice, latently infected with McKrae strain HSV-1, were treated with intraperitoneal injections of HBPG in a corn oil vehicle (200 mg/kg every 3 h for a total of ten doses), and subjected to hyperthermic stress to stimulate viral reactivation immediately before the third treatment. Three h after the last treatment, the mice were sacrificed, and the presence of infectious virus was determined by culture of ocular surface swabs and trigeminal ganglionic homogenates. Additionally, viral DNA in ganglionic extracts was analyzed by quantitative PCR. Controls included latently infected, stressed animals receiving injections of corn oil vehicle only, and latently infected, drug- and vehicle-treated, unstressed animals. HBPG had a statistically significant inhibitory effect on hyperthermia-induced viral reactivation. Homogenates of trigeminal ganglia and ocular surface swabs from HBPG-treated animals were less likely to contain infectious virus than those of infected, vehicle-treated, stressed controls (P < 0.005, ANOVA). Unstressed controls showed no reactivation. Quantitation of viral DNA in ganglionic extracts demonstrated a 100-fold reduction in the amount of viral DNA in the ganglia of HBPG-treated animals, compared with vehicle-treated controls (P < 0.05, ANOVA). The results indicate that HBPG has an inhibitory effect when given systemically for the suppression of herpes virus reactivation in mice.

Animals↗
PubMed1996

Effect of 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG), a thymidine kinase inhibitor, on clinical recurrences of ocular herpetic keratitis in squirrel monkeys.

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG) is a new viral thymidine kinase inhibitor that we tested for the ability to prevent recurrences of herpetic keratitis. Eighteen squirrel monkeys (Saimiri scuireus) were infected in both corneas with the Rodanus strain of herpes simplex virus type 1 (HSV-1). All corneas showed typical dendritic keratitis 3 days after infection, followed by spontaneous healing. On day 21, the monkeys were randomized into two coded groups and ocular examinations were begun. One group received intraperitoneal (i.p.) injections of HBPG, 150 mg/kg, in a corn oil suspension every 8 h, and the other group received i.p. injections of the corn oil vehicle only. On day 22, recurrences were induced by reducing the temperature of the room in the late afternoon so that a low of 18 degrees C was achieved during the night. After the morning treatment, room temperature was raised to the normal ambient temperature (24-27 degrees C), and treatment was discontinued. Treatment was reinstituted on day 27, the room temperature was lowered again on day 28, and treatment was again discontinued as before. Third and fourth cycles of treatment and cold stress were begun on days 34 and 69. Ocular examinations were continued until day 73, at which point the code was broken. We found that the HBPG treatment significantly reduced the number of corneas with recurrences during the treatment periods, compared with recurrences in untreated, cold-stressed animals (P = 0.01).

Animals↗
PubMed1996

Quantitation of herpes simplex virus type 1 DNA and latency-associated transcripts in rabbit trigeminal ganglia demonstrates a stable reservoir of viral nucleic acids during latency.

In this investigation we determined the dynamics of herpes simplex virus type 1 (HSV-1) DNA and latency-associated transcripts (LAT) in the latently infected rabbit trigeminal ganglion. Rabbit eyes were infected with either the McKrae strain or the l7Syn+ strain of HSV-1. Rabbits were sacrificed between 5 and 360 days after infection and their trigeminal ganglia were analyzed for the number of HSV DNA genomes and the number of neuronal cells expressing LAT. There was no statistically significant change in the number of HSV genomes or the number of neuronal cells expressing LAT in these ganglia between 20 and 360 days after infection. For both strains, the amount of HSV DNA averaged 16.8 genomes per 100 cells, and 9.2% of the neurons expressed LAT. There were 17 to 34 HSV genomes per LAT-expressing neuronal cell. The number of LAT-expressing neurons did not change over the 360 days. Spontaneous reactivation (HSV-1 recovery in tear film) and recurrence (HSV-1-specific epithelial lesions) occurred during the period of this study; however, these events did not alter the quantity of HSV-1 DNA or the number of LAT-expressing cells. These results suggest that after the latent infection is established, the viral DNA in the ganglia does not replicate to any measurable extent over long periods of latency, since no significant change in the number of HSV genomes occurs. The results also suggest that only a very small number of latently infected neuronal cells are needed to produce infectious HSV-1 during reactivation.

Actins↗
PubMed1996

Mechanisms of herpes simplex virus type 1 reactivation.

Primary cultures of trigeminal ganglion (TG) cells from herpes simplex virus type 1 (HSV-1) latently infected mice were used to study reactivation. Expression of HSV-1 latency-associated transcripts was noted in TG cell cultures. Infectious virus appeared in 75% of culture supernatants within 120 h after heat stress. Likewise, HSV-1 lytic-phase mRNA and proteins were detectable 24 h after heat stress. HSV-1 antigen first appeared in neurons after heat stress, indicating the neurons were the source of reactivation. The effect of heat stress duration on reactivation was determined. Reactivation occurred in 0, 40, or 67% of cultures after a 1-, 2-, or 3-h heat stress, respectively. However, 72-kDa heat shock protein expression was induced regardless of heat stress duration. Thus, reactivation was not a direct result of inducing the heat shock response. The capacities of several drugs to induce reactivation were also evaluated. While neither epinephrine, forskolin, nor a membrane-permeable cyclic AMP analog induced reactivation, dexamethasone did so in a dose-dependent manner. Furthermore, dexamethasone pretreatment enhanced the kinetics of heat stress-induced reactivation from TG cells. Collectively, the results indicate that TG cell cultures mimic important aspects of in vivo latency and reactivation. Therefore, this model may be useful for studying signalling pathways that lead to HSV-1 reactivation.

Animals↗
PubMed1996

Propranolol suppression of ocular HSV-1 recurrence and associated corneal lesions following spontaneous reactivation in the rabbit.

PURPOSE: Hyperthermia has been shown to induce HSV-1 ocular shedding in mice. Systemic administration of propranolol significantly reduced the recovery of infectious virus in the tears, cornea, and trigeminal ganglia of mice subjected to hyperthermia. The present study was performed to determine the effects of systemic propranolol on ocular shedding and recurrent corneal epithelial lesions in the rabbit model. METHODS: New Zealand white rabbits were infected with HSV-1 strain McKrae or 17Syn+. After latency was established, the animals were treated with systemic propranolol or saline (control) and examined by slit lamp biomicroscopy for corneal lesion. Tear film swabs were cultured to determine the frequency and duration of viral shedding. RESULTS: Propranolol caused a significant reduction in the frequency and duration of ocular HSV-1 shedding and a reduction in the frequency of recurrent corneal epithelia disease, compared with saline treatment. CONCLUSIONS: These results suggest that beta-adrenergic receptor blockers such as propranolol could be useful in suppressing HSV-1 ocular recurrences and corneal disease.

Adrenergic beta-Antagonists↗
PubMed1995

Cellular mechanisms involved in morphine-mediated suppression of CTL activity.

Based on a plethora of data from many laboratories, we have proposed the following mechanisms by which morphine alters immune homeostasis and immunocompetence in vivo (Fig. 2). Specifically, the administration of morphine subcutaneously via routing through blood interacts directly with opioid receptors on cells of the immune system or on receptors within the central nervous system. Although there is currently no evidence to support the direct involvement of morphine on lymphocyte opioid receptors, in vitro studies show the existence of functional, naloxone-sensitive opioid receptors (25). In addition, pharmacological and biochemical characterization of lymphocyte opioid receptors has been shown to be consistent in many instances, with the profile of neural-derived opioid receptors (25-27). Finally, recent molecular studies using oligonucleotide primers specific for the delta-class opioid receptor cloned from NG-108-15 cells (28) have been used in reverse transcription-polymerase chain reactions to generate a 400 bp product in SL which has 100% sequence homology with a published opioid receptor cloned from a brain library (35). However, future studies are necessary to establish the role of lymphocyte opioid receptors following the in vivo administration of opioids (e.g. fentanyl, methadone, and morphine). Since the administration of morphine subcutaneously appears to predominately interact with brain opioid receptors (3) located in the mesencephalon (5), other neuroendocrine systems become candidates for activation and subsequent direct modulation of immune function: (i) the HPA axis and (ii) the sympathetic nervous system (SNS).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Functional role and sequence analysis of a lymphocyte orphan opioid receptor.

Pharmacological evidence indicates that lymphocytes express opioid receptors, but this finding has been questioned. By DNA sequencing of reverse transcription-polymerase chain reaction products, we have found that mouse lymphocytes express mRNA encoding an orphan opioid receptor. These mRNA transcripts were detected in the CD4+, CD8+, and CD4- CD8- lymphocyte subpopulations. Northern blot analysis confirmed that splenic lymphocytes express a 1.5-kb orphan opioid receptor mRNA. Fifteen bases encoding Tyr71-Arg75 in the first intracellular loop are alternatively spliced, suggesting that orphan opioid receptor mRNA encodes two receptor subtypes. Treatment of lipopolysaccharide-stimulated lymphocytes with orphan opioid receptor antisense oligonucleotides suppressed polyclonal IgG and IgM production by 50%. Our results provide direct evidence that lymphocytes express an opioid-like receptor gene, and suggest that this receptor plays a functional role in immunocompetence.

Amino Acid Sequence↗
PubMed1995

Propranolol suppresses reactivation of herpesvirus.

Herpes simplex virus type 1 (HSV-1) reactivates from the nervous system and causes recurrent disease in end organs such as the eye and the lips. We found that the beta-adrenergic receptor blocker, propranolol, reduces HSV-1 reactivation in an animal model. Mice latent for McKrae strain HSV-1 were injected with propranolol or saline once a day for 3 successive days, and subjected to a brief period of hyperthermia on the second day to induce reactivation. Twenty-four hours after the third injection, swabs of the ocular surface and homogenates of the corneas and trigeminal ganglia were analyzed for the presence of infectious virus and viral DNA. Treatment with propranolol significantly decreased the appearance of infectious virus in the tear film, cornea, and trigeminal ganglia (P < 0.05, chi 2-test). The results suggest a possible new pharmacologic approach to suppressing herpesvirus reactivation in the nervous system and thereby preventing recurrent disease.

Adrenergic beta-Antagonists↗
PubMed1995

Collagen as a delivery system for hydrophobic drugs: studies with cyclosporine.

The time-honored approach to delivering drugs to the ocular surface is through the use of liquid drops and semisolid ointments. Such delivery systems, however, are not efficient at delivering therapeutic concentrations of drugs to the cornea and intraocularly. Over the past several years, we tested the biopolymer, collagen, as a means of delivering both hydrophilic and hydrophobic drugs to the ocular surface. This study summarizes results obtained using the hydrophobic drug, cyclosporine, incorporated into collagen shields and collagen particles. Corn oil drops containing cyclosporine were used as the control. Groups of anesthetized rabbits were fitted with collagen shields containing cyclosporine, treated topically with collagen particles containing cyclosporine suspended in an ocular surface lubricant, or given topical drops of corn oil containing cyclosporine. At intervals after a single treatment with one of the drug formulations, corneas, aqueous humor, and blood were collected for analysis of cyclosporine concentration. With either of the two collagen vehicles, peak concentrations of the drug were found in the cornea 4 hours after application. The corn oil vehicle yielded a significantly lower and earlier peak concentration (1 hour after application). By 8 hours, significant amounts of the drug were still present in the corneas of the collagen-treated animals, whereas drug levels in the corn oil treatment group had returned to baseline. Drug delivery profiles in the aqueous humor were similar, except that the amounts of drug were five times lower. No cyclosporine was detected in the blood from any of the treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cyclosporine in collagen particles: corneal penetration and suppression of allograft rejection.

The purpose of this study was to determine the pharmacokinetics of cyclosporine in the cornea and aqueous humor following topical application in collagen particles and to determine the capacity of cyclosporine in collagen particles to suppress corneal allograft rejection. Cyclosporine was prepared for topical application in three vehicles: collagen particles suspended in methylcellulose, collagen shields, and corn oil. Rabbit eyes were treated with a single application of one formulation. Corneas, aqueous humor samples, and blood samples were collected at 2, 4, 8, 12, and 24 hours. Peak concentrations of drug were reached between 2 and 4 hours in the cornea and between 4 and 8 hours in the aqueous humor. No drug was found in the blood. Collagen particles and shields produced significantly higher concentrations of cyclosporine in the aqueous humor and cornea, compared with corn oil drops, at virtually all time points. In the second part of the study, rabbits with vascularized corneas underwent corneal transplantation and were treated with one of the formulations beginning either at the time of transplantation (prophylactically) or at the first sign of an allograft immune reaction (delayed treatment). Cyclosporine delivered in collagen particles was effective in preventing primary corneal allograft rejection in nearly all of the grafts (96%) treated prophylactically, and in 67% of the grafts treated when signs of the first stage (grade 1) of immune graft reaction were observed. Grafts treated with cyclosporine in collagen shields showed similar survival rates (87% and 64%, respectively), whereas only 40% of the grafts treated prophylactically and only one (18%) receiving delayed treatment with the corn oil preparation survived. These results demonstrate the efficacy of collagen particles for delivery of hydrophobic drugs to the ocular surface and the potential for preventing corneal graft rejection with topically applied cyclosporine in a collagen-particle vehicle.

Administration, Topical↗
PubMed1995

Confocal microscopy of corneal graft rejection.

Corneal allografts were transplanted into inflamed and vascularized graft beds in rabbit eyes. The grafts were examined every 4 days by slit-lamp biomicroscopy and scanning confocal microscopy. Confocal images were recorded with a video camera and computer enhanced in real-time. Layers of the cornea were visualized in serial optical sections parallel to the epithelium. In the third postoperative week, signs of graft rejection were observed; slit-lamp examination revealed a circumferential line of epithelial rejection, along with cloudiness and edema. Vessels were observed growing into the graft. By confocal microscopy, infiltrating cells were seen in the graft stroma. Foci of cells were especially pronounced around the sutures. Scattered leukocyte infiltrates were prominent at capillary terminals. There was an accompanying reduction in the stromal keratocyte density in the region of the infiltrate. Additionally, various degrees of fibrosis were noted around each suture and at the host-graft interface. Confocal microscopy may provide a valuable clinical tool for determining the earliest indicators of an antigraft immune response, and as an aid in the differential diagnosis of other inflammatory conditions of the cornea.

Animals↗
PubMed1994

Chronic morphine treatment suppresses CTL-mediated cytolysis, granulation, and cAMP responses to alloantigen.

Exposure to opioid drugs (e.g., morphine) in vivo has been shown to suppress natural killer cell activity. However, the effects of in vivo exposure to opioids on cytotoxic T lymphocyte (CTL) activity has not been investigated. The administration of morphine (50.0 mg/kg, sc) to alloimmunized mice for 11 days resulted in a significant decrease in peritoneal and splenic CTL activity. Moreover, the intracellular content of serine esterases and esterase release by CD8+ effector cells from chronic morphine-treated mice was reduced compared to that of effector cells from vehicle-treated controls. In addition, the CD8+ cAMP response to alloantigen was diminished compared to CD(8+)-enriched cells from vehicle-treated animals. However, conjugate formation between effector and target and subsequent killing of target by effector cells did not reveal significant differences between vehicle- and chronic morphine-treated animals. Serum corticosterone and dehydroepiandrosterone levels were significantly lower in the chronic morphine-treated animals while proopiomelanocortin gene expression (exon 3) in splenic lymphocytes did not correlate with morphine-mediated suppression of CTL activity. These results indicate that CTL activity is sensitive to chronic morphine exposure, implicating opioids as important cofactors during viral infections in suppressing cell-mediated immunity.

Animals↗