Intracameral muramyl dipeptide-induced paracellular permeability associated with decreased glutamate transporter and gamma -glutamyltranspeptidase activities.

Muramyl dipeptide (MDP) (N -acetylmuramyl- L -alanyl- D - isoglutamine) was injected intracamerally to test if MDP applied to the aqueous side of the blood-aqueous barrier would increase paracellular permeability in association with diminished uptake of glutamate. The symptoms of anterior uveitis, i.e., increase in vascular dilatation, could be detected as early as 30 min post MDP injection while aqueous protein concentration did not increase at this time suggesting an initial dissociation between the circulatory and epithelial barrier responses. However, at 45 min, the aqueous protein concentration increased 10-fold (201+/-174 to 2094+/-1835 micrograms ml-1;P<0.001) rising progressively to 20-fold above the control eye at 60 min post injection (254+/-194 vs. 5038+/-2514 micrograms ml-1;P<0.001). Epithelial cell barrier paracellular permeability increased at 45 min as evidenced by the enhanced efflux of radiolabelled L -glucose out of the aqueous (8% and 13% faster than control at 45 and 60 min post MDP injection, respectively), coinciding with the accelerated protein influx. A near 50% reduction in efflux of both radiolabelled glutamate and D -aspartate was consistent with reduced glutamate uptake by the transport system X-AG. In addition, a 24% decline in aqueous glutamate, but not aspartate, was detected in the aqueous of the MDP-treated eyes in association with a 54% decrease in iris/ciliary body gamma-glutamyltranspeptidase activity consistent with reduced de novo glutamate formation from glutamine. The aqueous of MDP injected eyes also had 6-fold and 34-fold higher prostaglandin E2and F2alphaconcentrations, respectively (P

Inhibition of glutamate uptake causes an acute increase in aqueous humor protein.

Inhibition of glutamate transport has been shown to increase paracellular permeability of epithelial cell monolayers in vitro. To determine if blocking glutamate transport would affect tissue permeability in vivo, D-aspartate (D-Asp; 300 nmol 30 microliters-1) (a non-toxic competitive inhibitor of glutamate transport) or a placebo was injected into the anterior chambers of the fellow eyes of 15 adult rabbits. [14C]-L-glucose and/or [125I]-rabbit albumin were included in the injection vehicle as aqueous humor (AH) outflow markers. The specific inhibition of glutamate uptake by D-Asp was indicated by a 15% increase in AH glutamate (174 +/- 9 nmol ml-1 to 205 +/- 13 nmol ml-1; P = 0.03) at 1-1.5 hr post injection. Also, the efflux of [14C]-L-glucose and [125I]-rabbit albumin from the AH of D-Asp injected eyes was increased 22% over the placebo-injected control eyes (P < or = 0.02). Concomitantly, the total protein concentration in the AH from D-Asp injected eyes (517 +/- 35 micrograms ml-1) was 19% greater (P < 0.02) than the protein concentration in AH from placebo-injected control eyes (420 +/- 36 micrograms ml-1). In additional studies, an irreversible inhibitor of glutamate transport, threo-beta-hydroxyaspartate (THA; 30 nmol 30 microliters-1), was shown to increase the efflux of [14C]-L-glucose (22%; P < 0.05) from the anterior chamber and increase AH protein concentrations by 29% (484 +/- 112 micrograms ml-1 in control AH versus 686 +/- 117 micrograms ml-1 in THA AH, P = 0.08) at 1 hr post intracameral injection. SDS-PAGE analysis of the AH associated the protein increase in the D-Asp and THA injected eyes but not placebo-injected control eyes with a detectable increase in a 66 kDa protein (aligns with serum albumin) and several lower molecular weight (23-35 kDa) AH proteins. The results found suggest that inhibition of glutamate transport from the AH acutely increases intraocular epithelial/endothelial paracellular permeability.

Temporal association of interferon-alpha and p27 core antigen levels in sera of simian immunodeficiency virus infected monkeys.

We report the temporal association of interferon (IFN) and p27 core antigen production during experimental simian immunodeficiency virus Delta B670 (SIV) infection in rhesus monkeys. Peak serum IFN-alpha levels (10(2.8-5.0)U/ml) occurred 10 days post infection (p.i.) and peak p27 levels (3.1-34.4 ng/ml) occurred 10-14 days p.i. Acid-stable IFN-alpha (10(1.6-2.5)U/ml) was detected 3-5 days before p27 in sera from three monkeys and was detected with p27 (0.06-3.06 ng/ml) in four monkeys during the primary infection. Serum IFN-alpha and p27 levels became undetectable 24-40 days p.i. Two monkeys remained asymptomatic for SIV after the primary p27 antigenaemia, three monkeys had recrudescent (3-4 months p.i.) acid stable interferonaemias (10(1-2.5)U/ml) with p27 antigenaemias (0.06-2.7 ng/ml) that persisted until death, and two monkeys had acute SIV infections (died < or = 7 months p.i.) with persistent acid-stable interferonaemia (10(1.6-2.5)U/ml) and p27 antigenaemia (6-9 ng/ml). Our results indicate that the detection of acid-stable IFN-alpha in serum is closely associated with detection of p27 (P = 0.0001) and suggest that detection of acid-stable IFN-alpha and p27 core antigen is indicative of active SIV infection.

Inhibition of the enteroviruses that cause acute hemorrhagic conjunctivitis (AHC) by benzimidazoles; enviroxime (LY 122772) and enviradone (LY 127123).

Enviradone (EvirD, (E)-1-[(1-methylethyl) sulfonyl]-6-(1-phenyl-1-propenyl)-1 H- benzimidazole-2-amine) and Enviroxime (EvirX, 2-amino-1-(isopropyl-sulfonyl)-6-benzimidazole phenyl ketone oxime) inhibited enterovirus 70 (EV70) and coxsackievirus A24 variant (CA24v) infection of conjunctival and laryngeal cells. On average, the continuous presence of 1-3 micrograms of EvirD or EvirX/ml in cell cultures acutely infected with EV70 or CA24v inhibited virus production (> 2 log10 reduction) and 100% of the viral cytopathogenic effect (CPE). The 50% CPE inhibitory dose (ID50) for EvirD and EvirX against 11 EV70 and 15 CA24v isolates ranged from 0.01 to 0.3 microgram and 0.01-0.65 microgram/ml, respectively. The mean ID50 for EvirD and EvirX against the 26 AHC viruses was 0.17 +/- 0.12 microgram and 0.13 +/- 0.14 microgram/ml, respectively. Pretreatment for 15 min with 3 micrograms EvirX/ml or for 1-2 h with 3 micrograms EvirD/ml protected conjunctival cells against viral CPE. The cells were resistant to infection for 1-2 h at 33 and 37 degrees C after removal of EvirD and EvirX. The addition of 10 micrograms EvirD/ml up to 6 h or 10 micrograms EvirX/ml 1-2 h after low multiplicity infection inhibited viral CPE. Ten-fold less EvirD inhibited EV70 when added to glioma cells 2 h before infection than when added 2 h after infection. Our results indicate that EvirX and EvirD inhibit AHC viruses in vitro at concentrations that are not cytotoxic and suggest that EvirX or EvirD may be prove useful against AHC.

Analysis of neutralizing antibodies to Enterovirus 70 and Coxsackievirus A24 variant, levels of immunoglobulins and total protein in tears of patients with acute hemorrhagic conjunctivitis.

Tear samples were collected from 37 residents of the Dominican Republic <5 d post onset (p.o.) of symptoms (mean 1.73±1.17 d p.o.) of acute hemorrhagic conjunctivitis (AHC). Sixty-two percent (23/37) of the patients had bilateral infections. Anti-enterovirus 70 (EV70) tear neutralizing activity (TNA) (10(2->3.5) U/ml) and anti-Coxsackievirus A24 variant (CA24v) TNA (10(<1-3) U/ml), but no anti-poliovirus (PV) TNA was detected. The anti-EV70 TNA in pooled tear samples sedimented in sucrose density gradient fractions that corresponded to 19-7S serum anti-PV immunoglobulin (1g). Anti-CA24v TNA sedimented as 7S1g. 1gG levels (mean, 3.13±4.2mg/ml) were higher than 1gA levels (mean, 0.92±0.98 mg/ml) in 21 of 27 tear samples. 1gG levels in tears from six patients with bilateral AHC were associated with total tear protein (p=0.003), but not with the levels of TNA or interferon (IFN). The total protein in AHC tears (5.13±1.72 mg/ml) was two-fold less than the total protein in normal tears (11.2±3.25 mg/ml). 1gA levels increased from 0.31±.3 to 1.34±1.28 mg/ml in tears collected up to 3 d p.o. of AHC. 1gM was not detected (<0.01 mg/ml). EV70 was isolated from the tears of one patient. Taken together, our results suggest that EV70 and CA24v are endemic in the Dominican Republic and that the 1992 epidemic of AHC was due to EV70. The detection of 19S (IgM) and high levels of 7S (IgG) TNA to EV70<1 d p.o. of AHC indicate a rapid ocular immune response to EV70 and suggests that virus-specific TNAs inhibit AHC virus infection.

Acute hemorrhagic conjunctivitis.

Acute hemorrhagic conjunctivitis, an infection caused by enterovirus 70 and a variant of coxsackievirus A24, is characterized by the rapid onset of severely painful conjunctivitis and subconjunctival hemorrhage. The condition is usually benign and resolves in five to seven days; however, a polio-like paralysis (radiculomyelitis) develops in approximately one in 10,000 patients infected with enterovirus 70. No treatment is available. Information about acute hemorrhagic conjunctivitis should be provided to patients and the community in order to prevent undue alarm, discourage home remedies and control the spread of this highly contagious disease.

Antibodies may act synergistically or additively with interferon to inhibit poliovirus.

We investigated the protective effects of combinations of interferon-alpha (IFN-alpha) and neutralizing monoclonal antibodies (mAbs) and serum antibodies against poliovirus type 1 (PV-1) in vitro. Our results indicate that the antiviral effects of IFN-alpha and most neutralizing mAbs to PV-1 act synergistically to inhibit PV-1. However, the antiviral effects of IFN-alpha and one type specific mAb to PV-1 were additive. Further, the protective effects observed with combinations of IFN-alpha and rabbit, monkey or human serum containing effects observed with combinations strains Mahoney (Mah) and Sabin (Sab) were similar to those observed with combinations of IFN-alpha and mixtures of mAbs with synergistic and additive activities. Our studies suggest that the antiviral activity of neutralizing antibody acts with the antiviral activity of IFN to inhibit virus infection synergistically or additively and that the different antibody activities are associated with the mechanism of neutralization.

An assay for the detection of interferon dependent and interferon independent antibody activities.

A spectrophotometric assay is described for the detection of interferon (IFN) dependent antibody (IDA) activity (i.e., antibodies that act with IFN to synergistically inhibit virus infection) and IFN independent antibody (IIA) activity (i.e., antibodies that act additively with IFN to inhibit virus infection). Four neutralizing monoclonal antibodies (mAb) against poliovirus type 1 (PV-1) were tested. Three mAb exhibited IDA activity and one mAb exhibited IIA activity against PV-1 strain Sabin. Concomitantly, the respective IDA and IIA activities were confirmed by a yield reduction assay. Also, the spectrophotometric assay detected IIA activity against PV-1 and IDA activity against PV-2 and PV-3 in human serum. Interestingly, antibody to a synthetic peptide of PV-1 capsid protein VP2 exhibited IIA activity against PV-2 strain MEF. Thus, this assay can facilitate the identification and investigation of IDA and IIA activities. Further, the assay adds economic feasibility to studying the natural occurrence of these antibody activities in the general population and can be useful in assessing the therapeutic potential of vaccine induced and hyperimmune antibodies.

Interferon-gamma can remain on the cell surface during the induction of the antiviral state.

The antiviral activity of cell-associated, non-elutable recombinant human gamma interferon (rHuIFN-gamma) was neutralized by antibody. The neutralization of cell-associated rHuIFN-gamma was maximal through 2 h (60-100%) and declined through 8 h (20-40%). Concomitantly, the antiviral activity of cell-associated [Met-Gln-Asp-Pro]-rHuIFN-gamma was sensitive to trypsin digestion over the same time period. However, the cell-associated antiviral activity of [Cys-Tyr-Cys]-rHuIFN-gamma remained sensitive to trypsin through 8 h. Neutralization of cell-associated rHuIFN-gamma by antibodies to the N-terminal end of HuIFN-gamma suggests that the N-terminal end(s) of cell-associated rHuIFN-gamma is directed outward from the receptor. Further, immunoprecipitation of radio-labelled rHuIFN-gamma by antibody alone suggests that biologically active rHuIFN-gamma is an oligomer. Taken together, these studies suggest that neutralization of cell-associated rHuIFN-gamma is probably due to divalent binding of antibody to or between rHuIFN-gamma in receptors on the cell surface. Also, our studies indicate that rHuIFN-gamma can remain associated with the cell surface during the induction of the antiviral state (AVS) and that binding of antibody to cell-associated rHuIFN-gamma inhibits the molecular events responsible for induction of the AVS.

Cytopathogenicity, drug susceptibility, and thymidine kinase activity of a retinovirulent herpes simplex virus type 2.

We investigated some of the biological and biochemical characteristics of a neuroinvasive, retinovirulent herpes simplex virus type 2 strain SL (HSV-2[SL]) and compared them with those of a neurovirulent, nonretinovirulent HSV-2 (186). HSV-2(SL) was shown to spread rapidly and produce large syncytium in vitro. HSV-2(SL) and HSV-2(186) were equally susceptible to acyclovir (ACV) and thymine arabinoside (Ara-T). However, HSV-2(SL) was fourfold and 44-fold more susceptible than HSV-2(186) to iododeoxyuridine (IUdR) and bromovinyldeoxyuridine (BVDU), respectively. In addition, cytosolic TK from HSV-2(SL)-infected cells phosphorylated 4, 20, and 23,000 times more IUdR, iododeoxycytidine (IdCyD), and Ara-T than the TK of HSV-2(186), respectively. Further, HSV-2(186) TK did not phosphorylate Ara-T, but HSV-2(186) replication was inhibited by Ara-T. These studies indicate that the retinovirulent HSV-2(SL) has a syn phenotype and a TK with broad substrate activity.

Isolation of a herpes simplex virus type 2 that is retinovirulent in mice.

The virulence of a herpes simplex virus type 2 (HSV-2) isolated from the urine of a patient (SL) with acquired immunodeficiency syndrome (AIDS) and bilateral acute retinal necrosis (ARN), was investigated in mice. The ratio of plaque forming units (PFU) in fibroblasts to the 50% lethal dose (LD50) of HSV-2(SL) in mice was 10 fold more than the PFU to LD50 ratio of a neurovirulent HSV-2, strain 186. Further, HSV-2(SL) caused retinitis with and without lethal encephalitis in mice inoculated intracranially (i.c.). In contrast, mice inoculated with HSV-2(186) died of encephalitis without ocular disease. HSV-2(SL) was isolated from eye and/or brain tissue 1 to 15 days post i.c. inoculation. Ocular disease progressed from an initial mild chorioretinitis on day 8 to total retinal necrosis with panuveitis by day 11 in mice given 10 PFU of HSV-2(SL) i.c. HSV antigen was detected initially in the cells of the optic nerve and spread into the ganglial cells of the nerve fiber layer, the neurosensory cells of the inner nuclear layer, and the cells of the retinal pigment epithelium (RPE) between days 8 and 10. Thus, this study supports the concept that HSV neurovirulence varies between strains and presents a HSV-2 neurotransmission animal model of ARN.

Inhibition of epidemic isolates of coxsackievirus type A 24 by recombinant and natural interferon alpha and interferon beta.

Natural human interferons (IFN) and recombinant human IFNs (rIFN-alpha and rIFN-beta) inhibited the production of virus in Chang's human conjunctival cell cultures infected with epidemic isolates of acute hemorrhagic conjunctivitis virus, Coxsackievirus type A 24 (CA24). Generally, natural and rIFN-alpha and rIFN-beta were equally effective in inhibiting CA24 infection. However, rIFN-alpha A was more effective than rIFN-alpha C, D, I, J, and K in reducing virus infection, cytopathogenesis, and virus production. Recombinant IFN-alpha J was least effective in inhibiting CA24 in human conjunctival cell cultures. Also, the IFN titer was reduced 10- to 1,000-fold when cells were infected with greater than or equal to 0.3-0.5 CA24/cell, suggesting a dose-dependent IFN resistance by CA24. These results suggest that the antiviral activity of IFN against acute hemorrhagic conjunctivitis in vivo may vary with the CA24 isolates, the MOI, the type of IFN, and the time of infection with respect to beginning IFN treatment.

Nondetectable levels of interferon gamma is a critical host defense during the first day of herpes simplex virus infection.

Previous studies have demonstrated the importance of IFN alpha/beta in resistance to primary viral infections. However, the role of IFN gamma in primary infections is unclear. The present studies were undertaken to determine whether IFN gamma induction was an important early host defense against primary HSV infection. The approach was to block the IFN gamma response with antibodies to IFN gamma prior to infection and at various times post-infection (p.i.). The data indicates that treatment of mice with anti-IFN gamma prior to infection enhanced mortality (89% vs 37%). Anti-IFNs given at various times post HSV challenge proved most effective within the first 24 h of infection. The above results suggest for the first time that IFN gamma mediates important host defense(s) early during primary HSV infection. Similar results were obtained using antibody to IFN alpha/beta.

Histamine releasing activity (HRA) produced by leukocytes co-cultured with tumor cells.

Human stomach adenocarcinoma (AGS), astrocytoma (AST) and myelogenous leukemia (K562) cells were co-cultured with human peripheral blood leukocytes (PBL) through four days. Histamine releasing activity (HRA, lymphokines that stimulate degranulation of basophilic leukocytes with the release of histamine) and interferons alpha and gamma (IFN alpha and IFN gamma) were detected in the culture fluids. Maximal levels of HRA were detected by 24 hr in AST and K562-leukocyte co-culture fluids. Notably, only low levels of HRA was detected in AGS-leukocyte cultures. HRA was separated into two molecular weight species (60,000 and 25,000) by column chromatography. The IFN activity was shown to be a mixture of IFN alpha and IFN gamma (IFN alpha greater than IFN gamma). IFN did not cause histamine release from human PBL or affect HRA. Our results indicate that PBL from normal individuals produce HRA in response to tumor antigen and suggest that the basophilic leukocyte response to certain cancers may be related to HRA production.

Antibodies to the carboxyl terminus of mouse interferon-gamma neutralize its immunoregulatory and antiviral activities.

Antibodies to a synthetic carboxy-terminal peptide (Cys-Ser-Leu-Arg-Lys-Arg-Lys-Arg-Ser-Arg-Abu) (gamma-C-TP) of mouse interferon-gamma (MuIFN-gamma) were produced in rabbits. They neutralized the antiviral activity of MuIFN-gamma but not that of MuIFN-alpha/beta or human (Hu) IFN-alpha/beta or -gamma. They also inhibited the IFN-dependent enhancement of natural cytotoxic cells (NCC) and the in vivo plaque-forming cell (PFC) response to sheep red blood cells (SRBC). Thus, our results indicate that polyclonal antibodies specific for the nine carboxy-terminal amino acids of MuIFN-gamma can specifically inhibit the antiviral and immunoregulatory activities of this IFN in vitro. In addition, our findings indicate that endogenous production of MuIFN-gamma in vivo plays a role in development of the full antibody response to SRBC surface antigens.

Identification of a human monocyte cytotoxicity-inducing factor from T cell hybridomas produced from Sezary's cells.

Sezary's syndrome is a leukemic proliferation of OKT4+ lymphocytes. Sezary cells were isolated by differential centrifugation and fused to CEM.8azar.C, and HGPRTase-lacking clone of CEM. The hybrid cells were studied for their ability to produce soluble mediators of human monocyte cytotoxicity. The product of a single clone, FtF3, which bore the surface phenotype of Sezary cells, was characterized. Monocyte cytotoxicity-inducing factor (MCF) was found to be stable at pH 2 for 1 hr, unlike IFN-gamma, and was found to be more heat stable as well. Moreover, treatment of MCF with antisera to IFN-gamma, IFN-alpha or a combination of IFN-gamma and IFN-alpha failed to neutralize its biologic activity. MCF binds to matrix gel Red A. MCF eluted from this dye-ligand was found to have an apparent m.w. of 11,500 by gel filtration and 14,700 by SDS-polyacrylamide gel electrophoresis. MCF produced by hybridized Sezary cells appear to be neither IFN-gamma nor an altered molecular form of IFN-gamma, yet is a potent inducer of human monocyte cytotoxicity.

Conjunctivitis in rabbits caused by enterovirus type 70 (EV70).

A rabbit enterovirus 70 (EV70) model infection that closely mimics human enteroviral conjunctivitis was developed. Conjunctivitis occurred 24 hr following topical application of EV70. The conjunctivitis was characterized by tearing, redness, swelling of the eye lids, follicles in the superior palpebral conjunctiva, and dilatation of subconjunctival blood vessels. Histologic examination of conjunctival and corneal tissue taken 1 and 2 days after infection revealed numerous punctate areas devoid of squamous epithelium on the upper palpebral conjunctiva. Also, follicles without germinal centers were observed microscopically in the palpebral and tarsal conjunctiva. Fibroblast infiltration characteristic of wound healing and a sparse mononuclear infiltration was noted by the second day. Peak levels of virus [10(3) to 10(6.2) plaque forming units (PFU)/ml] were detected 1 to 2 days after infection and declined to undetectable levels after 3 to 5 days. Interestingly, antiserum to parental EV70 was less effective (8-10-fold) in neutralizing EV70 adapted to animal and tissue culture systems. This finding suggests that an antigenic variant of EV70 arose during adaptation. Fibroblast interferon (IFN beta), which is indicative of viral infection, was detected in tears from 6 of 16 rabbits and declined to undetectable levels 3 days after infection. Serum antibody to EV70 was detectable 8 to 10 days after infection. However, the level of serum antibody was highly variable. The results indicate that the clinical disease, virologic and immunologic courses were similar to that of the human infection. Results suggest that this animal model provides a system for studying the natural antigenic variation of EV70, the natural host defenses of the eye, and antiviral treatments against enteroviral conjunctivitis.

Activity of arildone with or without interferon against acute hemorrhagic conjunctivitis viruses in cell culture.

Arildone (WIN 38020; 4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione) inhibited the infectivity of acute hemorrhagic conjunctivitis viruses in tissue culture. Arildone did not inhibit interferon (IFN) production or IFN activity. Treatment of cultures with combinations of arildone and IFN resulted in an additive inhibition of acute hemorrhagic conjunctivitis virus production.