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Marlyn P Langford, Alexandra R Sebren, Maxwell A Burch, Thomas B Redens Department of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USACorrespondence: Marlyn P LangfordDepartment of Ophthalmology, Louisiana State University Health Sciences Center, 1501 Kings Hwy/PO Box 33932, Shreveport, LA 71130-3932, USATel +1 (318) 675-5018Fax +1 (318) 675-6000Email mlangf@lsuhsc.eduBackground: Acute hemorrhagic conjunctivitis (AHC) is a highly contagious eye disease caused by enterovirus type 70 (E70) and Coxsackievirus A24 variant (CA24v) with no clinically approved treatment. The antiviral activity of methylene blue (MB; a WHO essential medicine) against AHC viruses was investigated using human corneal epithelial cells (HCEC).Methods: Time and concentration-dependent MB accumulation by HCEC was determined colorimetrically and MB inhibition of virus production of 5 E70 and 3 CA24v AHC epidemic isolates in HCEC was determined by micro-plaque assay. AHC virus cytopathy inhibition by MB was detected by reductions in virus-induced caspase-3 activity and polymeric DNA fragments.Results: MB uptake by HCEC was rapid and concentration dependent. MB inhibition of E70 and CA24v production was concentration dependent. AHC virus yields were significantly lower (50 to > 10,000 fold) in HCEC pre-treated with 0.25– 1% MB than in placebo controls (p’s ≤ 0.01). MB pre-treatment significantly inhibited virus-induced caspase-3 activation and DNA fragmentation (p’s< 0.01). Virus-infected cells accumulate oxidized MB and MB application up to 6 h after infection inhibited virus production and virus-induced HCEC cytopathy.Conclusion: The results suggest MB treatment prior to and shortly after infection can inhibit AHC virus production and caspase-mediated HCEC cytopathy. The results support the therapeutic potential of ophthalmic solutions containing MB against AHC virus infection during epidemics.Keywords: Coxsackievirus A24, eye, infection, enterovirus 70, epidemic, caspase, apoptosis

Marlyn P Langford, Edwin A Anders, Maxwell A BurchDepartment of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Purpose: The purpose of this paper is to present the clinical course of a laboratory-acquired case of acute hemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24 variant (CA24v). Also, the anti-CA24v neutralizing activity and anti-CA24v immunoglobulin (Ig) G and secretory IgA (sIgA) in acute and convalescent tears and/or sera are presented.Case: A 60-year-old male presented with acute-onset left eyelid edema, tearing, conjunctival erythema, pain, foreign body sensation, and subconjunctival hemorrhage 24 hours after suspected laboratory exposure. Bilateral conjunctivitis presented 24 hours later and resolved in 10 days.Methods: Tear and blood samples were collected for virus isolation and neutralizing assays. CA24v-reactive IgG and sIgA in tear and/or serum samples were detected by immunofluorescent antibody analysis of ethanol-fixed virus-infected cells.Results: Peak tear neutralization titers (1,000–1,500 U/mL) against the isolated virus occurred 1 day post-onset (po) of AHC. Tear neutralization titers became undetectable by the sixth day as serum neutralization titers became detectable on the ninth day po (60 U/mL), peaked by 21 days (3,000 U/mL), declined by 1 year to 200 U/mL, and remained at 30 U/mL 5 years po. Antibody to human IgG, IgA, and secretory component (sIgA) reacted with CA24v-infected cells treated with pooled acute tears collected 1–4 days po. Predominantly, sIgA was detected in CA24v-infected cells treated with tears collected 4 years and 5 years post-AHC, while convalescent serum contained predominantly anti-CA24v IgG.Conclusion: AHC was confirmed by CA24v isolation, tear anti-CA24v neutralizing activity, and seroconversion. The detection of CA24v-reactive IgG, sIgA, and neutralizing activity in tears collected 1–4 days po of AHC supports plasma extravasation of IgG and suggests a defensive role for tear anti-CA24v sIgA. The results suggest that immunofluorescent antibody analysis of tears for persistent anti-CA24v sIgA may be useful in epidemiological monitoring of AHC. Keywords: neutralization, immunofluorescence, eye infection, enterovirus, seroconversion

Jordan M Burnham,1 Monali Sakhalkar,1 Marlyn P Langford,1 Chanping Liang,1 Thomas B Redens,1 Sushil K Jain21Department of Ophthalmology, 2Department of Pediatrics, Louisiana State University Health Sciences Center, Shreveport, LA, USABackground: Diabetes-related eye disease is due in part to oxidative stress. Gamma-glutamyl transpeptidase (GGT) is a γ-glutamyl cycle enzyme that protects against oxidative stress via glutathione recapture. This study investigates corneal and Schirmer tears GGT activity in diabetic and non-diabetic adults aged 50 to 83 years old.Methods: GGT activity was determined by colorimetric assay on 50 corneas from 14 diabetic (without keratopathy) and 20 non-diabetic donors and on Schirmer type 1 test strips (no anesthesia) of 14 diabetic and 14 non-diabetic subjects.Results: Type 1 (T1) diabetic cornea GGT activity was 40% lower than Type 2 (T2) diabetic cornea GGT activity (P = 0.04), but GGT activity was similar for corneas (without keratopathy) from diabetic and non-diabetic donors (P ≥ 0.44 for all). The number of endothelial cells/unit of GGT activity in diabetic corneas was 22% higher (P = 0.1) than in non-diabetic corneas. GGT activity per Schirmer strip and GGT activity per mm of tears were 36% and 50% higher (P ≤ 0.008 for all) for non-diabetic (tear volume dependent) than diabetic donors (tear volume independent), respectively. GGT activity per mm was 50% lower in T1 than T2 diabetics (P = 0.02). Higher tear GGT activity in non-diabetic than diabetic females (P ≤ 0.05) was due to higher GGT activity in the African American females.Conclusion: GGT activity was less in T1 than T2 diabetics, but comparable to non-diabetic corneas. Schirmer tear GGT activity in diabetic eyes was tear volume independent, less in T1 than T2, lower than in tear volume dependent, non-diabetic female eyes. Low cornea and tear GGT activity suggests loss of antioxidant potential and supports ocular antioxidant therapy for diabetic patients.Keywords: cornea, diabetes, endothelium, epithelium, eye, γ-glutamyl transpeptidase, tear, oxidative stress

John Carmody1, Raghunath P Misra1,2, Marlyn P Langford1, William A Byrd1, Lauren Ditta1, Bryan Vekovius1, Donald E Texada11Department of Ophthalmology, 2Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA, USAAbstract: A case of sporadic Burkitt lymphoma (sBL) presenting with jaw and lid involvement in a diabetic adult African American female and a review of adult orbital Burkitt lymphoma cases are presented. Lid edema, visual loss, ophthalmoparesis, proptosis, and sinusitis progressed over 4 weeks despite antibiotic and steroid treatment. Upper lid biopsy histopathological evaluation and immunophenotyping revealed a homogenous mass of atypical CD10 and CD20-negative B-cells and tingible body macrophages yielding a "starry sky" appearance. Cytogenetic analysis detected a minor variant c-MYC translocation, but no Epstein–Barr virus RNA. Detection of multiple lesions prompted a diagnosis of stage IV disease that totally regressed following radiation and chemotherapy. Review results of the six adult orbital sBL cases support a poor prognosis and a heightened suspicion of variant CD10, CD20 and BCL6 positive sBL in adults presenting with jaw pain and rapidly progressive orbital symptoms, particularly in female, African American, and diabetic patients.Keywords: B-cells, Burkitt lymphoma, cancer, diabetes, eye, Epstein–Barr virus, orbit, tumor

Noninfectious exudative conjunctivitis can be experimentally produced in rabbits by application of the apoptogenic bacterial cell wall peptidoglycan, muramyl dipeptide (MDP) to the ocular surface. The purpose of this study was to investigate the acute conjunctival cytopathology induced by unilateral ocular surface exposure to MDP. Hematoxylin and eosin staining assessed bilateral tear cytopathology and conjunctival histopathology. The caspases levels in conjunctival tissue and tears were measured in standard assays utilizing p-nitroanaline tagged caspase-specific substrates. Immunofluorescent antibody identified intracellular caspase-3, nuclear factor-κβ (NF-κβ), and oxidative DNA damage (8-OHdG; 8-oxo-2'-deoxyguanosine) in tear and conjunctiva cells. DNA extracted from conjunctival tissues and pooled tear fluids were visualized by ethydium bromide agarose gel electrophoresis. Onset of ipsilateral conjunctivitis was due to an epitheliopathy characterized by loss of conjunctival epithelial cell adherence, exuviation of conjunctival epithelial cells, and neutrophil infiltration. Caspase-3 levels were significantly higher in exuviated cells in ipsilateral than contralateral tear (p's ≤ 0.001) collected at 3-5 h post MDP. Significantly higher caspase-2, -3, -6, -8 and -9 (p's ≤ 0.03) levels were detected in ipsilateral than contralateral conjunctival tissue at 5 h. Polymeric DNA was detected in ipsilateral but not contralateral conjunctival tissue and tears. Caspase-3, NF-κβ, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κβ epithelial cells and polymeric DNA in conjunctival tissue and shedding of caspase positive cells and polymeric DNA into ipsilateral tears support MDP induction of acute programmed cell death in vivo. The results suggest that ipsilateral exudative conjunctivitis is due to acute caspase-mediated conjunctival epitheliopathy induced by topical exposure to the bacterial peptidoglycan MDP., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Peptidoglycan (PGN) recognition protein 2 (PGRP2; N-acetylmuramyl-L-alanine amidase (NAMAA)) activity in corneal epithelial cells is thought to inhibit corneal inflammation by reducing the PGN-induced cytokines. PGRP2 has not been reported in human retinal pigment epithelial (RPE) cells. RPE cell lysate NAMAA activity was measured densitometrically via cleavage of FITC-tagged muramyl dipeptide ( FITC MDP). RPE lysate degradation of the cytopathic activity of nucleotide-binding oligomerization domain (NOD) receptor agonists was assessed by caspase-3 activation and DNA ladder detection and quantitation. PGRP2/NAMAA protein was detected in RPE cells by immunofluorescent antibody assay. RPE lysate NAMAA cleaved FITC MDP in a dose- and time-dependent manner. RPE lysate selectively inhibited PGN cytopathic activity of NOD1 agonists containing D-γ-glutamyl-meso-diaminopimelic acid and NOD2 containing L-alanyl-D-isoglutamine. The results suggest RPE PGRP2 amidase selectively degrades PGN that stimulate NOD-mediated cytopathic activity. The failure of RPE NAMAA to degrade pro-inflammatory PGN may play a role in bacterial retinopathies., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

4-Hydroxyphenylpyruvate dioxgenase (HPPD) enzymes from rat and from several plants contained only about a single inhibitor-binding active site per dimer which matched the content of iron in the purified Arabidopsis thaliana and Avena sativa enzymes. The dimeric HPPDs were about 10 fold more catalytically active than the tetrameric P. fluorescens enzyme with k cat /K mHPP values ranging from 0.8 to 2.5 s -1 μM -1 . Most were also highly sensitive to herbicides with, for example, K i values for mesotrione ranging from 25 to 100 pM. Curiously HPPDs from cool climate grasses were much less herbicide-sensitive. When likewise expressed in Nicotinia tabacum, Avena sativa HPPD, K i value of 11 nM for mesotrione, conferred far greater tolerance to mesotrione (Callisto TM ) than did any of the more sensitive HPPDs. Targeted mutagenesis of the Avena HPPD led to the discovery of 4 mutations imparting improved inherent tolerance, defined as the ratio of K i to K mHPP , by about 16 fold without any loss of catalytic activity. The Nicotinia line with the highest expression of this quadruple mutant exhibited substantial resistance even up to a 3 kg/ha post-emergence application of mesotrione. The maximum observed expression level of heterologous plant HPPDs in tobacco was ca. 0.35% of the total soluble protein whereas the endogenous tobacco HPPD constituted only ca. 0.00075%. At such high expression even HPPDs with impaired catalytic activity could be effective. A quintuple mutant Avena sativa HPPD conferred substantial tolerance across a broad range of HPPD herbicide chemistries despite being only ca. 5 % as catalytically active as the wild type enzyme. Testing various wild type and mutant HPPDs in tobacco revealed that tolerance to field rates of herbicide generally requires about two order of magnitude increases in both inherent herbicide tolerance and expression relative to endogenous levels. This double hurdle may explain why target-site based resistance to HPPD-inhibiting herbicides has been slow to evolve in weeds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Maintaining the high glutathione (GSH; tripeptide of glutamate, cysteine and glycine) levels in the lens cortex promotes lens health. The role of glutamate/aspartate (Glu/Asp) transporters and the cystine (Cys)/Glu exchanger (Xc(-) exchanger) in maintaining GSH in transformed human lens epithelial cells (SRA 01/04) was investigated., Methods: Detection and differentiation of excitatory amino acid transporters (EAAT1-5) and the Xc(-) exchanger was performed by the uptake of radiolabeled l-Glu, d-Asp and l-Cys in the presence and absence of Na(+), substrate-specific inhibition studies and Western-blot analysis. Reductions in GSH levels post-inhibition of Xc(-) exchanger and EAAT activities by substrate inhibitors demonstrated the roles of EAAT and Xc(-) exchanger in maintaining GSH., Results: Glu and d-Asp uptake in HLEC was Na(+)-dependent. Strong inhibition by substrate-specific Glu/Asp uptake inhibitors and weak inhibition by kainic acid (KA) was consistent with Na(+)-dependent EAAT1/3/4/5 activity and weak EAAT2 activity, respectively. Na(+)-independency and Glu inhibition of Cys uptake were consistent with Xc(-) exchanger activity, but inhibition of Na(+)-dependent Cys uptake by N-acetylcysteine suggests Cys uptake by EAAT3. EAAT1-5 and xCT (Xc(-) exchanger light chain) immunoreactive peptides were detected by Western-blot analysis of HLEC lysates. EAAT and Xc(-) exchanger inhibition by substrate antagonists depleted GSH concentrations by 15-28% (p's ≤ 0.02), while GSH synthesis inhibition by buthionine sulfoximine depleted GSH by 33% (p = 0.008)., Conclusion: Inhibition of Glu and Cys uptake by EAAT and Xc(-) exchanger antagonists depletes GSH in human lens epithelial cells. These in vitro results support pivotal roles for EAAT and Xc(-) exchanger activities in maintaining GSH and protection against oxidative stress in cortical lens epithelium.

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5-5.0 × 10(6) cells/mL) were elevated 4-8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To investigate age-related effects on human corneal γ-glutamyltranspeptidase (GGT) (ectoenzyme important to maintaining corneal hydration and antioxidant potential via glutathione recapture)., Methods: Age-related differences between total, endothelial, and epithelial GGT activity and endothelial cell density were determined for corneas from 29 donors (mean age, 53 ± 17 years; age range, 13-83 years). GGT activity was determined using a standard colorimetric assay based on the transpeptidation reaction. Corneal GGT localization and expression was determined by immunohistochemistry., Results: Total corneal, endothelial, and epithelial GGT activities in the young (<50 years) donor corneas were 37% (P = 0.02), 44% (P = 0.001), and 36% (P = 0.06) higher, respectively, than in the senior (≥50 years) corneas. The age-related rates of decline for GGT activity were 1.0 unit per year for total cornea, 0.4 to 0.5 unit per year for endothelium, and 0.3 to 0.4 unit per year for epithelium. Notably, endothelial cell density in the young corneas was 14% (P = 0.001) higher than in the senior corneas declining about 100 cells per square millimeter per decade (0.3% per year). GGT activity per 10 endothelial cells decreased at about 0.2 units per year and GGT activity per 10 endothelial cells in the young corneas was 41% higher (P = 0.01) than in the senior corneas. Fewer immunoreactive GGT-positive epithelial cells were detected in senior cornea., Conclusion: The age-related loss of human corneal GGT activity was associated with reductions in endothelial and epithelial GGT activity, being because of reduced number of GGT-positive endothelial and epithelial cells and reduced GGT activity per endothelial cell.

The stimulus for caspase-mediated renal cell apoptosis in septic acute renal failure (ARF) is unclear. To demonstrate the nephrotoxic effects of bacterial cell wall components, the anti-cellular activity of bacterial muropeptides (muramyl dipeptides), peptidoglycans, and lipopolysaccharides was investigated in rabbit kidney cells. Changes in the cell membrane (APOPercentage™ dye uptake), caspase activities, and DNA degradation were quantified colorimetrically and using densitometric assays and their inhibition by caspase-specific and pan-caspase inhibitors was determined. The onset and levels of APOPercentage™ dye-positive rabbit kidney cells, caspase activities, and DNA degradation were closely associated. Specific caspase-1, -2, -3, -4, -8, -10, and -12 inhibitors reduced caspase-3 activity by ≥40%, but only caspase-3 and -8-specific inhibitors reduced apoptotic DNA levels. Pan-caspase inhibitor Q-VD-OPh was 10-fold more effective at inhibiting rabbit kidney cell death, caspase activation, and DNA degradation than caspase-family inhibitor Z-VAD-FMK. Apoptosis was inhibited effectively by both pan-caspase inhibitors when applied early during the stimulus-to-response period. Multiple initiator and effector caspases were activated suggesting extrinsic, intrinsic, and endoplasmic reticulum/stress apoptotic pathway stimulation in rabbit kidney cells treated with bacterial cell wall components. The results provide in vitro support for bacterial cell wall-induced apoptosis as a pathogenic mechanism of renal cell death in septic ARF and support the potential prophylactic use of pan-caspase inhibitors to suppress septic ARF.

Purpose: The viability and functions of the corneal epithelium are dependent in large measure on the active uptake of nutrients, growth factors, and amino acids from stroma and tear. The present study presents the cellular distribution(s) of glutamate, the Na(+)-dependent glutamate/aspartate transporters (excitatory amino acid transporters; EAAT1-5), Na(+)-independent glutamate/cystine exchanger (Xc(-) antiporter) subunits (xCT light chain and 4F2hc heavy chain), glutamine synthetase (GS), and gamma-glutamyltranspeptidase (GGT) in human corneal epithelium., Methods: Glutamate, EAAT1-5, xCT/4F2hc, GS, and GGT immunoreactive proteins were detected by immunofluorescence microscopy. Human corneal GGT activity was quantified using a standard colorimetric assay., Results: Glutamate, EAAT3>2>1, xCT/4F2hc, and GGT proteins were detected in the columnar and wing cells. Glutamate was reduced or absent in the EAAT negative, Xc(-) antiporter, and GS positive outer wing cell and flat superficial epithelial cell layers. All EAATs (EAAT3>4/5>1/2), xCT/4F2hc, GS, and GGT were detected in flat superficial epithelial cell layer., Conclusions: The localization of glutamate, multiple EAATs, Xc(-) antiporter proteins, and GGT to columnar and superficial epithelial cell layers suggests uptake of glutamate and cystine from the stroma and tear and supports their importance in regulation of glutamate/cystine and glutathione (GSH; a tripeptide of glutamate, cystine, and glycine) in the human cornea epithelium. In addition, the low glutamate levels in outer wing and flat superficial epithelial cells positive for Xc(-) antiporter and GS are consistent with exchange of glutamate by Xc(-) antiporter for extracellular cystine utilized in GSH synthesis and support coupling of ammonia detoxification with glutamate degradation by GS.

A case of orbital metastasis of cervical keratinizing squamous cell carcinoma is presented. The patient, in remission from primary cervical and ovarian cancers, presented with complaints of left eye ptosis and pain. Examination revealed the presence of a moderately tender mass along the left supra-temporal orbital rim and downward displacement of the left globe. Computed tomography revealed a poorly circumscribed mass with superior lateral wall bone loss. Excised tissue contained invasive, poorly differentiated nests of pan keratin and epithelial membrane antigen-positive squamous cells with numerous pleomorphic multinucleated giant cells. Multiple treatment regimes were unsuccessful, and the patient expired due to disease complications after 3 months.

The plasma levels of apoptotic DNA ladders (i.e., apoptosemia) and gamma-glutamyltranspeptidase (GGT) in diabetic outpatients and rats were investigated. Apoptotic DNA ladders were detected in plasma from 26.8% of type 1 (T1) and 18.5% of type 2 (T2) diabetic children 1-20 years of age, 25.7% of hospitalized children and 35.7% of adult RA outpatients, but in only 3.5% of adult pre-op patients. Plasma from 7.7% of young streptozotocin-induced diabetic but not control rats contained apoptotic DNA ladders. Apoptosemia was detected more often in male T1 (31%) and T2 (30.8%) diabetic outpatients than in female T1 (20.8%) and T2 (15.4%) diabetic outpatients. GGT in apoptosemic plasma was significantly higher than in nonapoptosemic plasma from T1 (P = 0.001) but not T2 diabetic children. The highest amounts of apoptotic DNA were detected most often in diabetic children > or =14 years of age. In vitro study results suggest that cell-free apoptotic DNA ladders appear prior to an increase in GGT activity in serum from human blood incubated at 37 degrees C. The results suggest that 24.7% of plasma samples from diabetic children contained apoptotic DNA ladders, the incidence and amounts of apoptotic DNA ladders were higher in the older diabetic children, and GGT was elevated in apoptosemic T1 diabetic children (P = 0.01). The results indicate that "silent" apoptosemia occurs in T1 and T2 diabetic children and suggest elevated GGT in diabetic children could be due to release from apoptotic cells.

LOC387715 is a hypothetical gene located on human chromosome 10q26.13 that is associated with the development of age-related macular degeneration (AMD). The native open reading frame (ORF) of LOC387715 cDNA - LOC387715(ORF), contains a large number of Escherichia coli (E. coli) rare codons (RC) including 5.6% and 15.0% Group-I and IIa translational problem causative (TPC) RCs, respectively, which forms 3 and 4 simple E. coli rare codon clusters (RCC) where RCs are spaced by 1 and 2 respective non-TPC codons and one complex E. coli RCC where RCs and RCCs are spaced by <5 non-TPC codons. We modified the entire 35 E. coli RCs (6, 16 and 13 Group I, IIa and IIb RCs, respectively) present in LOC387715(ORF) with their optimal or sub-optimal synonymous degenerate codons, and the resulted LOC387715(ORF)m was free from Shine-Dalgarno-like sequence (SDLS) and ribosome binding site complementary sequence (RBSCS). SDS-PAGE and Western blotting analysis demonstrated that LOC387715(ORF)m was capable of highly expressing the recombinant protein rLOC387715 in E. coli. Mass spectrometry analysis indicated that the bacterial expressed rLOC387715 contained the correct and expected amino acid (aa) sequence without aa misincorporation, aa missing or frame-shift. The results suggest that high and authentic expression of LOC387715 recombinant protein in E. coli was achieved by the synonymous modification of its native ORF cDNA sequence for all the 3 groups of bacterial RCs and the simultaneous elimination of SDLS and RBSCS sequences.

The distribution of glutamate (Glu), the Glu transporter GLAST-1, and glutamine synthetase (GS) in human and monkey anterior uveal tissue, as well as serum (S) to aqueous humor (AH) Glu and glutamine (Gln) gradients were investigated. Cross-linked Glu (xGlu), GLAST-1, and GS were detected using the immunofluorescent antibody technique. S/AH Glu, Gln, and alanine (Ala) concentrations were quantified by high performance liquid chromatography. xGlu immunoreactivity was detected in melanocytes, posterior pigmented epithelial/dilator muscle cells, vascular endothelial cells, and lymphocytes of the iris, as well as the pigmented (PE) and nonpigmented epithelial (NPE) cells and muscle cells of ciliary body. xGlu immunoreactivity was highly concentrated at the apices of GLAST-1, GS positive ciliary body NPE cells, and in GLAST-1 positive iris melanocytes and iris dilator muscle cells. AH Glu concentrations were lower (p < 0.001), while Gln was higher in monkey (p = 0.01) and human cataractous (p = 0.15) AH than serum. The results indicate that Glu is concentrated within GLAST-1, GS positive NPE cells and are consistent with the suggestion that Glu and Gln concentrations in AH may be due in part to GLAST-1 and GS activity in iris and ciliary body epithelial cells.

How the immune system recognizes endogenously arising tumors and elicits adaptive immune responses against nonmutated tumor-associated Ags is poorly understood. In search of intrinsic factors contributing to the immunogenicity of the tumor-associated Ag NY-ESO-1, we found that the NY-ESO-1 protein binds to the surface of immature dendritic cells (DC), macrophages, and monocytes, but not to that of B cells or T cells. Using immunoprecipitation coupled with tandem mass spectrometry, we isolated DC surface calreticulin as the receptor for NY-ESO-1. Calreticulin Abs blocked NY-ESO-1 binding on immature DC and its cross-presentation to CD8+ T cells in vitro. Calreticulin/NY-ESO-1 interactions provide a direct link between NY-ESO-1, the innate immune system, and, potentially, the adaptive immune response against NY-ESO-1.

Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), but not actinomycin D and guanidine.HCl (although guanidine.HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.

The uveitogenic bacterial cell wall component muramyl dipeptide (MDP) is apoptogenic in rabbit kidney cells. The purpose of this investigation was to assess the cytotoxic activity of MDP and staurosporine (STSP; induces cultured corneal and lens cells apoptosis) in rabbit ciliary body tissue. Anterior uveitis was determined by clinical symptoms and increased aqueous humor (AH) protein. Ciliary body tissue was assessed for histological changes, caspase-3 activity, dye uptake, distribution of immunoreactive caspase-3 and DNA ladders at 4 and 6 hours postinjection. Increases in caspase-3 activity, APOPercentage dye uptake, and localization of immunoreactive caspase-3 in ciliary epithelial cells were associated with ciliary cysts of detached nonpigmented epithelial (NPE) cells, as well as apoptotic and necrotic DNA ladders in ciliary body tissues from eyes injected with MDP and/or STSP. The results suggest that intracameral injection of the bacterial components MDP and STSP can induce acute endophthalmic changes in uveal tissue including formation of ciliary body, NPE and pigmented epithelial (PE) cell apoptosis, and ciliary body tissue necrosis.

Calreticulin (CRT) is a binding protein for apoptotic N-acetylmuramyl-L-alanyl-D-isoglutamine (L,D-MDP) or peptidoglycan in RK(13) cells. CRT on RK(13) cell surface (srCRT) forms complex(es) with tumor necrosis factor receptor 1 (TNFR1) and TNFR-associated death domain (TRADD) protein of the cell membrane. CRT polyclonal or monoclonal antibody binding to RK(13) srCRT dose-dependently inhibited L,D-MDP-induced apoptosis. In RK(13) cells, L,D-MDP up-regulated the TNFR1.TRADD complex of the plasma membrane and subsequently induced cytosolic TRADD-Fas-associated death domain protein complex. Biotinylated srCRT was capable of calcium-dependent binding of Sepharose-immobilized L,D-MDP or peptidoglycan. However, Toll-like receptors TLR-2 and TLR-4, Nod2, and CD14 of RK(13) cells did not specifically bind Sepharose-immobilized L,D-MDP. High concentrations (5-40 mm) of EGTA dose-dependently inhibited free L,D-MDP binding to purified RK(13) cell CRT and promoted free L,D-MDP dissociation from RK(13) cell CRT.MDP complex. Different concentrations of EGTA (0-40 mm) added to Dulbecco's modified essential medium with 1.8 mm calcium or phosphate-buffered saline with 0.18 mm calcium have different effects on medium free calcium concentrations but have identical inhibiting effects on L,D-MDP-induced apoptosis. More inhibition of the L,D-MDP-induced apoptotic DNA ladders and caspase-3 activity in RK(13) cells was obtained with EGTA pretreatment (83%) than just EGTA + L,D-MDP (47%). The knocking down of srCRT by antisense oligonucleotide CRTAS121 (250 nmol/ml) and stealth small interfering RNA CRT&#95;siR479 (150 pm/ml) for 2 days (44 and 66%, respectively), resulted in the inhibition of L,D-MDP-induced caspase-3 activity (47 and 65%, respectively). The results suggest that (a) the binding of L,D-MDP to srCRT is calcium-dependent, i.e. on srCRT-bound calcium, and (b) it is srCRT, not TLR-2, TLR-4, Nod2 or CD14, that mediates L,D-MDP-induced RK(13) cell apoptosis through activating the TNFR1. TRADD-Fas-associated death domain protein apoptotic pathway.

Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000-10,000 units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20-320units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.

The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.

Calreticulin (CRT) was isolated and identified as a protein in rabbit kidney RK(13) cells that binds the apoptogenic bacterial cell wall (BCW) components, muramyl dipeptide (MDP) and peptidoglycan (PG). Mannan-agarose purified RK(13) cell CRT (rCRT) selectively bound sepharose-immobilized L,D-MDP and PG, but not L,L-MDP or D,D-MDP. Purified rCRT and bovine CRT (bCRT) also bound free PG and L,D-MDP demonstrated in bioassays of RK(13) cell apoptosis. The results suggest that, in RK(13) cells, (a) CRT is a specific binding protein for both L,D-MDP and PG and (b) CRT binding L,D-MDP or PG is dependent on the stereoisomeric configuration of the dipeptide (L-alanyl-D-isoglutamine) moiety. In addition, the results also suggest that, in RK(13) cells, the binding of L,D-MDP, L,L-MDP, D,D-MDP, or PG to CRT correlates with their capacities of inducing apoptosis.

Purpose: Evaluate the immune response in rabbits injected with EV70, the agent of acute hemorrhagic conjunctivitis (AHC) and AHC associated neuropathy., Methods: Rabbits were injected intramuscularly with uv-light inactivated EV70 isolate J670/71. Neutralizing activity against EV70 was quantified in serum and tear samples and the immunoglobulin (Ig) classes of the neutralizing activity in serum identified by sucrose gradient ultra-centrifugation. Adjuvant muramyl dipeptide (MDP) was applied topically to assess the role of ocular inflammation on levels of neutralizing antibody, proteins and Ig in tears. The protective effects of human and rabbit sera and interferon-alpha (IFN-alpha) against EV70 were compared in human conjunctival and lens cells., Results: Sera collected at 6 and 13 d contained 19S IgM anti-EV70 neutralizing antibody, while serum collected 21 d post injection contained 19S IgM and 7S IgG anti-EV70 neutralizing antibody. Low titers of anti-EV70 activity (< or =30 U/ml) were detected in tears of seropositive rabbits. MDP induction of conjunctivitis in seropositive rabbits increased tear IgG concentration (3-fold) and anti-EV70 neutralizing antibody titers (> or =10-fold). The protective effect of the rabbit and human sera against EV70 infection in conjunctival, but not lens epithelial cells, was enhanced by the addition of IFN-alpha., Conclusions: Immunization with uv-light inactivated EV70 elicits a classical humoral immune response in rabbits. The protective activity of serum in EV70-infected human conjunctival cells, but not lens cells, was increased by IFN-alpha. Adjuvant MDP-induced conjunctivitis, increased blood-conjunctival barrier (BCB) permeability and anti-EV70 neutralizing activity in tear of seropositive rabbits. The results suggest immunization with inactivated EV70 could provide systemic as well as ocular protection during natural EV70 infection.

The cytotoxicity of bacterial cell wall components, muramyl dipeptide (synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine; L,D-MDP) and lipopolysaccharide (LPS), was investigated in several kidney cell lines. MDP and LPS were toxic to rabbit and monkey kidney cells, MDP was toxic to canine kidney cells, but not to human or porcine kidney cells. Notably, L,D-MDP was >100-fold more cytotoxic/microg than the D,D-MDP and L,L-MDP, as well as LPS. L,D-MDP and analogs containing L,D-MDP were the most widely cytotoxic of the MDP tested. The MDP-induced cytotoxicity was characterized as apoptosis by DAPI staining and DNA laddering. The acute rabbit kidney (RK13) cell apoptosis (cell death in < 5 h) induced by apical or basal application of MDP was associated with glutamate (Glu) release, decreased gamma-glutamyltranspeptidase (GGT) and acidosis and was suppressed by Indomethacin, Naproxen and Curcumin. The cytotoxic activity of L,D-MDP was decreased significantly by 24 h incubation in human sera. Aged (> 2 year-old) rabbits that apparently failed to quickly clear and excrete a uveitogenic dose of MDP within 24 h died in I week. The results indicate that minute amounts (5 ng/ml) of MDP containing L-alanyl-D-isoglutamine can induce renal cell apoptosis in vitro and support MDP-induced kidney cytotoxicity in rabbits. Also, the results indicate that MDP in sera can be detected utilizing the RK13 cell bioassay and that failure to rapidly clear and excrete L,D-MDP is associated with uveitis and death in aged rabbits.

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 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.

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.

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.

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, 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.

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.

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.

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.

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.

Acute hemorrhagic conjunctivitis (AHC) viruses--enterovirus type 70 (E70), and coxsackie-virus type A24 (CA24)--infect the superficial cells of the conjunctiva and cornea. We examined the growth of several E70 and CA24 isolates in monkey, rabbit, and mouse conjunctival and corneal (C/C) cells. We found that E70 isolates grew well in monkey (10(6.5) TCID50/ml), rabbit (10(4.5) to 10(5.5) TCID50/ml), and mouse (10(3.5) to 10(6.0) TCID50/ml) C/C cultures and caused cytopathic effects (CPE). In contrast, CA24 isolates replicated well and caused CPE only in monkey C/C cultures (10(6.5) TCID50/ml). These results show that E70 isolates grow readily in monkey, rabbit, and mouse C/C cells whereas CA24 isolates replicated in monkey C/C cells only. This finding is consistent with the suggestion that a lower animal reservoir may be a source of human infection by E70. These cell culture systems may be useful for studying virus-cell interactions and host defenses operating at the surface of the eye and for evaluating the feasibility of prophylactic or therapeutic use of interferon, interferon inducers, and/or antibody for controlling AHC virus infections.

We report the levels of coxsackievirus type A24 (CA24) and the levels and type of interferon produced early during naturally acquired picornavirus epidemic conjunctivitis. Virus levels ranging from 10(1.8) to 10(5.8) 50% tissue culture infective doses per ml were detected in 29 of 37 acute (collected 1 to 4 days after onset of conjunctivitis) tear samples. Interferon (10(1.5) to 10(3.3) U/ml) was detected in 12 of 29 tear samples collected on day 1, in 2 of 6 tear samples collected on day 2, and in 1 tear sample collected on day 3 after onset of conjunctivitis. The interferon activity in pooled tear samples was completely neutralized by antiserum against human fibroblast interferon, stable at pH 2.0, and active against different viruses. In addition, the interferon activity in tears, like human fibroblast and leukocyte interferons produced in vitro, protected human and rabbit, but not mouse, cells. This is the first report of production and identification of the antigenic type of interferon induced by an enterovirus during natural infection. The early appearance of fibroblast interferon suggests that it may be an important host defense at the local site of implantation against this and possibly other enterovirus infections.

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.

Human lymphocytes were treated with human alpha (IFN-alpha), beta (IFN-beta), or recombinant gamma (IFN-gamma) interferons separately or in combination to determine their ability to enhance natural killing against mouse L cell targets. Our results showed that recombinant IFN-gamma was approximately 50 times more active per unit of antiviral activity than either IFN-alpha or IFN-beta. Moreover, the levels of natural killing by lymphocytes treated with combinations of IFN-alpha and IFN-beta were additive, whereas combinations of recombinant IFN-gamma and IFN-alpha or recombinant IFN-gamma and IFN-beta were synergistic. The development of natural killing in lymphocytes treated with recombinant IFN-gamma did not occur more rapidly but reached higher levels (62%) than that observed with lymphocytes treated with IFN-alpha or IFN-beta (15%). The results suggest the importance of IFN-gamma and mixtures of IFN-gamma with IFN-alpha or IFN-beta in the enhancement of natural killing activity against virus infections and neoplasia.

Virus-specific, early appearing neutralizing activities (ENA) and interferon (IFN) were detected in tears collected from patients during epidemics of acute hemorrhagic conjunctivitis (AHC). In one study, ENA that neutralized enterovirus type 70 (EV70) was detected in tears collected from 114 of 130 AHC patients in Florida. In another study, ENA against coxsackievirus type A24 (CA24) was detected in tears collected from 39 of 57 patients in Singapore with AHC caused by CA24. No tear samples contained ENAs to both EV70 and CA24. Tear samples from uninfected eyes did not contain ENA to EV70 or CA24. ENA to EV70 was detected in 6 of 11 patients 1-6h before the onset of AHC. In addition, tears of 68% of patients seen on the day of onset produced tears that contained ENA to EV70. Thus, ENA to EV70 may be detected less than 24h after infection (based on 24h incubation period). IFN beta was detected in 30% of tear samples collected from patients on the day of onset of AHC caused by EV70. This finding suggested that ENA and IFN could act together to inhibit primary infections of AHC. It was found that the combination of ENA and IFN inhibited virus replication synergistically (greater than or equal to 300 fold reduction) in preinfected cells. Our findings suggest that ENA represents a previously unreported early defense mechanism of the eye, that endogenous ENA and endogenous IFN could inhibit viruses synergistically in vivo, and that ENA in tears could be useful in identifying the agent causing AHC.

Two Navajo Indian children with severe combined immunodeficiency disease (SCID) lost reconstituted immune function after virus infections. A serum factor which inhibited normal lymphocyte response to mitogens was found in one of them and led to the examination of sera from five other Navajos with SCID. Mean inhibition by six Navajo sera was 67%; no inhibitor was found in sera from normal adults and children. The inhibitor activity was nondialyzable and heat stable, yet partially sensitive to pH 2.0, suggesting that interferon(s) was present. Interferon (IFN) activity in patient sera ranged from 10 to 300 U/ml. Normal children had peak serum IFN levels of 100 and 30 U/ml in the acute and convalescent periods, respectively, of virus infections. IFN alpha, IFN beta, and IFN gamma were identified in SCID sera by specific antisera. Both inhibitor and IFN activities in three Navajo sera were 88-95 and 89-100%, respectively, removed with anti-IFN antisera. Similar patterns of inhibition of lymphoblastogenesis were seen with IFN standards. IFN levels in the SCID patients did not correlate with documented infections; elevated levels were present when no infections could be documented. The immunologic imbalances in some forms of SCID may be related to circulating inhibitors, possibly interferon.

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.

The mitogenicity, ability to induce immune interferon, and relationship between interferon synthesis and cell proliferative response were studied using human peripheral lymphocytes stimulated by staphylococcal enterotoxin A (SEA), phytohemagglutinin-P (PHA-P), and concanavalin A (ConA). Maximum cell proliferative responses ([(3)H]thymidine incorporation) and protein synthesis ((14)C-amino acid incorporation) occurred on days 3 and 4, respectively, after stimulation by each of the three mitogens. Maximal immune interferon levels were found 3 or 4 days after mitogen stimulation. SEA-treated cultures produced approximately three times more interferon than did cultures stimulated with PHA-P or ConA. Furthermore, SEA stimulated maximal cell proliferation over a much broader concentration range than did PHA-P and ConA (SEA, 10(-5) to 10(2) mug/ml; PHA-P, 10(1) to 10(2) mug/ml; ConA, 10(1) to 10(1.5) mug/ml). Interferon was also produced at maximal or near maximal levels over a broad concentration range of SEA (10(-2) to 10(2) mug/ml). Also, we found that inhibition of mitogen-induced DNA and protein synthesis to control levels by mitomycin C or cytosine arabinoside partially reduced interferon production. The DNA inhibitor studies indicate that immune interferon synthesis occurs maximally in association with at least some proliferative response and that submaximal levels of interferon production occur in mitogen-treated cultures in the absence of detectable proliferation. The ability of SEA to stimulate maximal DNA and immune interferon synthesis at concentrations of 3.5 x 10(-13) M and 3.5 x 10(-10) M, respectively, puts it in a potency range similar to that of hormones. Thus, SEA may play an important role in gut immunity and Staphylococcus aureus infections at concentrations well below those required for emetic effects.

Antibodies to an N-terminal synthetic peptide of mouse gamma interferon (MoIFN gamma) neutralized the antiviral activity of MoIFN gamma but not MoIFN alpha/beta, human IFN alpha (HuIFN alpha), HuIFN beta or cynomologus monkey IFN gamma (CynIFN gamma). Comparatively, antibodies to mouse N-terminal synthetic peptide showed only 10% reciprocal cross-reactivity in neutralization tests against heterologous HuIFN gamma and 13% cross-reactivity in the ELISA test against Hu N-terminal peptide. The predetermined specificity of these antibodies make them powerful tools for studying antigenic relatedness and biological properties of IFN gamma s.