Inhibition of Bacterial Peptidoglycan Cytopathy by Retina Pigment Epithelial PGRP2 Amidase.

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 (FITCMDP). 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 FITCMDP 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.

Conjunctival epitheliopathy induced by topical exposure to bacterial peptidoglycan, muramyl dipeptide.

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-κß (NF-κß), 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-κß, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κß 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.

Bilateral ptosis/blepharitis due to lepromatous leprosy.

PURPOSE: To underscore the importance of histopathological evaluation in cases presenting with a constellation of unusual ocular inflammation and physical findings. OBSERVATION: A 51-year-old male, presented with a chief complaint of worsening visual field loss due to droopy eyelids two months post excision of a right upper eyelid squamous cell carcinoma. His past medical history included chronic edematous facial features, chronic sinusitis, unexplained peripheral neuropathy, and worsening fatigue. Pre-blepharoplasty work-up revealed mechanical ptosis from lid edema, madarosis, a concave nasal bridge, pancytopenia, and numerous burn marks due to inadvertent injuries. Bilateral blepharoplasty was performed, and the excised tissue submitted for histopathological evaluation that revealed non-caseating granulomatous perineural inflammation with numerous acid-fast bacilli in dermal layers and nerves. These findings prompted a diagnosis of lepromatous leprosy with suspected bone marrow involvement. The source of the infection was unknown. The blepharoplasty restored his visual fields and multi-drug therapy (MDT) improved his general health and wellbeing with concomitant reductions of pancytopenia, fatigue, and facial edema. CONCLUSIONS AND IMPORTANCE: Biopsy histopathology, in patients with longstanding ocular adnexal inflammation, can facilitate diagnosis and treatment. To the authors' knowledge, this is an unusual ocular leprosy presentation and represents the first leprosy case diagnosed via blepharoplasty.

Methylene Blue Inhibits Acute Hemorrhagic Conjunctivitis Virus Production and Induction of Caspase-3 Mediated Human Corneal Cell Cytopathy.

BACKGROUND: 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.

Trigeminal hypertrophic interstitial neuropathy presenting as unilateral proptosis, ptosis, tearing, and facial neuralgia.

PURPOSE: To show the utility of MRI and histology in diagnosing rare cases of trigeminal hypertrophic interstitial neuropathy (HIN). OBSERVATIONS: A 57-year-old African-American woman presented with a 4-year history of right eye proptosis with tearing, headaches, and worsening right-sided trigeminal neuralgia symptoms and jaw pain. HIV and diabetes tests were negative and thyroid function was normal. MRI identified abnormal thickening of all trigeminal nerve divisions and proptosis secondary to right trigeminal nerve V1 division enlargement. The excised tissue contained S-100 positive Schwann cells in an onion-bulb pattern. Headaches resolved, but proptosis and mild trigeminal neuralgia remained 1 year post-surgery. CONCLUSIONS AND IMPORTANCE: Trigeminal HIN is very rare, but presents as chronic progressive ocular symptoms with trigeminal neuralgia. Trigeminal nerve hypertrophy is identified by MRI and confirmed histopathologically by detection of Schwann cells in an onion bulb formation.

Case Report of Proliferative Peripheral Retinopathy in Two Familial Lissencephaly Infants with Miller-Dieker Syndrome.

A complete ophthalmic examination is not routinely performed on infants with Miller-Dieker syndrome (MDS, chromosome 17p13.3 microdeletion). The authors present the cases of four cousins with MDS who also carried a 16p13.3 microduplication (not associated with Rubinstein-Taybi syndrome). Retinopathy of prematurity-like proliferative peripheral retinopathy (PPR) was detected in two male first cousins, but was not detected in the female half-cousins. PPR in the first infant resolved by 4 months, but the second infant's PPR progressed, requiring photocoagulation followed by lens-sparing vitrectomy. While ocular abnormalities are more prevalent and severe in other lissencephalopathies, the PPR in these MDS infants underscores the sight-saving potential of performing an ophthalmologic exam with early molecular testing for all lissencephaly infants.

EAAT and Xc⁻ Exchanger Inhibition Depletes Glutathione in the Transformed Human Lens Epithelial Cell Line SRA 01/04.

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.

Acute hemorrhagic conjunctivitis: anti-coxsackievirus A24 variant secretory immunoglobulin A in acute and convalescent tear.

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.

Recurrent annular peripheral choroidal detachment after trabeculectomy.

We report a challenging case of recurrent flat anterior chamber without hypotony after trabeculectomy in a 54-year-old Black male with a remote history of steroid-treated polymyositis, cataract surgery, and uncontrolled open angle glaucoma. The patient presented with a flat chamber on postoperative day 11, but had a normal fundus exam and intraocular pressure (IOP). Flat chamber persisted despite treatment with cycloplegics, steroids, and a Healon injection into the anterior chamber. A transverse B-scan of the peripheral fundus revealed a shallow annular peripheral choroidal detachment. The suprachoroidal fluid was drained. The patient presented 3 days later with a recurrent flat chamber and an annular peripheral choroidal effusion. The fluid was removed and reinforcement of the scleral flap was performed with the resolution of the flat anterior chamber. A large corneal epithelial defect developed after the second drainage. The oral prednisone was tapered quickly and the topical steroid was decreased. One week later, his vision decreased to count fingers with severe corneal stromal edema and Descemet's membrane folds that improved to 20/50 within 24 h of resumption of the oral steroid and frequent topical steroid. The patient's visual acuity improved to 20/20 following a slow withdrawal of the oral and topical steroid. Eight months after surgery, the IOP was 15 mm Hg without glaucoma medication. The detection of a shallow anterior choroidal detachment by transverse B-scan is critical to making the correct diagnosis. Severe cornea edema can occur if the steroid is withdrawn too quickly. Thus, steroids should be tapered cautiously in steroid-dependent patients.

Bilateral acute pyogenic conjunctivitis with iritis induced by unilateral topical application of bacterial peptidoglycan muramyl dipeptide in adult rabbits.

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.

Diabetic and non-diabetic human cornea and tear γ-glutamyl transpeptidase activity.

BACKGROUND: 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.

Age-related decrease in human corneal γ-glutamyltranspeptidase activity.

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.

Orbital sporadic Burkitt lymphoma in an adult diabetic African American female and a review of adult orbital cases.

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.

Multiple caspases mediate acute renal cell apoptosis induced by bacterial cell wall components.

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.

Glutamate, excitatory amino acid transporters, Xc- antiporter, glutamine synthetase, and gamma-glutamyltranspeptidase in human corneal epithelium.

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.

Orbital metastasis of keratinizing squamous cell cervical carcinoma with giant cells. A case report.

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.

Plasma levels of cell-free apoptotic DNA ladders and gamma-glutamyltranspeptidase (GGT) in diabetic children.

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.

Expression of recombinant protein encoded by LOC387715 in Escherichia coli.

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.

Apical localization of glutamate in GLAST-1, glutamine synthetase positive ciliary body nonpigmented epithelial cells.

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.

Dendritic cell surface calreticulin is a receptor for NY-ESO-1: direct interactions between tumor-associated antigen and the innate immune system.

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.