Microwave- and heat-based decontamination of N95 filtering facepiece respirators: a systematic review.

BACKGROUND: In pandemics such as COVID-19, shortages of personal protective equipment are common. One solution may be to decontaminate equipment such as facemasks for reuse. AIM: To collect and synthesize existing information on decontamination of N95 filtering facepiece respirators (FFRs) using microwave and heat-based treatments, with special attention to impacts on mask function (aerosol penetration, airflow resistance), fit, and physical traits. METHODS: A systematic review (PROSPERO CRD42020177036) of literature available from Medline, Embase, Global Health, and other sources was conducted. Records were screened independently by two reviewers, and data was extracted from studies that reported on effects of microwave- or heat-based decontamination on N95 FFR performance, fit, physical traits, and/or reductions in microbial load. FINDINGS: Thirteen studies were included that used dry/moist microwave irradiation, heat, or autoclaving. All treatment types reduced pathogen load by a log10 reduction factor of at least three when applied for sufficient duration (>30 s microwave, >60 min dry heat), with most studies assessing viral pathogens. Mask function (aerosol penetration <5% and airflow resistance <25 mmH2O) was preserved after all treatments except autoclaving. Fit was maintained for most N95 models, though all treatment types caused observable physical damage to at least one model. CONCLUSIONS: Microwave irradiation and heat may be safe and effective viral decontamination options for N95 FFR reuse during critical shortages. The evidence does not support autoclaving or high-heat (>90°C) approaches. Physical degradation may be an issue for certain mask models, and more real-world evidence on fit is needed.

Efficacy and safety of disinfectants for decontamination of N95 and SN95 filtering facepiece respirators: a systematic review.

BACKGROUND: Decontaminating and reusing filtering facepiece respirators (FFRs) for healthcare workers is a potential solution to address inadequate FFR supply during a global pandemic. AIM: The objective of this review was to synthesize existing data on the effectiveness and safety of using chemical disinfectants to decontaminate N95 FFRs. METHODS: A systematic review was conducted on disinfectants to decontaminate N95 FFRs using Embase, Medline, Global Health, Google Scholar, WHO feed, and MedRxiv. Two reviewers independently determined study eligibility and extracted predefined data fields. Original research reporting on N95 FFR function, decontamination, safety, or FFR fit following decontamination with a disinfectant was included. FINDINGS AND CONCLUSION: A single cycle of vaporized hydrogen peroxide (H2O2) successfully removes viral pathogens without affecting airflow resistance or fit, and maintains an initial filter penetration of <5%, with little change in FFR appearance. Residual hydrogen peroxide levels following decontamination were within safe limits. More than one decontamination cycle of vaporized H2O2 may be possible but further information is required on how multiple cycles would affect FFR fit in a real-world setting before the upper limit can be established. Although immersion in liquid H2O2 does not appear to adversely affect FFR function, there is no available data on its ability to remove infectious pathogens from FFRs or its impact on FFR fit. Sodium hypochlorite, ethanol, isopropyl alcohol, and ethylene oxide are not recommended due to safety concerns or negative effects on FFR function.

Decontaminating N95 and SN95 masks with ultraviolet germicidal irradiation does not impair mask efficacy and safety.

Inadequate supply of filtering facepiece respirators (FFRs) for healthcare workers during a pandemic such as the novel coronavirus outbreak (SARS-CoV-2) is a serious public health issue. The aim of this study was to synthesize existing data on the effectiveness of ultraviolet germicidal irradiation (UVGI) for N95 FFR decontamination. A systematic review (PROSPERO CRD42020176156) was conducted on UVGI in N95 FFRs using Embase, Medline, Global Health, Google Scholar, WHO feed, and MedRxiv. Two reviewers independently determined eligibility and extracted predefined variables. Original research reporting on function, decontamination, or mask fit following UVGI were included. Thirteen studies were identified, comprising 54 UVGI intervention arms and 58 N95 models. FFRs consistently maintained certification standards following UVGI. Aerosol penetration averaged 1.19% (0.70-2.48%) and 1.14% (0.57-2.63%) for control and UVGI arms, respectively. Airflow resistance for the control arms averaged 9.79 mm H2O (7.97-11.70 mm H2O) vs 9.85 mm H2O (8.33-11.44 mm H2O) for UVGI arms. UVGI protocols employing a cumulative dose >20,000 J/m2 resulted in a 2-log reduction in viral load. A >3-log reduction was observed in seven UVGI arms using >40,000 J/m2. Impact of UVGI on fit was evaluated in two studies (16,200; 32,400 J/m2) and no evidence of compromise was found. Our findings suggest that further work in this area (or translation to a clinical setting) should use a cumulative UV-C dose of 40,000 J/m2 or greater, and confirm appropriate mask fit following decontamination.

Decontamination interventions for the reuse of surgical mask personal protective equipment: a systematic review.

BACKGROUND: The high demand for personal protective equipment during the novel coronavirus outbreak has prompted the need to develop strategies to conserve supply. Little is known regarding decontamination interventions to allow for surgical mask reuse. AIM: To identify and synthesize data from original research evaluating interventions to decontaminate surgical masks for the purpose of reuse. METHODS: MEDLINE, Embase, CENTRAL, Global Health, the WHO COVID-19 database, Google Scholar, DisasterLit, preprint servers, and prominent journals from inception to April 8th, 2020, were searched for prospective original research on decontamination interventions for surgical masks. Citation screening was conducted independently in duplicate. Study characteristics, interventions, and outcomes were extracted from included studies by two independent reviewers. Outcomes of interest included impact of decontamination interventions on surgical mask performance and germicidal effects. FINDINGS: Seven studies met eligibility criteria: one evaluated the effects of heat and chemical interventions applied after mask use on mask performance, and six evaluated interventions applied prior to mask use to enhance antimicrobial properties and/or mask performance. Mask performance and germicidal effects were evaluated with heterogeneous test conditions. Safety outcomes were infrequently evaluated. Mask performance was best preserved with dry heat decontamination. Good germicidal effects were observed in salt-, N-halamine-, and nanoparticle-coated masks. CONCLUSION: There is limited evidence on the safety or efficacy of surgical mask decontamination. Given the heterogeneous methods used in studies to date, we are unable to draw conclusions on the most efficacious and safe intervention for decontaminating surgical masks.

Influence of race/ethnicity on prevalence and presentation of endometriosis: a systematic review and meta-analysis.

BACKGROUND: Understanding the impact of race/ethnicity on the prevalence and presentation of endometriosis may help improve patient care. OBJECTIVE: To review systematically the evidence for the influence of race/ethnicity on the prevalence of endometriosis. SEARCH STRATEGY: CENTRAL, MEDLINE, PubMed, Embase, LILACS, SCIELO, and CINAHL databases, as well as the grey literature, were searched from date of inception until September 2017. SELECTION CRITERIA: Randomised control trials and observational studies reporting on prevalence and/or clinical presentation of endometriosis. DATA COLLECTION AND ANALYSIS: Twenty studies were included in the review and 18 studies were used to calculate odds ratio (OR) with 95% confidence interval (CI) through a random effects model. Methodological quality was assessed using the Newcastle-Ottawa risk of bias scale (NOS). MAIN RESULTS: Compared with White women, Black woman were less likely to be diagnosed with endometriosis (OR 0.49, 95% CI 0.29-0.83), whereas Asian women were more likely to have this diagnosis (OR 1.63, 95% CI 1.03-2.58). Compared with White women, there was a statistically significant difference in likelihood of endometriosis diagnosis in Hispanic women (OR 0.46, 95% CI 0.14-1.50). Significant heterogeneity (I2  > 50%) was present in the analysis for all racial/ethnic groups but was partially reduced in subgroup analysis by clinical presentation, particularly when endometriosis was diagnosed as self-reported, CONCLUSIONS: Prevalence of endometriosis appears to be influenced by race/ethnicity. Most notably, Black women appear less likely to be diagnosed with endometriosis compared with White women. There is scarce literature exploring the influence of race/ethnicity on symptomatology, as well as treatment access, preference, and response. TWEETABLE ABSTRACT: Prevalence of endometriosis may be influenced by race/ethnicity, but there is limited quality literature exploring this topic.

Rainfall-induced release of fecal coliforms and other manure constituents: comparison and modeling.

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl-), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl-, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.

Effect of poultry diet on phosphorus in runoff from soils amended with poultry manure and compost.

Phosphorus in runoff from fields where poultry litter is surface-applied is an environmental concern. We investigated the effect of adding phytase and reducing supplemental P in poultry diets and composting poultry manures, with and without Fe and Al amendments, on P in manures, composts, and runoff. We used four diets: normal (no phytase) with 0.4% supplemental P, normal + phytase, phytase + 0.3% P, and phytase + 0.2% P. Adding phytase and decreasing supplemental P in diets reduced total P but increased water-extractable P in manure. Compared with manures, composting reduced both total P, due to dilution of manure with woodchips and straw, and water-extractable P, but beyond a dilution effect so that the ratio of water-extractable P to total P was less in compost than manure. Adding Fe and Al during composting did not consistently change total P or water-extractable P. Manures and composts were surface-applied to soil boxes at a rate of 50 kg total P ha(-1) and subjected to simulated rainfall, with runoff collected for 30 min. For manures, phytase and decreased P in diets had no significant effect on total P or molybdate-reactive P loads (kg ha(-1)) in runoff. Composting reduced total P and molybdate-reactive P loads in runoff, and adding Fe and Al to compost reduced total P but not molybdate-reactive P loads in runoff. Molybdate-reactive P in runoff (mg box(-1)) was well correlated to water-extractable P applied to boxes (mg box(-1)) in manures and composts. Therefore, the final environmental impact of dietary phytase will depend on the management of poultry diets, manure, and farm-scale P balances.

Nitrogen and phosphorus availability in composted and uncomposted poultry litter.

Poultry litter applications to land have been based on crop N requirements, resulting in application of P in excess of plant requirements, which may cause degradation of water quality in the Chesapeake Bay watershed. The effect of litter source (the Delmarva Peninsula and Moorefield, West Virginia) and composting of poultry litter on N mineralization and availability of P in two soil types (sandy loam and silt loam) was determined in a controlled environment for 120 d. Nitrogen mineralization (percent total organic N converted to inorganic nitrogen) rates were higher for fresh litter (range of 42 to 64%) than composted litter (range of 1 to 9%). The N mineralization rate of fresh litter from the Delmarva Peninsula was consistently lower than the fresh litter from Moorefield, WV. The N mineralization rate of composted litter from either source was not significantly different for each soil type (7 to 9% in sandy loam and 1 to 5% in silt loam) even though composting conditions were completely different at the two composting facilities. Litter source had a large effect on N mineralization rates of fresh but not composted poultry litter. Composting yielded a more predictable and reliable source of mineralizable N than fresh litter. Water-extractable phosphorus (WEP) was similar in soils amended with composted litter from WV and fresh litter from both sources (approximately 10 to 25 and 2 to 14 mg P kg(-1) for sandy loam and silt loam, respectively). Mehlich 1-extractable phosphorus (MEP) was similar in soils amended with WV fresh litter and composted litter from both sources (approximately 100 to 140 and 60 to 90 mg P kg(-1) for sandy loam and silt loam, respectively). These results suggest that the composting process did not consistently reduce WEP and MEP, and P can be as available in composted poultry litter as in fresh poultry litter.

Manure phosphorus extractability as affected by aluminum- and iron by-products and aerobic composting.

Shifts in manure phosphorus (P) chemical forms and pool sizes induced by water treatment residuals and industrial mineral by-products are largely undefined. We conducted a manure P fractionation study to determine mechanisms of reduction of dissolved reactive phosphorus (DRP) in poultry manure upon mineral by-product additions. The effects of composting on the P immobilization efficacy of the by-products were determined using laboratory self-heating composting simulators. The mineral by-products included an aluminum-water treatment residual (Al-WTR) and an iron-rich titanium-processing by-product. The noncomposted manure averaged 0.11 g g(-1) of total P as DRP forms. The by-products significantly reduced manure DRP, by an average of 39 and 48% in the Al- and the Fe-treated manure, respectively. The by-products also reduced the 0.5 M NH4F-extractable phosphorus (FEP) fraction. Shifts in P forms between FEP and 0.1 M NaOH-extractable phosphorus (SHEP) depended upon the Al and Fe contents of the by-products while the combined FEP + SHEP pool remained constant. Phosphate sorption measurements supported the observations that the Fe-rich by-product was more effective at reducing manure DRP and enhancing the formation of SHEP forms at the expense of FEP than the Al-WTR. Composting had no effect on the efficacy of either by-product to reduce DRP. Potential mechanisms of enhanced P stabilization in treated manure upon composting included chemical shifts from the DRP and FEP fractions to the citrate-bicarbonate-dithionite extractable P fraction. Thus, the choice of P immobilization agents affected the stability of immobilized P forms and should be taken into consideration in developing manure processing and nutrient stabilization methods.

Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells.

This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.

Compost age and sample storage effects on maturity indicators of biosolids compost.

Compost product safety and quality assurance are required to meet the needs of the horticultural, agricultural, and silvicultural user markets. At present, there exist no industry-wide sampling and testing protocols for compost products, thus limiting the production sector. The objective of this research was to test three methods for determining compost maturity. The study followed the composting process of a locally successful commercial composting operation that had been producing lime-stabilized biosolids compost in the Washington, DC metro region for 12 yr. Change over time in the dependent variables--Dewar flask self-heating capacity, oxygen uptake rate, and cation exchange capacity (CEC)-during a 57-d composting of lime-stabilized biosolids was studied. Because cold storage at 4 degrees C is recommended when compost samples cannot be tested for maturity immediately, cold storage of up to 11 wk was included as a variable. Mathematical models were developed that predict change in the Dewar flask self-heating capacity, oxygen uptake rate, and CEC with composting time and storage at 4 degrees C. The Dewar flask self-heating test was the most useful indicator of compost maturity. This test showed change throughout the 57-d biosolids composting period while oxygen respirometry did not change after 29 d. The CEC was found to increase with age and storage. Storage effects varied for the different tests. Except for Days 1 and 57, composts continued to stabilize during storage. Testing stored composts may produce erroneous results that suggest the compost is mature.

A method for detection of pseudobactin, the siderophore produced by a plant-growth-promoting pseudomonas strain, in the barley rhizosphere.

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10 CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif Nal selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 x 10 mol of ferric pseudobacting g (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.

Human melanoma-associated antigen expression on human neuroblastoma cells: effects of differentiation inducers.

We have described two human melanoma-associated antigens (HMAA), recognized by the murine monoclonal antibodies LS62 and LS109. LS62 recognizes the neuroglandular antigen (NGA), which is overexpressed in neoplastic melanocytes as well as in several tissues of neuroectodermal origin. These antibodies were used to screen six neuroblastoma cell lines and one neuroepithelioma cell line. A melanoma cell line, G361, known to express the two antigens, was used as the positive control. Variable expression of the two antigens was detected in neuroblastoma cells. The surface expression of NGA and of the LS109 antigen was modulated in parallel with the morphological differentiation induced by retinoic acid, 5-bromodeoxyuridine, or cyclic AMP analog/activators. The modulation of the expression of the two HMAA was detected in G361 melanoma cells and in one of the neuroblastoma cell lines, SK-N-SH. These results suggest altered expression of both antigens during melanoma and neuroblastoma cell differentiation in culture.

Effects of retinoic acid and bromodeoxyuridine on human melanoma-associated antigen expression in small cell lung carcinoma cells.

The dispersed neuroendocrine system includes cells with different embryological derivations, sharing a common neuroendocrine (NE) program, as indicated by the expression of NE markers, some of which are shared antigenic determinants. We report here that the small cell lung carcinoma cells NCI-H69 express the two human melanoma-associated antigens (HMAA) NGA/LS62 an LS109. Incubation of NCI-H69 cells with maturational inducers, such as retinoic acid and bromodeoxyuridine (BrdU), upregulated the expression of both HMAA. Exposure to BrdU for 4 weeks induced the appearance of a different phenotype in subpopulations of NCI-H69 cells, which became epithelioid, substrate-adherent, grew in monolayer and continued to express NE-associated antigens in variable amount. The shift in phenotype was not reversible after BrdU withdrawal and was maintained for at least 6 months in continuous culture. The substrate adhesion of NCI-H69 cells was paralleled by a change in NGA glycosylation pattern, thus suggesting a possible functional role for NGA in cell substrate adhesion/recognition.

Venous and fingertip blood to calculate plasma volume shift following exercise.

This study determined whether fingertip blood samples used to calculate percentage change in calculated plasma volume following exercise were in agreement with values obtained from venous blood samples. Twenty-five subjects engaged in two cycle ergometer exercises at 100 and 200 W, with percentage plasma volume shift (% PVS) determined after each from venous (VB) and fingertip (FT) blood. Values for % PVS were FT -6.25% compared with VB -8.04% (P less than 0.05), with the correlation between the two methods at r = 0.88. The following equation was established: corrected FT % PVS = (0.8662 * FT) - 2.625; SEE = 2.60%. In order to cross-validate this equation, fifteen additional subjects submitted to VB and FT. Corrected FT % PVS using the established equation resulted in a mean value of 9.53 compared with 10.53% for actual VB % PVS. Although these means were not significantly different, there was approximately a 25% chance that the corrected FT % PVS would be more than one standard error of estimate from the regression line. It was concluded that FT underestimates VB % PVS. However, when limited to group data, FT can be corrected to favorably represent VB % PVS following moderate to heavy cycle ergometer exercise.

Biosynthesis, glycosylation and intracellular processing of the neuroglandular antigen, a human melanoma-associated antigen.

Neuroglandular antigen (NGA) was identified as a human melanoma-associated antigen by a panel of murine monoclonal antibodies of both IgG2a (LS62, LS76, LS159) and IgG1 (LS113, LS140, LS152) subclasses, developed in this laboratory (L. Sikora, A. Pinto, D. Demetrick, W. Dixon, S. Urbanski, and L. M. Jerry, Int. J. Cancer, 39: 138-145, 1987). Monoclonal antibody LS62 was used to immunoprecipitate NGA from radiolabeled cultured melanoma cells, and it behaved as a heterogeneous glycoprotein "smear" on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (Mr 29,000-70,000). Radioactive pulse-chase time course experiments using human melanoma cells cultured in the presence or absence of inhibitors of protein glycosylation showed that the antigen consisted of a core protein with a molecular weight of 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule was modified by the addition of at least three N-linked oligosaccharide side chains (as revealed by limited N-glycanase digestion) to give a precursor form with a molecular weight of approximately 34,000. Subsequent processing steps yielded a heterogeneous family of glycoproteins with varying amounts of covalently attached carbohydrate. Much of this heterogeneity in both molecular weight and pI (as revealed by two-dimensional electrophoresis) could be removed by treatment of the antigen with neuraminidase, suggesting heavy sialylation of the glycoprotein. NGA could be detected on the surface of melanoma cells by fluorescence-activated cell sorter analysis, surface radioiodination, and, as previously shown, immunoperoxidase staining. However, there was a larger intracellular pool of the molecule and the antigen was rapidly released into the culture supernatant. The function of NGA remains unknown but its elevated expression in transformed melanocytes have prompted this characterization to understand its biochemical nature and relation to other melanoma-associated antigens.

Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10.

Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 x 10 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores.

Isolation and characterization of a heterodimeric surface antigen on human melanoma cells and evidence that it is the 4F2 cell activation/proliferation molecule.

Murine monoclonal antibody (MAb) LS109 was one of a series of MAbs produced by hyperimmunization of mice with detergent extracts of pooled melanoma cell lines and metastatic melanoma patient tumors. ELISA screening of extracts of individual cultured melanoma cell lines and single patient tumors with MAb LS109 gave an interesting pattern of reactivity. The antibody was strongly positive with some of these extracts, yet negative or weakly positive with others. In addition, there was strong reactivity with a restricted set of normal necropsy tissues and certain non-melanoma tumor extracts. Taken together, our data suggest that MAb LS109 recognizes a normal differentiation antigen which is perhaps aberrantly expressed or over-produced during certain stages of melanoma tumor progression. The antigen recognized by LS109 is a heterodimeric surface glycoprotein molecule, consisting of an 89-kDa "heavy" chain linked by disulfide bonds to an 83-kDa "light" chain. Under non-reducing SDS-PAGE conditions, the intact dimer migrates with an Mr of approximately 140kDa. The 89-kDa component appears to be heavily N-glycosylated whereas the 38-kDa component has little, if any, covalently attached carbohydrate. Our data show the biosynthesis, glycosylation and turnover of the LS109 antigen, as well as evidence of its surface localization. In addition, evidence is presented that the LS109 antigen is identical to the 4F2 cell activation/proliferation molecule previously described on a variety of normal and neoplastic cells.

Inhibition of neuroglandular antigen (NGA) glycosylation by phorbol ester in human melanoma cells.

NGA is a human melanoma-associated antigen recognized by a panel of murine monoclonal antibodies developed in this laboratory. NGA consists of a 23.5 kDa core protein which is glycosylated in vivo to give a family of glycoproteins (30-60 kDa). Treatment of human melanoma G361 cells with the phorbol ester PMA resulted in apparent partial inhibition of NGA glycosylation. After PMA treatment, NGA appeared as 3 different bands of 24, 29 and 34 kDa on SDS-PAGE. The 29 kDa band is similar to the one obtained by treatment with the ionophore monensin, which inhibits NGA O-glycosylation. PMA can modulate plasma membrane ion exchange, most likely by activating protein kinase C. In G361 cells PMA may produce the same net effect as monensin, by impairing transport in the Golgi complex and consequently inhibiting protein O-glycosylation through an ionophore-like effect. Treatment of G361 cells with both PMA and protein kinase C inhibitors re-established the usual NGA glycosylation pattern. Thus the observed effect of PMA on NGA glycosylation is reversible and appears to be mediated by protein kinase C activation.

Characterization of a novel neuroglandular antigen (NGA) expressed on abnormal human melanocytes.

Murine monoclonal antibodies (MAbs) were produced with reactivity to human malignant melanoma. Six MAbs, 3 of the IgGI (LS113, LS140, LS152) and 3 of the IgG2a (LS59, LS62, LS76) subclasses, were selected for their binding, with an identical pattern of reactivity, to a novel melanoma-associated antigen. As characterized by the enzyme-linked immunosorbent assay (ELISA), these MAbs were found to be positive on n-octyl-beta-D-glucopyranoside extracts of all 10 melanoma cell lines tested and on extracts of 22 metastatic melanoma tumors. The antibodies had minimal reaction with a panel of 14 normal adult tissue extracts. A degree of cross-reactivity was observed with 50% of 39 non-melanoma tumor extracts. The results obtained with the ELISA on cell line and tissue extracts were duplicated using the ABC method of peroxidase staining. The pattern of cross-reactivity, as demonstrated by the intense staining of paraffin-embedded and frozen tissue sections of normal, benign and malignant tissues, defines the recognized protein as a neuroglandular antigen (NGA). Immunoadsorbents made with the antibodies were used to purify the antigen shed from cultured melanomas. All 6 MAbs recognized this purified antigen while 5 other antimelanoma antibodies did not react with it. On gel electrophoresis this antigen is a highly glycosylated glycoprotein with a protein core of 21 kDa.