Renal pathological analysis using galactose-deficient IgA1-specific monoclonal antibody is a strong tool for differentiation of primary IgA nephropathy from secondary IgA nephropathy.

In several cases with IgA nephropathy (IgAN), differential diagnosis is difficult due to the complication with other systemic diseases which can induce secondary IgAN. Recently, we demonstrated that immunostaining with galactose-deficient IgA1-specific monoclonal antibody (KM55 mAb) specifically showed positive in primary IgAN cases. Here, we report four cases which we could make definitive diagnosis by immunohistological analysis using KM55 mAb. The underlying systemic diseases are rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), hepatitis C (HCV) and Crohn's disease (CD). Renal pathological findings in the four cases revealed mesangial proliferative glomerulonephritis with IgA and C3 deposits. Immunostaining with KM55 mAb was positive for three cases complicated with RA, SLE and CD, respectively. Thus, these three cases were diagnosed as primary IgAN and treated with tonsillectomy and steroid pulse therapy. These three cases finally achieved clinical remission. On the other hand, the case with HCV showed negative for KM55. Finally, we diagnosed as HCV-related nephropathy and successfully treated by antiviral agents. These cases suggested KM55 mAb is a strong tool to differentiate primary IgAN from secondary IgAN.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na+/H+ exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Salivary profile in diabetic patients: biochemical and immunological evaluation.

BACKGROUND: The aim of this study was to evaluate the biochemical and immunological characteristics of saliva from diabetic patients compared to non-diabetic adults. METHODS: Eighty-eight diabetic adults and 39 non-diabetic adults (control) were included in the study. Glucose, urea, calcium, total protein and amylase were determined by a colorimetric method. The levels of secretory IgA and the IgA anti-Streptococcus mutans and anti-insulin IgA antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Caries status was evaluated using the DMFT index. RESULTS: Glucose, urea, calcium, anti-S. mutans IgA, total IgA, and anti-insulin IgA were significantly higher in diabetic patients, whereas total protein and amylase levels were lower in these patients. There was no positive correlation between blood and salivary glucose levels in either group. Diabetic patients had a higher DMFT index. CONCLUSIONS: The present study showed for the first time that IgA levels in diabetic patients'saliva, shows correlation with systemic biochemical parameters. Thus the saliva is an useful tool to follow the systemic health status in these patients.

Cutaneous delayed-type hypersensitivity in patients with atopic dermatitis: reactivity to topical preservatives.

BACKGROUND: Patients with atopic dermatitis (AD) have chronic dry skin to which they frequently apply skin care products containing preservatives, and they are predisposed to developing cutaneous delayed-type hypersensitivity. OBJECTIVE: We sought to compare the rates of positive patch test reactions to allergens on the North American Contact Dermatitis Group (NACDG) standard tray among patients with and without AD and to assess whether atopic patients in our database were more likely to patch test positive to preservatives. METHODS: A total of 2453 patients underwent patch testing to the NACDG standard screening series. The incidence of positive patch test reaction among patients with AD (n = 342) and without AD (n = 2111) was assessed. Statistical analysis was done using a χ(2) test. RESULTS: Compared with nonatopic patients, patients with AD were statistically more likely to have positive patch tests. AD was associated with contact hypersensitivity to quaternium-15, imidazolidinyl urea, DMDM hydantoin, and 2-bromo-2-nitropropane-1,3-diol but not to parabens, formaldehyde, or diazolidinyl urea. LIMITATIONS: Only patients suspected of having allergic contact dermatitis were tested. Our population was geographically limited to metropolitan Kansas City, MO, and metropolitan New York City, NY. CONCLUSIONS: Patients with AD should avoid the use of skin care products preserved with formaldehyde releasers.

Cultivos discontinuos alimentados con urea de la cianobacteria Phormidium sp. en función de la salinidad y edad del cultivo / Urea fed-batch cultures of the cyanobacterium Phormidium sp. as a function of the salinity and age of cultures

Se comparó la eficiencia de sistemas de cultivos discontinuos alimentados versus cultivos discontinuos convencionales, en cuanto a concentración de nitrógeno, adicionando 0,2 mM de urea cada tres días al final de la fase exponencial, durante 21 días. Se realizaron cultivos con un volumen de 1500 mL a 15 y 35 UPS de salinidad, enriquecidos con medio ALGAL 8mM NaNO3, a 238 µmol q m-2 s-1, aireación constante, fotoperiodo 12:12 horas y temperatura de 29 ±3°C. Phormidium sp. posee la capacidad de hidrolizar la urea; mostrando una asimilación de 65±7,07% de la misma, con la mayor producción (p<0,05) de clorofila a, ficocianina y proteínas de 20,26±1,24; 203,47±12,83 y 707,87±28,47 µg mL-1en los cultivos alimentados. La producción de pigmentos vario en el tiempo, independientemente a la salinidad y sistema de cultivo, mientras que la producción de proteínas y carbohidratos totales fue directamente proporcional a la edad del cultivo, con valores máximos de 612,74 ± 5,41 µg mL-1 y 8,96±0,08 mg mL-1 respectivamente a los 31 días. La síntesis de lípidos y EPS fueron influenciadas (p<0,05) por la salinidad, presentando los máximos de lípidos a 15 UPS con 12,22±2,91µg mL-1, y los EPS se incrementaron a 35 UPS con 2,00 ± 0,26 y 2,03 ± 0,15 mg mL-1. Estos resultados determinan que los cultivos de Phormidium sp. alimentados con urea y a salinidades de 15 y 35 UPS, representan una alternativa económica para la producción de clorofila a, ficocianina y proteínas, incrementándose un 31,04; 40,72 y 31,94 % respectivamente en comparación con cultivos no alimentados.

Fed-batch system efficiency versus batch cultures was compared in relation to nitrogen concentration, adding 0,2mM urea at the end of the exponential phase, during 21 days. Cultures were carried out in 1500 mL to 1.5 and 3.5 UPS of salinity, enriched with Algal medium 8mM NaNO3, 238 mol q m-2 s-1, constant aeration, photoperiod 12:12 h. and 29 ±3°C. Phormidium sp. is able to hydrolyze urea; showing a total assimilation of 65±7.07%, with the highest (p< 0.05) chlorophyll a, phycocyanin and protein production of 20.26 ± 1.24, 203.47 ± 12.83 and 707.87 ± 28.47 µg mL-1 in the fed-batch cultures. On the other hand, pigment production varies in time, regardless salinity and culture system. Proteins and total carbohydrate production were directly proportional to the age of cultures, with maximum values of 612.74 ± 5.41 µg mL-1 and 8.96 ± 0.08 mg mL-1, respectively. Lipid and EPS were influenced (p< 0.05) by salinity, showing maximum of lipids at 15 UPS with 12.22±2.91 µg mL-1, and EPS at 15 and 35 UPS with 2.00 ± 0.26 and 2.03 ± 0.15 mg mL-1. These results determine that Phormidium sp. cultures fed with urea, to salinities of 15-35 UPS, represent an economic alternative for chlorophyll a, phycocyanin and protein production, with an increase of 31.04, 40.72 and 31.94% respectively in comparison with non-fed cultures.

Oral activated charcoal adsorbent (AST-120) ameliorates chronic kidney disease-induced intestinal epithelial barrier disruption.

BACKGROUND: Chronic kidney disease (CKD) impairs intestinal barrier function which by allowing influx of noxious products causes systemic inflammation. We have recently shown that intestinal barrier dysfunction in CKD is due to degradation of epithelial tight junction (TJ) which is, in part, mediated by influx of urea and its conversion to ammonia by microbial urease. We hypothesized that by adsorbing urea and urea-derived ammonia, oral activated charcoal (AST-120) may ameliorate CKD-induced intestinal epithelial barrier disruption and systemic inflammation. METHODS: Rats were randomized to the CKD or control groups. The CKD group was fed a chow containing 0.7% adenine for 2 weeks. They were then randomized to receive a chow with or without AST-120 (4 g/kg/day) for 2 weeks. Rats consuming regular diet served as controls. Animals were then euthanized, colons were removed and processed for Western blot and immunohistology, and plasma was used to measure endotoxin and oxidative and inflammatory markers. RESULTS: Compared with the controls, the untreated CKD rats showed elevated plasma endotoxin, IL-6, TNF-α, MCP-1, CINC-3, L-selectin, ICAM-1, and malondialdehyde, and depletions of colonic epithelial TJ proteins, claudin-1, occludin, and ZO1. Administration of AST-120 resulted in partial restoration of the epithelial TJ proteins and reduction in plasma endotoxin and markers of oxidative stress and inflammation. CONCLUSIONS: CKD animals exhibited depletion of the key protein constituents of the colonic epithelial TJ which was associated with systemic inflammation, oxidative stress and endotoxemia. Administration of AST-120 attenuated uremia-induced disruption of colonic epithelial TJ and the associated endotoxemia, oxidative stress and inflammation.

Influence of different length linker containing DHEA-7-CMO-enzyme conjugates on sensitivity and specificity of DHEA-17-CMO-antibody.

Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates.

Transient receptor potential vanilloid 1 agonists as candidates for anti-inflammatory and immunomodulatory agents.

We recently demonstrated that SA13353 [1-[2-(1-adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea], a novel transient receptor potential vanilloid 1 (TRPV1) agonist, inhibits TNF-alpha production through the activation of capsaicin-sensitive afferent neurons. In the present study, we investigated the effects of SA13353 on lipopolysaccharide (LPS)-induced cytokine production and a murine model of experimental autoimmune encephalomyelitis (EAE). SA13353 inhibited LPS-induced TNF-alpha and interleukin (IL)-1beta production while augmenting IL-10 production in mice. It also inhibited TNF-alpha and IL-1beta mRNA expression, and increased IL-10 mRNA expression in LPS-treated murine liver. These effects were not observed in TRPV1 KO and sensory denervated mice. Capsaicin and SA13353 increased serum neuropeptide levels, and calcitonin gene-related peptide fragment 8-37 (CGRP(8)(-)(37)), a CGRP antagonist, partially blocked the inhibitory effects of capsaicin and SA13353 on LPS-induced TNF-alpha production. These results suggest that the TPPV1 agonistic effects inhibit TNF-alpha production, at least partially, via neuropeptide release. SA13353 did not directly affect LPS-induced cytokine production in vitro using RAW264.7 macrophages, which do not express TRPV1. Therefore, we consider SA13353 to be a good tool for the investigation of the value of TRPV1 agonists for the treatment of chronic inflammation. In a murine EAE model, SA13353 attenuated clinical signs and histopathological changes. SA13353 attenuated cytokine levels, including TNF-alpha, IL-1beta, IL-12p40, IL-17, and interferon (IFN)-gamma, after proteolipid protein (PLP) immunization. In addition, SA13353 attenuated the increase of IL-17-producing cells. These results suggest that TRPV1 agonists may act as anti-inflammatory and immunomodulatory agents in vivo in certain inflammatory diseases.

Urea, the most abundant component in urine, cross-reacts with a commercial 8-OH-dG ELISA kit and contributes to overestimation of urinary 8-OH-dG.

Urinary 8-OH-dG is commonly analyzed as a marker of oxidative stress. For its analysis, ELISA and HPLC methods are generally used, although discrepancies in the data obtained by these methods have often been discussed. To clarify this problem, we fractionated human urine by reverse-phase HPLC and assayed each fraction by the ELISA method. In addition to the 8-OH-dG fraction, a positive reaction was observed in the first eluted fraction. The components in this fraction were examined by the ELISA. Urea was found to be the responsible component in this fraction. Urea is present in high concentrations in the urine of mice, rats, and humans, and its level is influenced by many factors. Therefore, certain improvements, such as a correction based on urea content or urease treatment, are required for the accurate analysis of urinary 8-OH-dG by the ELISA method. In addition, performance of the ELISA at 4 degrees C reduced the recognition of urea considerably and improved the 8-OH-dG analysis.

Uric acid nephrolithiasis: proton titration of an essential molecule?

PURPOSE OF REVIEW: The majority of uric acid nephrolithiasis in humans occurs in the absence of frank hyperuricosuria and is primarily a disease of excessively low urinary pH. Uric acid is substantially less soluble than urate salts so in low urine pH urate is protonated, thus favoring precipitation even under what is considered physiologic concentrations of total urinary uric acid/urate. This commentary examines the rationales behind the existence of uric acid in urine and body fluids in vertebrate evolution. RECENT FINDINGS: The purpose of uric acid in arthropod, avian and reptilian species is to enable nitrogen excretion in solid state without loss of water. The re-emergence of uric acid in higher primates as an end product of metabolism is intriguing since urea functions perfectly well as a nitrogenous waste. Uric acid must purvey important physiologic functions in primate biology. Numerous roles of uric acid as an antioxidant, immune signaling molecule, and a defender of circulatory integrity have recently been proposed. SUMMARY: There is little doubt that uric acid serves multiple important functions in higher primates. It is also conceivable, however, that this important molecule when present in the wrong concentration or context can lead to undesirable phenotypes.

Experimental elicitation of contact allergy from a diazolidinyl urea-preserved cream in relation to anatomical region, exposure time and concentration.

The elicitation potential of the cosmetic preservative diazolidinyl urea was studied in formaldehyde- and diazolidinyl urea-sensitized volunteer patients using a stepwise controlled exposure design. The test product was a facial moisturizer, preserved with varying concentrations of diazolidinyl urea, ranging from 0.05% to 0.6%. A repeated open application-like exposure test was performed on volunteers and a control group with the test product containing increasing preservative concentrations, on arm, neck and face, sequentially, for 2 weeks or until dermatitis developed. The preservative action in the cream at different test concentrations was tested in microbial challenge tests and was found effective at all concentrations tested. The study established a non-eliciting concentration of diazolidinyl urea of 0.05% in formaldehyde-sensitive patients and showed that the skin reactivity depends on the anatomical region, increasing from the upper arm to neck and, possibly, to the face. The study design, beginning on the upper arm and moving on to the neck and face seems to be relevant for the study of reactions to cosmetic products. A clear dose-response relationship was seen regarding preservative concentration in the product.

Anti-phospholipid antibodies in HIV infection and SLE with or without anti-phospholipid syndrome: comparisons of phospholipid specificity, avidity and reactivity with beta2-GPI.

Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent assay (ELISA) methods. Sera from 30 patients with systemic lupus erythematosus (SLE), but without features of anti-phospholipid syndrome (APS) (SLE/non APS), six with SLE and secondary APS, (SLE/APS) and 11 with primary APS (PAPS) were also evaluated as controls. The resistance of the aPL antibody binding to dissociating agents was evaluated by treating the ELISA wells, after serum incubation with 2 M urea or 0.6 M NaCl for 10 min. An anti-beta2-glycoprotein-I (beta2-GPI) ELISA was used to assess serum reactivity against beta2-GPI, a plasma protein considered as the true antigen of aCL antibodies occurring in APS and SLE patients. The prevalence of aCL, aPS, aPI and aPC antibodies in HIV-1 infection was 36%, 56%, 34% and 43% respectively, which was comparable to that found in SLE/APS and PAPS patients and significantly higher than that observed in SLE/non-APS patients. Anti-beta2-GPI antibodies occurred in 5% of HIV-1 infected vs. 17% in SLE/non-APS (P=0.11), 50% in SLE/APS (P=0.009) and 70% in PAPS patients (P=0.0014). A significant decrease of aPL binding after urea and NaCl treatment was observed in the sera of HIV-1-infected, compared to that of APS patients, indicating that aPL antibodies from HIV-1 infected individuals have low resistance to dissociating agents. In conclusion, aPL antibodies (1) occur in HIV-1 infection; (2) tend to recognize various phospholipids but not beta2-GPI; and (3) are of low resistance to dissociating agents-a finding probably reflecting low antibody avidity. Finally, these, like the autoimmune-type aCL antibodies, tend to recognize the oxidized CL-a finding probably indicating autoantibody generation as a result of neoepitope formation by oxidized PLs.

Production and characterization of monoclonal antibodies against urea derivatives.

A panel of monoclonal antibodies was generated against the urea-based hapten N-(2-N-chloroacetylaminobenzyl)-N'-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Cross reactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.

Diazolidinyl urea: incidence of sensitivity, patterns of cross-reactivity and clinical relevance.

Diazolidinyl urea (DIAZ) is a formaldehyde-releasing preservative used in cosmetics and personal-care products, which has been identified as a sensitizing agent in contact dermatitis. To determine whether DIAZ sensitization is secondary to formaldehyde release or due to its own allergenic properties, we reviewed 708 consecutive patch tests of patients with various dermatologic complaints. Profiles of the 58 individuals (8%) with DIAZ sensitivity were analyzed with respect to sex, age, exposures, and chronicity of dermatitis. Significant coexistent biocide reactivity was demonstrated for DIAZ and formaldehyde (81%); 12% reacted to DIAZ alone. We conclude that the primary mode of sensitization of DIAZ is via formaldehyde release and that independent contact allergy is less frequent.

Immunologic biomarkers associated with an acute exposure to exothermic byproducts of a ureaformaldehyde spill.

A community was exposed for several days to formaldehyde (HCHO), hexamethylenetetramine, trimethylamine, and paraformaldehyde emitted from an overheated tanker car containing ureaformaldehyde resin. Residents experienced acute HCHO symptoms at the time of the accident. Many developed chronic, multiple organ health complaints. Three years following the accident, exposed subjects were compared to residents of a nearby unexposed community for the following immunological parameters: white blood cell count, total lymphocyte count, percent and total lymphocyte subsets (CD5, CD4, CD8, CD19, CD25, and CD26 cells), prevalence of autoantibodies, and antibodies to HCHO-human serum albumin (HCHO-HSA) conjugate. The data were adjusted for gender, age, history of smoking, mobile home residency, and use of wood stoves. There was a statistically significant difference for the following: elevated percent and absolute numbers of CD26 cells (p less than 0.0001); autoantibodies (p less than 0.004), and greater titers of isotypes IgG (p less than 0.0005) and IgM (p less than 0.005) to HCHO-HSA. It is concluded that the exposed subjects had an activated immune system in addition to the elevated autoantibodies. Also, isotypes to HCHO-HSA resulted from the exposure and no other sources, such as smoking, mobile home residency, and use of wood stoves.

Host metabolites that phenotypically increase the resistance of Haemophilus influenzae type b to clearance mechanisms.

Unidentified low-molecular-weight factor(s) in serum or nasopharyngeal secretions were known to phenotypically increase the resistance of Haemophilus influenzae type b (Hib) to bactericidal and opsonic antibodies, and resistance was attributed to two hypothetical mechanisms. Serum components generating resistance were studied. Mechanism 1, present in some Hib strains and their capsule-deficient mutants and accompanied by apparent increases in lipopolysaccharide content, was reproduced with a mixture of glucose, lactate, urea, and bicarbonate. Mechanism 2, present only in capsulated Hib and accompanied by increased capsulation, was reported with a mixture of Ca++ and lactate. Hib incubated with these compounds in buffer or grown in serum filtrate was resistant, but Hib grown in conventional media containing the metabolites in serum filtrate was resistant, but Hib grown in conventional media containing the metabolites was not. The resistant phenotype, which resembles Hib in vivo, may depend on nutrient balance as well as the specific factors. Lactate apparently is an important energy source for Hib.

Contact allergy to diazolidinyl urea (Germall II).

4 cases of contact allergy to diazolidinyl urea (Germall II) in a "hypoallergenic" brand of cosmetics are described. 2 patients sensitized by these cosmetics were not allergic to formaldehyde. 2 other patients already sensitive to formaldehyde had exacerbations of dermatitis due to diazolidinyl urea. The following tentative conclusions were drawn. (i) Contact allergy to diazolidinyl urea may or may not be due to formaldehyde sensitivity. (ii) Patients allergic to formaldehyde may suffer contact allergic reactions from the use of cosmetics containing diazolidinyl urea. (iii) Patients sensitized to diazolidinyl urea may cross-react to imidazolidinyl urea and vice-versa. (iv) It is suggested that the sensitizing potential of diazolidinyl urea is greater than that of imidazolidinyl urea. (v) Aq. solutions may be preferable to pet. for patch testing with diazolidinyl urea.

Demonstration of anti-keratin antibodies by ELISA using keratin or thiol-containing compounds in urea as antigens.

During the development of an enzyme-linked immunosorbent assay (ELISA) for the demonstration of anti-keratin antibodies (AKA) it was observed that rabbit anti-keratin antisera also reacted with polystyrene surfaces treated with beta-mercaptoethanol in 8 M urea (ME-urea). Sera from non-immunized rabbits or rabbits immunized with antigens unrelated to keratin failed to react. The specificity of the reaction was further assessed by absorption experiments and by testing affinity-purified AKA. IgM activity against ME-urea could be demonstrated in 62.5% of sera from patients with infectious mononucleosis and in 37.5% of sera from patients with rheumatoid arthritis, and there was a good correlation to the presence of AKA. Coating of the solid phase with compounds containing free SH groups in 4-8 M urea generated the antigen of this ELISA. The exact molecular configuration of this presumptive synthetic antigen is obscure, but the ME-urea ELISA seems to provide a simple way to detect anti-keratin antibodies of a certain specificity.