Showed Cross Reactivity Research Articles

IRTA2 Protein on Lymphoma Cell Lines and Hairy Cell Leukemia Cells Detected by Novel Monoclonal Antibodies.

The immunoglobulin superfamily receptor translocation associated 2 (IRTA2) gene encodes a cell surface receptor homologous to the family of Fc receptors. Because of the restricted expression of the mRNA in B cell-linage cells, IRTA2 is a new potential target for the immunotherapy of B cell malignancies. To study the function and expression of the IRTA2 gene product, we produced monoclonal antibodies (MAbs) specific to IRTA2. We successfully made 36 MAbs from a mouse immunized with IRTA2-encoding plasmid DNA. All the MAbs reacted with IRTA2-extracellular domain-Fc fusion protein in ELISA and 293T cells expressing IRTA2 in a fluorescence-activated cell sorter (FACS) analysis. Binding of each MAb to a series of deletion mutants of IRTA2-Fc fusion protein and mutual competition of the MAbs to the binding to the whole extracellular domain of IRTA2-Fc fusion protein revealed that the panel of MAbs recognizes more than 10 epitopes on IRTA2. There are at least 1, 1, 2, 4, and 2 epitopes on extracellular domain 1 (ED1), ED3, ED1-3, ED4-9, and whole extracellular domain, respectively. Twenty five out of the 36 MAbs are specific to IRTA2 but 11 others showed cross reactivity to other members of the IRTA family. Among 36 MAbs produced, 3 MAbs (F25, F56 and F119) were used for further analysis based on their specific reactivity with recombinant IRTA2 expressed on 293T cells and lack of cross reactivity with other IRTA family members. In a FACS analysis, MAbs F56 and F119 detected IRTA2 expression on 6/7 B-cell non-Hodgkin's lymphoma and 1/6 Burkitt's lymphoma cell lines. Reverse transcriptase-polymerase chain reaction experiments and Western blotting using MAb F25 confirmed the expression profile. We also analyzed blood samples of 11 patients with hairy cell leukemia (HCL), 11 patients with chronic lymphocytic leukemia (CLL), and 4 patients with follicular lymphomas (FL) by FACS. We detected IRTA2 expression on 100% HCL (11/11), 55% CLL (6/11) and 0% of FL cells (0/4). Our results provide the first evidence that the IRTA2 is expressed on the surface of HCL and CLL cells as well as some human lymphoma cell lines. We expect IRTA2 will be established as a new target for immunotherapy.

Immunological Profile and Safety Evaluation in a 23-Week Single Dose Study in Cynomolgus Monkey with CHIR-12.12, a Fully Human Antagonist Anti-CD40 Antibody.

CD40 is highly expressed in B-cell malignancies and its activation is a growth and survival signal for these cells. We have generated a novel, highly potent, fully human anti-CD40 IgG1 monoclonal antibody, CHIR-12.12 using XenoMouse® mice (Abgenix, Inc), a strain of transgenic mice expressing fully human IgG antibodies. CHIR-12.12 has at least two mechanisms of cytotoxicity in vitro: it blocks CD40-ligand mediated CD40 activation and mediates B-cell killing by ADCC. In-vitro crossreactivity studies were performed and the antibody showed cross reactivity to non-human primate but not to rodent tissue. To investigate the potential long-term toxicity of the antibody and to observe the recovery of the induced B-cell depletion, a single-dose toxicity study was conducted in cynomolgus monkeys. Three animals per sex per group (two animals per sex in control group) were given a single intravenous dose of 0, 10, or 100 mg/kg CHIR-12.12 and were monitored for 23 weeks post dosing. In addition to a detailed physical examination, samples for clinical chemistry, hematology and coagulation parameters as well as urinalysis were taken every 2 weeks. FACS analyses were performed every two weeks to monitor changes in B-cell, T-cell subpopulation and NK-cell numbers. In addition, CD40 expression and antigen saturation were measured on the remaining B-cell populations over 23 weeks. To investigate T-cell activation and T-cell function after CHIR-12.12 dosing, CD25 and CD40 expression on T-cells were monitored and at Week 23 a Mixed Lymphocyte Reaction (MLR) test was performed. The results of the study showed that a single dose of CHIR-12.12 was well tolerated and did not elicit any adverse clinical signs or effect on body weights, clinical pathology and hematology including differential blood counts. Immunophenotyping analysis showed a dramatic reduction of B-cells after dosing. The remaining B-cells mostly consist of the CD20high CD40low population, which is a population uniquely found in cynomolgus monkey blood but not in human blood (Vugmeyster et al.; Cytometry. 2003 Apr;52A). Both treatment groups showed a recovery of B-cells to normal pre-dose values in the majority of animals 19 weeks after dosing. In contrast, the average CHIR-12.12 antigen saturation level returned to pre-dose values in the low dose group after 11 weeks and in the high dose group after 19 weeks. T-cell counts (CD3, CD4 and CD8 subpopulation) showed no treatment related changes in absolute cell numbers throughout the study. No changes in T-cell activation measured by CD25 and CD40 expression were detected. T-cell responsiveness was not altered as measured by MLR. The pharmacokinetic analysis showed that, consistent with other human IgG antibodies, CHIR-12.12 had a small volume of distribution, limited to the blood volume, and was slowly eliminated from the circulation. Dose-dependency was observed with the area under serum concentration-time curve (AUC) increasing in a greater than dose-proportional fashion. Likewise, the half-life also increased with dose, ranging from 10.3 days at 10 mg/kg to 16.8 days at 100 mg/kg. In conclusion, all animals remained healthy and no evidence of immune impairment other then the expected reversible B-cell depletion was detected with a single dose of CHIR-12.12 up to 100 mg/kg. These results support the clinical development of CHIR-12.12 antibody for treatment of B-cell malignancies.

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain.

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.

Commercially Available Antibodies to Human Tumour Necrosis Factor-α Tested for Cross-Reactivity with Ovine and Bovine Tumour Necrosis Factor-α using Flow Cytometric Assays

A thorough understanding of the immune system, including the role of different cytokines, during inflammatory diseases in ruminants could lead to the development of new diagnostic methods and treatments. Tumour necrosis factor-α (TNF-α) is an important cytokine in the onset of the inflammatory responses. Unfortunately, the number of studies on cytokines, like TNF-α, in ruminants is limited due to a lack of species-specific reagents. As cytokines have remained rather conserved during evolution, cross-reactivity between animal species may occur. Therefore, the aim of the present study was to investigate 5 commercially available antibodies against human TNF-α for their ability to cross-react with ovine and/or bovine TNF-α, using a bead-based flow cytometric method. Two of the antibody clones (Mab 11 and 6401.1111) showed cross reactivity with ovine recombinant TNF-α in concentrations above 2.5 ng/ml. However, none of the antibodies detected TNF-α in bovine milk, or serum containing known concentrations of bovine TNF-α, as earlier determined with ELISA. The results could be due to inability of the antibodies to cross-react between species, but quenching of the signal by matrix proteins might also have lowered the response.

イムノクロマト法検査キットチェックリアＯ‐１５７

Checlear O-157 is the immunochromatography test kit which is developed for easy and rapid detection of E.coli O-157 in food. Detection limited of E.coliO-157 by this method was 5 × 104cells/mL. Compare to the result of culture method (official method), data of Checlear O-157 correspond with culture method (official method). But Checlear O-157 showed cross reaction with some strains (Citrobacter freundii, Salmonella urbana, Bacillus cereus) other than E.coli. Considering those results, Checlear O-157 will be useful in preliminary screening of E.coli O157 in food.

Detection of hazelnut DNA traces in chocolate by PCR

SummaryBy use of the DneasyTM Plant Tissue kit (Qiagen Inc.) plant DNA could be extracted from chocolate and related matrices. The polymerase chain reaction (PCR) detection of mitochondrial plant DNA is directly correlated with the length of the amplified fragment indicating shearing of DNA during chocolate production. Hazelnut DNA could be specifically detected in chocolate matrices with primers derived from the intron between exon B and C of the mitochondrial gene nad1. Specificity was confirmed towards individual chocolate ingredients and in 20 hazelnut negative chocolates. From taxonomically closely related plant species, only Carpinus turczaninovii, Ostrya carpinifolia and Corylus americana showed cross reaction, this was because of the identical sequence of the nad1 fragment. Application of extra MgCl2 throughout the DNA extraction procedure and of a specially designed Mg2+ buffered PCR, increased the detection sensitivity of co‐processed hazelnut in chocolate to 0.001% or 10 ppm.

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake ( Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake ( Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.

Streptococcus pneumoniae from ophthalmic infections: serotype distribution and penicillin susceptibility

Streptococcus pneumoniae is one of the pathogens causing infection of the conjunctiva and the uveal tract. The present study began with the observation of two ophthalmic S. pneumoniae isolates showing intermediate resistance to penicillin. Among the 25 isolates of S. pneumoniae from 617 ophthalmic specimens, during the period of 14 months, four were found to exhibit an intermediate resistance to penicillin. Minimum Inhibitory Concentration values ranging from 0.125 μg/mL to 0.25 μg/mL was observed. No multidrug resistant strains were isolated. Serogrouping/typing of the S. pneumoniae revealed the following serogroups/types; 6A ( n = 3), 6B ( n = 2), 22 ( n = 3), 14 ( n = 3), 23A ( n = 2), and 1 each of 23B, 19A, 7B, 32, 9, 42, 21, 39, 10, 3, and 34. One strain showed cross reaction in pool 29, 35, and 47. These findings represent the first such observation of ophthalmic isolates from India.

A simple procedure for isolation of Bufo arenarum C 3 complement fraction and preparation of antiserum

Because of the need for antibodies in our studies involving the third component of complement in Bufo arenarum, we performed a simple procedure to purify C 3 from B. arenarum serum to use as antigen in the preparation of the antiserum. The strategy was based on the well-known ability of C 3 to bind to zymosan (Zy), a yeast cell wall extract comprised of polysaccharides. The Zy-bound fraction showed cross reactivity with a commercial antibody to human C 3 as well as a similar electrophoretic profile (SDS–PAGE) to C 3 from other species. The Zy-C 3 complex resulting from binding Zy to B. arenarum serum was injected into rabbits and the antiserum against this C 3-like fraction was purified by protein A-Sepharose chromatography. The purified C 3 antibody was found to be suitable for immunochemical studies.

Distribution of Connexin43 immunoreactivity in the retinas of different vertebrates

The distribution of Connexin43 (Cx43) was examined by immunoblotting and immunofluorescence microscopy in the retinas of five different vertebrates by using a C-terminal specific peptide antibody. The specificity of the antibody was proved on immunoblots, in which it showed cross reactivity with a 43-kDa protein in rat heart homogenates as well as in homogenates of rabbit, rat, chicken, turtle, and fish (carp and zebrafish) retinas. Immunofluorescence histochemistry with retinal cryosections revealed the presence of Cx43 in the retinal pigment epithelium cells of all tested species and in blood vessels of vascularized retinas (fish and rat). Cx43 immunoreactivity was further localized in the stria medullaris of rabbit retina, in the nerve fiber layer of rat retina, most likely in astrocytes, and in the area of the outer limiting membrane of the fish retina, most likely representing Cx43 in Müller glia cells. A punctate Cx43-immunoreactive pattern consistent with gap junctions was also detected in the outer plexiform layer of carp and zebrafish retinas, and a specific amacrine cell type, which ramified in two layers of the inner plexiform layer, was labeled in the zebrafish retina. The present results are in accordance with previous findings showing the abundance of Cx43 in astrocytes, endothelium, and epithelial cells. However, the presence of Cx43 immunoreactivity in a specific population of amacrine cells of the zebrafish retina might indicate that a Cx43-like protein is also expressed in neurons. J. Comp. Neurol. 396:310–321, 1998. © 1998 Wiley-Liss, Inc.

Distribution of Connexin43 immunoreactivity in the retinas of different vertebrates

The distribution of Connexin43 (Cx43) was examined by immunoblotting and immunofluorescence microscopy in the retinas of five different vertebrates by using a C-terminal specific peptide antibody. The specificity of the antibody was proved on immunoblots, in which it showed cross reactivity with a 43-kDa protein in rat heart homogenates as well as in homogenates of rabbit, rat, chicken, turtle, and fish (carp and zebrafish) retinas. Immunofluorescence histochemistry with retinal cryosections revealed the presence of Cx43 in the retinal pigment epithelium cells of all tested species and in blood vessels of vascularized retinas (fish and rat). Cx43 immunoreactivity was further localized in the stria medullaris of rabbit retina, in the nerve fiber layer of rat retina, most likely in astrocytes, and in the area of the outer limiting membrane of the fish retina, most likely representing Cx43 in Müller glia cells. A punctate Cx43-immunoreactive pattern consistent with gap junctions was also detected in the outer plexiform layer of carp and zebrafish retinas, and a specific amacrine cell type, which ramified in two layers of the inner plexiform layer, was labeled in the zebrafish retina. The present results are in accordance with previous findings showing the abundance of Cx43 in astrocytes, endothelium, and epithelial cells. However, the presence of Cx43 immunoreactivity in a specific population of amacrine cells of the zebrafish retina might indicate that a Cx43-like protein is also expressed in neurons.

Structure determination of the capsular polysaccharide from Vibrio vulnificus strain 6353.

Vibrio vulnificus is a pathogenic gram-negative bacterium, endemic to brackish waters, which is often isolated from sediments, from the water column or from shellfish. It is associated with wound infections and septicemia in humans and the virulence of V. vulnificus has been strongly associated with encapsulation. The capsular polysaccharide purified from a virulent strain of V. vulnificus 6353 did not show cross reactivity with antibodies to the capsular polysaccharide of a related pathogenic strain of V. vulnificus (MO6-24) the structure of which was recently reported. NMR spectroscopic analysis of the purified polysaccharide from strain 6353 showed that the polymer is composed of four sugar residues per repeating subunit including 2,6-dideoxy-2-N-acetylamino-alpha-D-glucose (QuiNAc), 2-deoxy-2-N-acetylamino-alpha-D-galactose (alpha-D-GalNAc), 2-deoxy-2-N-acetylamino-alpha-D-galcturonic acid (alpha-D-GalNAcA) and 2-N-acetylamino-alpha-D-glucuronamide (alpha-D-GlcNAcANH2). The 1H- and 13C-NMR spectra were completely assigned by homonuclear and heteronuclear NMR spectroscopy. Sugar types and anomeric configurations were determined from proton homonuclear coupling constants and glycosidic linkages were determined from 1H-13C heteronuclear multiple bond correlation spectra. Sugar identities were confirmed by high performance anion-exchange chromatography and absolute configurations were determined by gas chromatography in combination with molecular modeling and NMR spectroscopy. The structure of the polysaccharide repeating unit is: [-->4)-alpha-D-GalpNAc-(1-->3)-alpha-D-GalpNAcA-(1-->3)-alpha-D-++ +QuipNAc-(1-->]n alpha-D-GlcpNAcANH2 (1-->4)- -->. While there are some common features shared among the structures of the capsular polysaccharides of pathogenic strains of V. vulnificus, there are distinct differences in the detailed structures.

Simplified elimination of rabbit anti-rat acute phase protein IgG that shows cross reactivity with albumin

Antibody preparations against rat acute phase proteins were tested for cross reactivity with other serum proteins, including rat albumin. Rabbit anti-rat a alpha1-acid glycoprotein and ceruloplasmin IgG purified on protein A-Sepharose did not show any cross reactivity with rat albumin, hemoglobin or transferrin. Rabbit anti-rat haptoglobin and -macroglobulin IgG purified on protein A-Sepharose showed a 39% and 30% cross reactivity with rat albumin and a 20% and 19% cross reactivity with rat hemoglobin. Because these proteins in whole serum were not adsorbed on Cibacron Blue F3-GA Sepharose, the albumin would be adsorbed on Cibacron Blue F3-GA Sepharose by the use of whole rat serum. Rabbit anti-rat haptoglobin and alpha2-macroglobulin IgG showing cross reactivity with albumin was simply eliminated.

Basic evaluation of Chlamydia antigen detection by EIA using a dual amplification enhanced immunoassay method

Chlamydia trachomatis is one of the important pathogens of STD in our country. Therefore, rapid accurate, reliable and convenient tests for its detection are required. So far, IDEIA Chlamydia has been employed as a useful diagnostic kit. Now, IDEIA PCE Chlamydia, applied as a dual amplification EIA method, has been developed. In our present studies, the sensitivity, reproducibility, cross reactivity, and reliability of IDEIA PCE Chlamydia were investigated and compared with those of IDEIA Chlamydia and LCR Chlamydia. The sensitivity of IDEIA PCE Chlamydia showed 2.4 x 10(2) IFU/ml for C. trachomatis D, 1.2 x 10(2) IFU/ml for C. trachomatis E, 3.8 x 10 IFU/ml for C. trachomatis F, and 1.25 x 10(2) IFU/ml for C. trachomatis L2. With regard to reproducibility, more than 2.4 x 10(2) IFU/ml of all strains of C. trachomatis and negative samples gave highly reproducible values. Though no cross reactivity was recognized among three strains of Staphylococcus aureus with concentrations of more than 10(9) IFU/ml, non-heated samples of over 10(6) CFU/ml showed cross reactivity. In our observations, phosphate, Mg2+, Ca2+, and Fe3+ inhibited the efficacy of both IDEIA and IDEIA PCE Chlamydia. Ca2+ per se could be an inhibitor in the case of urine samples analyzed by IDEIA and IDEIA PCE Chlamydia. These results indicate that IDEIA PCE Chlamydia kit for detection of C. trachomatis may be clinically useful because of its improved sensitivity over IDEIA Chlamydia and its invariable specificity and reliability.

Allergic contact dermatitis due to eye drops. Their clinical features and the patch test results

We studied the clinical manifestations and causative agents in cases with allergic contact dermatitis due to eye drops who consulted the Department of Dermatology, Nippon Medical School during the period of 9 years between January, 1987 and December, 1995. Among 66,165 cases who visited the department during the studied period, 3,903 were suspected as contact dermatitis or drug eruption and underwent patch test. 141 of 3,903 cases (3.6%) were patch tested with eye drops and 49 cases (34.8%) reacted positively and were diagnosed as allergic contact dermatitis. Allergic ingredients were tested in 36 cases by the patch test with every ingredient. The 49 cases diagnosed as allergic contact dermatitis by eye drops, included 17 males and 32 females. 81.6% of the patients were aged over 40 years. The most frequent ages were between 60 and 69 years. The period from onset to the first visit to us was less than two weeks in 54.1% of the cases, but 3 cases consulted us after more than six months. The determined allergens are noted in order of frequency as follows: 1) fradiomycin sulfate in 14 cases, 2) ketotifen fumarate in 6 cases, 3) dibekacin sulfate in 3 cases, 4) befunolol hydrochloride in 3 cases, 5) phenylephrine hydrochloride in 3 cases and 6) benzalkonium chloride, the preservative of eye drops in 3 cases. Fradiomycin sulfate showed cross reactions to other aminoglycosides in the present cases. Every ingredient should be tested as much as possible, because there is a possibility that a preservative or a vehicle ingredient may be the allergen, though the incidence is low.

The ATP Synthase ofTrypanosoma bruceiIs Developmentally Regulated by an Inhibitor Peptide

TheTrypanosoma bruceiATP synthase, like those of other organisms, is composed of two moieties, the membrane bound F0and the catalytic F1with each of these parts comprised of multiple subunits. In addition, an endogenous inhibitor peptide of the ATP synthase has been identified from a variety of sources. Previous reports have suggested that theTrypanosoma bruceiATPase may not possess such an inhibitor. Recently, we have isolated an inhibitor peptide fraction from the procyclic form ofTrypanosoma bruceiby modification of a previously published procedure. This fraction is composed of two dominant polypeptides with estimated molecular weights of 14,000 and 12,000 and an additional polypeptide of 15,000 that may or may not be functionally required. Antibodies raised to the smallest polypeptide showed strong cross reactivity with the other two polypeptides, suggesting that they are related. Antibodies to rat liver inhibitor peptide show cross reactivity with the same polypeptides in crude fractions. The inhibitor peptide fraction strongly suppresses the ATPase activity of membrane bound ATPase in a Mg2+-dependent manner and is cold and heat stable. Using antibodies to the smallest polypeptide and rat liver inhibitor peptide we have shown in crude extracts from the three experimental life cycle stages ofT. bruceithat the inhibitor peptide(s) is developmentally regulated to a modest extent. The pattern of regulation is opposite of the pattern seen for the ATP synthase complex. This suggests that the ATP synthase is stringently controlled inT. bruceiin a unique way.

Purification and Partial Characterization of Arginine Decarboxylase from Brassica campestris

Arginine decarboxylase (EC 4.1.1.19) has been purified and characterized from Brassica campestris cv B-9. The enzyme was purified 1120 fold and the recovery was 9%. The mol wt of the enzyme determined by gel filtration was 240 kD with identical subunits of 60 kD. The pH and temperature optima for the enzyme were 8.0 and 30°C respectively. The Km was 0.31mM. Polyamines inhibited the enzyme activity significantly. Immunodiffusion with ADC-specific antibodies showed cross reactivity against purified ADC from Brassica.

Tumor associated antigens in human renal cell carcinoma: MHC restricted recognition by cytotoxic T lymphocytes.

Based on previous studies in human melanoma leading to the molecular cloning of genes encoding peptide antigens recognized by MHC-restricted cytotoxic T lymphocytes (CTL) we extended our efforts to renal cancer systems established in tissue culture. In two patients we obtained stable CD8+ CTL clones with high cytolytic activity for the corresponding autologous tumor cell line in vitro. Neither autologous EBV-transformed B lymphocytes or PHA-activated PBL nor natural killer targets K562 were lysed by these CTL clones. MZ1257-RCC CTL5-30 lysed autologous tumor cells as well as normal kidney cell cultures in an HLA-A2 restricted fashion. Further specificity analysis showed cross reactivity with HLA-A2 matched renal cancer cell lines, melanoma cell lines and tumor cell lines of other origins. HLA-A2 negative target cells were not lysed. The restriction element for T cell recognition in another renal cancer system, MZ1973-RCC, appeared to be HLA-B8 as tested with CTL 5-10. Crossreactivity of CTL 5-10 was documented with one HLA-B8 positive RCC line. Tumor cell lines of other origin were not lysed by CTL 5-10. For further study of CTL-defined epitopes, peptides were extracted from tumor cell lines. Peptides from cultured cell lines were acid-eluted and fractionated by reversed phase HPLC. The peptide fractions were tested in cytotoxicity assays for their ability to reconstitute RCC epitopes by addition to the HLA-A2.1 positive antigen processing mutant cell line 721.124xCEM.T2. One HPLC peak was identified containing an epitope for the HLA-A2 restricted MZ1257-RCC CTL 5-30. These peptide epitopes were present in the autologous renal cancer cell line MZ1257-RCC as well as in an allogeneic HLA-A2 positive RCC cell line MZ1973 and melanoma cell line SK29-MEL, but were absent in peptides eluted from autologous EBV-B cells. We conclude that renal cell carcinoma may induce autologous CTL. Epitopes recognized by MHC-restricted CTL on RCC may be shared by HLA-matched allogeneic renal cancer or even tumors of other origin, such as melanoma. The current challenge is the determination of the amino acid sequences of these CTL-defined peptides as a step to further characterize the nature of these tumor associated antigens.

Allergic contact dermatitis to an amine expoxy resin hardener showing cross reactivity to paraphenylenediamine

Allergic contact dermatitis to an amine expoxy resin hardener showing cross reactivity to paraphenylenediamine