Verneuil's disease, innate immunity and vitamin D: a pilot study.

BACKGROUND: Verneuil's disease is a chronic inflammatory skin disease of the follicles in apocrine glands rich area of the skin (axillary, inguinal, anogenital) and is associated with a deficient skin innate immunity. It is characterized by the occurrence of nodules, abscesses, fistulas, scars. Recently, vitamin D has been shown to stimulate skin innate immunity. OBJECTIVE: The primary objective of the study was to assess whether Verneuil's disease was associated with vitamin D deficiency. The secondary objective was to determine whether vitamin D supplementation could improve inflammatory lesions. METHODS: First, 25(OH) vitamin D3 serum levels in patients with Verneuil's disease followed at Nantes University Hospital were compared to those of healthy donors from the French Blood Bank. Then, a pilot study was conducted in 14 patients supplemented with vitamin D according to their vitamin D level at baseline at months 3 and 6. The endpoints at 6 months were decreased by at least 20% in the number of nodules and in the frequency of flare-ups. RESULTS: Twenty-two patients (100%) had vitamin D deficiency (level <30 ng/mL) of whom 36% were severely deficient (level <10 ng/mL), having correlation with the disease severity (P = 0.03268) vs. 20 controls with vitamin D deficiency (91%) of whom 14% were severely deficient. In 14 patients, the supplementation significantly decreased the number of nodules at 6 months (P = 0.01133), and the endpoints were achieved in 79% of these patients. A correlation between the therapeutic success and the importance of the increase in vitamin D level after supplementation was observed (P = 0.01099). CONCLUSION: Our study shows that Verneuil's disease is associated with a major vitamin D deficiency, correlated with the disease severity. It suggests that vitamin D could significantly improve the inflammatory nodules, probably by stimulating the skin innate immunity. A larger randomized study is needed to confirm these findings.

A new murine monoclonal antibody against Kx protein.

Mice immunized with a synthetic peptide located on an intracellular segment of the polytopic Kx protein (37 kDa) from human red blood cells (RBCs) produced a monoclonal antibody called C7B8. As expected, this antibody did not agglutinate common RBCs but reacted with permeabilized cells in flow cytometry. C7B8 recognizes the Kx protein on Western blots. Cross-reactivity of C7B8 with human calpain of human muscle extracts was demonstrated by Western blot analysis. This cross-reactivity precludes the use of C7B8 for Kx tissue distribution studies, but immobilized C7B8 was a convenient tool for purification of the Kell-Kx complex from RBC membrane extract by immunochromatography.

Epitope mapping of four novel CD44 monoclonal antibodies using surface plasmon resonance and soluble CD44.

CD44 is a ubiquitous multistructural and multifunctional cell surface adhesion molecule. The molecular diversity of this glycoprotein is generated by both post-translational modification and the differential use of alternatively spliced exons which play a critical role in determining the exact conformation of the molecule. CD44 isoforms are found in many tissues and in soluble form in plasma. Soluble CD44 was purified by a two-step purification combining ion exchange and immuno-affinity chromatography. Our aim was to check that all the known antigenic epitopes are present on sCD44, which could thus be used for the mapping of new anti-CD44 antibodies. Competitive binding assays using reference antibodies and novel anti-CD44 monoclonal antibodies were performed by real-time biospecific interaction analysis. Reference mAbs identified on soluble CD44 the three distinct epitopes previously defined using red blood cell membrane CD44. From the four novel CD44 mAbs, two mAbs (NaM198-6B5, NaM198-10B4) mapped to epitope group 1, whereas the others (NaM10-8F4, NaM77-9D6) mapped to epitope group 2. Immunopurified sCD44 obtained from the plasma of healthy donors appears to be a usable tool for the mapping of anti-CD44 mAbs.

A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin.

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.

Soluble CD44: quantification and molecular repartition in plasma of patients with colorectal cancer.

Based on the important role of CD44 in tumour progression and metastasis, we evaluated, in a prospective study, plasma-soluble CD44 (sCD44) as a serum marker in colorectal cancer. Blood plasma specimens from 89 patients with colorectal neoplasm, 22 patients with a gastrointestinal disease and 23 healthy donors were analysed for quantitation (ELISA assay) and purification of sCD44. The concentration of sCD44, indicating the concentration of all isoforms, was significantly higher in patients with colorectal cancer and intestinal disease than in normal individuals, but no significant differences were found between the two groups. We found no association between plasma levels and staging of the colorectal cancer patients according to Astler and Coller. A two-step batch purification combining ion exchange and immunoaffinity chromatography, followed by Western blot analysis, revealed a complex pattern with a major band corresponding to the standard form of CD44 and minor bands that may correspond to larger variant forms. No particular sCD44 isoform was clearly associated with anatomopathological or biological information.

Fine specificities of murine anti-Mg monoclonal antibodies.

The specificities of two murine anti-Mg monoclonal IgG1 antibodies, 3B10 and 2D5, were determined by pepscan analysis. The peptides which correspond to various fragments of amino-terminal portions of glycophorin A of group M (GPA-M), N (GPA-N) and Mg (GPA-Mg), and replacement analogues of some of these peptides, were synthesized on plastic pins and tested for binding of the antibodies. Both antibodies bound strongly to the N-terminal Mg octapeptide 1LSTNEVAM8, but they showed different subspecificities. The essential fragment of the epitope 2D5 are amino acid residues 2STNEV6. Replacement of any of these amino acid residues by Ala, and replacement of Glu5 residue by Gly, abolished or strongly reduced the antibody binding, but replacement of Asn4 by Thr gave only a moderate decrease of peptide activity. In contrast, the Leu1 and Asn4 residues were most essential components of the epitope 3B10, while Ser2, Thr3 and Glu5 seemed to be less important. Our present results and earlier ones on the specificity of human anti-Mg alloantibodies and monoclonal anti-M/Mg antibodies showed that antibodies reacting with Mg antigen recognize different fragments and/or different amino acid residues of the amino- terminal nonglycosylated domain of GPA-Mg. The knowledge of fine specificities of antibodies reacting with Mg antigen is interesting in view of the presence of anti-Mg alloantibodies in 1-2% of human sera.

The murine monoclonal antibody NaM26-4C6 identifies a common structure on band 3 and glycophorin C.

The murine monoclonal antibody NaM26-4C6 (IgM class), obtained from the splenocytes of a BALB/c mouse immunized with human umbilical cord red blood cells, was characterized by agglutination test and immunoblotting analysis. The structure of the NaM26-4C6 epitope was further elucidated by using a series of peptides synthesized on pins. The antibody agglutinated untreated and chymotrypsin-treated but not trypsin- or neuraminidase-treated human erythrocytes. Agglutination-inhibition test demonstrated that the antibody recognizes an epitope located on the N-terminal trypsin-sensitive portion of glycophorin C. The antibody bound on immunoblots to glycophorin C, and also to the band 3 protein and its 69-kDa N-terminal fragment but did not bind to desialylated and de-O-glycosylated glycophorin C. Peptide mapping allowed localization of the binding site on the 23-kDa N-terminal intracellular peptide of band 3. The antibody binds to the amino-acid sequences 22EDPDIP27 of band 3 protein and 15SLEPDPGM22 of glycophorin C, and residues D and P were found to be essential. The new epitope identified by NaM26-4C6 corresponds to a linear amino acid sequence located on the N-terminal intracellular portion of band 3 and to a more complex structure involving oligosaccharide chains on the N-terminal extracellular domain of GPC.

Characterization of a murine monoclonal antibody specific for swine beta1 integrin.

Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160-1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM160-1A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human beta 1 integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine alpha 2 integrin. Therefore, the NaM160-1A3 antibody was directed against porcine beta 1 integrin and allowed the purification of the complex alpha 2 beta 1, also termed Very Late Antigen 2 (VLA-2). It did not recognize human beta 1 integrin.

Immunochemical characterisation of monoclonal antibodies directed to glycophorins A and/or B.

Among sixty-nine monoclonal antibodies submitted to the workshop, 28 antibodies directed to glycophorins A and/or B but without blood group specificity were investigated by a series of methods involving agglutination, flow cytometry with CHO transfected cells expressing glycophorin A, ELISA with a carbohydrate-free peptide (residues 1-72) of glycophorin A, and immunoblotting. These MAbs were subdivided in several groups according to their specificity: N-terminal portion of GPA and GPB; N-terminal trypsin-sensitive portion of GPA; extracellular ficin-sensitive portion of GPA; intracellular domain of GPA; undetermined. Both flow cytometry with transfectant cells and ELISA with the synthetic peptide prove to be of value in order to determine subspecificities within these groups.

Flow cytometry and immunoblotting analysis of monoclonal antibodies directed to complement regulatory proteins.

A series of 18 monoclonal antibodies directed to complement regulatory proteins were investigated by flow cytometry and immunoblotting. Seventeen antibodies are directed against a phosphatidyl inositol-linked glycoprotein since they show a dual population of erythrocytes from a paroxysmal nocturnal haemoglobinuria (PNH) patient. From this group, 6 antibodies revealed a 65-70 kDa band on immunoblots allowing their identification as anti-DAF (Decay Accelerating Factor; CD55 antigen), and 11 bound to a 20 kDa molecule corresponding to MIRL (Membrane Inhibitor of Reactive lysis, CD59 antigen). One antibody revealed an homogeneous population from the PNH patient and bound to a 200 kDa band on immunoblot that might corresponds to the CR1 (Complement Receptor type 1; CD35).

Flow cytometry and immunoblotting analysis of monoclonal antibodies directed to CD-related antigens.

Thirteen monoclonal antibodies directed to red cell and white cell differentiation antigens have been analysed by flow cytometry and immunoblotting. Nine were identified as CD44 (2D3-1, -2, -3, -4), CD 47 (2D3-5 and -6), CD 58 (2D7 and -8), CD99 (2D3-9), whereas four (2D3-11, -12, -13, and 14) could not be characterised.

Production and properties of monoclonal antibodies against human IgG isotypes.

Several monoclonal antibodies (MAbs) against human IgG isotypes were obtained by the fusion of myeloma cells with splenocytes from mice immunized with IgG fractions extracted from human plasma. Four MAbs (F7H7, D4F8, B12A8, and E7E10) were selected by an ELISA technique on the basis of their ability to detect one of the four IgG subclasses. Their specificity was checked using a panel of pure myeloma proteins representative of the main allotypes present on IgG isotypes. In addition, two other MAbs (F3E12 and E6D6) were found able to detect specifically kappa or lambda light chains. The immunochemical properties of these MAbs were analyzed mainly in respect to their capacity to detect and to purify the different human IgG isotypes. The following data were obtained: (1) The ability of the MAbs F7H7, D4F8, B12A8, and E7E10 to measure the concentration of each IgG subclass in serum was estimated by an immunocapture ELISA. Results obtained with the new antibodies were compared with several other MAbs recommended by the IUIS/WHO human Immunoglobulins subcommittee. Similar or better results were obtained with the new anti-IgG1, anti-IgG3, and anti-IgG4, MAbs. (2) The same MAbs were tested for their ability to purify a single IgG subclass from IgG preparations and from normal and pathological sera. Fractions containing about 80% of purified IgG1, IgG3, and IgG4 were obtained after one-step immunoaffinity purification. Consequently, these MAbs proved to be useful to detect, to measure and to purify IgG subclasses.

Inheritance of abnormal glycophorin C of the Gerbich and Yussef type in a French family.

The discovery of a natural Gerbich antigen (anti-Ge2) in the serum of a propositus prompted us to study his red blood cells (RBCs) by using monoclonal anti-bodies (mAbs) directed against glycophorin (GP) C and GPD. An mAb directed against the Ge4 antigen (mAb NaM10-7G11) agglutinated both untreated and trypsin-treated cells, demonstrating the expression of a trypsin-resistant GPC (namely, GPC of the Gerbich type: GPCGe). Surprisingly, an anti-Ge3 antibody (mAb NaM19-3C4) agglutinated untreated cells, showing that they also express the Ge3 antigen that may be carried by normal GPC and CPD or by the abnormal GPC of the Yussef (Yus) type (GPCYus). Immunoblotting analysis performed with an mAb directed against the C-terminal portion of GPC showed that the propositus' RBCs do not contain normal GPC and GPD but both GPCGe and GPCYus. Analysis of RBCs from the family demonstrated that, like the propositus, 2 of the 3 sisters had inherited both the GYPCGe and the GYPCYus alleles from the parents, who carried either the GYPCGe or the GYPCYus allele. The third sister had inherited the normal GYPC alleles from her parents, whereas the child of the propositus had inherited the GYPCGe allele. Interestingly, natural anti-Ge2 antibodies were identified in the serum of 2 of the 3 Ge-negative individuals.

Influence of the size of the polar head of non-ionic detergents on membrane proteins immunoaffinity purification.

Nonionic polyoxyethylene type detergents (CxEy) are widely used to solubilize and purify membrane proteins. The detergent hydrophobic moiety (Cx) replaces phospholipids at exposed hydrophobic regions of the membrane proteins. During chromatography on an immobilized anti-Kell antibody to purify Kell protein (an integral erythrocyte protein), it was observed that the size of the polar head of an non ionic detergent added to the mobile phase appeared to influence the interaction of the detergent-protein complex with the immobilized antibody. Further studies were performed using another erythrocyte membrane protein, Glycophorin C and three anti-GPC monoclonal antibodies directed against three epitopes of the extracytoplasmic domain of the protein. The interaction of GPC with the three Protein A-coupled monoclonal antibodies was studied in the presence of three detergents C12E<9>, C13E<15> and C12E<23>. It was observed in batch mode and in column chromatography experiments that the adsorption of GPC to the immunoaffinity supports decreased as the size of the detergent polar head increased. Thus, the polyoxyethylene chain of a detergent might prevent the interaction of the detergent-protein complex with the immobilized antibody.

Characterization of new murine monoclonal antibodies directed against glycophorins C and D.

Six new murine monoclonal antibodies (mAbs) directed to the erythrocyte membrane glycophorins C (GPC) and D (GPD) were obtained from splenocytes of different BALB/c mice immunized with human red blood cells, and fully characterized. The mAbs were selected by agglutination tests with control and Gerbich-negative cells, and by immunoblotting analysis. They showed specificity for the N-terminal domain(s) of GPC (and GPD) and were classified into three categories by competitive analysis using 125I-labelled antibodies and real-time biospecific interaction. The first group (NaM10-7G11, NaM70-1G4 and NaM77-7B6) compete for epitope(s) located at the N-terminal portion of GPC. Agglutination-inhibition tests revealed that the 7G11 epitope involves the amino group of Met1 and sialic acid residue(s) whereas the 1G4 and 7B6 epitopes contain O-glycans. NaM89-2G11 belongs to a second group; its epitope is located in a region including Glu17, Asp19 and (an) O-glycan(s). The third group comprises mAbs NaM19-3C4 and NaM98-3C1 which bind to both GPC and GPD in proximity of the binding site of human anti-Ge:3 antibodies. In addition, mAb 3C4 (anti-GPC/GPD) was found to bind to approximately 125,000 sites per red cell. Considering that the ratio of the GPC to GPD is about 3-4 to 1, the number of GPC and GPD molecules was estimated as 95,000 and 35,000, respectively.

[Rapid diagnosis of paroxysmal nocturnal hemoglobinuria by gel test agglutination]. / Diagnostic rapide des hémoglobinuries nocturnes paroxystiques par agglutination en gel.

Murine monoclonal antibodies (MoAbs) directed against DAF (Decay Accelerating Factor, CD55 antigen) and MIRL (Membrane Inhibitor of Reactive Lysis, CD59 antigen) were used to identify the affected red cells (CD55-/CD59-) of PNH patients. MoAbs NaM16-4D3 (CD55, IgG2a) and NaM77-1E5 (CD59, IgG3) weakly agglutinate red cells and represent powerful tools to quantitate normal (PNHI) and abnormal (PNHII and PNHIII) cells from PNH patients by indirect flow cytometry. MoAbs NaM125-7H10 (CD55) and NaM123-6G12 (CD59), both IgM, were selected for their agglutinating properties and used for the separation of PNHI from PNHII and PNHIII red cells by the gel test technology. From analysis of artificial mixtures of DAF+ and DAF- cells, a direct relationship was established between fluorescent cells detected by flow cytometry, and erythrocytes agglutinated in microtyping cards. The method was further confirmed by analysis of ten blood samples from PHN patients and represent an alternative to classical hemolysis tests. On the basis of our experience we propose the following for the diagnosis of PNH: 1) agglutination test with NaCl microtyping cards using IgM CD55 and CD59; 2) flow cytometry analysis for accurate quantitation of CD55-/CD59- red cells.

[Characterization of murine monoclonal antibodies against fetal erythrocytes]. / Caractérisation d'anticorps monoclonaux murins dirigés contre les érythrocytes foetaux.

Balb/c mice were immunized against papain-treated fetal erythrocytes and splenocytes were fused with Sp2/0-Ag-14 myeloma cells. Several hybrids secreting antibodies directed against antigenic determinants predominantly exposed on fetal and cord cells were selected and cloned twice. Antibodies NaM61-1A2 and NaM61-768 (IgM class) were shown to be specific for an endo-beta-galactosidase-sensitive oligosaccharide chain. The antigen, strongly expressed on fetal and cord cells, was identified as the i blood group antigen. The antibodies represent powerful blood group reagents to be use in conventional agglutination techniques as well as in the gel typing system and in indirect flow cytometry. The antibody NaM46-4A8 (IgG class) is specific for an antigenic structure expressed on fetal cells and accessible only after papain, ficin, bromelin and endo-beta galactosidase treatment. The antigen was not identified.

A murine monoclonal antibody directed against the Gerbich 3 blood group antigen.

A murine monoclonal antibody (NaM19-3C4, IgG1, Kappa) was produced from splenocytes of mice immunized with red blood cells. The antibody agglutinated untreated Ge:2,3,4 and Ge:-2,3,4 erythrocytes in indirect antiglobulin test but failed to agglutinate trypsin-treated cells. Gerbich-negative erythrocyte of the Leach- (Ge:-2,-3,-4) and of the Gerbich- (Ge:-2,-3,4) types were not recognized by the antibody. Immunoblotting experiments showed that the antibody bound to glycophorins C and D from control erythrocytes and to the abnormal glycophorin C identified in the Gerbich-negative cells of the Yussef type (Ge:-2,3,4). No binding to the altered glycophorin C from Ge:-2,-3,4 erythrocytes was observed, indicating that the antibody specifically recognized the Ge:3 epitope localized within residues 40-50 of glycophorin C.

Characterization of murine monoclonal antibodies directed against the Kell blood group glycoprotein.

Murine monoclonal antibodies (MoAbs) were produced against the blood group KEL1 glycoprotein (93 kD component) immunopurified from human erythrocytes. One monoclonal antibody, 5A11 (IgGa, kappa), detects by immunoblotting a 93 and 184 kD component from KEL: 1,-2 or KEL: -1,2 red cell membrane preparations, separated by SDS polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions. The 184 kD component was not detected under reducing conditions, suggesting that it represented a dimer of the 93 kD KEL glycoprotein. Neither the 93 nor the 184 kD could be detected from K0 or McLeod erythrocyte membrane preparations, indicating that the monoclonal antibody reacts with the KEL glycoprotein previously identified as a 93 kD species. Since this antibody does not agglutinate native or protease-treated erythrocytes, it is likely that it reacts with the cytoplasmic domain of the KEL glycoprotein. This was also substantiated by showing that 5A11 could immunoprecipitate the 93 kD component from intact membranes and inside-out vesicles but not from right-side-out vesicles. Immunostaining of membrane proteins prepared from human blood cells (platelets, lymphocytes, monocytes and granulocytes) and non-human erythrocytes revealed that the 93 kD molecule was only present on human red cells. Several other murine monoclonal antibodies obtained from the same fusion experiment gave identical results, but competition analyses on immunoblots indicated that these antibodies reacted with distinct epitopes on the KEL glycoprotein.

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle.

Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twitch muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased 45Ca-binding. These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.