A pilot study to investigate the potential of mass spectrometry profiling in the discovery of novel serum markers in chronic renal disease.

PURPOSE: There has been significant criticism of how technologies such as SELDI have been used in biomarker discovery and how the data have been analysed. We initiated a proof-of-principle pilot study using SELDI with stringent pre-analytic and analytical procedures with robust statistical analysis, to determine whether, under such conditions, using different degrees of renal dysfunction as a model, useful data could be obtained. EXPERIMENTAL DESIGN: SELDI-TOF-MS profiling with stringent quality control measures was used to examine the proteomic profile of serum from healthy controls (n=30), patients with end-stage renal failure being treated by dialysis (n=30) and renal transplant patients (n=50) with varying degrees of graft stability. RESULTS: Principal component analysis of the data suggests that the continuum from normality to end-stage renal failure through 'stable' and 'unstable' transplant may be detected by SELDI profiling. Serum ß2 microglobulin was identified as a major component and this was validated using immunonephelometry. CONCLUSIONS AND CLINICAL RELEVANCE: This pilot study suggests that stringently controlled SELDI analysis is able to detect proteins which may be useful in the stratification of patients post-renal transplant. Further studies using a larger cohort of patients with chronic allograft dysfunction, defined by protocol biopsies, are indicated.

Effects of Clara cell 10 (CC10) protein on symptoms and signs of allergic rhinitis.

BACKGROUND: The Clara cell 10 (CC10) protein is produced by the airway epithelium. Reduced levels of CC10 are associated with allergic rhinitis and asthma. In experimental models, treatment with the CC10 protein may reduce features of airway inflammation. OBJECTIVES: To examine whether or not topical treatment with recombinant human CC10 (rhCC10) affects symptoms and signs of allergic rhinitis in a pollen season model. METHODS: Out of the pollen season, patients with allergic rhinitis received treatment with rhCC10, 0.56 mg per nasal cavity, once daily for 7 days in a double-blinded, placebo-controlled, crossover design. During this period, individualized allergen challenges were given once daily. Symptoms and peak nasal inspiratory flow (PNIF) were recorded daily in the morning, 10 minutes after challenge, and in the evening. Mean recordings of the last 3 days of the challenge series were used in the analysis. Nasal lavages were performed at the end of each challenge period, and eosinophil cationic protein, myeloperoxidase, and alpha2-macroglobulin levels were measured as indices of eosinophil and neutrophil activity and plasma exudation, respectively. RESULTS: Recombinant human CC10 did not affect allergen-induced morning, postchallenge, or evening symptoms compared with placebo. Morning, postchallenge, and evening PNIF were not improved by rhCC10. No statistically significant differences were observed between rhCC10 and placebo for any of the lavage fluid indices. CONCLUSIONS: Repeated nasal administrations of rhCC10 protein, in the present dose, do not exert antiallergic effects in seasonal allergic rhinitis.

[The role of macroglobulin family proteins in the regulation of inflammation].

The macroglobulin family represents a group of universal regulators involved into control of the inflammatory response to external and internal pathogens. Alpha-2-macroglobulin (MG), the major protein in the family, has 3 different binding sites and high affinity to an endocytosis receptor that allows MG to participate in recognition and phagocytosis of the foreign agents. The macroglobulin family proteins are most powerful apoptosis inhibitors: they bind autoaggressive hydrolases accumulating during inflammation. MG is the main transporter of cytokines and growth factors controlling the inflammatory response. At the same time, the macroglobulins are negative reactants of an acute phase of inflammation and the decrease of their synthesis at later stages is necessary for stimulation of collagenosis processes, coagulation, activation of thymulin, stimulating NK. At septic inflammation binding of macroglobulins to exotoxins can localize inflammation, and initiate systemic inflammatory response. Macroglobulin damage sharply reduces their subsequent utilization; this provokes accumulation of makroglodulin-proteinase complexes in biological fluids and may cause immune inflammation.

Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients.

Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.

An IgM anti-MBP Ab in a case of Waldenstrom's macroglobulinemia with polyneuropathy expressing an idiotype reactive with an MBP epitope immunodominant in MS and EAE.

In a previously described case of Waldenstrom's Macroglobulinemia, complicated by polyneuropathy, the IgM/lambda monoclonal antibody (mAb) was highly reactive with myelin basic protein (MBP). Given our demonstration that V lambda x, a recently described murine lambda variable region gene product, can itself bind MBP as well as confer MBP reactivity to an Ab, the possibility of a shared idiotypy between murine V lambda x and this human IgM/lambda anti-MBP was investigated. We characterized the epitope specificity of the macroglobulinemia patient's MBP-reactive IgM/lambda using indirect ELISA procedures with MBP, a citrullinated isomer of MBP termed C8, or peptide fragments of MBP as the coating antigens and monospecific Ab to V lambda x as the secondary Ab. The patient's MBP-reactive IgM/lambda was recognized by Ab specific for V lambda x and, like murine mAb containing V lambda x bound human MBP but not MBP-C8 nor other common autoantigens such as DNA, thyroglobulin, or actin. The anti-MBP reactivity was selective for MBP peptide 90-170 and preferentially recognized MBP peptide 84-96. Thus, the patient's macroglobulin and perhaps certain other human Ab with a 'V lambda x idiotype' bind to MBP peptide residues 84-96, an immunodominant peptide in multiple sclerosis patients. Such binding may be involved in the pathogenesis of neural damage in patients with neuroimmunologic disorders related to plasma cell dyscrasias or autoimmunity.

[Significance of monoclonal macroglobulinemia]. / Signification d'une macroglobulinémie monoclonale.

A monoclonal IgM protein in the plasma results from an expansion of a B lymphoid clone which encompasses a spectrum of different diseases: transient clones, monoclonal gammopathy of undetermined significance, Waldenström's macroglobulinemia and other B lymphoid malignancies, or primary amyloidosis. The main symptoms are related to the M component concentration and specific properties, the tumor development or both. Prognosis best correlates with the underlying histologic disorder.

A Waldenström's macroglobulin with anti-G3m(b1) antibody activity.

A human monoclonal macroglobulin (IgM, K) from a patient (KI) with Waldenström's macroglobulinemia was shown to have antibody activity against a human IgG (Gm) allotype. In hemagglutination tests, only one anti-D serum with G3m(b0b1) reacted with macroglobulin KI. Antiglobulin specificity of macroglobulin KI was determined to be an anti-G3m(b1) antibody by hemagglutination inhibition tests. Fab fragments from macroglobulin KI could react with human IgG3 protein possessing G3m(b1), but Fc fragments could not react. Gm phenotype in IgG isolated from serum KI was determined to be Gm(a,z,g,b0,s,t,u). This is the first report of a Waldenström's macroglobulin with antiglobulin specificity against a Gm allotype.

Venom resistance in the hedgehog, Erinaceus europaeus: purification and identification of macroglobulin inhibitors as plasma antihemorrhagic factors.

Hedgehog (Erinaceus europaeus) plasma contains factor(s) which neutralize the hemorrhagic activity of European viper (Vipera berus) venom. These antihemorrhagic factor(s) were purified by gel filtration on Sephacryl S-200 and chromatography on Cibacron Blue Sepharose. The macroglobulin fraction obtained was characterized for proteinase inhibiting activity, molecular weight and purity by polyacrylamide gel electrophoresis and immunological cross-reactivity and was found to contain the three macroglobulin proteinase inhibitors--alpha 2-macroglobulin, alpha 2-beta-macroglobulin and beta-macroglobulin. The purified macroglobulins were totally able to neutralize Vipera berus venom hemorrhagic activity, but no distinction between them in this respect was possible.

Immunochemical similarities between monoclonal antibacterial Waldenstrom's macroglobulins and monoclonal anti-DNA lupus autoantibodies.

Six monoclonal IgM from patients with Waldenstrom's macroglobulinemia that react with Klebsiella polysaccharides were tested for their ability to bind to nucleic acid antigens. One of the macroglobulins bound to the polynucleotide poly(G), and one bound to poly(G), poly(I), and single-stranded DNA. The reaction with the polynucleotides was specifically inhibited by the Klebsiella polysaccharide K30. A monoclonal lupus anti-DNA antibody (16/6) was found to react weakly with the Klebsiella polysaccharides K30 and K21. Five of the Waldenstrom macroglobulins shared an idiotypic determinant with the 16/6 anti-DNA antibody. The reaction between the macroglobulins and the antiidiotype serum was specifically inhibited by Klebsiella polysaccharides, an indication that the idiotypic marker was in the antigen-binding site of the macroglobulins. These results indicate the existence of widely dispersed conserved variable region genes that encode idiotypically related immunoglobulins with the capacity to bind to both bacterial polysaccharides and nucleic acids. Such genes can be expressed by patients with either Waldenstrom's macroglobulinemia or systemic lupus erythematosus.

A naturally occurring complement dependent human IgM macroglobulin inhibitory to Ehrlich ascites tumor cells.

Human serum and the sera of a number of other mammalian species contain a naturally occurring macroglobulin with antibody-like properties which, in the presence of complement, is cytotoxic to murine Ehrlich ascites tumor cells in vitro. The serum of guinea pigs, rats and mice lack this macroglobulin activity but serve well as complement sources. The cytotoxic activity was demonstrated in short-term tissue cultures of Ehrlich ascites tumor cells by the marked and immediate inhibition of nucleic acid and protein synthesis. The cytotoxic activity is independent of the lytic antibody system which reacts against neuraminidase-treated lymphocytes.

Human monoclonal macroglobulins with specificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6-pyruvylated-D-galactose.

Two human IgM myeloma proteins, IgMWEA and IgMMAY, were found to react with agar and Klebsiella polysaccharides that contain pyruvylated D-galactose (DGal). Quantitative precipitin data and precipitin inhibition studies with methyl alpha- and beta-glycosides of 4,6-pyruvylated-D-galactose showed their combining sites to be different, although each was directed against the pyruvylated-D-Gal, one reacting most specifically with Klebsiella polysaccharides with terminal nonreducing beta-linked 2,4 pyruvylated-D-Gal, whereas the other reacted equally well with Klebsiella polysaccharides that contain 3,4 beta-linked and 4,6 alpha-linked terminal nonreducing pyruvylated-DGal. Inhibition studies showed that both sites are directed toward one of the two space isomers of 3,4- or 4,6-pyruvylated DGal, the form in which the methyl group of the pyruvate is equatorial, or endo, and its carboxyl group axial, or exo, to the plane of the acetal ring. Coprecipitation studies showed the combining site of IgMWEA to be located on an (Fab')2 fragment and not on the (Fc)5mu fragment. The monoclonal peak in the serum of IgMMAY was specifically precipitated by Klebsiella polysaccharide. Myeloma proteins with specificities of this type may occur with reasonable frequency in humans and may be a consequence of clonal expansion from inapparent infection, carrier states, or disease produced by various Klebsiella organisms.

A monoclonal macroglobulin with antinuclear activity.

Serum containing a monoclonal IgM protein from a patient with Waldenstroms' macroglobulinaemia gave intense immunofluorescent staining of kidney nuclei. The Fab mu fragments of this immunoglobulin were obtained. The IgM and Fab fragments reacted in vitro with kidney nuclei using unfixed cryostat sections of rat or mouse kidney. After treatment of the patient with chemotherapy, the monoclonal IgM disappeared, and no more antinuclear activity could be detected in the serum. The results strongly suggest that this IgM protein had antinuclear activity.

Human macromolecular insoluble cold globulin (MICG). I. T-cell origin of T-MICG and null cell origin of N-MICG.

Although surface immunoglobulin characterizes B cells in man, there are few surface markers that distinguish T cells. We have described a new protein synthesized in human T cells, termed T-MICG. This protein is a macromolecule of 225,000 daltons, is insoluble in the cold, and migrates as a beta-globulin on electrophoresis. Separation of human peripheral blood lymphocytes into T and B-cell populations by rosette sedimentation and anti-human-Fab columns clearly demonstrated the T-cell origin of the 225,000 dalton component. Furthermore, null cells were shown to synthesize a protein of 185,000 daltons, termed N-MICG, with physical properties similar to T-MICG, T-MICG and N-MICG were shown to be antigenically dissimilar, employing antiserum to each of these proteins. The present studies demonstrate two novel cell surface markers, T-MICG and N-MICG, which characterize T cells and null cells, respectively.

Immunogenetics of the McB1 macroglobulin allotype in cattle.

The paper describes a cattle serum antigen (McB1) which is controlled by a dominant gene (McB1), independent from those controlling the two macroglobulin markers McA1 and McA2 as well as from that controlling the low-density lipoprotein marker Ld1A1. McB1 is located on a high-molecular-weight serum protein which, very likely, is an alpha2-macroglobulin.