Isolation and partial characterization of melanoma-associated antigens identified by autologous antibody.

The study of the autologous immune response to cancer avoids the difficulties encountered in the use of xenoantisera and may identify antigens of physiological relevance. However, the low titer and incidence of autologous antibody to melanoma have hampered further evaluation. By utilizing acid dissociation and ultrafiltration of serum, we have been able to augment the detectable autologous immune response to melanoma in the majority of patients studied. In autologous system Y-Mel 84:420, serum S150 demonstrated a rise in titer from 1:32 in native sera to 1:262,044 after dissociation. The antigen detected by S150 was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma, and head and neck carcinoma cell lines. It did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, or autologous cultured lymphocytes. Using polyacrylamide gel electrophoresis, S150 detects a 66,000-mol wt antigen in spent tissue culture media and serum ultrafiltrate. In cell lysate two bands between 20,000 and 30,000 mol wt are detected by S150. The 66,000-mol wt antigen is sensitive to trypsin digestion and but is resistant to pepsin and heat inactivation. Exposure of spent media to trypsin results in the development of a 24,000-mol wt band that appears to correspond to the antigen detected in the cell lysate. The difference between the antigens detected in the cell lysate as compared with spent media and serum ultrafiltrate may be due to degradation during cell lysis. We conclude that melanoma-associated antigens are present in the serum of patients with melanoma and are shed or secreted by their tumor cells.

Incidence of serum antibody reactivity to autologous head and neck cancer cell lines and augmentation of antibody reactivity following acid dissociation and ultrafiltration.

Serum antibody reactivity to squamous cell carcinoma of the head and neck (SCCHN) was evaluated in 41 autologous serum-tumor cell line combinations using the protein A hemadsorption assay. Autologous antibody reactivity (median titer of 1:4) was detected in sera from 24 of the patients tested. In 10 cases autologous antibody reactivity could be detected only in undiluted serum precluding further analysis. Analysis of higher titer sera from one patient revealed antibodies that define an antigen expressed on autologous tumor cells cultured from both the primary tumor (UM-SCC-17A) and from a metastasis (UM-SCC-17B). Absorption analysis showed that this antigen was also expressed on 6 of 10 allogeneic SCCHN cell lines but not on autologous fibroblasts or on allogeneic melanoma cell lines. Due to the low titer of autologous antibody reactivity in most sera, we sought to determine if dissociation of immune complexes through acidification and ultrafiltration of serum might enhance detectable antibody reactivity as has been done in previous studies in melanoma. Twelve serum samples from eight patients were subjected to acid dissociation and ultrafiltration (AD-U). Only six of the untreated sera had detectable antibody reactivity against the autologous SCCHN cell line whereas following AD-U all 12 sera had enhanced IgG reactivity against autologous SCCHN. Specificity analysis of one serum sample after dissociation revealed that the antibody detected an antigen common to SCCHN cell lines as well as melanoma, glioma, renal, and colon carcinoma cell lines. Circulating immune complexes may provide a reservoir of antibody with potential diagnostic and therapeutic applications.

Interleukin-2-induces development of denditric cells from cord blood CD34+ cells.

Dendritic cells (DC) have been shown to develop along a myeloid or lymphoid lineage of differentiation propagated from bone marrow or early thymic precursor cells with hematopoietic cytokines. In our study, we have induced growth and differentiation of DC from cord blood CD34+ cells initiated in interleukin-2 (IL-2) alone or in IL-2 + stem cell factor (SCF) + tumor necrosis factor alpha (TNF-alpha)-supplemented medium and cultured with IL-2 or IL-2 + SCF for 28-35 days. Dendritic morphology and antigenic phenotype of DC grown with IL-2 were characteristic for DC cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Growth and differentiation of DC was followed by an increase in expression of MHC II and co-stimulating molecules CD80 and CD86. We have also shown the expression of the IL-2 receptor (IL-2R) gamma-chain in CD34+ cells after 2-3 days of culture with IL-2 alone. The co-expression of the IL-2R alpha, beta, and gamma subunits in both DC cultured with IL-2- or GM-CSF-containing cocktail of cytokines was also shown. The time curve for induction of IL-2R demonstrated low levels of subunit expression at the beginning of culture. The number of CD1a cells co-expressing CD25, CD122, and CDgamma increased to about 24-68 and to 78-95% after 21 and 28-35 days, respectively. Development of natural killer cells was shown along with DC. The proportion of CD56+ cells and cytotoxicity increased in a time-dependent manner.

Native gel electrophoresis and isoelectric focusing of a 64-kilodalton eye muscle protein shows that it is an important target for serum autoantibodies in patients with thyroid-associated ophthalmopathy and not expressed in other skeletal muscle.

Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody-binding sites. We have used a gentle protocol for the preparation and purification of a 64-kilodalton (kDa) eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO) for use as antigen in immunoblotting. Pig EM membrane proteins were prepared from crude homogenates by high speed centrifugation and solubilized by hand homogenization. These native membrane proteins (NMprot) were then electrophoresed on an 8.5% polyacrylamide gel in the absence of SDS, reducing agents, or urea, and proteins from individual bands were eluted, applied to standard SDS-PAGE, and immunoblotted with selected TAO patient sera. A prominent 64-kDa protein, present in most of the bands, was recognized by autoantibodies in sera from 35% of the patients with TAO and 47% of those with Graves' hyperthyroidism without evident ophthalmopathy, but in only 4% of normal subjects. To further purify the 64-kDa protein and increase the sensitivity of immunoblotting, NMprot were separated by isoelectric focusing (IEF) in the absence of SDS, reducing agent, and urea. The 64-kDa protein appeared mainly in IEF fraction 7 and had an isoelectric point of 6.1-6.2. Similar results were found for a human EM protein of 64 kDa. Sera from groups of patients and normal subjects were tested in immunoblotting against a pig EM 64-kDa protein prepared from NMprot and purified in IEF. Tests were positive in 67% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 9% of normal subjects. The 64-kDa protein was not found in other skeletal muscle. The demonstration that a native 64-kDa protein that is specifically targeted by autoantibodies in the serum of patients with TAO is expressed in EM, but not other skeletal muscle, greatly enhances its possible significance in the pathogenesis of this eye disorder.

Re-examination of peripheral blood T cell subsets in dysthyroid orbitopathy.

Patients with dysthyroid orbitopathy (DO) were grouped according to a multifactorial assessment of disease severity and the rate of disease progression. Using this system and flow cytometric measurements of T cell subsets in the peripheral blood, a significant increase in the percentage of CD4+ lymphocytes correlated with disease severity in DO patients with progressive disease. These observations are consistent with the hypothesis that the CD4+ peripheral blood T helper cells play a significant role in the progression of DO.

New assays for the measurement of serum antibodies reactive with eye muscle membrane antigens confirm their significance in thyroid-associated ophthalmopathy.

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody binding sites. We have used nondenaturing methods for the purification of a 64-kDa eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO). Pig EM membrane proteins were prepared from crude homogenates by high-speed centrifugation and solubilized by hand homogenization. The 64-kDa protein was further purified by isoelectric focusing performed in the absence of SDS, detergents, reducing agents, and urea. Sera from patients with active TAO of recent onset and thyroid autoimmunity without ophthalmopathy were tested for reactivity against purified native 64-kDa protein in immunoblotting. Tests were positive in 64% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 13% of normal subjects. Many of the same sera were also tested for cytotoxic activity against human EM cells in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. ADCC tests were positive in 69% of patients with TAO but in no normal subject. The specificity and sensitivity of these two tests in TAO surpass those for all other published results for orbital tissue reactive autoantibodies. Although there was a tendency for a relationship between reactivity to the 64-kDa protein and cytotoxic activity against EM cells in ADCC there were many exceptions and overall the relationship between the two tests was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Powered mobility vehicles as aids in independent locomotion for young children. Suggestion from the field.

Both vehicles are good training devices for future mobility. The independent mobility provided by this equipment gave each child a boost of self-esteem and independence.

Who's rating you?

Web sites judging providers' quality are proliferating, and the number of employers, payers and consumers consulting them is ballooning. Critics say many sites are based on faulty conceptions, but if hospitals don't pay more attention, they risk losing their place in the quality debate and being judged by standards that they find neither valid nor fair.

Six sigma. The quest for quality.

Many hospitals are looking for a way to improve their quality performance to improve outcomes, increase patient satisfaction and lower costs. Frustrated by the slow gains from traditional quality programs, some executives have turned to Six Sigma, the statistical quality method made famous by Motorola and General Electric. But Six Sigma can be a difficult concept to grasp. Our toolkit will serve as a primer for health care executives to find out what Six Sigma is, how it can be applied to health care organizations, and how some hospital executives have made it work for them.

Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

Cardiac fibroblasts influence cardiomyocyte phenotype in vitro.

Cardiac fibroblasts impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby fibroblasts alter cardiomyocyte behavior. Fibroblasts and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned > or =72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of alpha-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means +/- SD: conditioned 46.3 +/- 6.0 vs. control 5.3 +/- 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1alpha, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-alpha) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor-beta and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions.

Preliminary investigation on humoral and cellular immune responses to selected food proteins in patients with Crohn's disease.

Crohn's disease is a chronic inflammatory disorder for which an immunologic etiology has been proposed. Food hypersensitivity may contribute to part of the pathogenesis of this disorder. In preliminary studies, we evaluated 11 Crohn's patients by history, skin testing (ST), total and specific(s) IgE and sIgE/sIgG4 levels to five food proteins [egg (E), milk (M), wheat (W), soy (S), and corn (C)] using a sensitive enzyme monoclonal antibody assay. Skin testing was also performed using grass and mold allergens. Lymphocyte concanavalin (Con A) mitogenic and antigenic responses to food proteins were also determined by tritiated thymidine incorporation. Mean sIgG4 values for four food proteins are listed below: (Table: see text). No patient reacted with elevated sIgE or sIgG4 to corn. All patients had low to negative sIgE levels to all foods and only three had increased total IgE. Three of eight were history and ST positive to M, E, and W. Six of eight had at least one positive ST to M, E, W, and S. All patients had negative sIgG4 to tested inhalants and two had elevated sIgE to grass pollen. Although mean lymphocyte Con A mitogenesis was significantly decreased in eight patients compared with controls (P less than .05), an increased food stimulatory response to milk protein was observed (P less than .05). Perhaps, decreased sIgE and cell-mediated mitogenic responsiveness may lead to an enhanced humoral IgG response. The increased sIgG4 humoral response to egg protein and cellular sensitivity to milk protein may indicate mucosal antigenic stimulation or leakage in patients with Crohn's disease in spite of negative sIgE levels.

Intermittent stimulation enhances function of conditioned muscle.

Skeletal muscle is highly adaptable in that its metabolic and contractile characteristics are largely regulated by its pattern of use. It is known that muscle phenotype can be manipulated via chronic electrical stimulation to enhance fatigue resistance at the expense of contractile power. Type 2A fibers are fatigue resistant, powerful, and considered most desirable for cardiac assist purposes. We have found that 12-wk of intermittent-burst stimulation produces a high percentage of 2A fibers and increases fatigue resistance and power in rabbit latissimus dorsi muscle. Fixed-load endurance tests were used to quantify fatigue resistance among normal and trained muscle groups. Control muscles were found to fatigue completely within 10-20 min. Muscles stimulated continuously for 6 wk retained 35% (71.5 +/- 19.5 g. cm) of their initial stroke work at 40 min. Muscles stimulated 12 h/day for 12 wk had the highest initial stroke work (449.7 +/- 92.4 g. cm) and the highest remaining stroke work (234.7 +/- 50.1 g. cm) at 40 min. Results suggest that employing regular resting periods during conditioning preserves strength in fatigue-resistant muscle.

Correlation of serum immunoglobulin E elevations with clinical stages of dysthyroid orbitopathy.

Total immunoglobulin E (IgE) was measured by an enzyme-linked immunoassay in serum samples from patients with dysthyroid orbitopathy and from a group of healthy volunteers. All the serum donors had no symptoms of allergy or infection and were not given any immunoregulative treatments for at least 6 months before the sampling. One hundred thirty-seven dysthyroid orbitopathy patients were rated clinically as belonging to one of the following groups: (1) stable dysthyroid orbitopathy; (2) active dysthyroid orbitopathy; (3) chronic or recurrent dysthyroid orbitopathy; or (4) dysthyroid orbitopathy characterized by limited myopathy. The serum IgE levels of all these groups were compared with 26 healthy, nonatopic volunteers. The mean IgE levels of groups 3 and 4 were significantly higher than the mean IgE level of the control group as well as that of the group with stable dysthyroid orbitopathy. Furthermore, serial readings on several patients were consistent with the hypothesis that serum IgE is elevated in connection with certain stages of rapid dysthyroid orbitopathy progression and also with two unusual clinical forms of dysthyroid orbitopathy.

Immunohistochemical evidence for IgA1 involvement in Graves ophthalmopathy.

Orbital muscle, adipose tissues, and periorbital muscle from 11 patients with Graves ophthalmopathy were studied with in situ assays using monoclonal antibodies for IgA1, IgA2, IgM, and IgG. Tissue biopsies were taken from varied extraocular muscles and orbital sites. All cases were from patients with severe disease or disease of long duration. Control specimens of extraocular muscle tissues were obtained from nine patients treated for unrelated orbital disorders. Only connective tissue associated with the extraorbital muscles and periorbital muscles showed any reactivity. Of the muscle tissue obtained from patients with Graves disease all exhibited IgA1 positive staining of the endomysium and perimysium, without staining of the muscle fibers themselves. Parallel sections of orbital muscles reacted with anti-IgA2 or anti-IgM antibody failed to demonstrate staining. Control extraocular muscle tissue did not stain with anti-IgM and one control muscle of seven reacted minimally with anti-IgA2. Some reactivity with anti-IgA1 was seen in four of the seven control muscles but this was qualitatively much less than that of muscle tissue from patients with Graves disease. Monoclonal anti-IgG did not stain tissue from the six Graves specimens and three control specimens tested.

Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat.

To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.

Serum antibodies reactive with eye muscle membrane antigens are detected in patients with nonspecific orbital inflammation.

PURPOSE: Nonspecific orbital inflammation, also called "orbital pseudotumor," has many of the features of thyroid-associated ophthalmopathy, especially when localized to the eye muscle. The purpose of this study is to test for circulating autoantibodies against eye muscle antigens and features of possible thyroid autoimmunity in patients with nonspecific orbital inflammation. METHODS: The authors studied eight patients with diffuse or localized nonspecific orbital inflammation. The presence of autoantibodies reactive with pig eye muscle membrane antigens and 1D, a recombinant 64 kilodaltons (kd) thyroid and eye muscle protein, were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The most frequently detected antibodies were those reactive with eye muscle membrane proteins of 55 and 64 kd, which were demonstrated in 62.5% and 62.5%, respectively, of patients with nonspecific orbital inflammation; antibodies against 95- and 45-kd proteins were each detected in 50% of patients. In health subjects, antibodies reactive with the 55- and 64-kd proteins were detected in 16% and 20% of patients, respectively; those reactive with the 95-kd protein were detected in 24% of patients and with the 45-kd protein in 20% of patients. On the other hand, antibodies to 1D were demonstrated in only one patient with nonspecific orbital inflammation and not at all in healthy subjects. The prevalence of positive tests were significantly greater in patients with nonspecific orbital inflammation than healthy patients only for antibodies reactive with a 55-kd protein. Of the four antigens, only the 55-kd protein was expressed in other (systemic) skeletal muscle. No patient had overt thyroid disease or detectable serum antibodies reactive with the thyroid-stimulating hormone receptor, and only one had antibodies reactive with the thyroid microsomal antigen. CONCLUSION: Serum autoantibodies reactive with eye muscle membrane proteins are demonstrated in the majority of patients with nonspecific orbital inflammation. Although the pathogenesis of this condition is unknown, autoimmunity against eye muscle antigens is a likely mechanism. While antibodies reactive with the thyroid microsomal antigen were detected in only one patient and anti-thyroid-stimulating hormone receptor antibodies in none of the patients, a possible association of nonspecific orbital inflammation with thyroid autoimmunity has not been excluded.