Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.     

Production and characterization of genus and species specific monoclonal antibodies against surface tegumental antigens of Schistosoma mekongi

Fourty-four primary brain tumors were studied by immunohistochemistry. 29 (67.2%) expressed HLA-Dr. The incidence and number of tumor cells positive for HLA-Dr correlated with the histological type of brain tumor and increased with the degree of malignancy. The mononuclear cells infiltrating in these tumors were mostly CD45ROT cells and macrophages. The former consisted mainly of CD4 and CD8 subsets. The numbers of CD45RO+ and CD8+ T cells in HLA-Dr-group were more than any of the HLA-Dr+, +2, +3 groups. The numbers of CD4 subset and macrophages were not affected by the level of HLA-Dr expression. These results suggest that the HLA-Dr expressed by brain tumor cells selectively inhibit CD8 subset which participates in immunoreaction against brain tumors in situ.     

Production and characterisation of polyclonal antisera against feline IgE

To confirm the characteristics of the stromal cells of Wharton's jelly, we investigated the morphological changes in these cells during each trimester of pregnancy. We evaluated the cytoskeletal features of these cells by examining immunohistochemically the localization of one of the contractile proteins, alpha-smooth muscle actin (ASMA). After the second trimester, the stromal cells of Wharton's jelly were stained with ASMA antibody, exhibited the ultrastructural characteristics of the myofibroblasts, and began to express numerous microfilaments in the cytoplasm. Postembedding immunogold labeling detected immunoreactivity for ASMA on these microfilaments. The finding indicated that the stromal cells of Wharton's jelly undergo a time-dependent maturation involving the differentiation of myofibroblasts during the last 6 months of pregnancy. These cells possess a contractile function that may help to protect the umbilical vessels from compression, considering that ASMA was detected in the microfilamentous bundles.     

Monoclonal antibodies directed against lipopolysaccharide of Legionella pneumophila serogroup 1

The monoclonal antibodies (MAbs) against lipopolysaccharide of virulent strain of Legionella pneumophila serogroup 1 were produced. Three most productive hybrid clones (5F4, 5F10 and 2C9) were selected from fusions of mouse myeloma cells with spleen cells from BALB/c mice, immunized with bacterial outer membrane antigens. All generated clones were IgG-secreting. The MAbs had narrow strain specificity and showed no cross-reactions with other unrelated bacterial species. These antibodies were tested in sandwich ELISA. The results suggest that the MAbs could be used for diagnostic purposes.     

Role of repetitive antigen patterns for induction of antibodies against antibodies

Antibody responses against antibodies, such as rheumatoid factors, are found in several immunopathological diseases and may play a role in disease pathogenesis. Experience shows that they are usually difficult to induce experimentally. Antibodies specific for immunoglobulin constant regions (anti-allotypic) or for variable regions (anti-idiotypic) have been investigated in animal models; the latter have even been postulated to regulate antibody and T cell responses via network-like interactions. Why and how such anti-antibodies are induced during autoimmune diseases, has remained largely unclear. Because repetitively arranged epitopes in a paracrystalline structure of a viral envelope cross-link B cell receptors efficiently to induce a prompt T-independent IgM response, this study used immune complexes containing viruses or bacteria to evaluate the role of antigen pattern for induction of anti-antibody responses. We present evidence that antibodies bound to strictly ordered, but not to irregularly arranged, antigens dramatically enhance induction of anti-antibodies, already after a single immunization and without using adjuvants. The results indicate a novel link between anti-antibody responses and infectious agents, and suggest a similar role for repetitive self-antigens such as DNA or collagen involved in chronic immunopathological diseases.     

Development and characterization of antibodies directed against the mouse D4 dopamine receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.     

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

A multiple beam technique was utilized to obtain angle independent Doppler color images (AIDCI) using an ultrasonic scanner with a linear transducer. A quantitative study using steady flow models has been performed to evaluate the accuracy of this method in velocity measurements. The results show that the velocity amplitudes measured with this method correlated with those calculated from the measured flow rates (r = 0.95-0.98). The flow angles obtained with this method also correlated with those calculated from the coordinates of the tube image (r = 0.93-0.96). To improve the interpretation of the angle independent results, a method for visualizing two-dimensional flow fields is presented and compared with two existing methods.     

Autoreactivity developing spontaneously in cultured mouse spleen cells. I. Evidence that cytotoxicity is directed against embryo-associated antigen

We have investigated the ability of male-mouse spleen cells before and after culture in the absence of deliberate antigenic stimulation to show specific cytotoxicity to syngeneic embryo-fibroblast cells. The data suggest that cytotoxicity which develops spontaneously in such spleen cell cultures is directed primarily against embryo-associated antigens. Syngeneic Con-A-stimulated spleen cells, which, unlike fresh normal spleen cells, are also lysed by rabbit anti-mouse embryo antisera, are also a suitable target to demonstrate spontaneously developing cytotoxicity.     

Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.     

Cytosolic proteins of 21-23 kDa are methylated by kidney cortex membrane-associated C-terminal carboxyl methyltransferases

The nursing home plays an increasing role as domicile in the last period of life. The following investigation is an assessment of the mortality after entering a nursing home, and an analysis of the mental and physical condition of the residents with the goal of being able to differentiate at admission to a nursing home between short-term and long-term "survivors". Of 317 nursing home residents, the initial physical examination upon admission was investigated retrospectively. The nursing home residents were divided into six groups according to their survival time: survival-time up to 1 week, > 1 week to 1 month, > 1 month to 6 month, > 6 month to 1 year, > 1 year to 5 years, and > 5 years. The mean age of the group studied at admission was 80 years; 75% of the residents were transferred from a hospital. On the average, six diseases or chronic disabilities per patient were diagnosed upon admission to the nursing home. The mean survival time was 2.7 years. The initial phase after admission was the most critical period with a high mortality in the first six months (41% of all nursing home residents); of these, 81% had died already within the first month. The assessment criteria, i. e., the level of communicative abilities, orientation, mobility, and urinary/fecal continence, were found to bear a close relation to survival time. Thus, an important prognostic role as "survival predictors" can be ascribed to these factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of a monoclonal antibody against the 128 kDa (CagA) protein of Helicobacter pylori

We report a case of herpes simplex encephalitis in which the patient was repeatedly examined by magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The patient was a 36 year-old woman who had been transferred to our institution 6 days after the onset of symptoms with mild consciousness disturbance, nuchal rigidity, and high fever. Cerebrospinal fluid examination revealed an elevated mononuclear cell count with normal sugar concentration. Intravenous aciclovir was started 7 days after the onset of symptoms. The initial plain computed tomography (CT) scans did not reveal any abnormal findings, but contrast enhanced CT the next day showed a slight enhancement effect around the affected middle cerebral artery. Serial MRI showed the initial high intensity lesion starting on the medial cortex of the temporal lobe, then spreading to throughout the entire temporal lobe. During this period SPECT showed a marked, broad hot spot in the temporal lobe. The medial temporal lobe was high density on the CT 15 days after the onset. As the encephalitic lesion spread more laterally, the hot spot on SPECT moved laterally and then decreased in activity. Eleven weeks after the onset, the MRI showed intracerebral vacuolization of the lesion and it appeared as a wide cold spot on SPECT. The cause of the hot spot seen in the acute period was thought to be vasoparalysis of the affected area rather than breakdown of the blood-brain barrier, or impaired washout of the isotope, because the SPECT images after acetazolamide administration showed the cold spot even in the subacute phase.     

Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen

We have shown previously that heat-killed bacillus Calmette-Guerin injected into the brain parenchyma becomes sequestered behind the blood brain barrier for months undetected by the immune system. However, independent peripheral sensitization of the immune system to bacillus Calmette-Guérin results in recognition of bacillus Calmette-Guérin in the brain and the induction of focal chronic lesions [Matyszak M. K. and Perry V. H. (1995) Neuroscience 64, 967 977]. We carried out ultrastructural studies of these lesions. Prior to subcutaneous challenge we used immunohistochemistry to detect bacillus Calmette-Guérin which was found in cells with the morphology of macrophages/microglia and in perivascular macrophages. Eight to 14 days after subcutaneous challenge there was a conspicuous leucocyte infiltration at the site of bacillus Calmette-Guérin deposits within the brain parenchyma. The majority of these cells were macrophages and lymphocytes, with some lymphocytes showing characteristic blast morphology. Dendritic cells in close contact with lymphocytes were prominent. Inflammatory cells were found in perivascular cuffs and within the brain parenchyma. The tissue was oedematous and some axons were undergoing Wallerian degeneration with associated myelin degeneration. Throughout the lesions, but more commonly at the edges, we detected macrophages containing myelin in their cytoplasm close to intact axons and axons with evidence of remyelinating sheaths, suggestive of primary demyelination. In older lesions, two to three months after the peripheral challenge, the oedema was less pronounced and there was little evidence of Wallerian degeneration. There were still many macrophages. lymphocytes and dendritic cells, although the number of these cells was lower than in earlier lesions. Late lesions also contained many plasma cells which were not present in early lesions. In these late lesions there were bundles of axons with no myelin or a few axons with thin myelin sheaths, suggestive of persistent or ongoing demyelination or remyelination. These observations show that, during a delayed-type hypersensitivity lesion in the CNS, the leucocyte populations change with time, and suggest that the mechanisms and type of tissue damage are different in the early and late stages of the lesion.     

Monoclonal antibody FC-5.01, directed against CD63 antigen, is internalized into cytoplasmic vesicles in the IIB-BR-G human breast cancer cell line

PURPOSE: Benign prostatic hyperplasia is common among men who may be candidates for prostate cancer screening using prostate-specific antigen (PSA) testing. Patterns of PSA testing among men with evidence of benign prostatic hyperplasia have not been studied. METHODS: We examined the prevalence and correlates of a self-reported history of PSA testing. In 1994, 33,028 US health professionals without prostate cancer aged 47 to 85 years provided information on prior PSA testing, lower urinary tract symptoms characteristic of benign prostatic hyperplasia, history of prostatectomy, and prostate cancer risk factors. In 1995, a subset of 7,070 men provided additional information on diagnosis and treatment of benign prostatic hyperplasia. RESULTS: From 39% of men in their 50s to 53% of men in their 80s reported PSA testing in the prior year (P <0.0001 for trend with age). Men were more likely to report PSA testing if they had lower urinary tract symptoms characteristic of benign prostatic hyperplasia (age-adjusted odds ratio for severe symptoms 2.2, 95% confidence interval 1.8 to 2.6), a prior history of prostatectomy (age-adjusted odds ratio 1.1, 95% confidence interval 1.02 to 1.2), or a physician diagnosis of benign prostatic hyperplasia (odds ratio 1.9, 95% confidence interval 1.7 to 2.2; adjusted for age, signs or symptoms of benign prostatic hyperplasia, and prostate cancer risk factors). CONCLUSIONS: These US health professionals reported preferential use of PSA testing among men least likely to benefit from early cancer detection (older men) and among men most likely to have a false-positive PSA result (men with benign prostatic hyperplasia). Physician and patient education are needed to promote more rational and selective use of this screening test.     

Detection of mosquito saliva-specific IgE and IgG4 antibodies by immunoblotting

IgE and IgG subclass antibodies against Aedes communis mosquito saliva were studied by immunoblotting in 12 adults with immediate and/or delayed skin reactions to mosquito bites. Four antigenic proteins, with molecular weights of 22, 30, 36, and 64 kd, were found in the mosquito saliva. Almost all subjects (11 of 12) had anti-mosquito saliva-specific IgE antibodies directed against the 36 kd protein. The IgG antibody response appeared to be restricted mostly to IgG4 (11 of 12) and IgG1 (8 of 11) subclasses against the same 36 kd antigen. Ten of the 12 subjects had both IgE and IgG4 antibodies to the 36 kd protein. No anti-mosquito antibodies were found in pooled sera of five infants never exposed to mosquito bites. These results show that most persons with immediate skin reactivity to A. communis mosquito bites have both IgE and IgG4 antibodies that recognize the 36 kd antigen present in the mosquito saliva, suggesting that anti-saliva antibodies may play a role in the pathogenesis of mosquito bite reactions.     

Rapid verification of the identity of questionable specimens using immunohistochemistry with monoclonal antibodies directed against HLA-class I antigens

PURPOSE: To evaluate the effect of substituting topical cyclosporin A 0.5% for topical corticosteroids in patients with postkeratoplasty glaucoma. METHODS: Topical cyclosporin A 0.5% was prospectively substituted for topical corticosteroids to treat 25 patients with postkeratoplasty glaucoma. RESULTS: Twenty-one (84%) of 25 patients showed a reduction in intraocular pressure (IOP) (range, 1-22 mm Hg; mean, 8.7 mm Hg). Follow-up ranged from 3 to 12 months (mean, 5.8). Graft clarity was maintained in all patients, with one allograft rejection episode. Thirteen patients were able to discontinue one or more glaucoma medication(s). CONCLUSION: Topical cyclosporin A 0.5% may be substituted for topical corticosteroids to aid in the management of postkeratoplasty patients with glaucoma. However, the resultant decrease in IOP may be associated with an increased risk for immune rejections.     

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.