Biomedical subjects

K Felgenhauer

Publications and source records attributed to K Felgenhauer.

At least 37 records · Page 2Linked to original sources

PubMed1997

Establishment of an efficient enzyme-linked immunosorbent assay for the determination of human choline acetyltransferase.

A specific and sensitive two-side enzyme-linked immunosorbent assay (sandwich-ELISA) was established for the reliable quantification of human brain and placental choline acetyltransferase (ChAT). In contrast to the radiometric assay developed by Fonnum, which is widely used for the measurement of enzyme activity, the sandwich-ELISA particularly recognized inactivated forms of the antigen. In the assay, affinity-purified polyclonal synthetic peptide antibodies adsorbed to the polystyrene surface of the microtiter plate were employed as capture reagent. Based on standard peroxidase protocols, immobilized ChAT was detected using monoclonal antibodies raised against human placental ChAT. By use of this ELISA, ChAT was determined at various purification stages of the enzyme, in body fluids, during recovery experiments and in sera of patients with severe brain damage.

Adolescent↗

PubMed1997

Detection of T cells reactive to intestinal alkaline phosphatase, an autoantigen in acute bacterial infections, and discrimination between autoantigen-specific CD5+ and CD5- B cells.

Autoantibodies of the IgG isotype, specifically directed against intestinal alkaline phosphatase (IAP), occur transiently in the majority of sera from patients with acute bacterial infections. Sometimes they are observed in autoimmune diseases. Using a T cell proliferation assay, it was found that isolated peripheral blood mononuclear cells (PBMC) from IAP autoantibody (IAPA)-positive patients (n = 18) responded significantly to IAP, whereas proliferation could not be induced in PBMC from healthy donors (n = 11). Significant stimulation of PBMC from patients (n = 11) was not obtained by use of transferrin, a common autoantigen in humans, indicating the specificity of stimulation of IAP-reactive T cells. Furthermore, T cell proliferation was observed when a highly purified IAP fragment (CNBr 21) spanning amino acids 334-462 of the primary structure of IAP was used as antigen. Thus, it was shown that an immunodominant T cell epitope resides within the CNBr 21 fragment which also contains a discontinuous B cell epitope as evaluated previously. Double immunocytochemical staining of T cell-depleted PBMC with IAP and an anti-human CD5 antibody allowed the detection of CD5+ B lymphocytes, which probably produce natural IAPA (nIAPA). These nIAPA-specific CD5+ B cells occurred with approximately the same frequency among T cell-depleted PBMC from healthy donors and those from patients. In contrast, IAPA-producing CD5- B cells were found in B cell-enriched preparations from patients, but not in those from healthy individuals.

Acute Disease↗

PubMed1997

Characterization of monoclonal and polyclonal antibodies to human choline acetyltransferase and epitope analysis.

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167-189 and 57-76, respectively.

Amino Acid Sequence↗

PubMed1996

A putative enzyme from various secretions specifically inhibits antibody-antigen interactions.

Various human secretions (intestinal secretion, saliva, nasal mucus, lacrimal fluid) have been found to inhibit the binding of antibodies to their antigens. Various characteristics (e.g. time, pH, temperature dependence, affinity and size exclusion chromatography) suggested that the inhibitory activity was attributable to an enzyme. Further investigations revealed that this enzyme reacted with the Fab portion of immunoglobulin G, specifically with the heavy chain. It is assumed that it represents a novel immunoglobulin-specific protease since similar results were not obtained with proteolytic enzymes from human digestive organs e.g. pepsin, trypsin and chymotrypsin. Finally, investigating saliva it was demonstrated that the putative protease was not identical to enzymes from periodontal bacteria which are proteolytic for the Fc portion of immunoglobulins. The findings could be of general importance in the design of immunoassays which are to be applied to human (and possibly animal) secretions.

Alkaline Phosphatase↗

PubMed1996

Choroid plexus: the major site of mRNA expression for the beta-trace protein (prostaglandin D synthase) in human brain.

Expression of beta-trace protein (beta-trace), recently identified as glutathion-independent prostaglandin D synthase (prostaglandin-H2 D-isomerase; EC 5.3.99.2), was localized in paraffin sections of the human brain by in situ hybridization using digoxigenin-labeled antisense cRNA probes. The mRNA for beta-trace was predominantly found in the epithelial cells of the choroid plexus. Hybridization signals were also obtained in some oligodendrocytes, particularly in the white matter. In the leptomeninges, specific signals were found in meningeal macrophages and in single cells of the arachnoid barrier layer. The cells exhibiting hybridization signals with the antisense cRNA probes for beta-trace were identified by counterstaining with antibodies directed against specific cell markers. Additionally, beta-trace mRNA was localized in tubular epithelial and basal cells of the human epididymis and in different cell types within the seminiferous epithelium of the testis.

Brain↗

PubMed1996

Definition of a discontinuous immunodominant epitope of intestinal alkaline phosphatase, an autoantigen in acute bacterial infections.

In patients suffering from acute bacterial infections specific autoantibodies of the immunoglobulin class G (IgG), directed against intestinal alkaline phosphatase (IAP), are transiently expressed in high titer. The epitopes on IAP which are recognized by these IAP auto-antibodies were investigated using chemical and enzymatic cleavage, followed by two-dimensional electrophoresis and immunoblotting of the fragments. Immunoreactive and nonreactive cleavage fragments were isolated and N-terminally sequenced. An epitope map was constructed by means of sequencing data, relative molecular mass of the fragments, and specific cleavage sites. To evaluate linear epitopes, the overlapping region of the immunoreactive fragments (amino acids 204-484 of the primary structure of IAP) was further analyzed by simultaneous synthesis of 95 peptides on an activated membrane. Four immunoreactive regions were revealed by immunodetection with IAP autoantibody-positive sera. Nine peptides comprising these regions were synthesized quantitatively and coupled to CH-Sepharose. However, specific IAP autoantibodies could not be isolated. This result indicated the absence of continuous epitopes at least in the analyzed region of the molecule. Binding studies with an IAP cleavage fragment suggested a discontinuous epitope involving amino acids 334-371. The results are indicative of an antigen-driven mechanism for the production of IAP autoantibodies initiated and maintained by self.

Acute Disease↗

PubMed1996

A novel enzyme-linked immunosorbent assay for determination of synaptophysin as compared with other quantification procedures.

A two-sided enzyme-linked immunosorbent assay (ELISA) has been established for reliable, specific and sensitive determination of synaptophysin (SYN), an intrinsic membrane protein of the small synaptic vesicles. This ELISA used a highly specific monoclonal antibody (SY 38) as capture reagent and a specific SYN antiserum in combination with a secondary peroxidase-conjugated antibody for detection. Calibration was carried out with immunoaffinity-purified SYN from porcine cortex. The sensitivity was found to be improved substantially when the ELISA was compared with previously used dot-immunobinding assays. This ELISA allowed rapid and reliable determination of SYN from detergent lysed homogenates, partially and highly purified preparations of rat, porcine and human brain. SYN was determined in highly purified small synaptic vesicles, and it was calculated to be 5.8% of total detergent solubilized protein.

Animals↗

PubMed1996

Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay.

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

Animals↗

PubMed1995

Cerebral endothelial cells are a major source for soluble intercellular adhesion molecule-1 in the human central nervous system.

Immunohistological analysis of tissue sections from human brain revealed that intercellular adhesion molecule-1 (ICAM-1) is mainly expressed on endothelial cells of small vessels, including the subependymal vessels of the choroid plexus. In addition, it is expressed on cerebrospinal fluid (CSF) cells in patients with inflammatory diseases of the central nervous system. Stimulation of confluent monolayers of adult human cerebral endothelial cells with lipopolysaccharide (LPS) or recombinant human tumor necrosis factor-alpha (TNF-alpha) could induce expression and secretion of soluble ICAM-1 in a dose dependent manner. In addition, sICAM-1 was also present in the supernatant from U251 glioma cells. No sICAM was detected in the culture supernatant from activated blood or CSF lymphocytes. Cerebral endothelial cells are therefore a likely source for sICAM-1 in the CSF.

Base Sequence↗

PubMed1995

A soluble form of tumour necrosis factor receptor in cerebrospinal fluid and serum of HTLV-I-associated myelopathy and other neurological diseases.

Paired samples of cerebrospinal fluid (CSF) and serum from 17 patients with human T-cell lymphotrophic virus I (HTLV-I)-associated myelopathy, 5 patients with multiple sclerosis and 11 controls with non-inflammatory disorders (migraine, idiopathic epilepsy and myelopathy of unknown aetiology) were examined by enzyme-linked immunosorbent assay for the presence of the 60-kDa soluble form of tumour necrosis factor receptor (sTNF-R). The results were compared with blood-CSF barrier function, cell count and the intrathecal synthesis of HTLV-I antibodies. No correlation could be demonstrated. High levels of sTNF-R were found in CSF of patients with HTLV-I-associated myelopathy and multiple sclerosis. In addition, intrathecal sTNF-R was also detected in the patients with non-inflammatory diseases, indicating that sTNF-R is definitively a normal constituent of CSF.

Blood-Brain Barrier↗

PubMed1995

Soluble forms of intercellular adhesion molecule-1 (ICAM-1) block lymphocyte attachment to cerebral endothelial cells.

Serum levels of circulating ICAM-1 are increased in various disorders including inflammatory diseases of the central nervous system (CNS). We recently described an association between high sICAM-1 levels in the serum of patients with multiple sclerosis and disease activity. The functional consequences of increased circulating adhesion molecules are not fully understood. This may simply arise as a consequence of inflammation or may have immune modulating properties. ICAM-1 plays an important role in the recruitment of activated lymphocytes to sites of inflammation within the CNS. We therefore tested the ability of soluble forms of ICAM-1 to prevent adhesion of activated lymphocytes to cerebral endothelial cells. Mitogen-activated blood mononuclear cells (PBMC) as well as PBMCs from patients with active multiple sclerosis adhered to cerebral endothelial cell cultures in vitro. This adhesion could be blocked if lymphocytes were preincubated with a recombinant form of soluble ICAM-1. In addition, serum from patients with active multiple sclerosis and high sICAM-1 levels blocked adhesion in a dose-dependent manner which was abrogated by pre-adsorption to an anti ICAM-1 antibody. Since soluble forms of ICAM-1 are able to block lymphocyte adhesion to cerebral endothelial cells, they may provide new therapeutic tools to interfere with the pathogenesis of inflammatory diseases of the CNS.

Cell Adhesion↗

PubMed1995

Origin and immunoregulation of autoantibodies against intestinal alkaline phosphatase.

In the sera of patients with acute bacterial infections specific autoantibodies (sIAPa) of the immunoglobulin class G (IgG) were found which bind to intestinal alkaline phosphatase (IAP) through the Fab portion. This was demonstrated using immunoaffinity (IA) isolation of sIAPa from patients' sera (particularly bacterial meningitis and ventriculitis) digestion with pepsin, purification of F(ab')2 fragments on protein A and subsequently binding on IAP coupled to CNBr (cyanogen bromide)-activated Sepharose. Immunoblots using specific anti-Fc and anti-Fab antibodies showed that the bulk of F(ab')2 fragments had bound. Additionally, binding of native IAP to the F(ab')2 fragments was observed after separation of F(ab')2 fragments using isoelectric focusing (IEF), blotting onto nitrocellulose and incubation with IAP. Moreover, we have demonstrated the occurrence of natural anti-IAP autoantibodies (nIAPa) which were isolated from sera of healthy individuals using IA chromatography. Investigation of isotype distribution revealed that IgG but not IgM or IgA were predominant even among nIAPa. The nIAPa fraction exhibited lower binding efficiencies on IEF blots than the sIAPa fraction, however, in contrast to sIAPa, cross-reactions with other autoantigens were observed for nIAPa. NIAPa and sIAPa did not show subclass restriction. As revealed by IEF the spectrotypes of sIAPa were found to be patient-specific, poly- to oligoclonal and stable during longer periods.

Acute Disease↗

PubMed1995

Specificity of intrathecal IgG synthesis for HTLV-1 core and envelope proteins in HAM/TSP.

INTRODUCTION: In patients with human T-cell lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP), we investigated the significance of HTLV-1 specific antibodies in the cerebrospinal fluid (CSF). MATERIAL AND METHODS: The quantities of HTLV-1 specific immunoglobulin G (IgG) in paired CSF and serum were evaluated by a sensitive enzyme immunoassay (EIA). The specificity of antiviral IgG was determined by radioimmunoprecipitation of HTLV-1 antigens. RESULTS: In 17 of 20 HAM/TSP patients, quantitative evaluation by EIA supplied evidence for antiviral IgG synthesis within the CNS. Radioimmunoprecipitation demonstrated IgG antibodies against HTLV-1 envelope and core proteins in all HAM/TSP CSF and sera tested. Regarding the 3 sample pairs indeterminate in EIA for intrathecal synthesis, 2 showed stronger precipitation of HTLV-1 antigens by CSF IgG than by equal amounts of serum IgG. CONCLUSION: Intrathecal antibody synthesis specific for both HTLV-1 core and envelope antigens is common in HAM/TSP, thus providing conclusive evidence for an immune response to HTLV-1 within the CNS.

Antibody Specificity↗

PubMed1995

HTLV-I associated myelopathy in patients from Brazil and Iran: neurological manifestations and cerebrospinal fluid findings.

We analysed sera and cerebrospinal fluid (CSF) of 20 cases of human T-cell lymphotropic virus type (HTLV-I) associated myelopathy/tropical spastic paraparesis (HAM/TSP) from Brazil and Iran and as controls, 16 Brazilian HTLVI seronegative individuals afflicted with other neurological diseases. It was observed in the HAM/TSP patients that: 1) all had an inflammatory reaction within the central nervous system (CNS); 2) 95% (19/20) showed oligoclonal bands reflecting intrathecal IgG synthesis; 3) 85% (17/20) presented a local synthesis of HTLVI antibodies; 4) 35% (7/20) had a measurable immunoglobulin (Ig) synthesis within the CNS. The CSF parameters of the HAM/TSP were compared with the clinical data (age at onset, duration of disease and disability level). Our data prove that CSF analysis is important for the diagnosis of HAM/TSP. There is no association between the severity of the disease and CSF findings.

Adult↗

PubMed1995

The value of CSF analysis for the differential diagnosis of HTLV-I associated myelopathy and multiple sclerosis.

Cerebrospinal fluid (CSF) and serum of 17 patients with HAM/TSP (HTLV-I associated myelopathy/tropical spastic paraparesis), six with multiple sclerosis and six with idiopathic epilepsy (non inflammatory control) from Brazil were analysed for the presence of intrathecal synthesis of virus-specific antibodies against measles, rubella, varicella zoster virus and herpes simplex virus by enzyme-linked immunosorbent assay (ELISA). All HAM/TSP and multiple sclerosis cases had an intrathecal immune response (oligoclonal IgG). In HAM/TSP, only 1/17 case showed a polyspecific intrathecal immune response against measles and rubella virus. In multiple sclerosis, specific antibodies against measles and rubella (MRZ response) were observed in all patients but not in the control with idiopathic epilepsy. The diagnostic and theoretical relevance of mono- and polyspecific immune responses is discussed for these chronic neurological diseases.

Brazil↗

PubMed1994

Specific diagnosis of progressive multifocal leukoencephalopathy by polymerase chain reaction.

Using polymerase chain reaction (PCR), 34 cerebrospinal fluid (CSF) samples from 28 patients with progressive multifocal leukoencephalopathy (PML) were analyzed. As controls, 116 samples were evaluated from 82 human immunodeficiency virus type 1 (HIV-1)-infected patients and 1 HIV-1-negative patient. Of the HIV-1-positive patients, 23 had cerebral toxoplasmosis, 10 had HIV leukoencephalopathy, and 49 had other neurologic complications. Detection of JC virus (JCV) DNA in CSF was increased 10-fold by the addition of carrier DNA before phenol-chloroform-isoamyl alcohol extraction. The primer pair JC 26/29, from the VP1/large T region, had a limit of detection of 10(5) JCV DNA molecules/100 microL. The primer pair JC 36/39, located in the large T gene region, had a 100-fold lower limit of detection. With JC 26/29, the sensitivity was 43% (12/28) and specificity was 100%. Using JC 36/39, sensitivity increased to 82% (23/28), and false-positive results were not observed. Diagnosis of PML is greatly aided by PCR analysis of CSF.

Base Sequence↗

PubMed1994

Human intestinal alkaline phosphatase-binding IgG in patients with severe bacterial infections.

Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither to the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation.

Alkaline Phosphatase↗

PubMed1994

Acute diffuse leukoencephalitis in HIV-1 infection.

The clinical, neuroradiological, and cerebrospinal fluid findings of a case with acute diffuse leukoencephalitis, a demyelinating disease associated with human immunodeficiency virus infection of the brain, is reported. The patient presented with acute tetraparesis as the primary manifestation of a previously symptom free HIV infection. Cerebrospinal fluid analysis showed enhanced inflammatory abnormalities with high concentrations of P24 antigen. MRI showed diffuse white matter hyper-intensities in both hemispheres. In the follow up over 22 months, the neurological deficits disappeared after antiretroviral treatment in good correlation with improvements in MRI as well as in inflammatory cerebrospinal fluid abnormalities.

AIDS Dementia Complex↗