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Biomedical subjects

E Felber

Publications and source records attributed to E Felber.

14 recordsLinked to original sources
PubMed1999

Detection of bone marrow micrometastasis.

The detection of metastasizing single tumor cells has so far been difficult. Using a monoclonal antibody (MAb A45-B/B3) recognizing human cytokeratin, we identified immunocytochemically single tumor cells and micrometastases in patients (n = 24) with nonsmall cell lung cancer at the time of surgery of the primary tumor. The cytokeratin-positive cells (1-14/5 x 10(5) cells) in the bone marrow samples of 9 (9/24) patients were found. We also found a garland-like cluster, which consists of seven cancer cells and two closely connected tumor cells from one bone marrow sample. These results indicate that this technique can be used as a early diagnostic technique of bone marrow micrometastasis in the patient with the nonsmall cell lung cancer.

Animals↗
PubMed1997

Occult epithelial tumor cells detected in bone marrow by an enzyme immunoassay specific for cytokeratin 19.

The presence of isolated carcinoma cells detected immunocytochemically in bone marrow has been shown to be of prognostic relevance for cancer patients. Unfortunately, the immunocytochemical method (ICC) is laborious and depends on the subjective interpretation of the individual investigator. Therefore, an immunoassay was designed for detection of cytokeratin 19 (CK19). By analyzing blood samples from 52 healthy volunteers and 40 bone-marrow aspirates from control patients, a cut-off point of 250 pg/ml CK19 was determined. Application of this cut-off point enabled a specificity of 95% to be shown for bone marrow and of nearly 100% for venous blood. The assay detected 10 HT-29 colon-carcinoma cells among 5 x 10(6) peripheral-blood leukocytes. In comparison with controls, bone-marrow samples of cancer patients were found to have significantly elevated levels of CK19 (p < 0.05). In the analysis of 386 marrow aspirates of cancer patients, a significant concordance of ELISA and ICC was observed (chi 2 = 18.3; p < 0.001). Both procedures, nevertheless, differed in 147 (38%) samples, of which more than two thirds (101) were only ELISA-positive. The CK status detected by ELISA did not correlate with the TNM stage and the histological grading. The established immunoassay allowed sensitive and specific detection of disseminated epithelial tumor cells and appeared to be faster, less laborious and more objective than ICC. Follow-up studies are required to assess the prognostic relevance of this ELISA before it can be applied as a routine method for monitoring of minimal residual epithelial cancer.

Biomarkers, Tumor↗
PubMed1994

Detection of autoantibodies against M2, LKM-1, and SLA in liver diseases by standardized uniform ELISA-techniques.

Antibodies directed against soluble liver antigen (SLA), liver kidney microsomal antigen (LKM-1-AG), and antimitochondrial antigen M2 (M2-AMA) are critical serological markers for the differential diagnosis of autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). The exact diagnosis of autoimmune hepatitis and PBC is of great clinical relevance, as it leads to different therapeutic strategies. In the present work, a simple and reliable ELISA test system is described, which applies the same test principle for the detection of three different species of autoantibodies important for the diagnosis of chronic liver disease. The ELISA assays are based on a competitive inhibition of binding of positive standard antibodies by patients sera containing antibodies of unknown specificity. The purified immunoglobulins of clinically and serologically clearly defined patients with SLA or LKM-1 positive AI-CAH and with M2 positive PBC were used as coating- and detection antibodies in the ELISAs. From homogenized rat liver the fractionated 100,000g supernatant was employed for the SLA ELISA, the microsomal preparation served as antigen for the LKM-1 ELISA and the mitochondrial preparation was used for the M2 ELISA. In 1,500 sera of patients with the differential diagnosis of a hepatobiliary disease, 17 gave a positive signal in the SLA-ELISA, 12 in the LKM-1-ELISA, and 72 in the M2-ELISA. The results of the ELISAs were compared with Western blotting and immunofluorescence staining pattern on cryostat sections and Hep2 cells. The antibody profiles of several patients are described in detail.

Adult↗
PubMed1994

Detection and characterization of residual disease in breast cancer.

Although micrometastatic tumor cell spread largely determines the prognosis of patients with operable breast cancer, it is usually missed by conventional tumor staging. Several groups (including ours) have therefore developed immunocytochemical and molecular assays that allow the specific detection and characterization of individual carcinoma cells disseminated to bone marrow, blood, and lymph nodes. These assays may improve the prognostic precision of the current classification systems and may provide a tool for the early assessment of the therapeutic effects of anticancer drugs on micrometastatic cells in individual patients. Another aspect of such methods is that they enable detection of tumor cell contamination in stem cell grafts and validation of the efficiency of purging techniques. The most extensive experience exists with immunocytochemical methods, some of which have the potential to serve as a benchmark for less validated molecular methods. Still, the specificity and sensitivity of immunocytochemical detection of single cancer cells are affected by several variables, which include the intricacies of antigen expression, the lack of distinct morphological characteristics, the size of the analyzed sample, and the staining techniques for visualization of antibody binding. This article provides a critical review of the opportunities and pitfalls related to new methods for the detection and monitoring of minimal residual disease in breast cancer.

Bone Marrow↗
PubMed1993

Classical pathway of complement activation in mammalian kidneys.

Two monoclonal antibodies (mAb M4d2 and M4d3) specific for the alpha 2-fragment (C4d), and one antibody (mAb M4c3) specific for the gamma-chain of human complement protein C4, have been tested for cross-reactivity against mammalian complement. These mAb have also been found to react with C4 from guinea-pig (mAb M4d2 and M4c3) as well as from cattle, baboon and rhesus monkey (mAb M4d3 and M4c3) in an activation ELISA. Since reactivity of mAb M4d2 and M4c3 included guinea-pig complement, the specific recognition of mammalian C4 could be confirmed with sera from C4-deficient (def') guinea-pigs. mAb M4d2 or M4d3, but not mAb M4c3, stained glomerular deposits within renal tissue sections from pig, cattle and guinea-pig. In the case of mAb M4d2, specificity of that staining could also be demonstrated in kidney specimens from C4-def' guinea-pigs. It can be concluded that, as in humans, the C4d fragment is also present in mammalian glomeruli. Compared with normal guinea-pigs, the C4-def' and C2-def' animals showed markedly increased glomerular deposits of IgM. It appears that glomerular deposition of complement C4d in mammals: (1) indicates activation via the classical pathway; (2) represents a general phenomenon of renal homeostasis; and (3) seems to be involved in the physiological clearance of immune complexes.

Animals↗
PubMed1991

Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection.

Complement activation in 73 renal transplant biopsies was investigated by indirect immunoperoxidase staining using MoAbs reactive with complement-split products. Intense deposition of complement fragments C4d and C3d in peritubular capillaries, indicating activation of the classical pathway, could be detected in the majority of transplanted kidneys with cell-mediated rejections. Abundant deposition of complement-split products was observed in 22 early biopsies from patients with high 'immunological risk' (i.e. previous, rejected transplants and/or circulating antibodies against HLA-antigens). Despite negative results in the crossmatch before transplantation and paucity of immunoglobulins in transplant biopsies, antibodies directed against endothelial cell antigens should be considered as a possible cause of classical complement activation.

Antibodies, Monoclonal↗
PubMed1990

Monoclonal antibodies to complement components without the need of their prior purification. II. Antibodies to mouse C3 and C4.

Rat monoclonal antibodies (MAbs) to mouse complement components C3 and C4 were produced by immunizing rats with cell-bound C3 and C4. This principle involves: a) using mouse thymocytes coated with syngeneic rat antibody isotypes that show high affinity to C1q, b) the intercalation of C1q from serum and c) the subsequent activation of the classical complement pathway leading to deposition of cell-bound complement components. Screening for anti-complement antibodies was performed on antibody coating microtiter plates with mouse serum as source of complement. The reactivity of the MAbs was determined by variations of the ELISA screening system using EDTA-serum to inhibit complement activation by C1 dissociation, serum rendered deficient of functionally active C3 by treatment with cobra venom factor (CVF) or serum of genetically C5-deficient mice. The specificity of the MAbs was confirmed by affinity chromatography followed by SDS-PAGE and immunoblotting. We were able to establish a panel of anti-C3 and anti-C4 MAbs of various isotypes.

Animals↗
PubMed1989

Deposition of complement activation products on plastic-adsorbed immunoglobulins. A simple ELISA technique for the detection of defined complement deficiencies.

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca2+ depletion, heat treatment (56 degrees C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def', C2 def', C6 def' human sera; C2 def', C4 def' guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.

Adsorption↗
PubMed1989

Classical pathway of complement activation in normal and diseased human glomeruli.

Monoclonal antibodies (mAb) reactive against complement components involved in the classical activation pathway were applied in an indirect immunoperoxidase technique for the histological study of normal and diseased human renal tissues. Prominent staining with antibodies against the C4d fragment was seen in all glomeruli and some renal arteriolar walls. The C4d staining was mesangial with light microscopy, whereas the subendothelial site of the glomerular basement membrane (GBM) also appeared to be positive in immunoelectron microscopy. In similar localization, albeit with distinctly weaker intensity, IgM and C4 binding protein (C4bp) were detected. In kidney biopsies from patients with various types of glomerulonephritis, C4d reactive antibodies stained the glomerular structures in a strong, diffuse or granular pattern in contrast to the more segmental distribution and weaker staining intensity in normal kidney specimens. Increased amounts of C4d, occasionally also of C4b, were paralleled in diseased kidney tissues by distinct deposits of IgM and/or IgG in the presence of C4bp. This study suggests that the C4d fragment in normal human glomeruli is indicative of a continuous, local complement activation via the classical pathway induced by the physiological deposition of IgM-containing immune complexes.

Animals↗
PubMed1989

Monoclonal antibodies to complement components without the need of their prior purification. I. Antibodies to mouse C1q.

Rat monoclonal antibodies (MAbs) reactive with mouse complement subcomponent C1q were raised applying a principle that requires minute amounts of serum and circumvents purification of serum-derived C1q. The principle involves a) using the high affinity of certain cell-bound antibody isotypes for intercalating C1q from serum of various species, b) selecting such antibodies as are syngeneic to the immunized animal species, thus avoiding the production of antibodies against the intercalating antibodies and c) screening for the anti-C1q MAb in microtiter plates coated with C1q-intercalating MAb isotypes that are heterogeneic to the immunized animal species. We could establish 3 MAbs of IgM subclass, whose reactivity to mouse C1q was shown by ELISA techniques. One of them (RmC13C9) crossreacted with human C1q and was shown by SDS-PAGE and subsequent immunoblotting to recognize the C-chain of mouse and human C1q. The other two MAbs are directed against SDS-sensitive epitopes on mouse C1q and were, therefore, further characterized by native agarose gel electrophoresis and immunohistochemical studies.

Animals↗
PubMed1989

Biosynthesis of complement C4 messenger RNA in normal human kidney.

Complementary DNA (cDNA) probes were used to investigate the extrahepatic production of the major histocompatibility complex (MHC)-linked complement components C4, factor B and C2 in various normal human tissues. The presence of the corresponding messenger RNA (mRNA) was tested by Northern blot analysis. Complement C4 mRNA was found in liver, and with high intensity also in normal kidneys. In contrast, no C2 mRNA and only very low amounts of factor B mRNA could be detected in the kidney. Slot blot hybridization was performed to quantitate the amount of C4 mRNA, and the intensity of C4 mRNA hybridization in the kidney samples was about 25% compared with liver RNA. C4-specific transcripts were not present in isolated glomeruli but in the renal interstitium. Other human tissues, such as tonsil, spleen, thymus, brain, lung and peripheral mononuclear cells, contained no C4 mRNA. Low amounts of C4 mRNA were found in colon, thyroid gland, lymph node and breast carcinoma. The results obtained with lung, where C2 mRNA was found but no C4 mRNA, further indicate an independent, tissue-specific regulation of the class III gene expression. The results, showing that the complement C4 genes are transcribed very efficiently in normal human kidney, suggest a direct role of complement C4 in renal pathogenesis.

Adult↗
PubMed1989

Complement activation in human lymphoid germinal centres.

The presence of complement activation products has been studied in morphologically normal human lymphatic tissue from tonsil, spleen and lymph node. Newly established monoclonal antibodies (mAbs) with reactivity against the C4 cleavage fragments C4a, C4b, C4c and C4d were applied on cyrostat sections in the indirect immunoperoxidase staining technique. Irrespective of organ type, C4d activation product could be detected in germinal centres of all secondary lymphoid follicles. To substantiate this finding, the complete sequence of complement activation products was investigated by a series of mono- and polyclonal antibodies to the complement proteins C1, C2, C3, factor B, C5, C9 to C5b-9 neoantigens and to the regulatory complement proteins C4 binding protein (C4bp), factor I, factor H and properdin. Similar to C4d, all secondary follicles exhibited a strong staining reaction for C3d antigens restricted to germinal centres. At the same site, albeit with distinctly weaker intensity, components of the membrane attack complex (MAC) C5b-9 were found. The simultaneous deposition of C1, C4b and C4bp in certain germinal centres indicates that complement activation is induced via the classical pathway. Concomitant deposition of IgM suggests IgM-antigen complexes that have been trapped on follicular dendritic cells (FDC) during normal immune response as the most likely candidates for activators of the classical pathway. Our data demonstrate that human lymphoid germinal centres as important sites of immune regulation closely interrelate with the complete cascade of complement-activation products, including the membrane attack complex (MAC).

Antigen-Antibody Complex↗