Manfred Eulitz

Identification and quantitation of human T-cell antigen by antisera purified from antibodies crossreacting with hemopoietic progenitors and other blood cells

Anti-T cell globulin (ATCG) prepared from antihuman thymocyte serum by absorption with kidney, cells from patients with chronic lymphatic leukemias, and several lymphoblastoid cell lines was shown to react specifically with human thymus-derived lymphocytes. While high activity against thymocytes and a T-lymphoblastoid cell line could be demonstrated, ATCG remained negative against several chronic lymphatic leukemias and B-lymphoblastoid cell lines. The ATCG was used in the cytotoxic test, electronmicroscopy, and immunoautoradiography for identification of T cells in thymus, tonsils, spleen, blood, bone marrow, lymphatic leukemias, and lymphoblastoid cell lines. A comparison of these results with the ability to form spontaneous SRBC-rosettes revealed remarkable deviations between both markers in leukemias. Absorption with human brain failed to remove specific activity of ATCG. Labeling experiments by immunoautoradiography and investigations by complement fixation permitted quantitation of relative T-cell antigen concentration on different cell populations. As further evidence for specificity it could be shown that ATCG was no longer toxic for hemopoietic progenitors, whereas unabsorbed globulin reduced the number of colonyforming cells considerably.

Classification of Amyloid Syndromes from Tissue Sections Using Antibodies Against Various Amyloid Fibril Proteins: Report of 142 Cases

To identify the chemical nature of amyloid fibril proteins in tissue sections, various formalin-fixed organs from 142 patients with amyloid were investigated with a panel of antisera directed against different purified amyloid fibril proteins from representative generalized amyloid syndromes.

Amino acid sequence elucidation of human acrosin‐trypsin inhibitor (HUSI‐II) reveals that Kazal‐type proteinase inhibitors are structurally related to β‐subunits of glycoprotein hormones

The amino acid sequence of the acrosin‐trypsin inhibitor HUSI‐II from human seminal plasma is presented which unequivocally identifies HUSI‐II as being of Kazal‐type. In addition, the HUSI‐II sequence shows a striking similarity to the middle part of glycoprotein hormone β‐subunits thus revealing a hitherto unknown structural and evolutionary relationship between Kazal‐type inhibitors and glycoprotein hormones.

Suppression of acute secondary disease by heterologous anti-brain serum

SummaryAntisera raised in rabbits against murine brain contained antibodies against thymocytes and bone marrow cells of mice. While cytotoxic tests revealed only low titer antibodies against B-cells, complement fixation, plaque-forming and colony-forming tests demonstrated little difference in antibody activity against T-or B-cells. Absorption of anti-brain serum with liver and plasmocytoma cells led to residual specific anti T-cell activity. Incubation of these absorbed anti-brain sera with parental spleen cells before transplantation into lethally irradiated H2incompatible F1 hybrids suppressed acute secondary disease. While all the controls died of acute secondary disease, about 90% of the recipients of spleen cells treated with anti-brain serum survived day 100 post transplantation, without showing any wasting.ZusammenfassungIn Kaninchen produzierte, gegen Mäusehirn gerichtete Antiseren enthalten Antikörper gegen Thymozyten und Knochenmarkzellen der Maus. Während in Zytotoxizitätstesten nur Antikörper mit niedrigen Titern gegen B-Zellen gefunden werden, zeigen Komplementbindungsreaktion,Jerne-Plaque-Test und CFU-Test wenig Unterschied in der Antikörperaktivität gegen T-oder B-Zellen. Durch Absorption von Anti-Hirn-Serum mit Lebergewebe und Plasmozytomzellen erhält man ein Serum, das nur noch spezifische T-Zellaktivität zeigt. Eine Inkubation von parentalen Milzzellen mit absorbiertem Anti-Hirn-Serum vor Transplantation auf letal bestrahlte F1-Hybride unterdrückt die akute Sekundärkrankheit. Während alle Kontrolltiere an Sekundärkrankheit starben, überlebten ungefähr 90% der Empfänger, die mit Anti-Hirn-Serum inkubierte Milzzellen erhalten hatten, den 100. Tag nach Transplantation ohne Zeichen einer Erkrankung.

N-Terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

SummaryIsolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant lowmolecular-weight polypeptide (approximately 3.5 kDa, designated ASc2 NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide α-hANP for the first 12 residues determined.

Demonstration of the Fc-receptor of blood cells by soluble peroxidase-anti-peroxidase (PAP) complexes

ZusammenfassungDer Fc-Rezeptor von normalen menschlichen Leukozyten, CLL-Zellen und hämatopoetischen Zell-Linien wurde mittels löslicher Peroxidase-anti-Peroxidase (PAP)-Komplexe untersucht. Etwa 9% normaler Lymphozyten zeigten eine starke, nahezu ununterbrochene Membranmarkierung. Einige dieser stark markierten Lymphozyten waren durch eine gemeinsame, auffällige Feinstruktur gekennzeichnet. PAP-positive Monozyten lagen in der Größenordnung von 25%, Neutrophile fast 100%, Eosinophile 0%, CLL-Zellen 10%. Markierte Membrananteile wurden von Monozyten ins Zellinnere aufgenommen. Die lymphoide Zell-Linie Daudi war kaum mit PAP zu markieren, die myeloide Zell-Linie K 562 war zu 75% stark positiv. Die PAP-Markierung war durch Vorinkubation mit Trypsin oder Neuraminidase nicht zu beeinflussen; Kontrollen mit PAP-F(ab)2 blieben vollständig negativ. Die Ergebnisse der PAP-Markierung stimmten mit EA-Rosetten und agg-Ig nicht immer überein.SummaryThe Fc-receptor of normal human leukocytes, of CLL-cells, and of hematopoietic cell lines was demonstrated with soluble peroxidase-anti-peroxidase (PAP) complexes. In about 9% of normal lymphocytes an almost continuous, strong labeling of the cell membrane was established. Some of these lymphocytes were characterized by a peculiar uniform fine structure. The percentage of PAP-labeled monocytes was in the range of 25%, neutrophils nearly 100%, eosinophils 0%, CLL-cells 10%. Labeled portions of the membrane were interiorized from monocytes. The lymphoid cell-line Daudi established from a Burkitts lymphoma appeared almost negative, the cell line K 562 established from a myeloid leukemia in 75% of the cells strongly positive. PAP-labeling was not influenced by preincubation with trypsine or with neuraminidase; it was negative when PAP-F(ab)2 was used. Results of PAP-labeling were not always in agreement with EA-rosettes or with agg-Ig.

Consequences of Antigen Self-Presentation by Tumor-Specific Cytotoxic T Cells

CD8-positive cytotoxic T cells (CTL) recognize antigenic peptides in combination with major histocompatibility complex (MHC) class I molecules on the surface of syngeneic antigen presenting cells (APC). In the present paper we show that cells from tumor antigen-specific CTL clones present their cognate antigenic peptide to other CTL from the same clone. Inter-CTL peptide presentation resulted in activation of the cells of one CTL clone to MHC-unrestricted lysis of bystander cells. In contrast to the behaviour of this clone, another CTL clone did not lyse bystander cells after incubation with the cognate peptide, but was activated to self-destruction. The human herpes virus Epstein-Barr virus is involved in the pathogenesis of a broad spectrum of human neoplasias. Using freshly established non-clonal T cells with specificity for a peptide derived from an Epstein-Barr virus encoded antigen we found again lysis of MHC mismatched bystander cells as a consequence of inter-CTL peptide presentation, indicating that bystander lysis following antigen self-presentation is not a phenomenon restricted to long-term in vitro cultured T cell clones. The potential implications for immunosurveillance against cancer and for tumor escape mechanisms are discussed.

ANTILYMPHOCYTIC ANTIBODIES AND MARROW TRANSPLANTATION: VI ABSENCE OF IMMUNOSUPPRESSION IN VIVO AFTER INJECTION OF MONOCLONAL ANTIBODIES BLOCKING GRAFT-VERSUS-HOST REACTIONS AND HUMORAL ANTIBODY FORMATION IN VITRO

The in vivo and in vitro effectiveness of several monoclonal antimouse T and B cell antibodies, of anti-Th-1 and of Iak serum, as well as of ATG were compared. The parameters were prolongation of skin graft survival, prevention of graft-versus-host disease (GVHD), antibody and primary and secondary plaque formation against sheep red blood cells (RBCs), and T cell depletion of lymphoid tissues. In general, in vitro effectiveness of the monoclonal antibodies exceeded their in vivo effectiveness. Skin graft survival was prolonged by ATG, but not by monoclonal anti-T, or anti-T plus anti-B antibody. GVHD was prevented by in vitro incubation of donor bone marrow with monoclonal anti-Th-1, but in vivo treatment of marrow donors was ineffective. Treatment with ATG was successful. Anti Iak antibody blocked plaque formation by spleen cells incubated with sheep RBCs, but had no effect on secondary plaque formation when given in vivo. Neither was there any in vivo effect of anti-Iak or anti-Th-1 on antisheep RBC agglutinin formation. ATG was effective in both of these assays, although its cytotoxic and complement-fixing titer did not exceed that of anti-Th-1 or anti-Iak. Although anti-Th-1 was cleared more rapidly from the serum of mice expressing the corresponding Th-1 alloantigen, than from mice with the noncorresponding alloantigen and although anti-Th-1 was shown to bind to the T cell areas of the lymphoid tissue, it did not—unlike ATG—deplete these areas of T cells. Possible reasons for the difference in effectiveness of in vitro and in vivo application of these monoclonal antibodies are discussed.

Idiopathic amyloidosis in the stone marten (Martes foina): Identification of amyloid fibril proteins in tissue sections using the immunoperoxidase technique

ZusammenfassungAmyloidfibrillen-Proteine, isoliert aus der Milz eines Steinmarders (Martes foina, Exleben) mit idiopathischer Amyloidose, zeigen Ähnlichkeit mit dem Amyloid-A-Protein. Ein Antiserum gegen diese Proteine kann die hier untersuchten Amyloid-Ablagerungen mit Hilfe der Immunperoxidase-Technik an formalin-fixierten und paraffin-eingebetteten Gewebeschnitten identifizieren.SummaryAmyloid fibril proteins isolated from a spleen of a wild stone marten (Martes foina, Exleben) with idiopathic amyloidosis show resemblance to protein AA by amino acid analysis. An antiserum directed against these proteins can be used to identify the martens amyloid in formalin-fixed tissue paraffin-embedded sections using the immunoperoxidase method.

Chemical Analysis of Beta2-Microglobulin Derived Amyloid in Patients on Long-Term Hemodialysis

Amyloid deposits of a patient (KRO) who underwent peritoneal and longterm hemodialysis were chemically, immunochemically and immunohistochemically investigated. The amyloid fibrils from bone marrow were concentrated, dissolved in 80% and separated in 60% formic acid by HPLC according to size. Several different protein peaks were recovered, having molecular weights of approximately 24, 18, 12, and 7–10 kd. The largest fraction represented the void volume peak. All fractions reacted strongly with an antiserum directed against β2m+). N-terminal amino acid sequence analysis showed three different molecules, all derived from β2m: one with the intact N-terminus, a second and a third with either isoleucine in position 6 or serine in position 11 as N-terminus, respectively.