Christian Klessen

Demonstration eines Alkali PAS-Effektes bei Verwendung von Perjodsäure in niedriger Konzentration

SummaryIt has been shown that an isolated KOH PAS-effect of epithelial mucosubstances in the colonic mucosae of the rat can be demonstrated also without the conventionally used preliminary oxidation/reduction step. The method is based on the use of strongly diluted periodic acid after previous treatment of tissue sections with ethanolic KOH. As the positive reacting material has been proven to be sensitive against treatment with neuraminidase the modified KOH PAS-reaction should be related to the presence of acylated sialic acid residues in mucosubstances of the colonic mucosae of the rat.ZusammenfassungEs wird über Untersuchungen berichtet, die zeigen, daß auch ohne Oxidation/Reduktion-Technik ein isolierter KOH PAS-Effekt an epithelialen Mukosubstanzen des Rattendickdarms nachweisbar ist. Die Methode beruht auf der Verwendung stark verdünnter Perjodsäure nach vorausgehender Schnittbehandlung mit äthanolischer KOH. Da sich das mit dieser modifizierten KOH PAS-Reaktion nachweisbare Material als Neuraminidasesensitiv erwies, dürfte die Reaktion auf acylierte Sialinsäurereste in bestimmten Mukosubstanzen des Rattencolon zu beziehen sein.It has been shown that an isolated KOH PAS-effect of epithelial mucosubstances in the colonic mucosae of the rat can be demonstrated also without the conventionally used preliminary oxidation/reduction step. The method is based on the use of strongly diluted periodic acid after previous treatment of tissue sections with ethanolic KOH. As the positive reacting material has been proven to be sensitive against treatment with neuraminidase the modified KOH PAS-reaction should be related to the presence of acylated sialic acid residues in mucosubstances of the colonic mucosae of the rat.

Cathepsins in basal cell carcinomas: activity, immunoreactivity and mRNA staining of cathepsins B, D, H and L

In the majority of neoplasms invasion is inevitably linked to metastasis and even small tumors have the dormant potential for metastasis. In basal cell carcinoma (BCC) invasion can be evaluated separately because local invasion but no metastasis occurs. Important proteases in invasion and metastasis are the cathepsins. Their activity and regulation has not yet been evaluated in BCC. We determined the activities, immunoreactivities and mRNA of cathepsins B, L and H in sections of different subtypes of BCC. BCC cells and peritumoral cells contained activities for cathepsins B and L. In all parts of the tumor, the reaction with cathepsin B and L substrate was stronger than in normal skin. The immunoreactive protein and mRNA for these proteases, in contrast, was elevated only occasionally in small tumor nodules. Immunoreactive protein and mRNA of cathepsin D was detected predominantly in the center of tumor nodules. Cathepsin H activities, immunoreactivities and mRNA in most BCCs were higher than in normal skin, and the reactive cells were located between and around tumor nodules, but not in the tumor nests. The results indicate that cathepsins B and L are involved in invasion of BCC cells. Cathepsin H of the peritumoral cells may either promote invasion of the tumor cells by degradation of the extracellular matrix or may reflect an elevated activity of the surrounding immunological cells. The pattern of cathepsin staining markedly differs from that observed in melanomas and may characterize locally invading non-metastatic tumors.

On testing the activity of proteases from human polymorphonuclear neutrophils on blood smears.

A cytochemical method is presented for the demonstration of proteases in human polymorphonuclear (PMN) neutrophils on fixed blood smears. This new technique is based on solubilization of proteases from PMN neutrophils by incubation with 0.25 M NaCl in borate buffer at pH 8.5 which leads to degradation of erythrocytes and plasma in a disclike zone (halo) around centrally situated PMN neutrophils, an effect that is visualized by staining smears using a modified colloidal iron reaction. Halo formation is inhibited by trypsin inhibitors of soya-bean as well as of chicken egg white mucoid and by phenylmethyl-sulfonylfluoride.

Enzymatic heterogeneity of bovine retinal pigment epithelial cells in vivo and in vitro

Abstract Background: The retinal pigment epithelium (RPE) contains a variety of enzymes that are involved in the metabolism of the neural retina. It has been shown that the photoreceptor densities display regional variations. We aimed to find out whether the enzymes in the RPE also display regional differences. Methods: Various enzymes were localized in RPE cells in situ and in cultured RPE cells. RPE cells of the entire tapetal region of adult and fetal bovine eyes were examined. The following enzymes were studied: Activities of aminopeptidases A and M, dipeptidylpeptidases I and II, and γ-glutamyltranspeptidase were localized by histochemistry. Alkaline phosphatase, dipeptidylpeptidase IV, and cathepsin B were studied by histochemistry and immunocytochemistry. In addition, activities of two enzymes were localized in one cell. Results: The distribution pattern of the enzymatic activities showed no marked regional differences. In contrast, pronounced cell-to-cell variability in the activities was detected for some of the enzymes tested. These intercellular differences were detected already in fetal retinae of early stages and persisted in RPE cultures. Conclusion: The heterogeneous distribution of enzymatic activities in RPE cells appears not to be caused exclusively by stimuli from the neural retina and from the choroid, because heterogeneity starts in the early fetal period and persists in culture.

Cytochemical investigation of neutral proteases in polymorphonuclear (PMN) neutrophils in acute inflammatory diseases

SummaryNeutral proteases can be released from PMN neutrophils in blood smears from healthy subjects by incubation with NaCl-borate buffer. The activity of the PMN proteases can be revealed by the degradation of erythrocytes and plasma within ring-shaped areas centered around each neutrophil (halo effect). During the acute stage of various inflammatory diseases (pneumonia, meningitis, cholecystitis, etc.) the activity of neutral PMN proteases is substantially reduced, as reflected by reduced halo formation. After recovery, halo formation returns to normal. Temporary lowering of neutral PMN proteases is thus one of a series of functional defects of PMN neutrophils which are detectable in the course of acute infectious diseases. These include reduced phagocytosis, altered chemotaxis and reduced bactericidal function. The cytochemical test for neutrophilic granulocyte function used in the present investigation is especially practical by comparison with the other techniques: it saves time and is simple to perform.

Zur Schnittmontierung mit Chrom-Gelatine und Formol-Gelatine bei Behandlung von Gewebsschnitten mit Perameisensäure

SummaryWhen tissue sections are treated with performic acid they usually become detached from their slides. This can be avoided, it has been found, by mounting the sections on slides coated with gelatin hardened with either chromium salts or formalin. In the mounting, it is of great importance to press the sections firmly to the slide. No wrinkles appear in sections mounted in this way after treatment with performic acid.ZusammenfassungDie Behandlung aufgezogener Gewebsschnitte mit Perameisensäure führt stets zu einem erheblichen Materialverlust durch Ablösung von Schnitten vom Objektträger. Es wird deshalb über ein einfaches Verfahren zur dauerhaften Schnittmontierung berichtet. Als Methode der Wahl erwies sich das Aufkleben der Schnitte mit Gelatine, die mit Chromsalzen oder Formalin gehärtet wurde. Wichtig ist, daß die Schnitte beim Aufkleben angedrückt werden. Derart aufgeklebte Schnitte haften auch nach Perameisensäurebehandlung zuverlässig und ohne Faltenbildung am Objektträger.

Zum Nachweis von Sialinsäure enthaltenden Glykoproteinen in den Mucoidzellen der Adenohypophyse der Ratte

SummaryBy means of the colloidal iron (CI) method strong reacting cells comprising thyrotropes, gonadotropes and an unidentified type of cells have been demonstrated in rat pituitary gland. Positive CI staining was weakened by prior treatment with neuraminidase thus indicating the presence of sialic acid residues. In view of the strong CI staining these cells may be regarded as basophilic.ZusammenfassungMit der Eisenbindungsreaktion (EBR) lassen sich in der Adenohypophyse der Ratte intensiv reagierende Zellen darstellen. Hierbei handelt es sich um gonadotrope und thyrotrope Zellen, sowie um einen nicht näher identifizierten Zelltyp. Nach Inkubation mit Neuraminidase fällt die EBR stark abgeschwächt aus. Die positive EBR ist dementsprechend im wesentlichen auf Sialinsäure zu beziehen. Die eisenpositiven Zellen sind deshalb auch vom cytochemischen Standpunkt aus als basophil zu bezeichnen.

Funktionsentwicklung der thyrotropen Zellen der Adenohypophyse des Goldhamsters

SummaryThe development of thyrotropin secreting cells in the pituitary gland of the golden hamster has been investigated in serial sections. With regard to the relationship between the beginning of thyrotropic activity in the fetal pituitary gland and onset of function in the fetal thyroidea the latter also has been investigated. Thyrotropic cells have been identified by staining with alicanblue (pH 0.3) or dichlorpseudoisocyanin after oxidation with performic acid or potassium permanganate. These reactions are based upon the relative high cystine content of TSH. Thyrotropic cells firstly are to be seen the 14th day of fetal life. These large cells of oval or angular form often have contact with the wall of a sinusoid via a long cytoplasmatic process. The grannies of the thyrotrophs show a marked tendency to be more concentrated at the cell borders giving the cell a characteristic hard outline. At the same time the thyroideaanlage shows first signs of specific function as represented by forming of follicles and production of colloid. During the further development the number of thyrotrophs increases. Form and size of the TSH-cells and the kind of granulation of these cells do not change. In the oldest stages examined (14th day p.p.) gonadotropic cells could not be identified.ZusammenfassungDie Entwicklung der thyrotropen Zellen in der Adenohypophyse des Goldhamsters wurde an Schnittserien untersucht. Zur Klärung der Frage, inwieweit zwischen dem Beginn der thyrotropen Aktivität und der Funktionsaufnahme der fetalen Schilddrüse ein zeitlicher Zusammenhang besteht, wurden die fetalen Schilddrüsen in die Untersuchung einbezogen. Ausgewertet wurden in erster Linie Präparate, die — nach Aufoxydation der für das thyrotrope Hormon charakteristischen Cystingruppen — mit Alcianblau (pH 0,3) und Dichlorpseudoisocyanin gefärbt worden waren.Thyrotrope Zellen treten in der fetalen Goldhamsterhypophyse erstmals am 14. Tag (untersuchtes Stadium: 13d 17 h) auf. Es sind große, längsoval-birnenförmige bis polygonale Zellen, welche häufig über einen sockelartig ausgezogenen Zellfortsatz mit einer Kapillarwand in Kontakt treten. Das färbbare granuläre Material ist hauptsächlich in der Zellperipherie konzentriert, wodurch es zu einer charakteristischen, scharfen Konturierung dieser Zellen kommt.Zu demselben Zeitpunkt zeigt die fetale Schilddrüsenanlage als erste Zeichen der spezifischen Funktionsaufnahme Follikel- und Kolloidbildung.Während der weiteren Entwicklung nimmt die Zahl der thyrotropen Zellen stetig zu. Zellform und -große sowie die Art der Granulation bleiben dabei nahezu unverändert. An den ältesten untersuchten Stadien (14 Tage p.p.) sind als gonadotrop zu charakterisierende Zellen noch nicht eindeutig nachweisbar.

Isolation of Bovine Retinal Pigment Epithelial Cells Using Adhesion to Agarose: Demonstration of Cellular and Regional Heterogeneity:

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.

Histochemischer Nachweis der insulären B-Zellen mit einer Perameisensäure-Eisenbindung/Sulfid-Silber Methode

SummaryB-cells of the pancreas are stained selectively with the performic acid-colloidal iron/sulfide-silver technique. A-cells and exocrine pancreas remain unstained. The rationale of the reaction depends on the oxidation of disulphide groups of (pro)insulin, or its precursors rich in cystine, to cysteine sulphonic acid groups. The free anionic groups so formed bind colloidal iron which in turn can be demonstrated by treatment with ammonium sulphide followed by “physical” development.ZusammenfassungDie B-Zellen des Inselapparates können mit einer Perameisensäure-Eisenbindung/Sulfid-Silber Methode elektiv dargestellt werden. A-Zellen und exokrines Pankreas bleiben ungefärbt. Die Reaktion verläuft, mehrstufig und beruht wahrscheinlich auf folgendem Prinzip: durch Perameisensäureoxidation werden Disulfidgruppen des (Pro)Insulins oder dessen cystinhaltiger Vorstufen in stark saure Cysteinsulfonsäuregruppen überführt, die kolloidales Eisen zu binden vermögen. Das gebundene Eisen wird mit Ammoniumsulfid und “physikalischer” Entwicklung in Form schwarzer Niederschläge dargestellt.