Peter Ruck

A New Rapid Immunohistochemical Staining Technique Using the EnVision Antibody Complex

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3′diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623–630, 2001)

Detection of elevated caspase activation and early apoptosis in liver diseases

Apoptosis has been implicated in the pathogenesis of many diseases including various forms of liver failure. The apoptotic process is essentially regulated by intracellular proteases, called caspases, which cleave several vital proteins. Despite the rapid elucidation of apoptotic signaling cascades, however, almost no information exists about the activation of caspases in situ. In the present study, a monoclonal antibody was employed which selectively recognized cleavage site-specific fragments of the caspase substrate cytokeratin-18. We demonstrate that this antibody labeled apoptotic hepatocytes in culture and, in addition, could be used to monitor caspase activation in formalin-fixed tissue biopsies. In liver sections of different liver diseases an increased number of early apoptotic cells was detected which were not found in normal tissue. Our data reveal that hepatobiliary diseases are characterized by elevated caspase activation and apoptosis, which can be specifically detected in situ by a cleavage site-specific antibody against cytokeratin-18.

Treatment of periprosthetic soft tissue infection of the groin following vascular surgical procedures by means of a polyvinyl alcohol-vacuum sponge system

Deep groin infections after prosthetic vascular surgical procedures represent a serious complication of surgical practice. Septicemia and/or erosive hemorrhage can both be consequences. In this situation, removal of the graft appears to be the only option. However, if the infection is detected early (type Szilagyi III), local treatment to eradicate the infection could serve as an alternative. Twenty‐four patients with confirmed infection of the soft tissue adjacent to the prosthetic material in the groin were treated locally by implantation of a vacuum sponge system. Duration of this treatment was 2 weeks. All patients showed excellent tissue granulation of the wound area and the microbial stains were negative at the end of therapy. In 21 patients the wound could be primarily closed after explantation of the sponge. Three patients underwent open treatment because of a skin defect. After 12 months, the wounds had healed well in all patients. Histologic evaluation revealed a physiological healing process. Deep soft tissue infections of the groin adjacent to prosthetic vascular material (type Szilagyi III) can be treated effectively and safely with the vacuum sponge system. The treatment is inexpensive, easy to perform, and the initial vascular reconstruction can be preserved. (WOUND REP REG 2003;11:104–109)

Collagenolysis in the lower uterine segment during parturition at term: correlations with stage of cervical dilatation and duration of labor.

OBJECTIVE The objective of this study was to quantify the extent of neutrophil infiltration and the concentrations of enzymes involved in collagenolysis in the lower uterine segment in relation to the degree of cervical dilatation and the duration of labor. STUDY DESIGN Biopsy specimens of the lower uterine segment were obtained from 62 women undergoing cesarean section at term. The number of extravascular neutrophils was assessed with enzyme histochemical evaluation, and the concentrations of matrix metalloproteinase-8, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 by were assessed by enzyme-linked immunosorbent assay. RESULTS The neutrophil count and the concentrations of matrix metalloproteinase-8, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 increased with increasing cervical dilatation. At >6 cm the neutrophil count and the concentrations of matrix metalloproteinase-8, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 were significantly higher than at <2 cm. An association with the duration of labor was found for the neutrophil count and the concentrations of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1. Multiple linear regression analysis showed that the degree of cervical dilatation is more closely related to the 4 laboratory parameters investigated than to the duration of labor. CONCLUSION The findings support the hypothesis that local changes (ie, collagenolysis) in the lower uterine segment unrelated to uterine activity play a crucial role in cervical dilatation at term.

Small epithelial cells in human liver cirrhosis exhibit features of hepatic stem-like cells: immunohistochemical, electron microscopic and immunoelectron microscopic findings.

Aims:  To investigate whether cells with features similar to those of the oval cells of rodents and the small epithelial cells (SEC) recently described in certain human liver diseases, i.e. hepatic progenitor cells, also occur in human liver cirrhosis.

Incidence and Pattern of Liver Involvement in Haematological Malignancies

The incidence and pattern of liver involvement in 127 liver specimens (2 biopsy and 125 autopsy specimens) from cases of acute myelogenous leukaemia (25), chronic myelogenous leukaemia (7), acute lymphatic leukaemia (5), chronic lymphatic leukaemia (9), multiple myeloma (25), low-grade non-Hodgkins lymphoma (25), high-grade non-Hodgkins lymphoma (24) and myeloproliferative diseases (7) were investigated histologically and immunohistochemically. Liver infiltration was found frequently in chronic leukaemia and myeloproliferative diseases (80-100%), acute leukaemia (60-70%) and non-Hodgkins lymphoma (50-60%), but was significantly less common in multiple myeloma (32%) than in any of the other diagnostic groups. Hepatomegaly was found in over 50% of cases in all the diagnostic groups, but was not always associated with infiltration. Diffuse, non-destructive infiltration was most common: in acute myelogenous leukaemia, both the portal triads and sinusoids were usually involved; in chronic myelogenous leukaemia, multiple myeloma and myeloproliferative diseases, infiltration was mainly sinusoidal; and in lymphatic leukaemia and non-Hodgkins lymphoma the portal triads were mainly involved. Nodular infiltration was seen in multiple myeloma and non-Hodgkins lymphoma. The primary tumours and liver infiltrates generally exhibited the same immunophenotype, although reactivity with the antibody L26 (CD20) was only found in the primary lesion in many high-grade B-cell lymphomas. Thus, liver involvement is common in haematological malignancies, but the incidence and pattern of infiltration vary amongst the different types.

Comparative analysis of the immunophenotypes of decidual and peripheral blood large granular lymphocytes and T cells during early human pregnancy.

PROBLEM: The functional role of the leukocytes in the decidua is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified.

Immunoreactivity of normal and neoplastic human tissue mast cells with macrophage-associated antibodies, with special reference to the recently developed monoclonal antibody PG-M1

There is increasing evidence in favor of the hypothesis that human tissue mast cells (MCs) are progeny of hemopoietic stem cells and are closely related to cells of the mononuclear phagocyte system. To test this hypothesis we investigated the immunoreactivity of normal/reactive MCs in 12 lymph node and tumor specimens and neoplastic MCs in 27 tissue samples from patients with various types of mastocytosis (urticaria pigmentosa, n = 13; cutaneous mastocytoma, n = 4; systemic mastocytosis, n = 6; and malignant mastocytosis, n = 4) with a panel of eight antibodies that stain macrophages or immune accessory cells and are reactive on routinely processed (paraffin-embedded, formalin-fixed) tissue. The MCs were stained by three of the macrophage-associated antibodies (namely, KP1 [CD68], Ki-M1P, and PG-M1 [CD68]), but were not stained by three other antibodies (namely, HAM56, MAC387, and LN5) or antibodies detecting immune accessory cells (DAKO-CD35 and anti-S-100 protein). While KP1 stained normal/reactive and neoplastic MCs in all the specimens investigated, Ki-M1P stained neoplastic MCs in nearly all the cases of mastocytosis but did not stain normal/reactive MCs. PG-M1 also failed to stain normal/reactive MCs and stained MCs in only approximately half of the specimens from cases of mastocytosis. Among these were most of the cases of systemic and malignant mastocytosis, but only a minority of the cases of cutaneous mastocytosis and a very few cases of urticaria pigmentosa. To summarize, (1) MCs display immunohistochemical staining properties resembling those of cells of the mononuclear phagocyte system but not those of macrophage derivatives belonging to the immune accessory cell compartment, and (2) PG-M1 and Ki-M1P are unique among the macrophage-associated antibodies investigated in that they do not stain normal/reactive MCs but exhibit preferential reactivity with the more atypical MCs in cases of systemic and malignant mastocytosis.

Congenital epulis and granular cell tumor : a histologic and immunohistochemical study

OBJECTIVES Although it is now reasonably certain that granular cell tumors derive from Schwann cells, the histogenesis of congenital epulis, which is largely isomorphic with granular cell tumor, remains unclear. A study was undertaken to compare the immunophenotype of these tumors with particular emphasis on the expression of matrix proteins and macrophage markers because such information is not available in the literature. STUDY DESIGN Four granular cell tumors and two congenital epulis were immunostained with a panel of 29 antibodies. Two congenital epulis and one granular cell tumor were investigated by electron microscopy, the latter also by immunoelectron microscopy. RESULTS Many similarities in immunostaining were found, for example, both tumor types were CD68+, Ki-M1P+, lysozyme-, vimentin+, fibronectin+, laminin+, lectin PHAE+, and lectin WGA+. However, differences were also noted, for example, granular cell tumor was always S100 protein+, but only one congenital epulis case was reactive (weak reactivity after microwave treatment), and staining with the proliferation markers anti-proliferating cell nuclear antigen and MIB 1 was found only in congenital epulis. Both tumor types exhibited pericellular and diffuse cytoplasmic staining for fibronectin and laminin. CONCLUSIONS The hypothesis that congenital epulis and granular cell tumor would exhibit similar reactivity for macrophage markers was confirmed: both were reactive with anti-CD68 and Ki-M1P and nonreactive with MAC387, anti-lysozyme, and 3A5. Intracytoplasmic staining for fibronectin and laminin, which has not been described previously in these tumors, appears to be a characteristic feature common to both tumors. This finding suggests that there could be a disturbance of synthesis and secretion of extracellular matrix proteins or a derangement of their receptor systems. This theory could be supported by the finding of intracytoplasmic CD49e-positive material in two cases.

Altered protease expression by periarterial trophoblast cells in severe early-onset preeclampsia with IUGR.

Abstract Adaptation of uteroplacental arteries in patients with early-onset preeclampsia combined with IUGR is compromized due to insufficient invasion of extravillous trophoblast cells (EVT) into the spiral artery wall. The underlying molecular mechanisms are widely unknown. We investigated expression and possible mechanisms of regulation of different matrix-metalloproteases (MMPs) by EVT in placental bed biopsies from patients with early onset preeclampsia combined with IUGR and healthy pregnant women. Expression of MMP-3 and MMP-7 by EVT was markedly reduced in preeclamptic patients, especially close to spiral arteries. In contrast to healthy pregnancies these cells strongly expressed the receptor for leukemia inhibitory factor (LIF). LIF is known to suppress MMP-expression and is produced by uterine natural killer (uNK) cells which we found to be present in higher concentrations in the placental bed of preeclamptic patients, and accumulating aside the spiral arteries. We speculate that in preeclampsia a maternal immune cell network accumulating and interfering in the placental bed leads to an altered cytokine environment, resulting in disturbed trophoblast cell function such as impaired MMP expression and reduced invasiveness.