Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets.

In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft. With improved isolation methods based on the original procedure published by Ricordi et al. yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.

CD143 in the development of atherosclerosis.

The expression of CD143 (angiotensin-I-converting enzyme, ACE) in cardiovascular diseases may be an important determinant of local angiotensin and kinin concentrations. Much of the experimental and clinical evidence suggests a crucial role for Ang II in fibrogenesis and the development of atherosclerosis. Therefore, we have studied the distribution of CD143 in atherosclerotic and non-atherosclerotic segments isolated from different parts of the human vascular tree, including aorta and coronary, carotid, brachial, renal, iliac and femoral arteries, and staged according to the AHA. Two hundred and thirty native and formalin-fixed specimens of 80 patients were analysed by sensitive APAAP-technique using ten different monoclonal and polyclonal antibodies to human CD143 and several controls. In non-atherosclerotic segments or intimal thickening, CD143 was found almost restricted to the endothelial cells of adventitial arterioles and small muscular arteries. In contrast, a striking accumulation of CD143 was detected in all early and advanced atherosclerotic lesions. This de-novo occurrence of CD143 within the intimal vascular wall was caused by spindle-shaped subendothelial cells with macrophagic/histocytic features, activated macrophages and foam cells. In addition, advanced lesions of atherosclerosis showed a marked neo-expression of CD143 in newly formed intimal microvessels. Hypocellular fibrotic plaques depleted in microvessels and macrophages showed only little CD143. The de-novo occurrence of CD143 was dependent on the stage of atherosclerosis but not on its particular localisation within the vascular system. This early and obligatory CD143 expression at an unusual vascular site may contribute to unusual tissue levels of angiotensins as indicated by co-localisation of immunoreactive Ang II. Thus, it may be an important pathogenetic step in the development of atherosclerosis and an established target for pharmacological prevention.

The expression of angiotensin-I converting enzyme in human atherosclerotic plaques is not related to the deletion/insertion polymorphism but to the risk of restenosis after coronary interventions.

Plasma and tissue concentrations of the angiotensin-I converting enzyme (ACE) have been shown to be associated with the ACE insertion/deletion (I/D) polymorphism. The purpose of this study was to examine the relation of ACE levels in atherosclerotic plaques to the ACE I/D polymorphism and to restenosis after balloon angioplasty and directional atherectomy (DCA). The study included 104 patients who underwent DCA and received angiographic follow-up at 12 to 18 months. The amount of ACE protein in various morphologically defined plaque components (fibrous, atheromatous, and complicated lesions) of the atherectomy specimens was determined by qualitative and semiquantitative immunohistochemistry. ACE levels were related to the ACE genotype, to plaque morphology and to the risk of restenosis. Sequential staining revealed that pathologic ACE overexpression of the atherosclerotic lesions occurred in intimal smooth muscle cells, fibrocytes/fibroblasts and macrophage/foam cells. The ACE content of the whole plaques and of the single plaque components was not associated with the I/D polymorphism, but with restenosis after coronary interventions. In addition, ACE levels in the atherosclerotic lesions correlated with the severity of vessel wall damage. The ACE phenotype might serve as an indicator for the risk of restenosis after coronary interventions.

Somatic isoform of angiotensin I-converting enzyme in the pathology of testicular germ cell tumors.

Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown in intratubular germ cell neoplasms (IGCN) and invasive germ cell tumors (GCT), suggesting the somatic isoform (sACE) as a characteristic component of neoplastic germ cells. We analyzed the distribution of sACE in 159 testicular GCT, including 87 IGCN. sACE protein was determined by immunohistochemistry (MAb CG2) on routinely formalin-fixed and paraffin-embedded tissue sections, supplemented by mRNA expression analysis using in situ hybridization. These data were compared with those obtained by germ cell/placental alkaline phosphatases (PIAP; MAbs PL8-F6 and 8A9) employing an uniform score system for the evaluation of immunoreactivity (IRS; possible values from 0 to 12). Expression of sACE and PIAP was found in all 87 analyzed IGCN (IRS > 4, median IRS of 12). Heterogeneous staining patterns were not related to the type of adjacent GCT but correlated with low expression in adjacent seminomas (P =.032 for sACE; P =.005 for PIAP). Both sACE and PIAP often showed a decreased and more heterogeneous but still moderate expression in 91 classic seminomas (median IRS of 8) and were completely absent in tumor cells of spermatocytic seminomas. Despite all similarities, we found sACE and PIAP differently regulated during GCT progression. This was documented by a well-preserved expression of either sACE or PIAP or both in all classic seminomas, low PIAP immunoreactivity in metastasis of seminomas, and completely diverging expression patterns in nonseminomatous GCT. Our findings underline the close molecular relationship between IGCN and seminoma, and suggest sACE as an appropriate marker for seminomatous differentiated tumors. HUM PATHOL 31:1466-1476.

Thromboangiitis obliterans: classic and new morphological features.

The clinical and pathological concept of thromboangiitis obliterans (TAO, Buerger's disease) is still controversial. While the clinical criteria of TAO are relatively well defined, the etiology is unknown and its diagnosis based on pathology is confusing, since there is no consensus on the precise pathological criteria for TAO. To investigate the morphological features that differentiate TAO from arteriosclerosis obliterans (ASO) or thromboembolism, and to clarify the morphological independence of TAO, we studied 94 amputated specimens of lower extremities, including 31 specimens from patients with a clinical diagnosis of TAO and 31 autopsy specimens as control cases. It was revealed that most of the classic morphological features described by Buerger and others are not helpful when considered independently in the differential diagnosis, except for intact internal elastic lamina. In addition, findings of intimal inflammation, intact media and absence of medial calcification were demonstrated to be common in both TAO and thromboembolism. Statistical analysis in the present study, the most comprehensive thus far, showed that novel findings of onion-like-shaped recanalizing vessels in the occluded arteries, adventitial fibrosis without medial fibrosis, swelling of the endothelium of the vasa vasorum and edema beneath the external elastic lamina were characteristic of TAO and would be helpful in a differential diagnosis. When a combination of these morphological features is present, diagnosis of a presumed overlap of TAO and ASO in the same site of the vessel concerned is possible. Furthermore, comparison of statistical evaluations based on morphological features performed in various diagnostic groups implies that the clinical diagnosis of TAO is currently underestimated because the results of the analysis of morphological features of specimens in which TAO was suspected or specimens selected on the basis of a broad and nonspecific definition of TAO were surprisingly similar to the results in strictly defined TAO cases. Our findings suggest that injury and regeneration of minute vessels such as recanalizing vessels and vasa vasorum play a part in the pathogenesis of TAO.

[Actinomycosis of the colon as a rare differential diagnosis of colonic carcinoma]. / Die Actinomykose des Colon als seltene Differentialdiagnose des Coloncarcinoms.

INTRODUCTION: Abdominal actinomycosis is an uncommon disease. Nevertheless it should be considered in case of unclear tumor-like abdominal masses. METHODS: We report a case of a 49-year-old patient with an intrauterine device. The patient was submitted with a solid and painful tumor in the upper abdomen. After sonography, computerized tomography, gastroduodenoscopy and colonoscopy the preoperative presumptive diagnosis was a carcinoma of the transvers colon invading the abdominal wall. Pathological examination after a right hemicolectomy surprisingly revealed an actinomycosis. RESULTS: Based on this case diagnostic tools and therapeutic options of actinomycosis of the colon are discussed. CONCLUSIONS: This case illustrates the importance to consider the possibility of actinomycosis when finding an unclear abdominal mass. After a surgical excision an abdominal actinomycosis requires antibiotic therapy.

[Embryonal germ cells and germ cell tumors]. / Embryonale Keimzellen und Keimzelltumoren.

Testicular germ cell tumors comprise of group of pluripotent tumors including seminomas and nonseminomas, arise from intratubular germ cell neoplasia and originate from the primordial germ cells/ gonocytes. Many well characterized markers of embryonic stem cells including CD9, PODXL and centromere-specific histone-H3-like protein CENPA are consistently expressed in TGCTs. In embryonic stem cells, pluripotency and self renewal capacities are provided by a network of OCT3/4, NANOG and SOX2. In testicular germ cell tumors, pluripotency genes OCT3/4 und NANOG are upregulated both, in seminomas and non-seminomas, while SOX2 is differentially upregulated in embryonal carcinomas only. Similar to embryonic stem cells, most histological elements of type II GCTs are sensitive to chemotherapy and irradiation. Furthermore, all invasive TGCTs show a consistent gain of the short arm of chromosome 12, as found in ES cells upon extensive in vitro culturing. Moreover, the genetic constitution of testicular germ cell tumors can also be linked to characteristics of embryonic stem cells, likely related to their specific inability to repair DNA damage and their high sensitivity to apoptotic cell death. In conclusion, testicular germ cell tumors represent embryonic cancers found in adults. Both the seminomas and nonseminomas have their specific population of stem cells representative of the primordial germ cells/gonocytes and for embryonic stem cells, respectively.

[Expression of angiotensin I converting enzyme in pulmonary hypertension]. / Expression des Angiotensin converting enzyme (ACE) bei pulmonaler Hypertonie.

We studied angiotensin I converting enzyme (ACE) expression in lung tissue from patients with different forms of pulmonary hypertension (PH) in comparison with that from morphologically normal lung specimens. ACE antigen expression was analysed by immunohistochemistry in morphologically normal lung tissue from 33 patients (19 males, 32-77 years; 14 females, 34-93 years) and compared to that in specimens from 94 patients (67 males, 30-97 years; 27 females: 27-90 years) with different clinically proven forms of PH (according to Venedig classification). Type specific vessel expression pattern as described for normal lung tissue was generally intensified in arteries, arterioles and capillaries of the lung specimens with PH. Specimens with PH due to left heart disease and chronic obstructive pulmonary disease (COPD) showed only very weak or no augmented arterial ACE expression, while PH due to collagenoses or interstitial lung disease showed significantly higher ACE expression. In human PH there is--comparable to animal models--a raised ACE expression in pulmonary lung vessels, with differences between the various forms of PH. These differences in ACE expression may be relevant for subtly differentiated therapeutic anti-ACE therapy regimes.

Metastasis of renal carcinoma colliding with glioblastoma. Carcinoma to glioma: an event only rarely detected.

This report presents the case of a 74-year-old woman who was simultaneously affected by two highly malignant neoplasms, a metastasizing renal cell carcinoma and a glioblastoma with sarcomatous component. Leptomeningeal metastasis of renal carcinoma is shown to invade the glioblastoma at its margin. Especially in gliomas, "cancer to cancer" phenomenomal are only rarely documented. Support by immunohistochemical data may prove those events to be more frequent than assumed.

Reappraisal of thromboangiitis obliterans--a pathological contribution.

Thromboangiitis obliterans (TAO), the Winiwarter-Buerger disease, is a vasoocclusive disease of unknown etiology which typically affects medium-sized extremital vessels of young male smokers. While the diagnosis of TAO is largely based on patients' presentation and clinical criteria, pathological substrates have been poorly defined and repeatedly disputed. Comparing the histology and immunohistochemistry of TAO especially with those of arteriosclerosis obliterans (ASO) and thromboembolism in two larger studies, we recently identified several features with significant meaning for the differential diagnosis. The unique tissue appearance of TAO is in favor of a general disorder of minute vessels indicating periarteritis rather than pure endarteritis, whereas an inflammatory reaction directed to the internal elastic lamina corresponds with the severity of disease. Since TAO or its syndromic equivalent is probably more common than currently diagnosed, the presented pathological criteria may help to identify latent cases, the overlap with ASO, and untypical organ involvement of TAO.

Unexpected immunoreactivities of intermediate filament antibodies in human brain and brain tumors.

Immunoreactivities of 35 different monoclonal antibodies (MAbs) that detect intermediate filaments were studied systematically on serial cryostat sections of 14 well-defined human gliomas (five astrocytomas, three oligodendrogliomas, six glioblastomas) and on normal brain. Glial fibrillary acidic protein (GFAP), vimentin, desmin, neurofilaments, and broad-specificity keratin MAbs, as well as MAbs that recognize several or only single keratin polypeptides, were used. Unexpected reactivities were surprisingly frequent. As these may lead to diagnostic confusion and misinterpretation on this material, the authors investigated these phenomena more thoroughly. Four major sources of artifactual staining were found: 1) positive staining attributable to the rabbit gamma G immunoglobulins used in the alkaline phosphatase anti-alkaline phosphatase technique; 2) certain desmin and keratin MAbs cross-reacted with astrocytic glia and with other brain-specific epitopes; 3) technical difficulties; 4) some MAbs directed against neurofilaments and keratins showed unexpected reactivities only on individual anaplastic gliomas. The implications of these findings for intermediate filament typing of neuropathologic material are discussed.

[CD 143 expression in benign and malignant vascular tumors]. / CD 143 Expression in benignen und malignen vaskulären Tumoren.

The somatic form of CD 143 (angiotensin-I-converting enzyme) has been detected in many endothelial cells of the human body. Recently, we could identify local CD 143 overexpression in various pathological conditions e.g. in capillary endothelial proliferations at the border of myocardial infarctions and in coronary atherosclerotic plaques. Using monoclonal antibodies against CD 143, CD 31 and CD 34 we evaluated, whether CD 143 can serve as a marker of endothelial differentiation in vascular/perivascular tumors. 39 benign and malignant tumors, formalin-fixed, paraffin-embedded were investigated by APAAP immunohistochemistry using epitope retrieval techniques. Expression patterns were separately qualitatively and semiquantitatively analysed. Positive detection of epitopes was registered if more than 10% of the tumor cells were stained. CD 143 was detected in the majority of benign and malignant vascular tumors. All benign and malignant haemangioperizytomas were CD 143 negative. None of the markers detected all haemangioendotheliomas or all angiosarcomas, but the combination of CD 143 and CD 31 turned out to be positive in all haemangioendotheliomas and angiosarcomas. We conclude that most of the benign and malignant endothelial tumors express CD 143. The best discrimination of intermediate grade and malignant endothelial tumors can be achieved by analysis of the CD 143 and CD 31 co-expression.

Angiotensin-converting enzyme (CD143) in neoplastic germ cells.

Angiotensin I-converting enzyme (ACE, CD143, Kininase II, EC 3.4.15.1) occurs in two isoforms; whereas the somatic isoform (sACE) appears in certain endothelial cells and some other cell types, the testicular isoform (tACE) was found in humans and various mammals only during spermiogenesis. An expression of ACE was reported formerly in some human seminomas, but its isoform type, cellular distribution, and pathogenetic meaning are not known. Therefore we analyzed normal human testes, 22 different testicular tumors, and 23 fetal and postnatal tissues of different stages of testicular development. By reverse transcriptase-polymerase chain reaction, ACE mRNA isoforms were assessed in homogenized tissue sections and in germ cells selectively isolated by laser-assisted cell picking. Immunohistochemistry was performed on consecutive sections using monoclonal antibodies specific to the human somatic isoform or both, sACE and tACE. In adult men, tACE was detectable in spermatids and spermatozoa, but normal spermatogonia and spermatocytes were not found to express ACE in any isoform. By contrast, both mRNA and protein of sACE were detectable in the cells of intratubular germ cell neoplasm, seminomas, and other testicular tumor types. Because sACE was also found in fetal germ cells, our findings point to profound differences in the regulation of ACE expression in fetal, mature adult, and neoplastic germ cells. They are in agreement with the concept that neoplastic germ cells phenotypically reflect an embryonic stage of cellular differentiation. Laser-assisted cell picking proved to be a reliable method to investigate differently regulated mRNA of cells which reside in close neighborhood within complex tissues.

Isoforms of angiotensin I-converting enzyme in the development and differentiation of human testis and epididymis.

Angiotensin I-converting enzyme (ACE; CD143, Kininase II, EC 3.4.15.1) is known to be crucial for male fertility in animal models. We therefore studied its testicular (tACE) and somatic (sACE) isoforms in foetal and adult human testis and epididymis using monoclonal antibodies and cRNA probes. During spermatogenesis, tACE was found only in differentiating germ cells and was the only isoform within the seminiferous tubules of adult men. Although tACE mRNA was present in spermatocytes, tACE protein was initially found in post-meiotic step 3 spermatids and increased markedly during further differentiation. The enzyme was strictly confined to the adluminal membrane site of elongating spermatids and was localized at the neck and midpiece region of released and ejaculated spermatozoa. In contrast, sACE was expressed heterogeneously in Leydig cells and endothelial cells of the testicular interstitium, and homogeneously along the luminal surface of epithelial cells lining the ductuli efferents, corpus and cauda of epididymis, and vas deferens. The cell- and site-restricted pattern of sACE corresponded to that found in foetal tissues except an additional and transient expression of sACE in foetal germ cells and foetal Sertoli cells. Our study documents for the first time in humans the regulation and unique cellular distribution of ACE isoforms during the ontogenesis of the lower male genital tract.

Monoclonal antibodies to denatured human ACE (CD 143), broad species specificity, reactivity on paraffin sections, and detection of subtle conformational changes in the C-terminal domain of ACE.

Two new mouse monoclonal antibodies (mAbs) were generated to denatured human angiotensin-converting enzyme (ACE, CD143). The clones 2E2 and 3C5, each of the IgG1 kappa chain isotype, detect ACE with high sensitivity, respectively, at 20 ng and 2 ng of protein per lane in Western blotting. They both recognize different epitopes on the C-domain of ACE located between amino acid residues 740 and 992. In formalin-fixed and paraffin-embedded human tissues, immunohistochemistry revealed all known expression sites of ACE, e.g. the epithelial brush borders of proximal kidney tubules, epithelial cells of epididymis, endothelial cells, activated macrophages as well as germ cells during spermatogenesis. In contrast to other mAbs to denatured human ACE, mAbs 2E2 and 3C5 demonstrate cross-reactivity with a broad spectrum of animal species such as monkey, rat, rabbit, cattle, dog, cat, and guinea pig. In addition, mAb 2E2 recognized mouse ACE in Western blotting and on paraffin sections. Our findings suggest that mAbs 2E2 and 3C5 are useful for identifying even subtle changes in ACE conformation resulting from denaturation. These mAbs are also sensitive tools for the detection of minimal amounts of ACE in biological fluids and tissues using proteomics approaches. Their reactivity in routinely processed tissues of various species may prove useful for correlation of ACE expression in animal models to human diseases.

[Angiotensin converting enzyme (ACE, CD143) in the regular pulmonary vasculature]. / Angiotensin-I-converting-Enzym (ACE, CD143) in regelrechtem humanem Lungengewebe.

Angiotensin I converting enzyme (ACE, CD143) is an endothelial transmembrane Zn2+-dipeptidylpeptidase. By formation of angiotensin II and degrading bradykinin it acts as a vasoconstrictor. We examined endothelial ACE expression in human pulmonary vessels in specimens from 20 female and 19 male patients (age: 34-76 years) by immunohistochemistry. In all specimens, capillary endothelial cells showed the strongest expression, followed by those in arterioles and arteries. Venules and veins showed next to no staining. The differences in staining intensities were significant ( P<0.001). Sex affected neither the expression intensity nor the expression pattern. Summarizing, we demonstrate the existence of a vessel-type specific ACE expression pattern for pulmonary vessels. The nearly exclusive endothelial ACE expression in capillaries and arterial vessels points to ACE as an immunohistochemical marker for these vessels in normal lung tissue.

[Violations of chemotherapy protocols for ovarian cancer--reasons and consequences]. / Verletzungen des Therapieplans und des Chemotherapieprotokolls beim Ovarialkarzinom--Gründe und Konsequenzen.

In times when colony-stimulating factors were not available, delays of treatment or dose reductions were necessary, to assure that chemotherapy could be safely administered. In a retrospective analysis the effects of chemotherapy protocol violations on the survival of patients with ovarian cancer was evaluated. The serial courses of leukocyte counts were often the determinants for a protocol adequate chemotherapy in contrary to the thrombocytes but the serial platelet counts had no influence on protocol violations. Only time-related protocol violations have been found in 7.6% of the cases. There seems to be no apparent influence on patients' survival. However, accomplishment of treatment schedules, which may be regarded as a reaction towards unsatisfactory tumour response, at the initial visit alone or in combination with time-related protocol violations as well as tumour stage, course of tumour markers (CA 125) had a strong impact on survival while higher dosage levels produces only a trend towards improved survival. The use of growth factors will probably reduce the percentage of protocol violations caused by neutropenia, but it is questionable if it will reduce mortality due to tumour progression.

MUC1 in normal and impaired spermatogenesis.

The MUC1 mucin [also known as episialin, epithelial membrane antigen (EMA) or polymorphic epithelial mucin (PEM)] is a component of the mucosal glycocalyx, contributing to anti-adhesive and protective cell functions. MUC1 has been shown in a variety of epithelial cell types in the reproductive tracts of males and females, but this is the first report of its expression in human testis and non-epithelial cells of the germ cell lineage. Analysing 65 testes with normal or impaired spermatogenesis, we identified MUC1 protein in maturing germ cells by immunohistochemistry using the monoclonal antibodies HMFG1, HMFG2 and SM3 binding to different glycosylation variants. MUC1 expression was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis on tissue extracts of human testis, and RT-PCR of selected germ cells after UV laser-assisted cell picking. MUC1 glycosylation variants were selectively distributed during normal spermatogenesis. Whereas HMFG1 labelled certain groups of pachytene spermatocytes, HMFG2 labelled only spermatids. Low glycosylated forms of MUC1 mucin, recognized by SM3, were not found. In contrast to its weak expression during normal spermatogenesis, the HMFG1 glycosylation variant accumulated markedly in all spermatocytes showing abnormal or arrested maturation. These results suggest a variable glycosylation of MUC1 mucin in differentiating germ cells, which is aberrant in pathological conditions.