Vera Samoilova

Vascular smooth muscle and nitric oxide synthase

The concept of endothelium‐derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP‐dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS isoforms depending on the blood vessel type. These findings suggest an alternative mechanism by which local NOS expression may modulate vascular functions in an endothelium‐independent manner.—Buchwalow, I. B., Podzuweit, T., Böcker, W., Samoilova, V. E., Thomas, S., Wellner, M., Baba, H. A., Robenek, H., Schnekenburger, J., Lerch, M. M. Vascular smooth muscle and nitric oxide synthase. FASEB J. 16, 500–508 (2002)

Non-specific binding of antibodies in immunohistochemistry: fallacies and facts

The current protocols for blocking background staining in immunohistochemistry are based on conflicting reports. Background staining is thought to occur as a result of either non-specific antibody (Ab) binding to endogenous Fc receptors (FcRs) or a combination of ionic and hydrophobic interactions. In this study, cell and tissue samples were processed according to routine protocols either with or without a blocking step (goat serum or BSA). Surprisingly, no Abs in samples processed without a blocking step showed any propensity for non-specific binding leading to background staining, implying that endogenous FcRs do not retain their ability to bind the Fc portion of Abs after standard fixation. Likewise, we did not find any non-specific Ab binding ascribable to either ionic or hydrophobic interactions. We determined that traditionally used protein blocking steps are unnecessary in the immunostaining of routinely fixed cell and tissue samples.

Differentiation and histogenesis of syringomatous tumour of the nipple and low-grade adenosquamous carcinoma: evidence for a common origin.

Syringomatous tumour of the nipple and low‐grade adenosquamous carcinoma (LGAdSC) of the breast are regarded as distinct entities. To clarify the nature of these two lesions, we compared the expression of different lineage/differentiation markers in 12 syringomatous tumours of the nipple, nine LGAdSCs, and normal breast epithelium.

K5/K14-positive cells contribute to salivary gland-like breast tumors with myoepithelial differentiation

Salivary gland-like tumors of the breast show a great variety of architectural patterns and cellular differentiations such as glandular, myoepithelial, squamous, and even mesenchymal phenotypes. However, currently little is known about the evolution and cellular differentiation of these tumors. For that reason, we performed an in situ triple immunofluorescence lineage/differentiation tracing (isTILT) and qRT-PCR study of basal (K5/K14), glandular (K7/K8/18), and epidermal-specific squamous (K10) keratins, p63, and smooth muscle actin (SMA; myoepithelial marker) with the aim to construct and trace different cell lineages and define their cellular hierarchy in tumors with myoepithelial differentiation. isTILT analysis of a series of 28 breast, salivary, and lacrimal gland tumors, including pleomorphic adenomas (n=8), epithelial-myoepithelial tumors (n=9), and adenoid cystic carcinomas (n=11) revealed that all tumor types contained K5/K14-positive progenitor cells in varying frequencies from a few percent up to 15%. These K5/K14-positive tumor cells were found to differentiate to glandular- (K8/18-positive) and myoepithelial-lineage (SMA-positive)-specific cells and were also shown to generate various heterologeous cell differentiations such as squamous and mesenchymal progenies. p63 was co-expressed with K5/K14 in basal-like progenitor cells, myoepithelial, and squamous cells but not in glandular cells. Our results show that the corresponding counterpart tumors of breast and salivary/lacrimal glands have identical cellular compositions. Taken together, our isTILT and RNA-expression data indicate that look-alike tumors of the breast represent a special subgroup of basal-type tumors with benign or usually low malignant potential.

L-arginine-NO-cGMP signalling pathway in pancreatitis

The role of nitric oxide (NO) in the human pancreas and in pancreatitis still remains controversial. Furthermore, conflicting conclusions have been reached by different laboratories about the localization of the NO-generating enzyme (NO synthase, NOS) in the pancreas. Here, we investigated the co-expression of NOS with enzymes involved in regulation of NO signalling in the normal human pancreas and in pancreatitis. We found that the whole NO signalling machinery was up-regulated in pancreatitis, especially within the exocrine compartment. Furthermore, the exocrine parenchymal cells revealed higher levels of oxidative stress markers, nitrotyrosine and 8-hydroxyguanosine, in pancreatitis, which reflects the exceptional susceptibility of the exocrine parenchyma to oxidative stress. This study provides a direct link between oxidative stress and the enzymatic control of the NO bioavailability at the cellular level and endows with further insight into fundamental mechanisms underlying pancreatic disorders associated with disruptions in the L-arginine-NO-cGMP signalling enzyme cascade.

Squamous/epidermoid differentiation in normal breast and salivary gland tissues and their corresponding tumors originate from p63/K5/14-positive progenitor cells

A small group of tumors of breast and salivary glands contains squamous/epidermoid elements as a constitutive feature (e.g., squamous carcinoma, syringomatous tumors, and mucoepidermoid carcinoma). Other tumors (e.g., pleomorphic adenoma, adenomyoepithelial tumors, and adenoid cystic carcinoma) may show occasionally squamous differentiation. Furthermore, squamous metaplasia may be observed in non-neoplastic breast and salivary tissues. However, the histogenesis of these squamous differentiations is far from being understood. Based on our earlier in situ triple immunofluorescence and quantitative reverse transcription (RT)-PCR experiments for basal keratins K5/14 and p63 as well as for glandular keratins (K7/K8/18), squamous keratins (K10 and K13), and myoepithelial lineage markers (smooth muscle actin, SMA), we here traced the squamous/epidermoid differentiation lineage of 60 tumors of the breast and/or salivary glands, cultured tumor cells of 2 tumors, and of 7 squamous metaplasias of non-neoplastic breast and salivary tissues. Our results indicate that both the neoplastic lesions as well as the non-neoplastic squamous metaplasia contain p63/K5/14+ cells that differentiate toward K10/13+ squamous cells. Thus, cells with squamous/epidermoid differentiation undergo a transition from its original p63/K5/14+ precursor state to K10/13+ squamous lineage state, which can be pictured by triple-immunofluorescence experiments. Given the immunophenotypic similarity of p63/K5/14+ tumor cells to their physiological p63/K5/14+ counterparts in normal breast and salivary duct epithelium, we suggest that these cells provide an important histogenetic key to understanding the pathogenesis of squamous differentiation both in normal breast/salivary gland tissues and their corresponding tumors.

Origin and differentiation of breast nipple syringoma

Similarities in morphology and in glandular and squamous differentiation patterns amongst syringomas of the breast nipple and of the skin suggest a common nature, but the origin of nipple syringoma remains undefined. Using triple immunofluorescence analysis, we found that cells immunopositive for basal keratins K5 and 14 undergo differentiation into glandular and squamous cell lineages. Both tumour types expressed K10, indicative of squamous lineage, but there were specific differences in their glandular lineage. In contrast to the breast nipple syringoma, which expressed glandular keratins K8/18/19, syringoma of the skin only expressed the glandular keratin K19. Therefore, syringomas of the breast nipple and of the skin resemble glandular lineages of the breast nipple duct or eccrine duct epithelium, respectively. From these results we conclude that K5/14-positive cells of the breast nipple ducts are the putative cells of origin for syringomas of the nipple, which highlights the organotypic glandular differentiation potential.

Characterization of mast cell populations using different methods for their identification

Mast cells are ubiquitous throughout the human tissues and play an essential role in physiology and pathology. For evaluation of patients with pathological conditions, mast cells were primarily detected using metachromatic staining with toluidine blue. In the last decades, the staining arsenal of pathologists was enriched with enzyme histochemical and immunohistochemical methods, and it was established that depending on species and tissue localization mast cells are not similar both in appearance and function. The aim of this study was to characterize different mast cell populations using the up-to-date methods of their identification. We compared standard metachromatic method for mast cells with enzyme histochemical detection of chloroacetyl esterase and with immunohistochemical detection of tryptase and chymase in human and rodent tissues. Combination of these methods allowed us to assay quantitatively mast cell populations in different organs of humans and rodents. Furthermore, we assessed the appropriate implementation of each of these methods for mast cell identification in diagnostic labs.

Tryptase as a polyfunctional component of mast cells

Mast cells are haematopoietic cells that arise from pluripotent precursors of the bone marrow. They play immunomodulatory roles in both health and disease. When appropriately activated, mast cells undergo degranulation, and preformed granule compounds are rapidly released into the surroundings. In many cases, the effects that mast cells have on various inflammatory settings are closely associated with the enzymatic characteristics of tryptase, the main granule compound of mast cells. Tryptase degranulation is often linked with the development of an immune response, allergy, inflammation, and remodelling of tissue architecture. Tryptase also represents an informative diagnostic marker of certain diseases and a prospective target for pharmacotherapy. In this review, we discuss the current knowledge about mast cell tryptase as one of the mast cell secretome proteases. The main points of the reviewed publications are highlighted with our microscopic images of mast cell tryptases visualized using immunohistochemical staining.

Multicolor immunofluorescence reveals that p63- and/or K5-positive progenitor cells contribute to normal breast epithelium and usual ductal hyperplasia but not to low-grade intraepithelial neoplasia of the breast

We contend that knowledge about the cellular composition of normal breast epithelium is a prerequisite for understanding proliferative breast disease. Against this background, we used multicolor immunofluorescence to study normal breast epithelium and two types of intraepithelial proliferative breast lesion for expression of the p63, basal keratin K5, glandular keratin K8/18, SMA, ER-alpha, and Ki67. We studied eight normal breast epithelium samples, 12 cases of usual ductal hyperplasia, and 33 cases of low-grade intraepithelial neoplasia (9 flat epithelial atypia, 14 low-grade ductal carcinoma in situ and 10 cases of lobular neoplasia). Usual ductal hyperplasia showed striking similarity to normal luminal breast epithelium including p63+ and/or K5+ luminal progenitor cells and the full spectrum of luminal progeny cells. In normal breast epithelium and usual ductal hyperplasia, expression of ER-alpha was associated with lack of expression of the proliferation antigen Ki67. In contrast, we found in both types of low-grade intraepithelial neoplasia robust expression of keratin K8/18 and a positive association between ER-alpha and Ki67 expression. However, these lesions were consistently negative for p63 and/or K5. Our observational study supports the view that usual ductal hyperplasia and low-grade intraepithelial neoplasia are different entities rather than part of a spectrum of the same disease. We propose a new operational model of cell differentiation that may serve to better understand correlations between normal breast epithelium and proliferative breast diseases. From our data we conclude that p63+ and/or K5+ progenitor cells contribute to maintenance of normal epithelium and usual ductal hyperplasia, but not to low-grade intraepithelial neoplasia of the breast.