Chloroacetate esterase reaction combined with immunohistochemical characterization of the specific tissue microenvironment of mast cells.

This study provides a combined histochemical method for detecting enzyme activity of chloroacetate esterase simultaneously with immunolabeling of the components of a specific tissue microenvironment on formalin-fixed, paraffin-embedded specimens. Chromogenic detection of the molecular targets within and outside the mast cells provides novel options in determining the histoarchitectonics of organ-specific mast cell populations, studying the functional significance of chloroacetate esterase and specifying the immune landscape of the tissue microenvironment.

The Remodeling of Dermal Collagen Fibrous Structures in Mice under Zero Gravity: The Role of Mast Cells.

Mechanisms of adaptive rearrangements of the fibrous extracellular matrix of connective tissues under microgravity practically remain unexplored, despite the most essential functions of the stroma existing to ensure the physiological activity of internal organs. Here we analyzed the biomaterial (the skin dermis) of C57BL/6J mice from the Rodent Research-4 experiment after a long stay in space flight. The biomaterial was fixed onboard the International Space Station. It was found that weightlessness resulted in a relative increase in type III collagen-rich fibers compared to other fibrous collagens in the skin. The number of mast cells in the skin did not change, but their secretory activity increased. At the same time, co-localization of mast cells with fibroblasts, as well as impregnated fibers, was reduced. Potential molecular-cellular causes of changes in the activity of fibrillogenesis under zero-gravity conditions and the slowdown of the polymerization of tropocollagen molecules into supramolecular fibrous structures, as well as a relative decrease in the number of fibrous structures with a predominant content of type-I collagen, are discussed. The data obtained evidence of the different sensitivity levels of the fibrous and cellular components of a specific tissue microenvironment of the skin to zero-gravity conditions. The obtained data should be taken into account in the systematic planning of long-term space missions in order to improve the prevention of undesirable effects of weightlessness.

Protease Profile of Tumor-Associated Mast Cells in Melanoma.

Mast cells (MCs) produce a variety of mediators, including proteases-tryptase, chymase, and carboxypeptidases-which are important for the immune response. However, a detailed assessment of the mechanisms of biogenesis and excretion of proteases in melanoma has yet to be carried out. In this study, we present data on phenotype and secretory pathways of proteases in MCs in the course of melanoma. The development of melanoma was found to be accompanied by the appearance in the tumor-associated MC population of several pools with a predominant content of one or two specific proteases with a low content or complete absence of others. Elucidation of the molecular and morphological features of the expression of MC proteases in melanoma allows us a fresh perspective of the pathogenesis of the disease, and can be used to clarify MCs classification, the disease prognosis, and evaluate the effectiveness of ongoing antitumor therapy.

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.

Identification of autofluorescent cells in human angioimmunoblastic T-cell lymphoma.

Endogenous cell autofluorescence is a common nuisance that complicates the use of fluorescence microscopy. When using fluorescence-labeled antibodies for specific cell labeling in tissue sections of human angioimmunoblastic T-cell lymphoma (AITL), we encountered with a problematic autofluorescence of multiple cells. These cells emitted fluorescence signals in the green, red and deep-red spectral range. Characterization of these autofluorescent cells solely on the basis of their autofluorescence failed. To identify these enigmatic cells residing the lymphoma tissue, we combined two imaging techniques-fluorescence and brightfield microscopy. Combined fluorescence/brightfield imaging of cells immunolabeled with a panel of CD antibodies raised against diverse cellular components allowed us to identify the autofluorescent cells in the AITL as eosinophils. These cells tended to accumulate in the vicinity of capillaries and arterioles apparently mediating the process of angiogenesis resembling other angiogenesis-associated diseases.

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.

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

AIMS: 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. METHODS AND RESULTS: Using triple immunofluorescence labelling and quantitative RT-PCR for keratins, p63, and smooth muscle actin, we demonstrated that syringomatous tumour and LGAdSC contain p63+/K5/14+ tumour cells, K10+ squamous cells, and K8/18+ glandular cells, with intermediary cells being found in both lineages. Identical p63+/K5/14+ cells were also found in the normal breast duct epithelium. CONCLUSIONS: Our data provide evidence that syringomatous tumour of the nipple and LGAdSC are identical or nearly identical lesions. They contain p63+/K5/14+ cells as the key cells from which the K10+ squamous lineage and the K8/18+ glandular lineage arise. On the basis of our findings in normal breast tissue and associated benign lesions, we suggest that p63+/K5/14+ cells of the normal breast duct epithelium or early related cells might play a key role in the neoplastic transformation of both syringomatous tumour and LGAdSC. We propose that the differentiation patterns found in both lesions reflect the early ontogenetic stages of the 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.

Carboxypeptidase A3-A Key Component of the Protease Phenotype of Mast Cells.

Carboxypeptidase A3 (CPA3) is a specific mast cell (MC) protease with variable expression. This protease is one of the preformed components of the secretome. During maturation of granules, CPA3 becomes an active enzyme with a characteristic localization determining the features of the cytological and ultrastructural phenotype of MC. CPA3 takes part in the regulation of a specific tissue microenvironment, affecting the implementation of innate immunity, the mechanisms of angiogenesis, the processes of remodeling of the extracellular matrix, etc. Characterization of CPA3 expression in MC can be used to refine the MC classification, help in a prognosis, and increase the effectiveness of targeted therapy.

Expression of CD38 in Mast Cells: Cytological and Histotopographic Features.

The biological significance of the CD38 molecule goes beyond metabolic, enzymatic, and proliferative functions. CD38 possesses the functions of an exoenzyme and receptor, and is actively involved in the mechanisms of adhesion, migration, intercellular signaling, formation of immune synapses, and modulation of the activity of a wide range of immune and non-immune cells. The aim of this study was the immunohistochemical assessment of the cytological and histotopographic characteristics of CD38 expression in mast cells. CD38 expression was found in a minority of the mast cell population. It is characterized by wide variability from low to high levels. The intensity of CD38 expression in mast cells has organ-specific features and depends on the development of pathological processes in a specific tissue microenvironment. The mechanisms of intercellular interaction between mast cells and CD38+ cells foster new understanding of the protumorigenic or antitumor potential of tryptase.

Identification of CTLA-4-Positive Cells in the Human Tonsil.

CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) was originally defined as a T-lymphocyte antigen and was used as a target in cancer immunotherapy. Unfortunately, the existence of CTLA-4 in cells other than T-lymphocytes is often overlooked. The goal of the present study was to analyze the distribution pattern of CTLA-4 in the human tonsils using a panel of anti-CTLA-4 antibodies of different clones. We found that CTLA-4 was expressed in T-lymphocyte cells of various geneses, including hematopoietic cells and their derivatives (monocytes, macrophages, dendritic, plasma cells, mast cells, and neutrophils), as well as stromal cells of mesodermal (mesenchymal) origin and reticular epithelial cells of ectodermal origin. The expression of CTLA-4 in cells of different origins supports the proposition that CTLA-4 is not restricted to the lymphoid cell lineage and can provide broader effects of CTLA-4 on immune regulation.

Immunophenotyping of the PD-L1-positive cells in angioimmunoblastic T cell lymphoma and Hodgkin disease.

OBJECTIVE: Programmed death-1 (PD-1) and its ligand PD-L1 are now used as predictive biomarkers to guide clinical decisions. Precise characterization of PD-L1-positive cells may contribute to our knowledge of which patients derive benefit from the PD-L1 blockade therapy. RESULTS: To address this issue, we performed immunophenotyping of PD-L1-positive cells in Hodgkin lymphoma and in angioimmunoblastic T cell lymphoma (AITL) employing multiple immunofluorescent immunolabeling. We found that PD-L1-positive cells and PD-1-positive cells both in Hodgkin lymphoma and in AITL belong to two completely different cell lineages. In both lymphomas, PD-1 was found exclusively in T-lymphocytes, whereas PD-L1 was revealed in the tumor microenvironment cells including macrophages. PD-L1 was also detected in CD30-positive cells in Hodgkin lymphoma but not in AITL. The marker of B-cell lineage, CD20, was not detectable in PD-L1-positive cells both in AITL and in Hodgkin. Our study highlights the importance of comprehensive assessment of PD-1/PD-L1 regulatory pathways for employing PD-L1 as a predictive biomarker in clinical practice. PD-L1-antibody therapy is proven in Hodgkin lymphoma. Comparative immunophenotyping of the PD-1/PD-L1 axis provides a support for attempts to prove this principle also for AITL.

The role of arterial smooth muscle in vasorelaxation.

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.

Multiple immunolabeling with antibodies from the same host species in combination with tyramide signal amplification.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies. This obstacle can however be avoided with the use of striping buffers eluting the primary/secondary antibody complex. After elution of the previous primary/secondary antibody complex, the next primary antibody from the same host species can be applied. Recently, a group from VENTANA (Tucson, AZ, USA) presented a fully automated multiplex protocol for fluorescent immunohistochemistry on the platform of VENTANA's BenchMark ULTRA slide stainer using the same species antibodies in combination with tyramide signal amplification. We adapted the automated protocol of VENTANA for the use in a routine histochemical laboratory and present here a standard procedure with a manual mode of operation for simultaneously detecting two or more antigens from the same host species.

New Insight into the Role of Nitric Oxide Pathways in Pancreas.

Nitric oxide (NO) is generated by a family of enzymes termed NO synthases (NOS) that convert L-arginine to NO and citrulline. The role of NO as an important biological mediator and recognition of the pathophysiological significance of superoxides/NO interaction has led to an intensive research and development of therapies based on the interception of the NO signaling cascade in the pancreatitis course. However, the presence and localization of the NO-generating enzymes in various organs including pancreas are subject to controversy. We assumed that this controversy might reflect rather the diversity of experimental approaches and an insufficient sensitivity of the methods used. Applying tyramide signal amplification (TSA) immunohistochemical technology, we were able detect all three NOS isoforms both in exocrine and endocrine compartments and in the vasculature in the normal pancreas and in pancreatitis. This also allowed us to demonstrate that oxidative stress runs ahead of NOS up-regulation, which implies that the NO enhancement in the course of pancreatitis is likely to be an adaptive mechanism aimed at maintaining the homeostatic cellular level of the bioactive NO. The aims of this minireview are to describe normal intrapancreatic NO pathways and the role of NO in the pancreatitis course.

Oxidative stress and NO generation in the rat pancreatitis induced by pancreatic duct ligation.

The interaction between nitric oxide (NO) and superoxides is critical in the development of an acute pancreatitis. Previously, we reported that the expression of superoxides and of the NO-generating enzyme (NO synthase, NOS) was up-regulated in the human pancreatitis, especially within the exocrine compartment indicating an exceptional susceptibility of the exocrine parenchyma to oxidative stress. The aim of the present study was to compare the regulation of NO signalling pathways in the human pancreatitis and in an animal model of an acute pancreatitis induced by pancreatic duct ligation (PDL) in rats. In the PDL-induced rat pancreatitis, we revealed a similar pattern of oxidative stress and NOS up-regulation in acinar and in ductal compartments, like in the human pancreatitis. This demonstrates that the PDL-induced rat pancreatitis is a proper model for further studies of acute pancreatitis development in humans.

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.

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.

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.

Cellular control of nitric oxide synthase expression and activity in rat cardiomyocytes.

Potential ortho- and pathophysiological roles for nitric oxide synthases (NOS) in cardiac functions have been and are continuing to be described. However, cellular signaling mechanisms controlling nitric oxide (NO) production in the heart remain obscure. The aim of this study was to investigate signaling mechanisms involved in regulation of NOS expression and NO generation in cardiomyocytes. Using immunocytochemical methods in conjunction with western blotting, we have found that cultured neonatal rat cardiomyocytes express constitutively all three NOS isoforms targeted predominantly to the particulate component of cardiomyocytes - mitochondria and along contractile fibers, as well as along plasma membrane including T-tubules. Biochemical assay of NO generation has shown that exposure of cultured neonatal rat cardiac cells to isoproterenol (beta-adrenergic stimulation), iloprost [stable prostaglandin I(2) (PGI(2)) analogue], as well as inflammatory cytokines and dibutyryl adenosine-3',5'-monophosphate (db-cAMP), resulted in a marked up-regulation of NOS expression by cardiomyocytes. In db-cAMP-stimulated cells, inhibition of protein kinase A (PKA) and protein kinase C (PKC) reduced immunolabeling of NOS and concomitantly lowered NO production. Taken together, these data point to an involvement of beta-adrenergic mechanisms, cytokine and PGI(2) receptors, adenylyl cyclase, PKA, and PKC in the control of NO generation and expression of NOS in rat cardiomyocytes.