Combined histochemical approach in assessing tryptase expression in the mast cell population.

To increase the efficiency of interpretation of mast cell's contribution to the state of a specific tissue microenvironment, it is necessary to detail the molecular composition of their secretome and analyze the pathways of degranulation. Developed method of combining immunomorphological and histochemical staining protocols contributes to the most objective detection of the integral level of tryptase expression in the intraorgan population of the skin mast cells. Novel technique for tryptase detection expands the possibilities of morphological analysis, provides researchers with additional data on the structure of the mast cell population and helps visualize the processing and cytological features and structural targets of tryptase during the development of adaptive and pathological reactions. Objective determination of the tryptase profile for organ-specific mast cell populations is in great demand in clinical practice for the interpretation of pathological processes, including inflammation and oncogenesis.

Initial Characterization of Transgenic Mice Overexpressing Human Histamine H2 Receptors.

In an integrative approach, we studied the role of histamine H2 receptors in the mouse heart. We noted that histamine, added cumulatively to the organ bath, failed to affect the force of contraction in left atrial preparations and did not change spontaneous heart rate in right atrial preparations from wild-type mice. By contrast, in the same preparations from mice that overexpressed the human H2 receptor in a cardiac-specific way, histamine exerted concentration- and time-dependent positive inotropic and positive chronotropic effects. Messenger RNA of the human H2 receptor was only detected in transgenic mice. Likewise, immunohistology and autoradiography only gave signals in transgenic but not in wild-type cardiac preparations. Similarly, a positive inotropic and positive chronotropic effect was observed with histamine in echocardiography of living transgenic mice and isolated perfused hearts (Langendorff preparation). Phosphorylation of phospholamban was increased in atrial and ventricular preparations from transgenic mice, but not in wild-type animals. The effects of histamine were mimicked by dimaprit and amthamine and antagonized by cimetidine. In summary, we generated a new model to study the physiologic and pathophysiologic cardiac role of the human H2 receptor.

Metabolic profile and nitric oxide synthase expression of skeletal muscle fibers are altered in patients with type 1 diabetes.

We investigate muscle fiber composition, fiber-specific glycolytic and oxidative enzyme capacity and nitric oxide synthase (NOS) expression in skeletal muscle of patients with type 1 diabetes (T1D) compared to individuals with normal glucose tolerance (NGT). Vastus lateralis muscle was obtained by percutaneous biopsy from 7 T1D patients and 10 healthy controls with similar characteristics. Using cytophotometry, muscle fiber composition and fiber type-specific glycolytic and oxidative enzyme activities were measured in slow oxidative (SO), fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers. In addition, NOS 1-3 protein expression was mea-sured. The glycolytic fiber fraction was 1.4 fold higher, whereas FOG and SO fiber fractions were significantly reduced by 13.5% and 6.2% in skeletal muscle from T1D patients. Glycolytic enzyme activities and fiber-specific ratio of glycolytic relative to oxidative enzyme activity were significantly higher in all fiber types of T1D patients and correlated with HbA (1c). Expression of NOS1-3 isoforms was reduced in skeletal muscle of T1D subjects. Increased glycolytic enzyme activity in muscle of T1D patients is most likely due to both a higher number of fast glycolytic fibers and a shift towards increased glycolytic metabolism in all fiber types. Alterations in muscle fiber distribution and enzyme activities seem to be due to impaired long-term glycemic control.

Fibre-related nitric oxide synthase (NOS) in Duchenne muscular dystrophy.

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The loss of NO synthase (NOS) from the sarcolemma was assumed to be associated with development of Duchenne muscular dystrophy (DMD). We have, however, recently reported that, in contrast to the commonly accepted view, NOS expression in DMD myofibres is up-regulated. This poses the question of the fibre type-specific NOS expression in DMD muscles and how the NOS expression is related to the regeneration or degeneration status. To address this issue, we examined localization of NOS isoforms I, II and III in skeletal muscles of DMD patients employing immunohistochemical labelling with tyramide signal amplification complemented with enzyme histochemistry. We found that NOS immunolabelling as well as metabolic enzyme activity in DMD muscles were heterogeneously distributed along the fibre length of DMD muscle fibres revealing regenerating and degenerate (hypercontracted) fibres as well as normal segments. Like in normal muscles, positive NOS immunoreactivity was found to be associated with fast-oxidative glycolytic (FOG) phenotype. The regeneration status of NOS-positive segments was deduced from the presence of neonatal and developmental myosin heavy chains. High NOS expression in regenerating DMD muscle fibres can be well reconciled with reports about the protective role of endogenous NO in inflammatory diseases and in muscle repair.

Ck5-positive cells are precursor cells of glandular and myoepithelial cell lineages in the human breast epithelium. A new cell concept as a basis for a better understanding of proliferative breast disease?

The question of whether stem cells exist in the human breast has never been satisfactorily resolved. Using a double immunofluorescence technique for simultaneous demonstration of cytokeratin subgroups 5, 8/18/19 and the differentiation marker smooth muscle actin, we provided direct evidence of the existence of a Ck5-positive cells that differentiate to either the glandular or myoepithelial cell lineage via intermediary cells. We postulate that the Ck5-positive cells are phenotypically and behaviourally committed adult stem cells. We further provide evidence that benign proliferative breast disease lesions resemble normal breast epithelium, whereas most breast carcinomas phenotypically consist of glandularly differentiated cells. The results were corroborated biochemically by Western blotting techniques. This cell biological model provides a new tool which helps to distinguish benign and malignant breast lesions. Furthermore this model provides a basis to unravel the regulatory mechanisms that govern normal and pathological cell growth.

Increased importin alpha protein expression in diabetic nephropathy.

BACKGROUND: Importins transport kinases, transcription factors, and viral proteins into the nucleus. Since the expression of several genes is increased in diabetic nephropathy, we tested the hypothesis that importin protein expression is increased in diabetic kidneys. METHODS: Immunohistochemistry and Western blotting were used in kidneys from streptozotocin-treated diabetic rats and from spontaneously diabetic Goto-Kakizaki rats. The effects of high glucose and mannose also were tested in cell culture experiments. RESULTS: In normal rat kidneys, importin alpha isoforms were differentially expressed in glomerular cells and tubular segments, while importin alpha1/Rch1 was expressed only in tubules and peritubular cells. In diabetic rat kidneys from both models, the importin alpha isoform expression was markedly up-regulated. Western blotting revealed strong up-regulation of importin alpha7 and minor up-regulation of other isoforms. Exposure of various cell types to high glucose or mannose (25 mmol/L) led to increased expression of importins alpha3, alpha5/hSRP1, and alpha7 in different cultured cells, while up-regulation of other importin alpha isoforms was less consistent. CONCLUSIONS: A specific importin alpha isoform up-regulation takes place in kidneys of diabetic rats. Diabetes is a stimulus for increased importin alpha7 expression. Thus, nuclear transport in diabetes may be increased in glomerular and tubular cells. The signaling pathways appear differentially regulated in glomeruli, proximal, and distal tubules. The enhanced nuclear transport may participate in increased gene expression and nephrosclerosis in diabetes.

Nitric oxide synthase isoforms I, III and protein kinase-Ctheta in skeletal muscle fibres of normal and streptozotocin-induced diabetic rats with and without Ginkgo biloba extract treatment.

The expression of nitric oxide synthase (NOS) isoforms I, III and protein kinase-Ctheta (PKCtheta) in rat vastus lateralis muscle was demonstrated immunohistochemically and then correlated to the physiological metabolic fibre types: SO (slow-oxidative), FOGI, FOGII (fast-oxidative glycolytic; I more glycolytic, II more oxidative), and FG (fast-glycolytic). NOS expression in muscles from different experimental groups (normal and diabetic rats, with and without Ginkgo biloba extract treatment) was assayed by Western blotting. Generally, NOS I and PKCtheta were co-expressed in fibres with predominantly oxidative metabolism (SO, FOGII). This suggests an interplay of PKCtheta and NOS I in nitric oxide production by oxidative fibres. NOS III was more highly expressed in fibres with predominantly glycolytic metabolism (FOGI, FG). A somewhat lower NOS I immunoreactivity was also found in NOS III positive fibres suggesting that NOS III and NOS I are co-expressed in these fibres. Western blotting revealed that NOS I as well as NOS III expression in the vastus lateralis muscle was down-regulated in diabetes and increased after Ginkgo biloba extract treatment. These effects may be associated with a diminished glucose uptake by myocytes of diabetic musclesand with an improved muscle function after Ginkgo biloba treatment.

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

Inducible nitric oxide synthase in the myocard.

Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin.

The CD4+ natural killer (NK)T cells in the liver are potent IL-4 producers and hence may promote Th2 cell development. Following Mycobacterium bovis bacillus Calmette Guérin (BCG) infection, IL-4-producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70% Percoll) by flow cytometry. The present study shows that M. bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL-4 to IFN-gamma. The number of CD4+ NK1+ TCR alpha/beta(intermediate) cells increased in the low-density fraction (<40% Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density. The number of IL-4-producing cells, however, was small and high frequencies of IFN-gamma-secreting cells were identified in the low-density fraction after TCR/CD3 ligation. Accordingly, selected low-density CD4+ NKT cells encompassed high numbers of IFN-gamma producers and minute numbers of IL-4-secreting cells. Induction of low-density CD4+ NKT cells by M. bovis BCG was abrogated by endogenous IL-12 neutralization which also caused increased bacterial growth in the liver. We assume that M. bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL-4 to IFN-gamma through IL-12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN-gamma-producing Th1 cells.

Adenylyl cyclase in the heart: an enzymocytochemical and immunocytochemical approach.

This review provides a discussion of the localization of adenylyl cyclase (AC) in normal mammalian heart tissue employing enzymocytochemistry (detection of the catalytic activity of AC by a metal precipitation technique) and immunocytochemistry (immunolabeling of the enzyme protein with antibodies against AC subtypes). By the metal precipitation technique, AC activity was localized in adult guinea pig cardiomyocytes along the sarcolemma and the T-tubule membranes. This reaction can be enhanced by hormones and guanylyl imidodiphosphate, fluoride, and forskolin. With this technique, no precipitates were detected at the sarcoplasmic reticulum. However, under ischemic conditions, AC activity was also found in the junctional sarcoplasmic reticulum of rat cardiomyocytes. Immunocytochemistry revealed AC in the plasma membrane of rat cardiomyocytes. Detection of AC in the perinuclear space of cardiomyocytes might reflect initiation of synthesis and processing of the enzyme protein. Colocalization of AC with cytoskeleton fibers of non-cardiomyocytes emerging in the cell culture of neonatal rat cardiocytes imply a direct cytoskeletal-AC interaction. Finally, it can be stated that the immunolabeling pattern of AC in cryosections of adult and new-born rat hearts reveals a good correspondence with the localization of AC activity in cardiomyocytes demonstrated by enzymocytochemistry.

Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture.

Recognition of the role of nitric oxide (NO) in cardiovascular regulations raised an acute interest in NO-generating enzymes-nitric oxide synthases (NOS). Nevertheless, the subcellular localization of inducible isoform of NOS (NOS II) and regulation of its expression in the cardiomyocyte still remains to be elucidated. Therefore, we focused this study on the subcellular localization of NOS II in cultured neonatal rat cardiomyocytes using immunocytochemical techniques at the light and electron microscopic level as well as the demonstration of NADPH-diaphorase activity and the Griess assay for NO measurement. Cultivation of neonatal cardiomyocytes during 2 and more days induced a moderate increase in the NOS II immunolabeling in defined cytoplasmic structures and a nuclear NOS II staining in some cells. Exposure of the cell cultures to exogenous cAMP markedly stimulated NO production with a concomitant enhancement of NOS II immunolabeling of cardiomyocytes. cAMP-induced changes were significantly attenuated by dexamethasone. This report provides evidence for the localization of NOS II in the perinuclear space, Golgi complex, mitochondria, plasma membrane and along contractile fibers of cardiomyocytes, as well as for the appearance of NOS II staining of the cell nuclei in the course of cultivation. In non-cardiomyocytes contaminating the cell culture, positive immunoreaction was detected in the Golgi complex and endoplasmic reticulum. Our data point to a notable constitutive expression of NOS II in rat cardiomyocytes apparently dependent on the developmental stage.

Involvement of tubulin and inhibitory G proteins in the interaction of Listeria monocytogenes with mouse hepatocytes.

Intracellular and cell-to-cell spread of Listeria monocytogenes has been considered exclusively actin dependent. By immunocytochemical techniques, we provide evidence for an involvement of inhibitory G proteins and tubulin in "comet tail" formation in L. monocytogenes-infected mouse hepatocytes.

Signal transduction and phagosome biogenesis in human macrophages during phagocytosis of Mycobacterium bovis BCG.

Downstream signal transduction via heterotrimeric GTP-binding proteins to protein kinase C has been reported to be a central event in induction of rapid phagocytosis of extracellular particles by macrophages. However, the signalling pathway involved in mycobacterial uptake and phagosome biogenesis is poorly understood, and there is lack of information about in situ localization of PKC, cytoskeletal proteins, and G-proteins in mycobacterial vacuoles. Employing immunocytochemical methods, we provide evidence that alpha-subunits of stimulatory and inhibitory G-proteins and PKC beta as well as two major cytoskeletal components, microfilaments and microtubules, participate in uptake of Mycobacterium bovis BCG by human macrophages and co-localize in phagosomes. This implies that cellular signalling via G-proteins and PKC beta may occur not only at the level of the plasma membrane; rather, the alpha-subunit of G-proteins and PKC beta may be translocated to the effector proteins involved in phagosomal biogenesis. A similar pattern of accumulation of G-proteins, PKC, and both microfilamental and microtubular cytoskeleton around vacuoles containing internalized latex beads indicates their general role in phagocytosis.

Localization of G-proteins in macrophages and E. coli during phagocytosis.

Heterotrimeric GTP-binding proteins (G-proteins) have been shown to play an important role in cellular signalling. However, G-protein involvement in the intracellular spreading of bacterial pathogens is still poorly understood. In this study, antibodies, that recognize G-protein alpha-subunits (anti-G alpha), were used to investigate the localization of G-proteins in the macrophage-like cell line P388D1 and E. coli, also in their L-forms, during phagocytosis. In E. coli, anti-G alpha-binding sites were detected preferably in the cell wall and septa of the whole bacterial forms as well as in the cytoplasm of L-forms. Western blotting of bacterial lysates demonstrated protein bands with positive immunoreaction to antibodies against Gs alpha, Gi alpha, and Gcommon alpha with a higher affinity to the antibody against Gs alpha. Immunoreaction with the anti-Gs alpha-antibody was markedly higher in pathogenic strains of E. coli. Because of the conserved structure in all GTP-binding proteins which seem to derive from a single primordial protein involved in signal transduction mechanisms, it is reasonable to assume that some anti-Ga-positive proteins in E. coli might be related to G-proteins of higher organisms. A putative candidate for bacterial G-proteins seems to be a 36 kDa protein. Enhancement in G-protein immunostaining in the cytoplasm of macrophages around the internalized bacteria testifies to the involvement of G-proteins in mediation of endocytosis responses of phagocytes.

Immunocytochemical studies of the Gi protein mediated muscarinic receptor-adenylyl cyclase system.

The localization of three key signal transduction components was indicated in rat heart tissue by immunocytochemical and histochemical experiment. It was shown that: 1. The M2 muscarinic receptors are localized along outer cell membranes and T-tubule membranes of cardiomyocytes but additionally at membranes of endothelial cells and fibroblasts. 2. Gia was found along outer cell membranes of cardiomyocytes and other cells of the heart and also inside the cells of the perinuclear space in close contact to the nuclei envelope and the endoplasmic reticulum membranes. Goa were found to be associated mainly in atrial tissue, especially at the nerval (neuronal) endings located among the cardiac muscle cells. This was shown in parallel incubation with specific neuronal antibody as a marker for these structures. 3. Adenylyl cyclase was localized along the sarcolemma and the T-tubule membranes in normal cardiomyocytes of rat and guinea pig hearts. Under ischemic conditions, the adenylyl cyclase was also seen in junctional sarcoplasmic reticulum membranes. The reasons for this changed localization need further elucidation. Binding of the adenylyl cyclase within the molecular structure of the membrane or variation of the marker penetration remain to be clarified.

Ultrastructure of circulating immune complexes isolated from the blood plasma of patients suffering from infectious diseases.

An original procedure which permits to isolate circulating immune complexes (CIC) from the blood plasma in a form of a dense pellet was developed. This procedure was applied for the ultrastructural analysis of CIC isolated from blood of healthy blood donors and patients suffering from Yersinia enterocolitica (YE) and Yersinia pseudotuberculosis (YP) infections. The here reported method of CIC isolation from blood plasma permitted to visualize CIC electronmicroscopically as amorphous masses of low, middle, and high electron density with inclusions of cell debris. In contrast to CIC of healthy blood donors, CIC of infected patients contained various bacteria and fungiformic structures. For the first time, this method made possible an ultrastructural demonstration of bacterial destruction outside of phagocytes in vivo. This method also permits to visualize and identify bacteria in cases of lingering forms of infection when hemoculture tests fail. Therefore, electronmicroscopic examination of CIC preparations from the blood plasma might be a very informative indicator of bacteriemia in the course of an infection process and serve as an indicator of therapeutic effects. In lingering forms of an infection process, ultrastructural analysis of CIC preparations can be of prognostic value and serve as an indicator of therapeutic effects. This method might be also advantageous as an additional test for the exposure of latent bacterial persistence in diagnostically complicated cases.

Comparative immunocytochemical demonstration of G proteins in rat heart tissue.

Localization of G proteins in the rat heart tissue was investigated using primary affinity-purified antibodies against synthetic peptides with amino acid sequences corresponding to alpha-subunits (alpha i common and alpha i 1, 2) of G proteins. Detection of immunoreactivity was performed with the peroxidase-anti-peroxidase complex (PAP), avidin-biotin complex (ABC) and fluorescein-labelled secondary antibodies for light microscopy and the protein A-gold technique for electron microscopy. In ventricles and atria, immunostaining for G proteins was detected in the sarcolemma and perinuclear space of cardiomyocytes. In endotheliocytes and fibroblasts, immunoreactivity was present also in the endoplasmic reticulum. All four immunocytochemical methods permit to demonstrate the same localization of G proteins in heart tissue. The ABC method and fluorescein labelled secondary antibodies technique showed the same sensitivity which is higher than that of the PAP method. Nomarski contrast microscopy enhanced the visualization of the final reaction product formed by the peroxidase reaction developed with diaminobenzidine in the ABC method. The results are discussed in terms of the role of G proteins in signal transduction via plasma membrane and membranes of the intracysternal space of the cell.

Adenylate cyclase activity of cultural strains of Neisseria gonorrhoeae and Staphylococcus aureus.

Adenylate cyclase of gonococci and staphylococci from patients with gonorrhoeae and pyoderma was studied ultracytochemically. Adenylate cyclase activity was mainly revealed on the inner side of the plasma membrane and septa of dividing cells. The enzymic activity is markedly higher in penicillinase producing strains and dividing bacteria. These data may be useful for the diagnostics and treatments of the infections concerned.

Localization of nucleoside phosphatases (ATPase and 5'-nucleotidase) and nuclear ribonucleoproteins in the human endometric glandular cells during the secretory phase.

Endometrial curettings from 3 healthy women between 16th and 19th d of the menstrual cycle were investigated employing ultracytochemical methods. In endometrial glandular cells ATPase and 5'-nucleotidase activities were demonstrated in the nuclei, nucleoli, and nuclear channel system (NCS), i.e., mainly in structures characterized ++as ribonucleoproteins according to the Bernhard method. The functional relationship between the NCS and the molecular biology of the endometrial glandular cell is discussed.