DHA Supplementation Attenuates MI-Induced LV Matrix Remodeling and Dysfunction in Mice.

OBJECTIVE: Myocardial ischemia and reperfusion (I/R) injury is associated with oxidative stress and inflammation, leading to scar development and malfunction. The marine omega-3 fatty acids (ω-3 FA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are mediating cardioprotection and improving clinical outcomes in patients with heart disease. Therefore, we tested the hypothesis that docosahexaenoic acid (DHA) supplementation prior to LAD occlusion-induced myocardial injury (MI) confers cardioprotection in mice. METHODS: C57BL/6N mice were placed on DHA or control diets (CD) beginning 7 d prior to 60 min LAD occlusion-induced MI or sham surgery. The expression of inflammatory mediators was measured via RT-qPCR. Besides FACS analysis for macrophage quantification and subtype evaluation, macrophage accumulation as well as collagen deposition was quantified in histological sections. Cardiac function was assessed using a pressure-volume catheter for up to 14 d. RESULTS: DHA supplementation significantly attenuated the induction of peroxisome proliferator-activated receptor-α (PPAR-α) (2.3 ± 0.4 CD vs. 1.4 ± 0.3 DHA) after LAD occlusion. Furthermore, TNF-α (4.0 ± 0.6 CD vs. 1.5 ± 0.2 DHA), IL-1ß (60.7 ± 7.0 CD vs. 11.6 ± 1.9 DHA), and IL-10 (223.8 ± 62.1 CD vs. 135.5 ± 38.5 DHA) mRNA expression increase was diminished in DHA-supplemented mice after 72 h reperfusion. These changes were accompanied by a less prominent switch in α/ß myosin heavy chain isoforms. Chemokine mRNA expression was stronger initiated (CCL2 6 h: 32.8 ± 11.5 CD vs. 78.8 ± 13.6 DHA) but terminated earlier (CCL2 72 h: 39.5 ± 7.8 CD vs. 8.2 ± 1.9 DHA; CCL3 72 h: 794.3 ± 270.9 CD vs. 258.2 ± 57.8 DHA) in DHA supplementation compared to CD mice after LAD occlusion. Correspondingly, DHA supplementation was associated with a stronger increase of predominantly alternatively activated Ly6C-positive macrophage phenotype, being associated with less collagen deposition and better LV function (EF 14 d: 17.6 ± 2.6 CD vs. 31.4 ± 1.5 DHA). CONCLUSION: Our data indicate that DHA supplementation mediates cardioprotection from MI via modulation of the inflammatory response with timely and attenuated remodeling. DHA seems to attenuate MI-induced cardiomyocyte injury partly by transient PPAR-α downregulation, diminishing the need for antioxidant mechanisms including mitochondrial function, or α- to ß-MHC isoform switch.

Optimized Ki-67 staining in murine cells: a tool to determine cell proliferation.

The reliable analysis of the cell cycle status has become increasingly relevant for scientific and clinical work, especially for the determination of tumor cell growth. One established method to characterize the proliferation activity of cells is the analysis of the Ki-67 protein. Ki-67 is expressed in the nucleus during the whole cell cycle except for the G0 phase. Several different protocols exist for the examination of the Ki-67 protein in tissue and cell culture, but most of them are defined for human cells. For the analysis of the Ki-67 protein in murine tissue and cell culture there is a variety of protocols existing which recommend different fixation and permeabilization reagents or special kits. In this study, we established a reliable protocol for Ki-67 staining in murine cells and tissue based on PFA fixation, which can be used not only for flow cytometry but also for immunofluorescence microscopy analysis. We tested our protocol successfully with three different Ki-67 anti-mouse antibodies in cell culture, regenerating liver tissue and mouse melanoma tumor to demonstrate the general applicability.

Regulation of p53 under hypoxic and inflammatory conditions in periodontium.

OBJECTIVES: Different studies suggest that inflammation as well as hypoxia leads to an increase of p53 protein levels. However, the implication of p53 during oral inflammatory processes is still unknown. The aim of this study was therefore to investigate the effect of hypoxia and inflammation on p53 regulation in human periodontium in vitro and in vivo. MATERIALS AND METHODS: Under hypoxic and normoxic conditions, human primary periodontal ligament (PDL) fibroblasts (n = 9) were stimulated with lipopolysaccharides (LPS) from Porphyromonas gingivalis (P.g.), a periodontal pathogenic bacterium. After different time points, cell viability was tested; p53 gene expression, protein synthesis, and activation were measured using quantitative RT-PCR, immunoblotting, and immunofluorescence. Moreover, healthy and inflamed periodontal tissues were obtained from 12 donors to analyze p53 protein in oral inflammatory diseases by immunohistochemistry. RESULTS: LPS-P.g. and hypoxia initially induced a significant upregulation of p53 mRNA expression and p53 protein levels. Nuclear translocation of p53 after inflammatory stimulation supported these findings. Hypoxia first enhanced p53 levels, but after 24 h of incubation, protein levels decreased, which was accompanied by an improvement of PDL cell viability. Immunohistochemistry revealed an elevation of p53 immunoreactivity in accordance to the progression of periodontal inflammation. CONCLUSIONS: Our data indicate that p53 plays a pivotal role in PDL cell homeostasis and seems to be upregulated in oral inflammatory diseases. CLINICAL RELEVANCE: Upregulation of p53 may promote the destruction of periodontal integrity. A possible relationship with carcinogenesis may be discussed.

Hypoxia and P. gingivalis synergistically induce HIF-1 and NF-κB activation in PDL cells and periodontal diseases.

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1ß, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1ß, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1ß, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.

LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to periodontitis.

Oxidative stress is characterized by an accumulation of reactive oxygen species (ROS) and plays a key role in the progression of inflammatory diseases. We hypothesize that hypoxic and inflammatory events induce oxidative stress in the periodontal ligament (PDL) by activating NOX4. Human primary PDL fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (LPS-PG), a periodontal pathogen bacterium under normoxic and hypoxic conditions. By quantitative PCR, immunoblot, immunostaining, and a specific ROS assay we determined the amount of NOX4, ROS, and several redox systems. Healthy and inflamed periodontal tissues were collected to evaluate NOX4 and redox systems by immunohistochemistry. We found significantly increased NOX4 levels after hypoxic or inflammatory stimulation in PDL cells (P < 0.001) which was even more pronounced after combination of the stimuli. This was accompanied by a significant upregulation of ROS and catalase (P < 0.001). However, prolonged incubation with both stimuli induced a reduction of catalase indicating a collapse of the protective machinery favoring ROS increase and the progression of inflammatory oral diseases. Analysis of inflamed tissues confirmed our hypothesis. In conclusion, we demonstrated that the interplay of NOX4 and redox systems is crucial for ROS formation which plays a pivotal role during oral diseases.

Imaging of the hypoxia-inducible factor pathway: insights into oxygen sensing.

The transcription factor complex hypoxia-inducible factor (HIF)-1 controls the expression of most genes involved in adaptation to hypoxic conditions. HIF-1 is a heterodimer composed of oxygen-labile HIF-alpha and constitutively expressed HIF-beta subunits. The oxygen-dependent regulation of HIF-alpha is a multistep process that includes degradation under normoxia but stabilisation, translocation into the nucleus and activation under hypoxic conditions. The present paper summarises the contributions of optical methods to the understanding of oxygen-dependent regulation of the HIF-1 pathway. The tissue- and cell-specific distribution of HIF-alpha was visualised immunohistochemically and by immunofluorescence. Transcriptional activity of HIF-1 was monitored using green fluorescent protein as a reporter under control of hypoxia response elements in living cells, spheroids and tumour tissues in living mice. With cyan and yellow variants of green fluorescent protein fused to HIF subunits and regulatory proteins, subcellular distribution, migration and interaction were imaged in vivo by means of fluorescence recovery after photo-bleaching and fluorescence resonance energy transfer. Noninvasive imaging of these cellular and molecular processes by laser scanning microscopy complements ex vivo molecular biology assays and provides an additional spatial and temporal dimension to the understanding of the HIF-1 pathway.

Antagonism of lipopolysaccharide-induced blood pressure attenuation and vascular contractility.

OBJECTIVE: Aim was to assess whether lipopolysaccharide (LPS)-induced decrease of total peripheral resistance depends on Toll-like receptor (TLR)4 signaling and whether it is sensitive to NO-synthase or TLR4 antagonists. METHODS AND RESULTS: C3H/HeN mice (control), expressing a functional, and C3H/HeJ mice, expressing a nonfunctional TLR4, were compared. LPS (20 mg/kg) was injected i.p. 6 hours before hemodynamic measurements. L-NAME and SMT, inhibitors of NO production, and Eritoran, a TLR4 antagonist, were tested for their impact on vascular contractility. Aortic rings were incubated for 6 hours with or without LPS (1 microg/mL), or with LPS+Eritoran (2 microg/mL) and their phenylephrine-induced contractility was measured using a myograph. The expression of cytokines in aortic tissue was examined by real-time polymerase chain reaction. In control mice LPS induced a significant decrease of blood pressure and an increase of heart rate, whereas C3H/HeJ remained unaffected. LPS induced an increase of cytokine expression and a depression of vascular contractility only in control mice but not in C3H/HeJ. L-NAME and SMT increased contractility in all rings and restored LPS-dependent depression of contractility. Eritoran prevented LPS-induced loss of contractility. CONCLUSIONS: LPS upregulates cytokine expression via TLR4 and induces attenuation of smooth muscle contractility which can be effectively antagonized.

[Hight resoluton optical microscopy of the cellular cycle in psoriasis patients]. / Estudio con microscopia optica de alta resolucion (MOAR) del ciclo celular en pacientes con psoriasis.

Skin tissue from patients with Psoriasis was analyzed using HROM (High Resolution Optical Microscopy), studying epithelial differentiation and possible structural alterations of the queratinocytes. The samples were taken from 10 patients with histopathologic diagnosis of Psoriasis. This tissue samples where affixed with glutaraldehide buffer-collidine for 48 hours. Later processed with the HROM technique and colored with toluidine blue, metilene blue, basic Fuscine, and silver metenamine. The basal epithelial elements presented ovoid nucleus and most of them had prominent nucleolus. In 7 of the studied cases, the granulose stratus was absent, and thinner in the rest, with nucleus and nucleolus retention. At this level queratinocytes where observed with perinuclear anfofilia, as well as linfocitic and macrophagic infiltrate and union complex where elongated.

Estudio con microscopia optica de alta resolucion (MOAR) del ciclo celular en pacientes con psoriasis / Hight resoluton optical microscopy of the cellular cycle in psoriasis patients

La Psoriais se caracteriza por incremento del ciclo celular a nivel epidérmico, evidenciado a nivel histopatológico por un intensa hiperparaqueratosis, acantosis, papilomatosis e inflamación crónica. Materiales y métodos: Se estudió con microscopia óptica de alta resolución (MOAR), la epidermis de pacientes portadores de Psoriasis, analizando, la diferenciación epitelial y las posibelas alteraciones estructurales de los queratinocitos. Resultados: Los especímenes correspondieron a 10 pacientes con diagnóstico clínico e histopatológico de psoriasis. Las biopsias de piel fueron fijadas en solución de glutaraldehido buffer-collidina durante 48hs, procesados con la técnica para MOAR y coloreados con azul de toluidina, azul de metileno, fuscina básica y metenamina-plata. Los elementos epiteliales basales presentaban núcleos ovoideos, varios de ellos con nucleólos prominentes. En 7 de los casos estudiados el estrato granuloso estuvo ausente y en los 3 casos restantes observamos retención de núcleos y nucleólos. Conclusiones: Seobservaron queratinocitos con anfofilia e infiltrado linfocitario y de macrofagos.

Skin tissue from patients with Psoriasis was analyzed using HROM (High Resolution Optical Microscopy), studying epithelial differentiation and possible structural alterations of the queratinocytes. The samples were taken from 10 patients with histopathologic diagnosis of Psoriasis. This tissue samples where affixed with glutaraldehide buffer-collidine for 48 hours. Later processed with the HROM technique and colored with toluidine blue, metilene blue, basic Fuscine, and silver metenamine. The basal epithelial elements presented ovoid nucleus and most of them had prominent nucleolus. In 7 of the studied cases, the granulose stratus was absent, and thinner in the rest, with nucleus and nucleolus retention. At this level queratinocytes where observed with perinuclear anfofilia, as well as linfocitic and macrophagic infiltrate and union complex where elongated.

Vascular endothelium express CS-1 fibronectin in allergic contact dermatitis.

BACKGROUND: Allergic contact dermatitis (ACD) is a common human dermatosis in which not all the mechanisms involved in its pathogenesis have been elucidated. OBJECTIVE: To study the expression of CS-1 fibronectin, TARC and Th1-associated chemokine receptors in biopsies from allergic patch test reactions. MATERIAL AND METHODS: Thirteen patients already diagnosed with ACD were challenged on the back with the antigen responsible of the disease and macroscopic responses and biopsies taken after 48 h. Skin biopsies from negative control challenge sites, AD and ICD were also taken. Samples were fixed, embedded in paraffin wax and processed in order to perform histological and immunohistochemical studies. RESULTS: All subjects with ACD showed a positive clinical response and a perivascular mononuclear cell infiltration at 48 h, which was not seen in the negative controls. The majority of skin-infiltrating cells were CD4+ and CD8+ and up to 54% or 40% of them expressed CXCR3 or CCR5, respectively. We also showed expression of CS-1 fibronectin in inflamed endothelial cells not only in ACD but also in AC and ICD. In contrast TARC was only expressed in ACD and AC. CONCLUSION: We showed for the first time that CS-1 fibronectin is expressed in dermal vessels from allergic patch tests positive reactions, as well as irritant and atopic skin lesions.

A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold.

Cultured CO2-sensitive neurons from the ventrolateral medulla of newborn rats enhanced their bioelectric activity upon intracellular acidification induced by inhibition of the Na+/H+ exchanger type 3 (NHE3). Now we detected NHE3 also in the medulla oblongata of adult rabbits. Therefore, this animal model was employed to determine whether NHE3 inhibition also affects central respiratory chemosensitivity in vivo. Seven anesthetized (pentobarbital), vagotomized, paralyzed rabbits were artificially ventilated with O2-enriched air. From the phrenic nerve compound discharge, integrated burst amplitude (IPNA), respiratory rate (fR), and phrenic minute activity (IPNA. fR) were taken as measures of central respiratory rhythm and drive. Effects of potent NHE3 inhibition with the novel brain permeant substance S8218 were studied by comparing respiratory characteristics before and after up to 9.2 +/- 1.1 mg/kg cumulative drug application, yielding average plasma concentrations of 0.9 +/- 0.2 microg/ml. In response to S8218, the baseline level of IPNA. fR was significantly enhanced by an average of 51.0 +/- 6.4% (n = 27, p < 0.0001). The influence of NHE3 inhibition on the respiratory CO2 response was studied at plasma concentrations of S8218 maintained in the range of 0.3 microg/ml (10(-6) M). Although the metabolic acid-base status thereby remained widely unchanged, the group mean apneic threshold PaCO2 was significantly lowered by 0.45 +/- 0.11 kPa (n = 7, p < 0.01), whereby in four of seven animals even strong hyperventilation failed to suppress phrenic nerve rhythmicity completely. Likewise, S8218 significantly augmented IPNA. fR, in the range of PaCO2 between 1 and 6 kPa above threshold, by an average of 38.0 +/- 8.5% (n = 35, p < 0.0001). These in vivo results are compatible with the effects of NHE3 inhibition on chemosensitive brainstem neurons in vitro. Moreover, rhythmogenesis is supported through NHE3 inhibition by lowering the threshold PCO2 for central apnea.

[Granulosa cell tumors: structural and ultrastructural analysis]. / Tumores de células de la granulosa: análisis estructural y ultraestructural.

UNLABELLED: These ovarian neoplasm derive from the ovarian stromal component constituting around the 5 to 12% of all ovarian tumors. OBJECTIVE: To examine the histopathological and ultrastructural morphologic characteristic of the neoplastic cells and the patognomonic element of these tumors: Call Exner's Bodies MATERIALS AND METHODS: The materials corresponded to 2 women of 52 and 55 years. The syntomatology was abdominal tumor that went in increase. The materials were fractioned for the histopathological conventional study and for ultrastructural analysis. For this last one, they were fixed in Karnovsky, refix in osmio and included in Araldita. RESULTS: By means of the different observations it was determined in both cases the nuclear atipia, indentations nuclei and prominent nucleoli. In one of the cases the presence of Bodies of Call-Exner was detected, and ultrastructurally was compound by whirled fibrils and amorphous material, with dense structures electron to its around. It was interest the infiltrated of plasmatic cells around the tumors cells. These neoplasms are of interest due to their impredictible behavior and to the hormonal production that can originate alterations in other organs of the genital apparatus.

Tumores de células de la granulosa: análisis estructural y ultraestructural. / [Granulosa cell tumors: structural and ultrastructural analysis]

These ovarian neoplasm derive from the ovarian stromal component constituting around the 5 to 12

of all ovarian tumors. OBJECTIVE: To examine the histopathological and ultrastructural morphologic characteristic of the neoplastic cells and the patognomonic element of these tumors: Call Exners Bodies MATERIALS AND METHODS: The materials corresponded to 2 women of 52 and 55 years. The syntomatology was abdominal tumor that went in increase. The materials were fractioned for the histopathological conventional study and for ultrastructural analysis. For this last one, they were fixed in Karnovsky, refix in osmio and included in Araldita. RESULTS: By means of the different observations it was determined in both cases the nuclear atipia, indentations nuclei and prominent nucleoli. In one of the cases the presence of Bodies of Call-Exner was detected, and ultrastructurally was compound by whirled fibrils and amorphous material, with dense structures electron to its around. It was interest the infiltrated of plasmatic cells around the tumors cells. These neoplasms are of interest due to their impredictible behavior and to the hormonal production that can originate alterations in other organs of the genital apparatus.

Nutritional immunomodulation leads to enhanced allograft survival in combination with cyclosporine A and rapamycin, but not FK506.

BACKGROUND: Recently, specific immunonutrients were found to increase experimental allograft survival when combined with cyclosporine A (CsA). This study compared the effect on rat cardiac allograft survival when nutritional immunomodulation was used with CsA, rapamycin (Rapa), or tacrolimus (FK506). METHODS: Intra-abdominal ACI to Lewis cardiac allografts were performed and assessed daily by palpation. Study groups included untreated controls and those receiving CsA, Rapa, or FK506. Rats were fed ad libitum with Impact diet (fortified with fish oil, arginine, and RNA) or standard rat food. Further study groups were transplanted that received a donor-specific transfusion in addition to immunosuppression and diet. RESULTS: Allograft survival was extended by combining Impact with CsA (45.3+/-19 days) and Rapa (165.3+/-52 days), but not FK506 (12.4+/-3.2 days). Mean graft survival in the Rapa/Impact group met criteria for functional tolerance. The addition of a donor-specific transfusion did not lead to graft survival advantages over similar groups not receiving a donor-specific transfusion. CONCLUSIONS: The use of immunonutrients improves transplant outcome in animals treated with short courses of CsA and Rapa, but not FK506. These findings highlight the potential differences in the effects of nutritional immunomodulation with different immunosuppressive drugs in the treatment of transplant patients.

Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro.

Clinical and experimental evidence suggests that granulocyte-colony stimulating factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Excessive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sepsis. Therefore, the aim of the present study was to investigate the influence of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and NO synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantitative analyses of iNOS cDNA revealed that G-CSF significantly reduced interferon-gamma/lipopolysaccharide (IFN-gamma/LPS) dependent iNOS gene expression (P < 0.05) following 6, 18, 24, and 48 h incubation periods. In addition, the co-application of G-CSF resulted in a decreased IFN-gamma/LPS mediated iNOS protein generation as detected by immunoblotting methods after 24 and 48 h. Measurement of the stable NO metabolites showed a significant reduction of nitrite/nitrate concentrations following co-incubation of VSMC with G-CSF + IFN-gamma/LPS (242.57 +/- 10.73 nmol NO2-/NO3-/mg cell protein, n = 8) as compared to IFN-gamma/LPS treatment (306.20 +/- 19.26 nmol NO2-/NO3-/mg cell protein, n = 8, P < 0.05) following a 24-h incubation protocol. This inhibitory effect of G-CSF was still present after a 48 h incubation period (G-CSF + IFN-gamma/LPS: 319.56 +/- 6.26 nmol NO2-/NO3-/mg cell protein; IFN-gamma/LPS: 489.20 +/- 27.15 nmol NO2-/NO3-/mg cell protein (P < 0.05), n = 8, respectively). The present findings suggest that inhibition of iNOS gene expression and NO generation in VSMC might be one of the protective anti-inflammatory effects of G-CSF during sepsis.

Role of tumor necrosis factor alpha in hyperthermia-induced apoptosis of human leukemia cells.

We used the human myelomonoblastic leukemia cell line PLB-985 to study the effects of temperatures ranging from 37 degrees C to 43 degrees C for 1 h on the induction of apoptosis and cell cycle distribution in leukemia cells. The threshold temperature for the onset of apoptosis was 42 degrees C. Whereas hyperthermia exerted no effect on the expression of Bcl-2 and Bax, heat induced a >30-fold increase of tumor necrosis factor (TNF) alpha mRNA expression and a significant increase in TNF-alpha protein secretion. This endogenous production of TNF-alpha correlated directly with the temperature-induced apoptode effect. Blocking TNF-alpha expression via treatment with pyrrolidinedithiocarbamate or blocking TNF-alpha activity with neutralizing antibodies abrogated heat-provoked apoptosis. In addition, exposure of cell culture supernatant of heat-treated PLB-985 cells to untreated cells induced an apoptotic effect. These data indicate a TNF-a-mediated self eradication of the leukemia cells after heat exposure. Inducing apoptosis with wild-type TNF-alpha or p55 and p75 protein muteins demonstrated that this effect was mediated by the p55 receptor. Interestingly, the autocrine suicidal loop found in immature leukemia cells was lost after granulocytic differentiation with 0.5% N,N-dimethylformamide. These data should be of critical importance for the understanding of the biological impact of fever as well as for developing therapeutic approaches to malignant diseases

[Differential expression of mucin carbohydrates in human endometria]. / Expresión diferencial de los carbohidratos de mucina en endometrios humanos.

The use of Lectins to identify oligosaccharides in mucin substances has been increased by the role played by cell surface carbohydrates in invasion and metastasis processes. We studied in this work normal endometrial tissue, with benign and malignant entities in search for the presence of the Galactose beta 1-3 N Acetylgalactosamine(Gal beta 1-3 GalNAC alpha and Galactose beta 1-3 N Acetylgalactosamine (Gal beta 1-3 alpha and beta) using the Lectins: Agaricus bisporus (ABL) and Arachis hipogea (PNA) respectively. The specific control were baths with galactose for PNA and with porcine stomach mucin for ABL. The use of these two Lectins allowed to differentiate substances bonded or non bonded to Sialic Acid, since PNA fails to label when the oligosaccharide is bonded to this acid Sialic. Significant differences were noticed on the bonding patterns of both Lectins on tissues with benign, malignant and normal entities. In this latter case the labelling was always continuous in both Lectins whereas it was irregular in the carcinoma.

Expresión diferencial de los carbohidratos de mucina en endometrios humanos. / [Differential expression of mucin carbohydrates in human endometria]

The use of Lectins to identify oligosaccharides in mucin substances has been increased by the role played by cell surface carbohydrates in invasion and metastasis processes. We studied in this work normal endometrial tissue, with benign and malignant entities in search for the presence of the Galactose beta 1-3 N Acetylgalactosamine(Gal beta 1-3 GalNAC alpha and Galactose beta 1-3 N Acetylgalactosamine (Gal beta 1-3 alpha and beta) using the Lectins: Agaricus bisporus (ABL) and Arachis hipogea (PNA) respectively. The specific control were baths with galactose for PNA and with porcine stomach mucin for ABL. The use of these two Lectins allowed to differentiate substances bonded or non bonded to Sialic Acid, since PNA fails to label when the oligosaccharide is bonded to this acid Sialic. Significant differences were noticed on the bonding patterns of both Lectins on tissues with benign, malignant and normal entities. In this latter case the labelling was always continuous in both Lectins whereas it was irregular in the carcinoma.

Neopterin-induced tumor necrosis factor-alpha synthesis in vascular smooth muscle cells in vitro.

Synthesis and secretion of proinflammatory mediators like tumor necrosis factor-alpha and neopterin are common events in severe systemic inflammatory disorders, e.g. sepsis and septic shock. Recent data suggest that both substances show similarities with respect to their bioactivities. In the present study we investigated the potential interactions of neopterin and tumor necrosis factor-alpha on inducible nitric oxide synthase gene expression and nitric oxide generation in rat vascular smooth muscle cells. In addition, we studied the influence of neopterin on tumor necrosis factor-alpha synthesis in this cell type. Single stimulation of smooth muscle cells with either neopterin or tumor necrosis factor-alpha caused inducible nitric oxide synthase gene expression and nitric oxide production. Coincubation of cells with both compounds resulted in at least additive effects on nitric oxide synthesis. Quantification of tumor necrosis factor-alpha cDNA revealed a dose-dependent effect of neopterin on tumor necrosis factor-alpha gene expression. Similar results were obtained concerning the detection of tumor necrosis factor-alpha protein and the assessment of tumor necrosis factor-alpha bioactivity. These data suggest that neopterin and tumor necrosis factor-alpha are closely associated with regard to synthesis and effects, respectively. The interactions of both inflammatory mediators in vascular smooth muscle cells might contribute to the excessive release of nitric oxide observed during sepsis, thus triggering cellular destruction and multiple organ failure.

Neopterin induces nitric oxide-dependent apoptosis in rat vascular smooth muscle cells.

Numerous studies indicate that proinflammatory substances like tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) as well as macrophage-derived neopterin are increased in atherosclerotic tissue and thus are potentially involved in the process of atherogenesis. Since apoptotic death of vascular smooth muscle cells (VSMC) is reported to occur in atherosclerotic lesions, we investigated the effects of neopterin, TNF-alpha, and IFN-gamma on apoptosis in cultured VSMC. Morphological changes characteristic of apoptosis as well as DNA fragmentation were detected in cells treated with neopterin, TNF-alpha/IFN-gamma, and neopterin + TNF-alpha/IFN-gamma. Simultaneously, neopterin, TNF-alpha/IFN-gamma, and neopterin + TNF-alpha/IFN-gamma led to inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) synthesis. NO generation was significantly reduced when cells were cotreated with the competitive iNOS inhibitor aminoguanidine. This was accompanied by decreased percentual apoptosis as detected by FACS analysis using all kinds of stimuli. We conclude that neopterin as well as TNF-alpha/IFN-gamma are potent mediators of apoptotic death in VSMC which is at least in part triggered by NO synthesis induced by these proinflammatory mediators.