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Influence of CO2 pneumoperitoneum on systemic and peritoneal cell-mediated immunity.

Port site metastases could be due to mechanical reasons or impairment of host defenses. As it is known that carbon dioxide is toxic for lymphocytes in vitro we decided to investigate lymphocyte stress during laparoscopy. Blood samples and peritoneal fluids were obtained before and after pneumoperitoneum from 16 patients undergoing laparoscopic cholecystectomy. Lymphocyte subsets were determined by flow cytometry. Propidium iodide was used as a lymphocyte vitality test. Cytokines were measured by an ELISA system. Significant falls in the absolute lymphocyte count and T3 and T4 lymphocytes occurred on postoperative day 1 with a quick return to the preoperative value on day 2. T8, natural killer cells, T4/T8, and T4+/T8+ counts were stable. Interleukins 1 beta and 6 and tumor necrosis factor-alpha were depressed during the two postoperative days. Peritoneal lymphocytes were not destroyed by pneumoperitoneum as demonstrated by the propidium test, nor were they locally impaired by carbon dioxide. The circulating lymphocyte subpopulation decrease favors moderate, brief immunodepression. The origin of port site metastases is not immunologic depression but, rather, facilitated implantation of malignant cells by hyperpressure into raw tissues.

Carbon Monoxide↗

Thyroid hormone responsive protein (THRP) mediates thyroid hormone-induced cytotoxicity in primary neuronal cultures.

The thyroid hormone responsive protein (THRP) is a novel gene product that remains responsive to thyroid hormone (TH) in the cerebral cortex of adult rats. To study the effects of THRP on neuronal cell survival, primary neurons cultured from rats at embryonic day 19 were treated with either 10(-7) mol L(-1) 3,5,3'-triiodothyronine (T(3)), or 10(-7) mol L(-1) L: -thyroxine (T(4)). This resulted in decreasing neuronal cell number starting 48 h after treatment. T(3) -related cytotoxicity was also documented by measurement of lactate dehydrogenase release into the medium and by propidium iodide staining. Treatment of cells with 10(-7) mol L(-1) T(3) resulted in a significant increase in THRP mRNA levels as early as 24 h of treatment in a concentration-dependent manner. T(3) treatment did not alter glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA levels. Exogenous expression of THRP by transfecting cells with a THRP expression construct (pSVL-THRP) was associated with a significant increase in cell death as measured by the increased number of propidium iodide staining cells (18.0+/-2.1 cells per field) compared with mock-transfected cells (3.3+/-0.2), P<0.002. To further document THRP-induced cytotoxicity, the cells were either transfected with pSVL (empty vector)+pSV2neo (neomycin resistance vector for cell labeling), pSVL-THRP+pSV2neo, or pSVL-THRP+pc-Abl (cAbl tyrosine kinase expressing vector)+pSV2neo. After 24 h the cells were treated with 500 microg mL(-1) G418 (a congener of neomycin) to eliminate the non-transfected cells. Transfection with pSVL-THRP reduced neuronal survival relative to cells transfected with pSVL (356+/-15.6 compared with 145+/-16.9, P<0.05). Co-transfection of THRP with wild-type c-Abl did not alter the effect of THRP on cell survival. It is concluded that THRP is an important factor in TH-induced neuronal cell death.

Animals↗

Methylene blue is more toxic to erythroleukemic cells than to normal peripheral blood mononuclear cells: a possible use in chemotherapy.

Methylene blue (MB) is a phenothiazine with radio and photosensitizing properties and anti-tumoral activity. Our group has shown that MB was capable of inhibiting the in vitro growth of erythroleukemic cells with multidrug resistance (MDR). However, there are no studies comparing the cytotoxicity of this molecule for normal and tumoral cells. In this work, the cytotoxicity of MB was measured by MTT method in erythroleukemic and melanoma lineages, comparing it with that of normal cells:lymphocytes and melanocytes. MB was more cytotoxic for tumoral cells; however, there was no difference between erytroleukemic cells with or without MDR phenotype. Lymphocytes and erythroleukemic cells were much more sensitive to the effects of MB than melanoma cells and melanocytes. The proliferation of phytohemagglutinin-activated lymphocytes was inhibited when 3H-thymidine incorporation to DNA was measured. We tried to analyze whether the cells were dying, via apoptosis or necrosis, using Anexin-V and propidium iodide. Despite higher levels of Anexin-V, it was not possible to distinguish necrosis from apoptosis, as the fluorescence of MB is in the same channel as propidium iodide. The production of hydrogen peroxide was measured by cytometry using dihydrorhodamine 123 (DHR). Despite the erythroleukemic cells and lymphocytes being capable of producing free radicals, there was no relation between the production and the sensitivity of various cells to MB. Our results suggest that MB should be used as a chemotherapeutic agent, because of its preferential cytotoxic effects over tumor cells, considering the fact that MDR cells are also sensitive, and due to its radio and photosensitizing activities.

Antineoplastic Agents↗

Antitumor activity of a phenoxazine compound, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one against human B cell and T cell lymphoblastoid cell lines: induction of mixed types of cell death, apoptosis, and necrosis.

PURPOSE: We studied the antitumor activity of 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx), which was synthesized by the reactions of 2-amino-5-methylphenol with bovine hemoglobin, on human B cell lymphoblastoid cell lines, P3HR-1 and Raji derived from African Burkitt's lymphoma, and the human T cell lymphoblastoid cell line Molt-4. We also studied whether Phx might cause apoptosis and necrosis in these cells. METHODS: We evaluated cell viability and apoptosis and necrosis of the cells in the presence of Phx, by using agarose gel electrophoresis, flow cytometry, and fluorescence microscopy. RESULTS: Phx suppressed the viability of P3HR-1, Raji, and Molt-4 cells, though the suppression patterns were different, i.e., Phx suppressed the viability of P3HR-1, Raji, and Molt-4 cells at higher concentrations, while the drug enhanced the viability of Raji cells, but not those of P3HR-1 and Molt-4 cells at lower concentrations. To investigate which type of cell death - apoptosis or necrosis - is induced by Phx, induction of DNA ladder, phosphatidylserine externalization, and propidium iodide-permeable cells were examined in Phx-treated cells. Although Phx did not induce DNA ladder formation, it induced the phosphatidylserine externalization and propidium iodide-permeable cells, suggesting that Phx caused a mixed type of cell death, both apoptosis and necrosis. The population of early stage apoptotic cells was dominant in Raji cells, and that of the late stage apoptotic/necrotic cells was dominant in Molt-4 cells after 72-h treatment with Phx. The population of the early stage apoptotic cells and the late stage apoptotic/necrotic cells was almost equal in P3HR-1 cells in the presence of Phx, though the population of both types of cells increased with time. The nuclear morphological analysis of Phx-treated Raji, P3HR-1, and Molt-4 cells also showed that Phx induces apoptosis. CONCLUSIONS: The present results suggest that Phx shows antitumor activity against human B cell-derived and T cell-derived lymphoblastoid cell lines, in vitro, causing apoptosis and necrosis.

Annexin A5↗

A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.

Animals↗

Noncolorimetric measurement of cell activity in three-dimensional histoculture using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide: the pixel image analysis of formazan crystals.

We describe a novel system for measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction in three-dimensional histoculture which is no longer dependent on colorimetric determination of extracted formazan, but rather is based on a pixel image analysis of formazan crystals, and which allows intratumor heterogeneity to be taken into account. The MTT test is based on the enzymatic reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide to formazan crystals by living, metabolically active cells, but not in dead cells. The reaction was carried out in situ in six-well plates on gel-supported histocultured human tumors. After a 24-h incubation with different drugs the tumors were incubated with a solution of MTT. Frozen sections of the tumor pieces were made and the slides were then stained with a propidium iodide solution, whose fluorescence is proportional to the number of cells present. We demonstrate here that the formazan crystals, formed by MTT reduction, reflect polarized light and that this can be quantified by using an image analysis system based on bright-pixel quantitation directly on a frozen section of the original tissue. Combined with the use of the fluorescent dye propidium iodide, also measured by pixel analysis, we can express a ratio between the total amount of MTT reduction and the total number of cells present in the specimen that expresses the effect of drugs on the histocultured tumors. Since histology is well maintained in histoculture it is possible to take into account the heterogeneity present in the tumor with regard to drug response.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗

Induction of cytoskeletal rearrangements and loss of volume regulation in epithelial cells by Treponema denticola.

The early responses of oral epithelial cells to the adhesion of the oral spirochaete Treponema denticola were studied as a model of microbial perturbation of the plasma membrane. KB cell (ATCC CCL 17) monolayers were incubated with T. denticola (ATCC 35405) in alpha-MEM (minimal essential medium) for periods of 1-4 h at 37 degrees C without serum. Control cultures were exposed to bacteria-conditioned alpha-MEM without serum or bacteria or to alpha-MEM alone. At the end of each incubation, detached and attached epithelial cells were harvested and analysed separately. Compared with controls, T. denticola induced in 25% of cells a two-fold, time-dependent increase of detachment by 4 h. Detached cells in both T. denticola-exposed and control cultures exhibited 25% reductions in modal diameter, did not exclude propidium iodide, did not readhere, and did not form colonies. In T. denticola-exposed cultures, a larger subset (75%) of cells remained attached to the substratum, demonstrated no significant reduction of colony-forming efficiency and excluded propidium iodide. However, these cells exhibited a 21% reduction in diameter (p < 0.05), a 60% decrease of F-actin (p < 0.001), and a 74% reduction in the proportion expressing desmoplakin II (p < 0.01) after exposure to T. denticola. Flow cytometry showed a small (14%) but significant (p < 0.001) reduction in mean fluorescence intensity due to keratin expression in T. denticola-treated cultures. Exposure of cells to anisosmotic media demonstrated that, in contrast to controls, cultures challenged by bacteria failed to undergo compensatory volume regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

Electroporation of extraneous proteins into CHO cells: increased efficacy by utilizing centrifugal force and microsecond electrical pulses.

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.

Animals↗

A rapid micromethod for evaluating T cell subsets in blood using monoclonal antisera.

A simple accurate method is described for enumerating T cell subsets in whole blood. The method, which depends on indirect immunofluorescence using biotin-coupled monoclonal antisera and fluorescein-coupled avidin, and propidium iodide for nuclear counterstaining, was compared with the conventional method based on initial separation of lymphocytes by density flotation and exposure to monoclonal antisera. Accurate identification of mononuclear cells in whole blood by nuclear staining with propidium iodide was established. The whole blood method gave numbers for T cell subpopulations generally comparable with those obtained by the conventional method, but slightly higher numbers of Leu2a+ cells were found by the whole blood method, and shown to be higher because of selective loss of Leu2a+ plastic-adherent cells in the conventional method. The whole blood method is quicker, uses only 0.5 ml blood and is economical in use of monoclonal reagents.

Antibodies, Monoclonal↗

A flow cytometric technique for simultaneous analysis of human mononuclear cell surface antigens and DNA.

A method for dual staining of mononuclear cells for lymphocyte phenotypic markers and DNA is described. The cells were stained with fluorescein-conjugated monoclonal antibodies and then rendered permeable to propidium iodide using saponin. Propidium iodide stains DNA and, using flow cytometry, cell cycle analysis of individual lymphocyte subpopulations can be determined. Saponin acts within 1 min, preserves expression of surface antigens and is effective at all concentrations from 0.001% to 1%. This technique is simple, rapid and gives reproducible results.

Antigens, Surface↗

Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods.

The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a 'sub-G1' peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.

Animals↗

Production of frameshift mutations in Salmonella by phenanthridinium derivatives: enzymatic activation and photoaffinity labeling.

The effect of metabolic activation on the mutagenic potential of some phenanthridinium compounds was examined in Salmonella typhimurium strains TA1538 and TA1978 . All of the compounds tested were mutagenic in TA1538, a DNA excision-repair-deficient strain, when metabolizing enzymes were included in the assay. Reversions were not detected when these compounds were examined under the same conditions in TA1978 , the isogenic strain of TA1538 proficient in DNA repair. The mutagenic activity of an azido analog of propidium iodide was also examined using photoactivation and enzymatic activation, and with both conditions, reversions were observed in TA1538 but not in TA1978 . Furthermore, the ranking of mutagenic activity of propidium azide relative to ethidium azide analogs was comparable for both types of activation. The evidence from several studies suggests that the structural requirements for mutagenic activity for this series of phenanthridinium compounds appear to be the same whether mutagenesis is induced via photoactivation or metabolic activation. The interaction with DNA resulting in covalent alteration of the DNA is implicated as the mutagenic mechanism whether the active species is generated by metabolic- or photo-activation.

Affinity Labels↗

A method for specific transmitter identification of retrogradely labeled neurons: immunofluorescence combined with fluorescence tracing.

In the present article a method is described which allows the delineation of the projections of a single neuron as well as the identification of one or more of its chemical components. The technique is a combination of retrograde tracing and fluorescent dyes based on the work of Kuypers and collaborators and indirect immunofluorescence histochemistry as originally described by Coons and collaborators. The crucial parameters including the selection of the dyes, the injection technique and tissue processing as well as the appropriate immunohistochemical fluorescent markers and filter combinations are discussed. The method of choice involves the use of the retrogradely transported dyes Fast Blue, True Blue or Propidium Iodide, and in addition, for double labeling experiments, Diamidino Yellow or Primuline. They are combined with FITC (Propidium Iodide) or TRITC (Fast Blue, True Blue, Diamidino Yellow, Primuline) as immunofluorescence markers.

Animals↗

Ischemia-induced death of astrocytes and neurons in primary culture: pitfalls in quantifying neuronal cell death.

We have demonstrated that both astrocytes and cerebellar granule cell neurons die during an ischemic insult only when there is complete loss of mitochondrial membrane potential. This was determined by comparing the ability of mitochondria to sequester rhodamine 123 with the ability of cells to exclude propidium iodide. We have also demonstrated that in astrocyte cultures cellular LDH loss correlates directly with propidium iodide uptake, i.e. cell death, and inversely with rhodamine 123 uptake. Thus, both LDH loss and rhodamine 123 uptake can be used to quantitatively measure astrocyte cell death in culture; however, this is not the case with neuronal cultures. It was demonstrated that even in highly enriched cerebellar granule cell neuron cultures where astrocytes comprise less than 10% of the total culture protein, approximately 40% of total culture LDH was in the astrocytes. This was also the case with rhodamine 123 sequestration. Caution must therefore be used when using the LDH technique to determine the proportion of neurons that have died in such enriched neuronal cultures.

Animals↗

Adenosinergic modulation of CA1 neuronal tolerance to glucose deprivation in organotypic hippocampal cultures.

Glucose deprivation produced neuronal degeneration of CA1 pyramidal neurons in hippocampal slice cultures. The effects of the adenosine agonist cyclohexyladenosine (CHA) and antagonist cyclopentylxanthine (CPX) on CA1 neuronal loss following hypoglycemia was examined using propidium iodide fluorescence as an indicator of cell death. The intensity of propidium iodide fluorescence in hippocampal area CA1 was quantified using Optimas image analysis software. Following 2 or 3 h of glucose deprivation, CPX significantly enhanced injury in the CA1 region while CHA provided significant protection. These results suggest that adenosine plays an important role in endogenous neuronal protection during hypoglycemic injury, and also supports a role for the use of adenosine agonists as neuroprotective agents.

Adenosine↗

Substance P-containing primary sensory neurons projecting to the inferior mesenteric ganglion: evidence from combined retrograde tracing and immunohistochemistry.

The origin of substance P-containing fibres in the inferior mesenteric ganglion of the guinea-pig was studied by combining retrograde tracing and indirect immunofluorescence techniques. 'True Blue' or propidium iodide was injected into the inferior mesenteric ganglion. Four days later the animals were perfused with formalin and the dorsal root ganglia L2 and L3 were dissected out. Sections of the spinal ganglia were cut on a cryostat and analysed in a fluorescence microscope with appropriate filter combinations and subsequently processed for indirect immunofluorescence using antiserum to substance P. Several ganglion cells containing both dye (True Blue or propidium iodide) and substance P-like immunoreactivity were observed, strongly supporting the view that at least some of the substance P fibres in the inferior mesenteric ganglion are of sensory nature. Taken together with physiological results from other workers, the present findings suggest that substance P can be released from peripheral, sensory nerve endings, inducing changes in the membrane potential of sympathetic neurons.

Animals↗

Differential radioprotection of cultured human diploid fibroblasts and fibrosarcoma cells by WR1065.

The present studies were performed to determine whether WR1065, the dephosphorylated, free-thiol active metabolite of WR2721, could provide differential radioprotection of normal and tumor cell lines in vitro and secondly to investigate potential mechanisms for the selective nature of the radioprotection at the cellular and molecular level. When 4 mM WR1065 was administered 30 min prior to and during irradiation, a protection factor of 1.9 was obtained in clonogenic assays performed with normal human diploid fibroblasts (AG1522) while no protection of fibrosarcoma cells (HT1080) was observed. Some radioprotection of fibrosarcoma cells was observed with higher drug concentrations (10-40 mM), but the increase in survival was considerably less than the plateau level reached with the diploid fibroblasts (3-fold vs 24-fold at 6 Gy). The observation of such a selective effect in vitro with WR1065 indicates that differences in tissue-specific variables such as blood flow, pH, pO2, and drug dephosphorylation cannot solely account for the selective nature of the radioprotection afforded by WR2721 in vivo. Incubation of nucleoids with increasing concentrations of the DNA intercalating dye propidium iodide was used to titrate the ability of DNA to undergo supercoiling changes. The relaxation and rewinding of supercoiled DNA loops in isolated nucleoids serves as an indicator of both the presence of DNA damage and inherent differences in DNA loop characteristics. Fibrosarcoma cells had a much larger propidium iodide-relaxable DNA loop size than fibroblasts. The rewinding phase of the DNA supercoiling response is impaired by the presence of radiation-induced DNA strand breaks. Four mM WR1065 resulted in a significant reduction in the amount of rewinding inhibition observed after a dose of 10 Gy in diploid fibroblasts (protection factor = 1.43) but did not alter the response of irradiated fibrosarcoma cells. These results, indicating that WR1065 had a preferential radioprotective effect in vitro on both survival and the manifestation of DNA damage at the nucleoid level, are consistent with the hypothesis that cell type differences in chromatin organization and DNA-drug associations could play a role in the selective radioprotection.

Cell Survival↗

Cholinergic systems in the rat brain: II. Projections to the interpeduncular nucleus.

The cholinergic innervation of the interpeduncular nucleus was investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Following propidium iodide or Evans Blue infusion into the interpeduncular nucleus, brains were processed for co-localization of transported fluorescent label and ChAT and AChE. Control infusions of tracers were made into the ventral tegmental area. In order to delimit the course of putative cholinergic afferents to the interpeduncular nucleus from extra-habenular sources, knife cuts surrounding the habenular nuclei were performed. Somata containing propidium iodide that were highly immunoreactive for ChAT were found primarily in the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, and the dorsolateral tegmental nucleus, also referred to as the laterodorsal tegmental nucleus. A few such co-labeled somata were also detected in the medial septal nucleus, substantia innominata, nucleus basalis, and pedunculopontine tegmental nucleus. A good correlation was observed between intensely-staining, AChE-containing and ChAT-positive neurons projecting to the interpeduncular nucleus from the aforementioned structures. Although the medial habenula contained numerous cells demonstrating transported label following interpeduncular infusion of fluorescent tracers, the ChAT-positivity associated with somata in that nucleus was weak compared to ChAT-like immunoreactivity in known cholinergic neurons in the basal forebrain and brainstem. Knife cuts that separated the habenular nuclei from the stria medullaris and neural regions lateral and posterior to those nuclei while leaving the fasciculus retroflexus intact resulted in a reduction of ChAT-like immunoreactivity in the medial habenular nucleus, fasciculus retroflexus, and interpeduncular nucleus. These data suggest (1) that the cholinergic innervation of the interpeduncular nucleus derives primarily from ChAT-positive cells in the basal forebrain and dorsolateral tegmental nucleus and (2) that putative cholinergic fibers having their origin in the medial habenula, if they exist, constitute a minor portion of the cholinergic input to the interpeduncular nucleus.

Acetylcholinesterase↗