In vitro culture of human peritoneal fluid cells

The author has studied the behaviour of cells from human ascitic fluid in long-term culture (5-6 months). Three cellular types are described with different morphological features, namely the cellular shape, the fashion in which the cell spreads on the glass, the nucleocytoplasmic ratio, the chromatin appearance and the abundance of mitochondria. The three cellular types can phagocytose, but each one in a different way. The first one phagocytoses exclusively erythrocytes 'by contact' without emission of pseudopods; the second one phagocytoses degenerating nucleated cells in the same way as the first; the third type phagocytoses degenerating nucleated cells by emission of long pseudopods. The origin of these three cellular types is discussed; it is felt that they are transformed mesothelial cells. According to this study, it cannot be excluded, especially for the second and the third type, that they are histiocytes coming from serous membranes. The life in vitro of the three cellular types is depending upon the composition of nourishing medium. Cells can divide by mitosis only during the first 10 - 15 days of culture (mitotic index 0.1-3.0(0/00). Nuclear amitosis, nucleolus expulsion into cytoplasm and cytoplasmatic DNA synthesis can be observed in healthy cells.     

Function of peritoneal exudate cells after abdominal surgery

Peritoneal macrophages and polymorphonuclear neutrophils are key cells in the repair of postoperative injury. Increased numbers of macrophages migrate into the peritoneal cavity after operation and the function of these cells changes over the postoperative interval. Macrophage activities, such as respiratory burst, arachidonic acid metabolism, monokine secretion, and plasminogen activator inhibitory activity, are elevated by peritoneal operation. However, the secretion of plasminogen activator activity is decreased after operation. The kinetics with which each of these functions changes varies with the parameter examined, indicating a complex regulation of the differentiation of leukocytes after operation. In addition, the activity of postoperative macrophages can be modulated in vitro by exposure to cytokines and conditioned media from polymorphonuclear neutrophils and macrophages. Thus, cell-cell interactions and factors secreted within the peritoneal cavity may regulate the contribution of postoperative leukocytes to peritoneal repair after operation.     

The in vitro effect of hydrocortisone on endocytosis in cultured mouse peritoneal macrophages

Three juvenile patients with cerebellar astrocytomas which have seeded the spinal subarachnoid space are presented. Histologic verification of the similarity between the posterior fossa tumor and its spinal implant was obtained in two of the three patients. The cerebellar tumors in all cases have been benign (grade I),and the behavior, other than their seeding has also been indolent. Review of pertinent literature discloses no similar experience with cerebellar astrocytomas. Aggressive therapy is advocated for the rare patient with subarachnoid seeding from this benign lesion.     

Characterization of cell growth-inhibitory factor in inflammatory peritoneal exudate cells of rats

We characterized the nature and reaction mode of the cell growth-inhibitory factor (here designated CGIF) from rat peritoneal exudate cells (PEC). The soluble fraction separated from the lysate of Enterococcus faecalis-induced 24 hr PEC completely inhibited Con A-induced thymocyte mitogenesis. Gel filtration chromatography showed that CGIF has a molecular weight of approximately 23-25 kDa. Isoelectric focusing with Rotofor indicates that the factor has an isoelectronic point of 5.8-6.4. CGIF was inactivated by treatment at 70 C, for 30 min or by tryptic digestion, but the activity was not destroyed by the reduction with dithiothreitol. As well as thymocyte proliferation, CGIF completely suppressed 3H-thymidine incorporation of splenocytes which were stimulated by either Con A or LPS, suggesting the factor is effective on both T and B cells. The acting point of the inhibitor appeared to be a later stage of the lymphocyte activation sequence, since it was still effective when added 28.5 hr after the addition of Con A. CGIF also reduced the viability of these cells when added with mitogens such as Con A or LPS. CGIF thus appears to be distinct from interleukin-1 receptor antagonist or transforming growth factor-beta.     

In vitro evaluation of medicinal plant extracts against Pestalotiopsis mangiferae

A serious leaf-spot disease of Mangifera indica was noted during the last 10 years in Satpura plateau of India. On the basis of characteristic symptoms and cultural characters, the pathogen was identified as Pestalotiopsis mangiferae which is hitherto not reported from Satpura plateau of India. Screening of 17-medicinal plants against the test pathogen revealed 14 antimycotic whereas 3-plants, viz., Argemone mexicana, Caesalpinia bonducella, and Casia fistula acclerated the growth of the pathogen. The maximum activity was shown by Eucalyptus globulus (88%) and Catharanthus roseus (88%) followed by Ocimum sanctum (85.50%), Azadirachta indica (84.66%), Ricinus communis (75%) and Lawsonia inermis (74.33%) while the minimum activity was exhibited by Jatropha curcas (10%).     

In vitro effect of imipenem on Acinetobacter baumannii

In Salmonella Typhimurium, the stationary phase sigma factor RpoS regulates the expression of genes associated with adaptation, survival, and virulence. Expression of rpoS is known to be under environmental control and yet, to date, there have been no studies that assess the expression of this global regulator in real food systems. Skim milk represents a useful model food; using an spvRA::luxCDABE reporter construct, we have determined RpoS availability from an inoculum of Salmonella Typhimurium LT2. We report that RpoS activity increases exponentially at the end of the logarithmic phase of growth, consistent with data from nutrient media.     

In vitro culture of hamster ovarian primary interstitial cells: effect of serum

The function of ovarian interstitial cells has been largely addressed using rat theca-interstitial cell culture. However, this preparation is primarily enriched with theca and secondary interstitial cells, which make it difficult to address selectively the function of the primary interstitial cells. We have developed an in vitro culture of hamster ovarian primary interstitial cells. Cells were isolated from postnatal hamster ovaries by collagenase digestion and purified over a Percoll gradient. The preparation contained 90% viable, pure interstitial cells, which anchored to the plastic and glass culture surface in the presence of fetal bovine serum. Cell proliferation was noted in the presence of serum dosages higher than 0.2%; however, reduction of serum concentration to 0.1% or complete serum starvation did not affect cell viability but almost completely abolished cell proliferation as determined by [3H]thymidine incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage immunoreactivity, which corresponded to basal progesterone and androstenedione accumulation. Replacement of serum to starving cells resulted in the induction of the "S" phase and "M" phase specific cyclins, and resumption of cell proliferation. Our results indicate that hamster primary interstitial cells can be cultured in vitro as a monolayer, and the anchorage and proliferation of these cells depend on serum supplement; however, a viable monolayer can be maintained for several days without serum. This model will be useful for addressing the mechanisms of differentiation of ovarian interstitial cells.     

In vitro effect of disodium cromoglycate on phagocytic activity of polymorphonuclear cells of healthy asthmatic patients

Disodium chromoglicate interferes cellular membrane transport, inhibits mediators release and activation of polymorfonuclear cells (PMN) by blocking intracellular free calcium increment. In order to assess if DC reduces in vitro phagocytic activity of PMN, these cells obtained from allergic asthmatic patients and healthy subjects were incubated with DC or Hanks solution (HS). Their phagocytic activity was measured with chemoluminiscence technique. There were not significant differences in phagocytic activity between the cells incubated with DC and those incubated with HS, nor between cells from asthmatic patients and healthy subjects. We conclude that DC has not in vitro effect on phagocytic activity of PMN from healthy and asthmatic subjects. There are no difference in PMN phagocytic capacity between healthy and asthmatic subjects.     

In vitro antioxidant and cytotoxic activity of extracts of Baccharis coridifolia DC

Onchocercal keratitis (river blindness) is one of the leading worldwide causes of blindness. Light microscopic analysis of human specimens and corneal tissue from experimental models has implicated the eosinophil as an important cell in the inflammatory response. Our previous studies in experimental murine onchocercal keratitis have demonstrated that the inflammatory infiltrate is composed primarily of eosinophils displaying ring shaped or bilobed nuclei. However, a number of cells were not characterizable by light microscopy, presumably due to mechanical distortion. To more fully characterize the inflammatory cell infiltrate, we examined corneal specimens by transmission electron microscopy. In addition to typical eosinophils with bilobed and ring shaped nuclei, this approach revealed cells with variable nuclear morphology and cell shape which contained the dense cored granules characteristic of eosinophils. Hence, the degree of pleomorphism of eosinophils is broader than appreciated and underscores the importance of this cell in experimental murine onchocercal keratitis.     

The effect of mesothelium damage on peritoneal transport functions: in vitro studies

Bidirectional transport across rabbit parietal peritoneum of urea, uric acid, gentamycin and albumin were examined in control conditions and after mechanical or chemical mesothelium damage. The transport mean values, exprerssed as transport coefficients, of intact peritoneum amounted 1.37; 1.18; 4.30; 0.20 [10(-4) cm s-1] respectively. The destruction of mesothelial barrier increased, in similar range, both absorption and excretion component of the transport of urea, uric acid and albumin but not gentamycin. In the latter case, mesothelium injury enhanced peritoneal excretion by 86% and absorption by 162%. An asymmetry in gentamycin transport was observed which can be unfavourable for peritoneal dialysis patients.     

Cyclooxygenase and lipoxygenase arachidonate metabolites synthesized by mouse peritoneal macrophages: in vitro effect of N-phenyllinoleamide from toxic oil samples

N-phenyllinoleamide (NPLA) has been detected as extraneous compound in adulterated cooking oils associated with a unique epidemic disease known as the Toxic Oil Syndrome (TOS). In this communication we report on the action of NPLA on the endogenous cyclooxygenase and lipoxygenase arachidonate metabolism. Results show that mouse peritoneal macrophages (MPM) exposed to 1 mM NPLA for 2 h undergo significant increases of 6-keto prostaglandin F1a, prostaglandin E2, leukotriene B4, 12- and 15-hydroxyeicosatetraenoic acids. MPM prelabelled with 3H-AA showed an enhanced release when exposed to NPLA. Thus, it is concluded that NPLA potentiates AA release from cell membrane phospholipids and the subsequent cyclooxygenase and lipoxygenase oxidative metabolism of this precursor to various eicosanoids. This is in agreement with the implication of peroxidative process mediated by fatty acids anilides in TOS.     

Effect of peritoneal fluid and serum from patients with endometriosis on mouse embryo in vitro development

BACKGROUND AND OBJECTIVES: The purpose of this study was to determine the effect of pregnancy care within family practice on medical students' choices of family practice as a career and to determine the effect the degree of emphasis on pregnancy care has on students' choices of specific residency programs. METHODS: Eight hundred and ten randomly selected student members of the American Academy of Family Physicians (AAFP) and 805 randomly chosen members of the American Medical Student Association (AMSA) were sent an 11-item survey that asked how their career (specialty) and specific residency program choice would be affected if family practice residencies included more (or less) training in pregnancy and delivery management. RESULTS: Fifty-one percent of AAFP members and 37% of AMSA members would be less likely to enter family practice if pregnancy care was eliminated from the specialty. Six percent of AAFP members and 9% of AMSA members stated that they would be more likely to enter family practice if the specialty ceased this training. Students who plan to enter family practice favor a residency program with a stronger pregnancy care experience over a residency program with less emphasis on this training by a 10:1 ratio. CONCLUSION: This study shows significant medical student interest in a high level of pregnancy care experience in family practice training programs.     

In vitro effect of parachlorophenol and camphorated parachlorophenol on macrophages

The purpose of this study was to investigate the "in vitro" effect of parachlorophenol and camphorated parachlorophenol, used in endodontics for the disinfection of root canals, on the substrate adherence capacity of macrophages. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. As a test of macrophage phagocytic function, the adherence capacity of macrophages to a plastic surface was determined. Assays were conducted in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. Results showed that parachlorophenol and camphorated parachlorophenol significantly decreased the substrate adherence capacity of inflammatory macrophages. Taking into account that adhesion is the first step in the phagocytic process of macrophages and in antigen presentation, parachlorophenol and camphorated parachlorophenol could inhibit macrophage function and modulate immune and inflammatory reactions in periapical tissues.     

In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/paracrine role of leptin

The present study has examined the effects of the adipocyte-derived hormone, leptin, on lipolysis in fat cells of different types of mice. Exposure to leptin (1.25.10(-6) M to 1.25.10(-12) M) increased (P < 0.01) the lipolytic activity of fat cells obtained from lean mice. A greater stimulation was observed when adipocytes from ob/ob mice were examined. Throughout the concentrations tested, the leptin-induced lipolysis observed in fat cells of lean animals was smaller than that obtained in ob/ob mice. The maximal lipolytic effect in obese animals was observed with 10(-8) M of OB protein. The lipolytic activity following the addition of 1.25.10(-10) M to 1.25.10(-6) M was significantly increased (P < 0.01) in ob/ob mice compared to lean animals. Adipocytes from ob/ob mice responded in a dose-dependent manner to the OB protein, while the leptin-induced lipolysis observed in lean animals was dose-independent. In contrast to lean and ob/ob mice, leptin did not stimulate lipolysis in adipocytes from db/db mice, which have a mutation in the leptin receptor gene. These in vitro studies suggest an autocrine/paracrine action of leptin on white fat cells and envisages the involvement of the OB protein, not only in centrally mediated pathways, but also in physiological functions which take place peripherally.     

In vitro effect of garlic powder extract on lipid content in normal and atherosclerotic human aortic cells

In the present study, the mechanism of the in vitro effect of garlic powder extract (GPE) on lipid content of cultured human aortic cells was investigated. The addition of GPE abolished atherogenic blood serum-induced accumulation of free cholesterol, triglycerides, and cholesteryl esters in smooth muscle cells derived from uninvolved (normal) intima. In cells isolated from atherosclerotic plaque, GPE lowered these lipids. GPE inhibited lipid synthesis both in normal and atherosclerotic cells. It inhibited acyl-CoA:cholesterol acyltransferase activity that participates in the cholesteryl ester formation and stimulated cholesteryl ester hydrolase that degrades cholesteryl esters. This may explain the lipid reduction caused by GPE in atherosclerotic cells. GPE inhibited the uptake of modified low density lipoprotein and degradation of lipoprotein-derived cholesteryl esters, thus considerably reducing the intracellular accumulation of cholesteryl esters. This suggests the mechanism responsible for the prevention of lipid accumulation in aortic cells caused by atherogenic blood serum.     

In vitro activation of mouse macrophages by rat lymphocyte mediators

Macrophage-activating factors (MAF)3 were released by presensitized rat lymphocytes stimulated in vitro with the appropriate antigens. Different supernatants of presensitized rat lymphocytes specifically stimulated in vitro with several different mouse, dog, and rat tumor or normal cells were capable of rendering normal rat and mouse macrophages nonspecifically cytotoxic in vitro to their respective syngeneic tumor cells. The release of active mediators by rat lymphocytes sensitized in vivo was dependent upon immunologically specific recognition of an antigen in vitro. When rat lymphocytes were incubated in vitro with antigens unrelated to the in vivo sensitizing antigens, no release of MAF occurred. Once rat MAF was released, it activated both syngeneic (rat) and xenogeneic (mouse) macrophages to kill tumor cells in vitro. These activated marcophages destroyed all syngeneic tumor targets. Such cytotoxicity was obtained even when the cells used to elicit release of MAF were totally unrelated to the target tumor cells. The data thus demonstrated that MAF can cross strain and even species specificities and can activate macrophages to kill tumors in a nonspecific manner. The cytotoxicity mediated by in vitro activated mouse macrophages decreased with time once the macrophages were removed from MAF; and by 7 days postactivation, the macrophages were not cytotoxic. However, when incubated again with MAF, significant reactivation was observed. This suggested that activation of macrophages in vivo may be a continuous process of lymphocyte-macrophage interaction.     

In vitro cytotoxicity of paclitaxel-transferrin conjugate on H69 cells

Transferrin is a serum glycoprotein involved in iron transport. Transferrin acts also in cell growth regulation through membrane receptors. The number of transferrin receptors is increased in tumor and other rapidly dividing cells. This renders transferrin suitable for use in cytotoxic drugs targetting tumor cells. Paclitaxel was derivatized on 2' carbon and coupled with trasferrin using glutaraldehyde. The cytotoxicity of the conjugate was evaluated on small cell carcinoma of the lung cell line (H69). As compared to paclitaxel, the conjugate exhibited a slight decrease in cytotoxicity.     

Actions of capsaicin on mouse dorsal root ganglion cells in vitro

The effects of capsaicin were investigated on different populations of dorsal root ganglion cells in the in vitro mouse spinal cord-dorsal root ganglion preparation using intracellular electrodes. Dorsal root ganglion cells were characterised by the conduction velocity of their propagated action potential evoked by electrical stimulation of the dorsal root, and by the shape of their action potential. All cells with C-fiber characteristics (conduction velocity < 0.6 m/s; broad action potential with shoulder on the descending slope) were depolarised and generated action potentials when capsaicin (100-700 nM) was added to the bathing solution for 30 s. At these concentrations the membrane potential of DRG cells with myelinated fibers (conduction velocity > 2.0 m/s) was unaffected. Concentrations of capsaicin of 1.0-5.0 microM depolarised 50% of cells with conduction velocity > 10 m/s. During the depolarization of the membrane no action potentials were generated. In 50% of the capsaicin-sensitive neurons with conduction velocity faster than 10 m/s there was an initial hyperpolarization. Electrical stimulation of the dorsal root failed to evoke action potentials during the depolarization in 38% of the DRG cells with myelinated fibers and in all C-fibers tested within 10 min of the onset of the capsaicin effect. Passive depolarization of the membrane by intrasomal current injection mimicked the conduction block in neurons with large myelinated fibers. These observations confirm that capsaicin applied directly to the dorsal root ganglion affects, in a dose-dependent manner, both myelinated and unmyelinated primary afferents with a higher potency for C-neurons. Capsaicin evoked action potentials in C-neurons but not in neurons with myelinated fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro 31P-magnetic resonance spectroscopy of muscle extracts in malignant hyperthermia-susceptible patients

BACKGROUND: It was recently suggested that malignant hyperthermia-susceptible (MHS) patients could have an elevated peak of phosphodiesters in leg muscles using in vivo phosphorus magnetic resonance spectroscopy. In the current study, analysis of the phosphodiesters of muscle extracts of MHS and malignant hyperthermia-negative patients was performed using in vitro phosphorus magnetic resonance spectroscopy to chemically identify and to compare the muscle concentrations of water-soluble compounds between the two groups with respect to the muscle fiber type composition. METHODS: Perchloric acid extracts of the vastus medialis muscle of seven MHS patients and ten malignant hyperthermia-negative patients on the basis of the European malignant hyperthermia contracture test were subjected to in vitro phosphorus magnetic resonance spectroscopy carried out at 9.4 T. In addition, chemical identification of the phosphodiester region and histologic examination of the muscle specimens were performed. RESULTS: The peak in the phosphodiester region was assigned to glycerophosphorylcholine. Muscle perchloric acid extracts of MHS patients had a significantly (P < 0.05) higher glycerophosphorylcholine to the sum of phosphocreatine and inorganic phosphate (glycerophosphorylcholine/ [phosphocreatine +inorganic phosphate]) value than those of malignant hyperthermia-negative patients. Neither a difference in the fiber type composition between the two groups nor any specific myopathy were found. CONCLUSIONS: In the absence of histologic differences between muscle specimens of MHS and malignant hyperthermia-negative patients, these results could suggest that glycerophosphorylcholine could be a marker of an impairment in the phospholipid metabolism in the skeletal muscle of MHS patients.