Non-stimulated Cultures Research Articles

Production of hemopexin by TNF-α stimulated human mesangial cells

Plasma hemopexin has been shown to induce proteinuria after intrarenal infusion in rats, as well as glomerular alterations identical to those seen in corticosteroid-responsive nephrotic syndrome (CRNS). The question emerged whether also renal cells are potentially able to release hemopexin. Normal human mesangial cells (HMC) were incubated overnight in serum-free medium with or without tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL). Parallel cultures were supplemented with prednisolone (10-3 mol/L). Concentrated supernatants were analyzed by Western blotting, using antihemopexin immunoglobulin G (IgG). Antitransferrin IgG served as control antibody. In addition, cytospins were stained using polyclonal or monoclonal antihemopexin IgG. A part of the cells was used for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR), to study hemopexin mRNA. Eighty five kD bands were exclusively detected by antihemopexin IgG in the Western blots in supernatants from TNF-alpha-stimulated cultures and to a lesser extent in prednisolone-treated cultures. Cells from TNF-alpha-stimulated cultures stain positive for hemopexin in contrast to those from prednisolone-treated or nonstimulated cultures. RT-PCR data suggest that mRNA for hemopexin is up-regulated in TNF-alpha-treated versus prednisolone-treated HMC. Stimulated HMC are able to release hemopexin in vitro in a corticosteroid-dependent manner. As preliminary data indicate that mesangial hemopexin is able to affect glomerular anionic sites, it is conceivable that stimulated mesangium may contribute to enhanced glomerular permeability in CRNS through local hemopexin release.

Alendronate and etidronate do not regulate interleukin 6 and 11 synthesis in normal human osteoblasts in culture.

Bisphosphonates exert a potent inhibitory effect on bone resorption. Several studies have been performed, with contradictory results, to ascertain whether the effect of bisphosphonates on osteoclasts could be produced, at least in part, by modulation of the synthesis of resorption-promoting factors by osteoblasts. The aim of this study was to evaluate the effect of etidronate (10-4-10-9 M) and alendronate (10-7-10-12 M) on the production of IL-6 and IL-11 using human osteoblast cultures. Cytokines were quantified by ELISA, and mRNA expression was tested. Treatment with alendronate and etidronate had no effect on the synthesis of IL-6 or IL-11, and IL-6 and IL-11 mRNA levels. These results were obtained both in nonstimulated cultures and in cultures stimulated by means of TNF-a, IL-1b, and TNF-a+IL-1b, with or without FCS. In conclusion, a possible indirect osteoclast-mediated effect of alendronate and etidronate on bone resorption would not be exerted through reduction in osteoblastic synthesis of IL-6 and IL-11.

Biodegradation of the synthetic pyrethroid cypermethrin in used sheep dip.

To investigate the breakdown of cypermethrin synthetic pyrethroid (SP) insecticide-based used sheep dip (USD), with its indigenous microbial community and two previously isolated SP-degrading microorganisms. Cultures of USD (50 ml) containing 250 ml l(-1) cypermethrin were inoculated with the SP-degrading organisms and incubated at 25 degrees C with agitation at 80 rev min(-1) for 14 days. The viable cell counts and concentration of cypermethrin were monitored. A non-stimulated control was also carried out. The previously isolated bacteria were the most effective at degrading cypermethrin, leaving approximately two-thirds the concentration of SP as was found in the control. The non-stimulated cultures showed negligible breakdown of SP over the experimental period. The previously isolated SP-degrading bacteria could have a use in the treatment of SP USD. In situ treatment of SP-based USDs to detoxify the active ingredient before disposal could be very useful in helping to deal with agricultural pesticide waste. Such an approach, or by ex situ treatment would be more preferable to current methods, such as those of incineration and disposal to land.

Biological effect of varying peptide binding affinity to the BoLA-DRB3*2703 allele

MHC class I and II molecules are immunoregulatory cell surface glycoproteins, which selectively bind to and present antigenic peptides to T-lymphocytes. Murine and human studies show that variable peptide binding affinity to MHC II molecules influences Th1/Th2 responses by inducing distinctive cytokine expression. To examine the biological effects of peptide binding affinity to bovine MHC (BoLA), various self peptides (BoLA-DQ and fibrinogen fragments) and non-self peptides from ovalbumin (OVA), as well as VP2 and VP4 peptides from foot and mouth disease virus (FMD-V) were used to (1) determine binding affinities to the BoLA-DRB3*2703 allele, previously associated with mastitis susceptibility and (2) determine whether peptide binding affinity influences T-lymphocyte function. Peptide binding affinity was determined by a competitive assay using high affinity biotinylated self-peptide incubated with purified BoLA-DRB3*2703 in the presence of various concentrations of competing peptides. The concentrations of non-self peptide required to inhibit self-peptide binding by 50% (IC50) were variable, ranging from 26.92 to > 320 μM. Peptide-specific T-lymphocyte function was determined by measuring DNA synthesis, cell division, and IFN-γ production in cultures of mononuclear cells from a BoLA-DRB3*2703 homozygous cow. When compared to non-stimulated control cultures, differences in lymphocyte function were observed for all of the assessed parameters; however, peptide-binding affinity did not always account for the observed differences in lymphocyte function.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs.

To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Stimulation of Apoptosis in Human Granulosa Cells from in Vitro Fertilization Patients and Its Prevention by Dexamethasone: Involvement of Cell Contact and Bcl-2 Expression

Human granulosa cells obtained from in vitro fertilization patients are highly luteinized, but can still be stimulated by LH/cAMP for production of progesterone. This stimulation involved enhancement of apoptosis. Incubation of the cells with dexamethasone (Dex) reduced the apoptotic incidence compared with nontreated cells and completely abolished the increase in apoptosis stimulated by LH or forskolin, concomitantly with a pronounced increase in progesterone production. Organization of the actin cytoskeleton was dramatically reduced after LH/forskolin stimulation. In contrast, Dex prevented disorganization of the actin filament networks. LH and forskolin also decreased the organization of gap junctions, which could be prevented by Dex. However, the intracellular level of connexin 43 was elevated in the presence of LH, forskolin, and Dex. Endogenous levels of the survival gene protein Bcl-2 were significantly elevated in all cultures treated with Dex compared with either nonstimulated cultures or cultures stimulated with LH and forskolin. Our data suggest that LH/cAMP can stimulate steroidogenesis even during the initial stage of apoptosis of human granulosa cells, whereas Dex, which blocks apoptosis, could further elevate progesterone production. Moreover, the integrity of gap junctions and the actin cytoskeleton as well as elevated levels of Bcl-2 may play an important role in the suppression of apoptosis of human granulosa cells.

Nitric oxide production as an indication of Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus).

White-tailed deer (Odocoileus virginianus) are reservoirs for Mycobacterium bovis in northeast Michigan, USA. Production of nitric oxide (NO) by activated macrophages is a potent mechanism of mycobacterial killing. The capacity of macrophages to produce NO, however, varies among mammalian species. The objective of this study was to determine if mononuclear cells from white-tailed deer produce nitrite as an indication of NO production and, if so, is NO produced in response to stimulation with M. bovis antigens. Supernatants were harvested from adherent peripheral blood mononuclear cell (PBMC) cultures that had been stimulated with either Mannheimia haemolytica lipopolysaccharide (LPS) or media alone (i.e., no stimulation). Nitrite levels within M. haemolytica LPS-stimulated culture supernatants exceeded (P < 0.05) those detected within supernatants from non-stimulated cultures as well as those detected within supernatants from cultures receiving an inhibitor of NO synthase in addition to M. haemolytica LPS. In response to stimulation with M. bovis antigens, nitrite production by PBMC from M. bovis-infected deer exceeded (P < 0.05) the production by PBMC from non-infected deer. The response of PBMC from infected deer to M. bovis antigens exceeded (P < 0.05) the response of parallel cultures from the same deer receiving no stimulation. The response of PBMC from M. bovis-infected deer to M. avium antigens did not differ from that of PBMC from M. bovis-infected deer to no stimulation or from that of PBMC from non-infected deer to M. avium antigens. These findings indicate that adherent PBMC from white-tailed deer are capable of NO production and that mononuclear cells isolated from M. bovis-infected white-tailed deer produce NO in an antigen-specific recall response.

Production of oxytocin receptor and cytokines in primary uterine smooth muscle cells cultivated under inflammatory conditions.

We studied the production of the oxytocin receptor and interleukins in human uterine smooth muscle cells cultured in vitro in the presence of cytokines that were shown to be elevated in gestational diseases such as intrauterine infections and chorioamnionitis. Human uterine smooth muscle cells were cultured in the absence or presence of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS). Additionally, cells were cultivated under hypoxic conditions (3.5% oxygen). After 6, 12, 24, and 48 hours of incubation, oxytocin receptor mRNA was measured from total RNA using quantitative, competitive reverse transcriptase-polymerase chain reaction. Secreted cytokines (IL-1beta, IL-6, or IL-8) were quantitated from supernatants after 6, 12, 24, and 48 hours of stimulation by commercially available enzyme-linked immunosorbent assay. In nonstimulated cultures basal secretion of IL-1beta, IL-6, and IL-8 was detectable. Supplementation of IL-1beta induced a statistically significant decrease in oxytocin receptor mRNA abundance, whereas IL-6, TNFalpha, LPS, or hypoxia did not significantly affect oxytocin receptor gene expression. The cytokines IL-1 and TNFalpha induced IL-6 and IL-8 release, whereas secretion of the two interleukins was not altered in the presence of LPS or hypoxia. Expression of IL-1beta was not significantly induced under inflammatory or hypoxic culture conditions. The constitutive and cytokine-inducible expression of interleukins from uterine smooth muscle cells suggests that the myometrium may contribute to the overall production of inflammatory mediators in the uterus that are thought to govern term- or infection-induced preterm labor. Down-regulation of the oxytocin receptor under IL-1beta in myometrial cells may indicate that initiation and maintenance of labor could be partially limited under severe inflammatory conditions such as chorioamnionitis.

Production of Pro- and Anti-Inflammatory Cytokines of Human Placental Trophoblasts in Response to Pathogenic Bacteria

We studied the production of cytokines in purified cultures of human term trophoblasts in the presence of pathogenic strains of Escherichia coli, Bacteroides fragilis, Mycoplasma hominis, Staphylococcus aureus, and Streptococcus agalactiae, which have been identified in intrauterine infections. Human villous trophoblasts were isolated from term placentas after cesarean section and purified by several steps. After 6, 12, and 24 hours of incubation with the different heat-inactivated bacteria, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10) as well as tumor necrosis factor-alpha (TNF-alpha) were measured from supernatants by commercially available enzyme-linked immunosorbent assay. Expression of cytokine mRNAs was determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). In nonstimulated cultures, low (IL-1beta and TNF-alpha) and high (IL-6, IL-8, and IL-10) basal secretion of cytokines was detectable. The pathogenic microorganisms induced a dose- and time-dependent release of IL-1beta, IL-6, IL-8, and IL-10, whereas TNF-alpha secretion was not elevated. E coli was the most potent inducer followed by B fragilis, S agalactiae, S aureus, and M hominis. Transcripts encoding IL-1beta, IL-6, IL-8, or IL-10 were elevated in the RT-PCR reactions, suggesting that transcriptional mechanisms contribute to elevated cytokine expression. Pathogenic microorganisms stimulated mRNA expression and polypeptide release of pro- and anti-inflammatory cytokines from placental trophoblasts. Induction of both inflammation-promoting and inflammation-inhibiting cytokines by bacterial products could play a role in modulating the inflammatory response associated with chorioamnionitis.

Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3–5) making the interpretation difficult. We modified the assay by the addition of interferon-α and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-α were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4 +, CD8 +, and γδ TCR + T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4 + T cells and γδ TCR + cells were the predominate subsets of lymphocytes responding to PPD. γδ TCR + cells also proliferated in non-stimulated cultures; however, the γδ TCR + cell proliferative response of infected cattle was significantly ( p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4 + but not γδ TCR + cells of infected cattle. Administration of PPD for CCT also boosted interferon-γ (IFN-γ) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-γ response in cells isolated from non-infected cattle. These data indicate that CD4 + and γδ TCR + cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4 + cell response is boosted by intradermal injection with PPD for CCT.

Specific in vitro proliferative immune responses and lymphokine production in Ethiopian children with and without tuberculosis.

We investigated the profile of some in vitro parameters of cellular immune responses in non-HIV-infected Ethiopian children and young adults with and without tuberculosis (TB) as compared to healthy Ethiopian and non-Ethiopian controls. The in vitro proliferative responses of peripheral blood mononuclear cells (PBMC) to purified protein derivative (PPD) were determined in 15 Ethiopian children and young adults with TB, 12 healthy Ethiopian children who were contacts of TB patients, 20 Ethiopian children without contact with TB and ten non-Ethiopian controls. All TB patients and contacts had a positive Mantoux skin test. The PBMC proliferative response to PPD of the Ethiopian children with TB was significantly higher than that of the Ethiopian children without TB, while all Ethiopian children demonstrated stronger proliferative response as compared to non-Ethiopian healthy controls. Interleukin 2 (IL-2), interferon gamma (IFN-gamma), interleukin 4 (IL-4) and interleukin 6 (IL-6) were measured by ELISA assays performed on the supernatant of PPD-stimulated and non-stimulated PBMC cultures of seven Ethiopian children with TB, ten Ethiopian children without TB and eight non-Ethiopian controls. IFN-gamma and IL-4 were undetectable and IL-2 levels in unstimulated supernatants were low in all groups. PPD stimulation induced a significant rise in IL-2 levels in Ethiopians with TB as compared to all other groups. There was no increase above baseline in IL-6 levels in any group studied. Ethiopian children with TB exhibit a strong cellular immune response as expressed by Mantoux tests and lack of stimulation of IL-4 and IL-6 production. This pattern suggests a Th1 type effective cellular immune response to mycobacteria in a cohort of young Ethiopians with TB.

Effects of MA 956 superalloy and alpha-alumina particles on some markers of human osteoblastic cells in primary culture.

One of the problems associated with the modern biomaterials used in prostheses is osteolysis, which, although its exact origin is unknown, has been associated with wear particles. Osteoblasts seem to participate directly in this phenomenon. This paper investigates in vitro cellular response to the wear particles from the metal substrate and ceramic covering (alpha-alumina) of a new titanium yttrium aluminum alloy, MA 956, that has been proposed as a biomaterial because of its exceptional mechanical and electrochemical properties. The effect of different sizes (10 and 80 microm) of MA 956 and alpha-alumina particles on osteoblast function was studied in primary human bone cell cultures. Cells were harvested from trabecular bone fragments obtained during knee arthroplasty. Osteoblastic cell response to the particles was measured by assaying C-terminal type I procollagen (PICP), alkaline phosphatase, and osteocalcin secretion, with and without 1.25(OH)(2)D(3) stimulation, in the cell-conditioned medium. Both sizes of MA 956 and alpha-alumina particles decreased PICP secretion in nonstimulated osteoblastic cells, but this secretion was not affected in the cultures stimulated with 1.25(OH)(2)D(3). Only the 10 microm alpha-alumina particles inhibited alkaline phosphatase activity in 1.25(OH)(2)D(3)-stimulated and nonstimulated cultures. The rise in osteocalcin levels after 1.25(OH)(2)D(3) stimulation was lower in the presence of the 10 microm MA 956 particles than in the presence of alpha-alumina particles. Although both materials seem to have directly affected in vitro osteoblastic cell function, the increase in osteocalcin levels after 1.25(OH)(2)D(3) stimulation was lower after exposure to MA 956 particles than the increase observed after exposure to alpha-alumina particles. Therefore, it does not seem that osteocalcin stimulated bone resorption, suggesting that MA 956 would be less likely to provoke osteolysis.

Expressions of epidermal growth factor family and its receptor in hepatocellular carcinoma cell lines: relationship to cell proliferation.

In 6 HCC cell lines, clear expressions of EGFR and TGF-alpha were found in flow cytometry, while expressions of EGF, HB-EGF and AR were quite low. TGF-alpha secretion into culture supernatants became measurable when TPA 0.5 microM was added. TPA accelerated the proliferation of KYN-3 cells, and anti-TGF-alpha neutralizing antibody suppressed this proliferation in a dose-dependent manner. Addition of exogenous TGF-alpha, EGF, AR, or HB-EGF with heparin accelerated cell proliferation. In non-stimulated cultures, cell proliferation was suppressed by anti-EGFR neutralizing antibody, but not by the antibodies for EGF, TGF-alpha, AR and HB-EGF. HCC may possess a paracrine system regulated by these 4 ligands, and an autocrine system, under a certain condition, via TGF-alpha and EGFR.

T lymphocytes that infiltrate nasal polyps have a specialized phenotype and produce a mixed T H1 /T H2 pattern of cytokines

Background: Nasal polyps (NPs) are inflammatory reactions in the nasal mucosa the etiology and pathogenesis of which remains unknown. Objective: The purpose of this study was to study in detail the phenotype and function of T lymphocytes infiltrating NPs by analyzing the expression of surface markers and cytokine secretion. Methods: NP tissue samples and peripheral blood were obtained from 18 patients. Mononuclear cells were purified from these samples, and their phenotype was investigated by triple-color immunofluorescence and flow cytometric analysis. Cytokine production was determined in cultures by using an ELISA technique. Results: NP lymphoid cells mainly consisted of T lymphocytes. These T lymphocytes showed a CD45RO+CD45RA– phenotype and expressed pan-T cell molecules; the CD8+ subset was predominant. NP T cells showed a lower density of CD28, CD3, and TCR-αβ compared with T cells from peripheral blood. NP T lymphocytes expressed the activation markers DR and CD69 and exhibited the adhesion molecule profile CD54+, CD62L–, and CD103+ CD49d low . Virtually all NP T cells bore CD95 (FAS), but they did not undergo apoptosis, either spontaneously or induced by CD95 cross-linking with the mAb CH11. The pattern of cytokines secreted by NP T lymphocytes was characterized by the spontaneous and simultaneous production of IFN-γ and IL-5. Neither IL-2 nor IL-4 were detectable in nonstimulated cultures. Conclusion: This study defines the T lymphocytes that infiltrate NPs as memory T cells in an activated status, with homing properties related to the mucosal immune system. They are resistant to anti-CD95-mediated apoptosis and produced a mixed T H1 /T H2 cytokine pattern as defined by the simultaneous production of IFN-γ and IL-5. (J Allergy Clin Immunol 1998;102:953-60.)

Production of Interleukin-6 in Cultures of Peripheral Blood Mononuclear Cells from Children with Primary Protein-Calorie Malnutrition and from Eutrophic Controls

The spontaneous as well as mitogen-induced in vitro production of interleukin-6 (IL-6) was studied in cultures of peripheral blood mononuclear cells (PBMC) from 14 children with marginal protein-energy malnutrition, 43 children with definite protein-energy malnutrition and 38 eutrophic controls of similar age, sex, race and socioeconomical condition. PBMC were cultured without added mitogen or stimulated with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA). After 48 h incubation, cell-free culture supernatants were collected and stored at –70°C. The amount of IL-6 in the supernatants was determined by a specific bioassay based on the proliferation of B9 hybridoma cells using human rIL-6 as standard. The mean level of IL-6 was significantly increased in supernatants from nonstimulated PBMC cultures from definitely malnourished children as compared with that observed in those of the controls. Stimulation with either LPS or PHA induced a rise in cytokine bioactivity in the supernatants of PBMC cultures from the different nutritional groups tested. Interestingly, IL-6 was significantly increased in the supernatants of PHA-stimulated cultures from malnourished children as compared with those of the controls.

Src protein and tyrosine-phosphorylated protein profiles in marrow stroma during osteogenic stimulation.

Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiation and function. We followed temporal changes of Src protein abundance in marrow stromal cells induced to mineralize by dexamethasone (DEX), growth in cold temperature, or both. Given the tyrosine kinase function of Src and its numerous substrates, profiles of phosphotyrosine-containing proteins were followed as well. On day 11 of stimulation, specific alkaline phosphatase (ALP) activity at 30 degrees C decreased under DEX relative to 37 degrees C cultures, in accord with increased cell counts. Mineralization per well under DEX increased by 25% at 37 degrees C, whereas at 30 degrees C it increased by more than threefold regardless of the DEX stimulation. At 30 degrees C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respectively. Cultures at 37 degrees C showed a general drop per cell of many phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyrosine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell of Src protein (pp60src) followed a similar trend, and in addition a truncated Src molecule, p54/52src, was detected as a putative cleavage product presumably representing its carboxy terminus. The pp60src was most abundant, relative to its truncated product, in day 7 nonstimulated cultures, whereas under DEX stimulation the truncated species pp54/52src showed the highest relative abundance on days 7. At 30 degrees C, DEX stimulation accentuated the increase in Src protein on day 3, showed no change on day 7, and returned to increase Src protein on day 10. Potassium ionophorvalinomycin, considered to select against mineralizing osteoprogenitors at 30 degrees C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomycin. On day 7 of DEX stimulation, the presence of valinomycin resulted in low p54/52src. Among phosphotyrosine-containing proteins, a 32-34 kDa band, as yet unidentified, showed the most concordant changes with mineralization induction. P32-34 decreased by DEX on days 2 and 8 and increased by low temperature alone or combined with DEX on day 3. On day 7, p32-34 did not change under DEX, but valinomycin selected cells with less phoshpotyrosine-containing p32-34. Taken together, high Src abundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent induction of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (which affects mineralization). The reported binding of amino-terminal Src oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membrane raises the question of its possible involvement in mitochondria-regulated mineralization.

Conservation of Apoptosis as an Immune Regulatory Mechanism: Effects of Cortisol and Cortisone on Carp Lymphocytes

This is the first study to show that apoptosis as an immune regulatory mechanism is conserved in fish, demonstrating its importance in maintaining immunological homeostasis. The data further show that this mechanism is subject to control by glucocorticosteroids. Carp plasma cortisol concentrations increase from 20 to 434 ng/ml and cortisone from 5 to 50 ng/ml within 9 min of the onset of handling stress. At basal steroid concentrationsin vitro,cortisol, but not its conversion product cortisone, inhibits proliferation of peripheral blood lymphocytes (PBL), as measured by [3H]thymidine incorporation. Induction of apoptosis in activated PBL is the apparent mechanism of cortisol action. In nonstimulated PBL cultures, apoptosis is induced by neglect (a lack of stimulating signals). Stimulation with LPS or PHA rescues lymphocytes from this type of apoptosis. Stimulated PBL populations, however, are sensitive to cortisol-induced apoptosis. Culture supernatants from activated PBL protect PBL from apoptosis by neglect, probably by supplying a growth signal. These supernatants, however, have no effect on cortisol-induced apoptosis.

Possible role for CD40-CD40L in the regulation of interstitial infiltration in the kidney

Interstitial infiltration by mononuclear cells is a hallmark of most inflammatory kidney diseases, and the degree of infiltration is associated with disease progression. It has been demonstrated that proximal tubular epithelial cells (PTEC) are an important source of different cytokines/chemokines and thereby play a central role in the regulation of the local inflammatory response. CD40 is a cell surface receptor involved in immune regulation for which the ligand is expressed on activated T cells. By different staining methods, CD40 was found expressed in cryosections on the basolateral side of tubuli, as well as on the surface of an SV40-transformed PTEC line (PTEC-TRL) and on primary PTEC cultures. Cross linking CD40 receptor on these cultured cells, using a CD40L-transfected mouse fibroblast, resulted in strong up-regulation of the production of the chemokines IL-8, MCP-1 and RANTES. For IL-8 and MCP-1 production, the stimulation index after CD40 activation ranged from two- to sevenfold. Much stronger effects were observed for RANTES production, where levels remained undetectable (< 0.1 ng/ml) in non-stimulated cultures, whereas CD40 activation resulted in a strong production reaching 5 ng/ml in a 72-hour culture period. These data suggest that CD40L-CD40 interactions between infiltrating activated T cells and PTEC might be an important factor in the regulation of interstitial infiltration within the kidney.

Vitamin E Exposure Modulates Prostaglandin and Thromboxane Production by Avian Cells of the Mononuclear Phagocytic System

The production of Prostaglandin E2 (PGE2) and Thromboxane B2 (TXB2) by turkey blood monocytes and a chicken mononuclear phagocytic cell line MQ-NCSU after exposure to vitamin E (VE) was examined. Turkey embryos were exposed in ovo to 0 and 10 international units (IU) of VE; blood monocytes were collected at 2 weeks of age and cultured. MQ-NCSU macrophage monolayers were exposed to 0, 0.1, 0.25, and 0.5 IU VE. The monocyte/macrophage cultures were exposed to 1 μg/mL bacterial lipopolysaccharide (LPS). Non-stimulated parallel cultures were maintained as controls. The PGE2 and TXB2 levels were quantitated in culture supernatants by a competitive ELISA. Blood monocytes from the 10 IU VE poults produced lower PGE2 levels as compared with the 0 IU VE controls. Upon stimulation with LPS, monocytes from the 10 IU VE group exhibited levels of PGE2 that were higher than the 0 IU VE group. Levels of TXB2 were not quantitated in the poult blood monocyte culture supernatants. The PGE2 and TXB2 levels in the supernatant of the VE treated MQ-NCSU macrophage cultures were lower than the 0 IU VE controls. Stimulation with LPS resulted in increased PGE2 and TXB2 production by the VE-exposed macrophages. The results from this study suggest that in ovo or in vitro exposure with VE may either upregulate or downregulate PGE2 and TXB2 production by monocytes/macrophages, and that this production may be dependent upon the exposure to a variety of external stimuli and/or the state of macrophage activation.