Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line.

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system.

Murine gamma interferon activates the release of a macrophage-derived Ia-inducing factor that transfers Ia inductive capacity.

In this report we demonstrate that when the murine macrophage tumor cell line P- 388D1 is incubated for 48-72 h with either concanavalin A-stimulated rat spleen cell supernatant or cloned murine immune interferon (IFN-gamma), the cultured cells release a cell-free factor activity that in turn induces the cell surface expression of Ia antigen on the murine monocyte cell line WEHI-3. This IFN-gamma-stimulated, Ia-inducing activity cannot be blocked with an anti-IFN-gamma heteroantiserum that does block the induction of Ia expression on WEHI-3 by both cloned murine IFN-gamma and rat Con A supernatant. The Ia-inducing factor ( IaIF ) generated from P- 388D1 after stimulation by IFN-gamma does not demonstrate any antiviral activity. The P- 388D1 -derived IaIF is not shed plasma membrane Ia glycoprotein molecules, as demonstrated by the inability of the active component to bind specifically to an anti-I-Ad affinity column or to a protein A column after the active supernatant is first treated with an excess of anti-I-E/Cd,k monoclonal antibody.

The pH dependence of photosensory responses in Stentor coeruleus and model system.

1. Live Stentor coeruleus exhibits a substantially red-shifted fluorescence maximum, corresponding to the anionic species of the photoreceptor chromophore. No change was observed in either the absorption or fluorescence excitation spectrum, indicating an efficient deprotonation of the photoreceptor pigment upon excitation by light. 2 Changes in external pH exhibit a dramatic effect on the pulmonary response of Stentor. Phototaxis is specifically inhibited at pH less than 6, with loss of photosensory perception which is restored when the pH is returned to pH greater than 6. 3. Fluorescence changes of 9-aminoacridine in suspensions of live Stentor indicate the generation of a pH gradient upon irradiation with light. Both pH gradient and phototaxis were inhibited by the addition of nigericin and p-tri-fluoromethoxy carbonyl cyanide phenylhydrazone (FCCP). 4. Incorporation of the Stentor photoreceptor protein in to artificial liposomes demonstrates the ability of the system to generate pH gradients across model membranes as monitored by the quenching of 9-aminoacridine fluorescence. The effect of external pH on net proton movement in the model system is strikingly similar to the pH dependent of the liver Stentor, thus lending support for transient proton flux being an important mode of light signal processing for photosensory transduction.

Spectroscopic characterization of the Stentor photoreceptor.

1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.

Cellular control of in vitro progression of murine myeloid leukemia: progression accompanies acquisition of independence from growth factor and stromal cells.

The ability of bone marrow stroma cells of normal WCB6F1 (+/+) mice versus their congenic Sl/Sld stromal-defective littermates to support sustained proliferation and leukemic transformation of the growth factor-dependent myeloid cell line FDC-P1 was studied. Extensive proliferation of factor-dependent cells occurred on (+/+) normal long-term marrow culture stroma without the addition of growth factor, whereas factor-dependent cells dissipated from Sl/Sld stromal cultures after addition. The sustained proliferation that occurred on +/+ stromal layers later resulted in the appearance of factor-independent cell lines that were no longer dependent upon stroma. Factor-independent cell lines were cloned by limiting dilution and analyzed for expression of cell surface antigens to prove their origin from FDC-P1. Factor-independent cells, but not factor-dependent cells, formed tumors in syngeneic mice. These studies demonstrate a critical role for marrow stroma in the stepwise development of murine leukemia and are concordant with the previous data obtained in in vivo studies by McCool et al. that the splenic stroma of irradiated Sl/Sld mice do not support growth of Friend virus-induced preleukemic cell colonies. The present data demonstrate in a preleukemia model not induced by Friend virus complex that normal (+/+) stromal cells promote the in vitro proliferation of factor-dependent preleukemic cells and their subsequent transition to factor-independent leukemia cells, but Sl/Sld defective stroma do not efficiently promote this transition.

Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis.

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogeneous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and gamma 2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.

A novel mast cell growth factor (MCGF-3) produced by marrow-adherent cells that synergizes with interleukin 3 and interleukin 4.

A clonal marrow-adherent stromal cell line, +/+-1 LDA11, was derived and found to produce hemopoietic stimulatory activity for an interleukin 3 (IL-3)-dependent mast cell line, NFS/N1. This factor-dependent mast cell line displayed restricted growth factor responsiveness to only IL-3, interleukin 4 (IL-4), and the stromal cell-produced factor. The factor produced by stromal cells was distinguished from IL-3 and IL-4 and was characterized biochemically. This factor appears to be a novel mast cell growth factor (MCGF-3) capable of synergizing with IL-3 and IL-4. It may have broader reactivity in hemopoiesis than simply IL-3-dependent mast cells, and it may prove relevant to stromal cell-mediated hemopoiesis.

Differences among mononuclear cell subpopulations in HIV seropositive or seronegative homosexual and heterosexual men as determined by four-color flow cytometry.

Four-color cell surface immunofluorescence and flow cytometry analysis was used to quantitate mononuclear cell subpopulations from HIV seropositive (HIV+) and seronegative (HIV-) homosexual men and heterosexual men. HIV+ men were divided into two groups based on peripheral blood CD4/mm3 of greater than 500 or less than 500. CD4+ cells that were simultaneously CD45R-, CDw29-, and 13- were significantly less in HIV+ men with less than 500 CD4/mm3 (17%) compared to heterosexual men (34%). This lower percentage of "CD4 only" cells in HIV+ males with less than 500 CD4/mm3 correlated with a significantly higher percentage of CD4+ cells that were CD45R+, CDw29+, and 13+ in these individuals. CD8+ cells that were CD45R+, 13+, but CD38-, were significantly less in HIV+ men with less than 500 CD4 as compared to HIV- homosexual men. In contrast, a second CD8+ subpopulation that was CD45R-, CD38+, and either 13+ or 13- was significantly greater in less than 500 HIV+ men as compared to both HIV- homosexual men and heterosexual men. A significant difference in this subpopulation was observed between the less than 500 and greater than 500 HIV+ groups and correlated with seropositivity for viral p24 antigen. Interestingly, CD8+ cells that were CD45R+, as well as CD38+, and either 13+ or 13- were significantly greater in the less than 500 HIV+ group compared to the greater than 500 HIV+ group, and did not correlate with p24 seropositivity. The percentage of monocyte/macrophages that were CD4- or expressed dim CD4 immunofluorescence, but were 13+, was significantly greater in HIV+ men (43%) compared to HIV- homosexual men (27%). In summary, we have identified previously undescribed mononuclear cell subpopulations that were altered with HIV infection and, in some cases, correlated with the stage of disease.

Photoreactivity of lysergic acid diethylamide and its possible utility as a photoaffinity labeling reagent.

Aqueous solutions of lysergic acid diethylamide (LSD) are extremely sensitive to light in the near-ultraviolet region of the spectrum. This rather efficient photoreaction yields a variety of products which have very low affinity for LSD-binding sites on plasma membranes from Fasciola hepatica. Since this photoreaction may be elicited by normal white fluorescent lighting in the laboratory, it represents a potential source of error in determining the binding affinity of LSD. Utilizing this photoreactivity advantageously, [3H]LSD was used to photolabel membrane proteins. Covalent binding of [3H]LSD was shown to be a function of the duration of illumination and was inhibited by 5-hydroxytryptamine and nonradioactive LSD. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of [3H]LSD labeled membranes from F. hepatica showed two proteins which were selectively labeled by the photoreactive [3H]LSD. This method of direct photolabeling with non-derivatized [3H]LSD may allow identification of LSD-binding proteins in a variety of systems.

In situ evidence for DNA fragmentation in Huntington's disease striatum and Alzheimer's disease temporal lobes.

To test the hypothesis that apoptosis is involved in human brain neurodegenerative disorders, we investigated whether DNA fragmentation occurs in Alzheimer's disease (AD). Huntington's disease (HD) and Parkinson's disease, as well as in temporal lobe epilepsy, using neurologically normal post-mortem human brain tissue as a control. Using in situ end labelling of DNA, we found evidence of DNA fragmentation in cells in temporal cortex and hippocampus from patients with AD and in striatum from those with HD. In contrast, only scattered DNA fragmentation positive cells were detected in the pial surfaces of some of the neurologically normal human brains. Thus, cells in the HD striatum and AD temporal cortex exhibited DNA fragmentation, suggesting that apoptosis may be involved in these disorders.

Loss of A1 adenosine receptors in human temporal lobe epilepsy.

Using quantitative receptor autoradiographic methods we have examined A1 adenosine receptors, adenosine uptake sites, benzodiazepine receptors, NMDA, AMPA, and kainic acid receptors in temporal lobes removed from patients suffering from complex partial seizures and in normal control post-mortem temporal cortex. Binding to A1 adenosine receptors and NMDA receptors was reduced in epileptic temporal cortex, while the other neurochemical parameters were unchanged. The reason for this A1 receptor loss is unclear as it occurred in both idiopathic and symptomatic cases and thus may be a consequence rather than an initial cause of seizures. However, because adenosine is a powerful anticonvulsant substance, loss of anticonvulsant A1 receptors may contribute to the human epileptic condition. It is also possible that the observed differences in A1 binding are due to autopsy vs. biopsy changes in the levels of A1 adenosine receptors.

Postoperative analgesia after peripheral nerve block for podiatric surgery: clinical efficacy and chemical stability of lidocaine alone versus lidocaine plus ketorolac.

BACKGROUND AND OBJECTIVES: The purpose of this study was to determine whether the addition of ketorolac tromethamine to local anesthesia for ankle block alters the quality or duration of analgesia after podiatric surgery. The second aim was to determine the chemical stability of ketorolac tromethamine when added to local anesthetic solutions. METHODS: The study design was double-blinded, placebo-controlled, and randomized. Seventy-nine American Society of Anesthesiologists (ASA) class I or II patients scheduled for bunionectomy or hammer toe repair, or both were randomized to 1 of 4 groups. Group L received plain 1.73% lidocaine for their ankle block. Group K received 1.73% lidocaine with ketorolac (4 mg/mL) added to the local solution. Group Kiv received 1.73% plain lidocaine for ankle block and 20 mg of ketorolac intravenously. Group E received 1.73% lidocaine with .67% ethanol added. The final concentration of lidocaine for all groups was 1.73%. The block performed in each patient was a 5-point ankle block. Beginning at 1 hour after the completion of the block and every 30 minutes thereafter, visual analogue scale (VAS) and verbal pain scores were recorded. The time from performance of the block to the initial pain and time to the first oral pain medication intake were also recorded. The time and amount of postoperative oral analgesics in the first 9 hours after the block were recorded. Adverse events were also recorded for each group. RESULTS: There were significantly lower overall VAS and verbal pain scores for group K compared with groups E and L and group Kiv compared with group E. Group K also had a significantly longer time to the first reported pain and first oral pain medications than groups E and L, but not with Group Kiv. The same group had significantly fewer average doses of pain medications postoperatively than Groups E and L. Group E had significantly shorter times to first report of pain and first pain medications and higher mean dose of postoperative oral analgesics than group K and Group Kiv. There were no untoward side effects reported from any group. Chemical analysis by gas chromatography (GC) and capillary electrophoresis (CE) showed no significant change in composition of the solutions when ketorolac was mixed with lidocaine and/or bupivacaine and stored at 37 degrees C for 1 week. CONCLUSIONS: The addition of ketorolac to lidocaine for ankle block contributed to longer duration and better quality analgesia after foot surgery compared with plain 1.73% lidocaine or 1.73% lidocaine plus intravenous ketorolac. The ethanol vehicle is unlikely responsible for the analgesic effects of ketorolac. Ketorolac retains its chemical stability when placed in local solutions of lidocaine or bupivacaine.

Detection of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro.

A progenitor cell CFU-B1 (blast cell colony forming unit) present in human bone marrow and capable of producing blast cell containing colonies in vitro was detected using a serum containing semisolid culture system. The CFU-B1 has the capacity not only to undergo self-renewal, but also commitment to a number of hematopoietic lineages. This progenitor cell therefore has characteristics which suggest that it is identical to or closely related to the human pluripotent hematopoietic stem cell. Pretreatment of marrow cells with 5 fluorouracil facilitated detection of CFU-B1 derived colonies. The formation of CFU-B1 derived colonies was dependent upon the addition of media conditioned by the human bladder carcinoma cell line 5637. The ability of 5637 CM (conditioned media) to support blast cell colony formation was in part but not totally ablated by pretreatment of the CM with an IL-1 alpha (interleukin-1) neutralizing antibody. This data suggests that IL-1 alpha plays a role in the regulation of primitive events occurring during human hematopoiesis. IL-1 alpha might be exerting these effects by either acting directly on the CFU-B1, causing marrow accessory cells to elaborate other cytokines or by synergizing with cytokines already present in 5637 CM.

Characterization of a T-cell subset prevalent in immunoregulatory disorders in humans.

T cells from individuals with certain autoimmune diseases (rheumatoid arthritis, graft-versus-host disease, acquired immunodeficiency syndrome) express high levels of a cell surface sialoglycoprotein with a molecular weight of 140 kDa (gp140). Although a low frequency of gp 140+ T cells was detected in the blood of normal individuals, upon stimulation with autologous EBV-transformed B cells (AMLR), the frequency of expression of gp140 was increased threefold. To further characterize gp 140+ T cells, rosetting techniques with ox erythrocytes coated with monoclonal anti-gp 140 antibody were used to isolate T-cell subsets for phenotypic, cell cycle, and functional analysis. The majority of gp140+ T cells expressed cytotoxic/suppressor (CD8+) phenotype in both normal and AMLR-activated states. Unstimulated gp140+ T cells had significantly greater nucleic acid content, as measured by acridine orange and flow cytometry, than gp140- T cells. Surprisingly, the gp140+ T-cell subset had a less proliferative response in vitro to pokeweed or phytohemaglutinin mitogens. These results suggest that gp140+ T cells in normal individuals and in patients with autoimmune diseases may have been activated previously in vivo and that they are relatively resistant to reactivation in vitro.

The same cloned murine B lymphoma cell lines can be selectively induced to release either interleukin-1- or interleukin-2-like factor activity.

A series of cloned murine B lymphoma cell lines including WEHI-5, WEHI-231, 2PK-3 and L10A/2J have been studied previously for their ability to present soluble protein antigen in a major histocompatibility complex (MHC) restricted fashion. These B cell lines have been shown to be effective accessory cells in the in vitro stimulation of antigen-specific, MHC-restricted, continuous T cell lines; and in the in vitro stimulation of antigen-specific, MHC-restricted T-T hybridoma cell lines. Using 2PK-3 and L10A/2J as examples of this group of B cell lymphomas we demonstrate in this study that these tumor cell lines constitutively release an interleukin-1 (IL-1) like factor activity as determined by the ability of the conditioned medium from these cultures to support the synergistic stimulation of thymocyte proliferation in the presence of concanavalin A (Con A). Conversely, these same constitutive supernatants will not stimulate the proliferation of IL-2 dependent cell lines such as CTLL-2 or HT-2. Stimulation of 2PK-3 and L10A/2J by lipopolysaccharide (LPS) results in the release of increased levels of the IL-1 like factor activity. By contrast, stimulation of the same cloned 2PK-3 and L10A/2J cell lines with the polyclonal activator Staph. aureus results in the release of a soluble factor activity which functionally acts like IL-2 since conditioned medium from S. aureus stimulated 2PK-3 and L10A/2J cultures will support a CTLL proliferation response as well as stimulate thymocyte proliferation. Thus, the same cloned B cell lines can be differentially stimulated to release lymphokine activity with either IL-1 or IL-2 like functional properties.

Characterization and functional properties of tumor cell lines in accessory cell replacement assays.

This study reports the initial phenotypic and functional characterization of a series of cloned, murine, myelomonocytic tumors and their parent cell line WEHI-3, and a group of murine B lymphoma tumors. The tumor cell lines of the myelomonocytic lineage demonstrated the ability to reconstitute a macrophage-depleted, primary in vitro anti-SRBC PFC response, but only marginally enhanced an accessory cell-depleted, ova-primed, lymphocyte proliferation in vitro response. The B lymphoma tumors displayed exactly the reverse functional profile, being highly efficient in reconstitution of the proliferation response, but not supporting the SRBC PFC response. Detailed analysis of the cell surface phenotype of the various B lymphoma tumors used in this study show they display cell surface markers characteristic of normal B lymphocytes but are heterogeneous in their various stages of differential arrest. Future work will concentrate on the orchestration of Ia-mediated and soluble factor-mediated (IL 1) modalities of antigen-triggering of lymphocytes by these B lymphoma and myelomonocytic tumor cell lines.

A comparison of the effects of lipopolysaccharide and ceramide on arachidonic acid metabolism in THP-1 monocytic cells.

Ceramide has been shown to be an important second messenger for signal transduction in cells of myeloid lineage. Studies have suggested that lipopolysaccharide (LPS) may activate signaling pathways by mimicking the action of ceramide. We explored this hypothesis with THP-1 cells in terms of the effects of LPS, C2 ceramide, and sphingomyelinase on arachidonic acid metabolism as measured by the release of radiolabeled eicosanoids. Arachidonic acid metabolism was activated by both LPS and ceramide. However, the ratio of prostaglandin E2 to leukotriene C4 was 10 times higher in cells treated with LPS than with ceramide. Unlike LPS, prior exposure to ceramide did not desensitize the cells to subsequent challenge with either LPS or ceramide, nor could LPS desensitize the cells to challenge with ceramide. The results suggest that, although LPS and ceramide may share signaling components, the signaling pathways are not identical.

Efficacy of subarachnoid meperidine for labor analgesia.

Meperidine is an opioid agonist with known weak local anesthetic properties. To determine the efficacy of subarachnoid meperidine as a labor and delivery analgesic, 20 term parturients were given 10 mg meperidine via continuous spinal catheter. Visual analog pain scores on a ten-point scale and patient satisfaction scores on a four-point scale were measured before and after establishment of the block and one hour after maximum block was achieved. Time to pain relief and return of pain was recorded. Additional doses of 7 mg meperidine were given subarachnoid via the catheter when patients requested additional analgesia. Follow-up assessment 24 hours postpartum was used to determine overall patient satisfaction. Visual analog pain scale scores (mean +/- SD) were 8.57 +/- 1.43 before block, 0.62 +/- 0.89 immediately after block, and 0.33 +/- 0.57 at one hour after block (p less than 0.0001). Patient satisfaction scale scores (mean +/- SD) were 0.83 +/- 0.88 before block, 3.90 +/- 0.37 immediately after block, and 3.85 +/- 0.31 at one hour after block (p less than 0.0001). At follow-up, 14 of 18 patients rated satisfaction as excellent, with the remaining 4 rating it as good. Expulsive efforts were excellent in 14, good in 3, and fair in 1; 2 patients had cesarean sections. Mean time to onset of pain relief was 3.9 minutes (range, 2-12), with analgesia lasting a mean of 83 minutes (range, 38-180). Two patients developed slight motor block. Side effects appeared insidiously and are similar to those observed with other neuraxial opioids.