Rapamycin and FK506 differentially inhibit mast cell cytokine production and cytokine-induced proliferation and act as reciprocal antagonists.

We have previously demonstrated that cyclosporine (CSA) and FK506 are able to selectively inhibit cytokine production by murine mast cell lines at concentrations comparable to those observed with thymus-derived lymphocytes (T cells). The selectivity of these effects were demonstrated by the failure of CSA and FK506 to inhibit cytokine-induced mast cell proliferation at equivalent or higher concentrations. In this report, we examined the ability of rapamycin (RAP) to inhibit cytokine production and cytokine-induced proliferation by a factor-dependent murine mast cell line and compared its activity to that of the structurally related macrolide FK506. The mast cell clone, MC/9, was stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187, or to proliferate in response to exogenous cytokines such as interleukin-3 and interleukin-4, produced by the helper T cell clone D10.G4. RAP did not inhibit cytokine production by MC/9, even at concentrations greater than 1000 nM. FK506 and CSA inhibited cytokine production with IC50 of 0.8 and 16.2 nM, respectively. In contrast to its lack of effect on cytokine production, RAP potently inhibited cytokine-induced proliferation of MC/9 cells with an IC50 of 1.9 nM. Because RAP and FK506 are structurally related and yet have divergent biological effects, we examined the ability of RAP to antagonize inhibitory effects of FK506 on mast cell cytokine production and the ability of FK506 to antagonize inhibitory effects of RAP on cytokine-induced mast cell proliferation. The addition of RAP in molar excess reversed inhibition of mast cell cytokine production mediated by FK506, but not that of CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporine and FK506 inhibition of murine mast cell cytokine production.

The ability of cyclosporine (CSA) and FK506 to inhibit cytokine production by factor-dependent murine mast cell lines was investigated. The mast cell clone, MC/9, and two mast cell lines, MCIII and MCVI, were stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187. The production of cytokines by stimulated mast cells cultured in the presence or absence of drug was monitored by bioassay of culture supernatants for induction of proliferation by factor-dependent cell lines and inhibition of these responses by neutralizing monoclonal antibodies. Both CSA and FK506 inhibited mast cell cytokine production at concentrations comparable to those observed with T cells. However, the degree of inhibition of cytokine production varied among the mast cell lines as well as between different cytokines produced by a given mast cell line. For example, CSA completely inhibited interleukin-2 (IL-2), IL-3, IL-4 and granulocyte-macrophage colony stimulating factor secretion by all three lines, with the exception that IL-2/IL-4 production by MCIII was partially resistant to inhibition by CSA. Similarly, FK506 completely inhibited cytokine production by MC/9, partially inhibited cytokine production by MCIII and had differential effects on IL-3/granulocyte-macrophage colony-stimulating factor and IL-2/IL-4 production by MCVI. Consistent with their ability to selectively inhibit cytokine gene transcription in T cells, neither CSA nor FK506 inhibited factor-dependent proliferation by these mast cell lines. In view of the putative role of cytokines in inflammation and late phase asthmatic reactions, these observations may be of particular significance in development of methods of pharmacologic intervention.

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Patterns of lymphokine production by primary antigen-specific/MHC restricted murine helper T cell clones.

In this study we examined a panel of CD4+ antigen specific/MHC restricted T cell clones for their ability to secrete IL-2, IL-4, and IFN-gamma upon stimulation with con A, three lymphokines which are diagnostic for the TH1 and TH2 subtypes of helper T cells. Eight of the twelve clones we analyzed did not fit the classical TH1/TH2 patterns of lymphokine secretion. Seven of these clones secreted both IL-2 and IL-4 and two of these also produced IFN-gamma. The remaining non-classical clone secreted IL-4 and IFN-gamma but not IL-2. Data from the subcloning of the IL-2/IL-4/IFN-gamma triple producers were not consistent with the parental lines being a mixture of TH1 and TH2 cells. The IL-2/IL-4 double producers (IFN-gamma negative) cannot be explained by the parental lines being a mixture of the TH1 and TH2 subtypes. Nevertheless, these double producers were subcloned and the results provided convincing evidence that clones which secrete both IL-2 and IL-4 do exist. Lymphokine loss variants involving IL-2, IL-4 or IFN-gamma were observed among subclones derived from the double and triple producers as well as in several parental lines maintained in continuous culture. We also observed the appearance of inducible IFN-gamma production in some subclones derived from parental clones where production of IFN-gamma was not detectable. The phenotypes of these variants failed to indicate an obvious trend toward the TH1 and TH2 subtypes. Thus, our results suggest that more heterogeneity in the population of CD4+ helper T cells exists than can be explained by the TH1 and TH2 subtypes of these cells.

Mupirocin: a topical antibiotic with a unique structure and mechanism of action.

The chemistry, mechanism of action, antimicrobial activity, pharmacokinetics, clinical efficacy, adverse effects, dosage, and administration of mupirocin are reviewed. Mupirocin, formerly termed pseudomonic acid A, is a topical antibiotic under investigation for the treatment of impetigo and other superficial primary and secondary skin infections. Mupirocin (Bactroban, Beecham Laboratories) is currently formulated as a 2% ointment in a water-miscible polyethylene glycol base. The drug is a unique antimicrobial agent because of its structure and mechanism of action. Mupirocin apparently exerts its antimicrobial activity by reversibly inhibiting isoleucyl-transfer RNA, thereby inhibiting bacterial protein and RNA synthesis. Mupirocin has excellent in vitro activity against staphylococci and most streptococci but less activity against other gram-positive and gram-negative bacteria. The drug will only be used topically because of its rapid and extensive systemic metabolism. Several controlled clinical trials documented that mupirocin was significantly better than the polyethylene glycol vehicle alone or ampicillin and as effective as cloxacillin, dicloxacillin, or erythromycin in producing clinical and bacteriological cures in patients with impetigo and wound infections caused by gram-positive pathogens. Limited studies suggest that mupirocin may also have a role in eradicating nasal carriage of staphylococci. Reported adverse effects are local and may be related to the polyethylene glycol vehicle base. Mupirocin should be useful for treating patients with impetigo and wound infections caused by Staphylococcus aureus. However, additional controlled, comparative clinical studies are needed to identify the role of mupirocin in treating other primary and secondary skin infections and for eliminating nasal carriage of staphylococci.

Beta adrenoceptor modulation of the generation of murine cytotoxic T lymphocytes in vitro.

The effects of stimulating beta adrenoceptors on lymphocytes during the generation of cell-mediated immunity were examined. In an in vitro system for the generation of murine cell-mediated cytotoxicity, addition of isoproterenol (10(-7) M), epinephrine (10(-6) M) or norepinephrine (10(-4) M) enhanced the number of lytic units generated compared to control cultures. This increase was blocked by dl-propranolol (5 X 10(-6) M). I-Propranolol (10(-11) to 10(-7) M) blocked the isoproterenol-induced increase in lytic units per culture, but d-propranolol (10(-11) to 10(-7) M) did not. Terbutaline (10(-5) M), a relatively selective beta-2 agonist, similarly augmented the generation of cell-mediated cytotoxicity, with the increase again blocked by propranolol. Butoxamine (5 X 10(-6) M), a beta-2 antagonist, but not atenolol (5 X 10(-6) M), a beta-1 antagonist, blocked the epinephrine-induced increase in cell-mediated cytotoxicity. Addition of phentolamine (5 X 10(-6) M) had no effect on the epinephrine-induced increase in lytic units per culture. However, in the presence of phentolamine, norepinephrine increased lytic units per culture to a greater degree than that seen with norepinephrine alone, suggesting a balance between positive beta effects and inhibitory alpha effects upon simultaneous alpha and beta stimulation. These data provide further evidence for an immunoenhancing role of beta receptor stimulation during the generation of immune responses.

Documenting workload to better integrate clinical and distributive services.

Use of workload and work-flow documentation in two pharmacy satellites to evaluate pharmacists' productivity is described. Workload was evaluated during 14 consecutive days in May 1983 and 7 consecutive days in May 1984. All pharmacists and technicians working in the satellites recorded times for their activities on a form that listed every possible activity; interruptions such as answering the telephone, responding to walking requests, replacing stock, and lunches and break times were also recorded. Concurrently, the clinical coordinator observed and evaluated work performed for four-hour time blocks at least once each day. Analysis of workload and work-flow information identified problems with scheduling, priorities, interruptions, and pharmacists' knowledge of clinical pharmacy practice. Based on these observations, the following changes were implemented: pharmacists were scheduled to work either inside or outside the satellites for two-week or one-month time periods, priorities were assigned to certain tasks performed inside or outside the satellites, job assignments were made based on the pharmacist's capabilities and the needs of the patient-care areas, a form for documenting potential problem orders was created, and pharmacists were evaluated monthly and given one-on-one instruction by the clinical coordinator. Documentation of time use identified problems and led to changes in assignments that better integrated clinical, educational, and distributive responsibilities for the purpose of providing more efficient and effective services.

Alterations in lymphocyte subpopulations in copper-deficient mice.

Analyses of cell surface determinants of splenocytes from copper-deficient C58 mice indicate alterations in lymphocyte subpopulation characteristics. Both the absolute number and the relative percentage of surface immunoglobulin-bearing (B) cells from copper-deficient mice were significantly greater than those from copper-supplemented controls. The relative percentage of Thy 1.2-positive (T) cells was decreased, and the decrease was most prominent within the Lyt 1-positive (helper) T-cell subset. The functional responsiveness of both B cells and T cells was decreased in copper deficiency.

The immunomodulatory action of frentizole, a novel immunosuppressive agent.

The in vitro effects of frentizole and methylprednisolone on human PBL were examined. Both drugs exhibited differential effects on lymphocyte subpopulations. Frentizole was more effective in suppressing human lymphocytes responding to Con A and PWM, than it was in cells activated by PHA, specific antigen, or alloantigen. Methylprednisolone, on the other hand, was more inhibitory for cells stimulated by PHA, specific antigen, or alloantigen. These data suggest that frentizole has a preferential effect on the cytotoxic/suppressor subpopulation of human T lymphocytes while methylprednisolone appears to preferentially affect the helper/inducer subset. Differences in potency between the two drugs were also observed. Methylprednisolone was better able to maintain its inhibitory effects during longer periods of culture than frentizole. Both agents were less effective if their addition to the culture was delayed 24-48 hrs and neither drug was very effective in overcoming the adjuvant action of poly A:U on mitogenically activated human PBL.

Use of clinical pharmacists to reduce cefamandole, cefoxitin, and ticarcillin costs.

The financial impact of using cefamandole and cefoxitin rather than cefazolin and of using ticarcillin rather than carbenicillin in one institution was assessed; the effectiveness of clinical pharmacists in reducing the costs associated with these drugs also was determined. During Phase 1 (July 1, 1980-March 31, 1981), the numbers of intravenous piggyback cefazolin, cephalothin, cefamandole, cefoxitin, carbenicillin, and ticarcillin doses prepared were recorded. Quarterly purchase data for each drug were determined from invoice records. During Phase 2 (April 1, 1981-September 30, 1981), eight clinical pharmacists reviewed all patient charts for cefamandole, cefoxitin, and ticarcillin orders. If the indication for these orders was missing or considered inappropriate, the pharmacist contacted the prescriber and recommended substituting appropriate doses of cefazolin for cefamandole and cefoxitin and of carbenicillin for ticarcillin. The number of doses prepared and quarterly purchase data were collected as in Phase 1. The projected savings resulting from clinical pharmacist input relating to these drugs was calculated. Based on Phase 1 data, the total theoretical expense resulting from cefamandole and cefoxitin use instead of cefazolin and from ticarcillin use in place of carbenicillin was projected to be $233,448 annually. Cefamandole and cefoxitin accounted for 59.8 and 39.7% of total cephalosporin use in Phases 1 and 2, respectively. Ticarcillin accounted for 77.1% of the total ticarcillin and carbenicillin doses in Phase 1, and 16.6% in Phase 2. A projected annual savings of $156,756 was achieved because of clinical pharmacist input at a cost of $16,000 for time devoted to the effort. Clinical pharmacists were effective in reducing the use of cefamandole, cefoxitin, and ticarcillin in situations where cefazolin or carbenicillin could be substituted.

Hiring drug information personnel.

An analysis of the functions performed at a drug information center (DIC) is described as the basis for hiring personnel. A functional job analysis demonstrated a division in the duties of DIC personnel. The workload demanded a drug literature specialist (DLS) to access, store, and retrieve drug information, and a practitioner in drug information (PDI) to analyze this information and make the appropriate clinical judgements. The DIC hired a DLS with a background in library science and experience with computer searching techniques to work with the PDI. It is suggested that the combination of a qualified librarian and a drug information pharmacist can provide the DIC with the optimal personnel. The analysis was a valuable tool in selecting personnel who possess the appropriate skills, job understanding, attitude, and innovative abilities.

Synergistic effects of lipopolysaccharide on phytohemagglutinin- and concanavalin A-induced deoxyribonucleic acid synthesis in human peripheral blood lymphocytes: participation of T lymphocytes.

Lipopolysaccharide induces a synergistic uptake of tritiated thymidine in peripheral blood lymphocytes (PBL) when cultured with phytohemagglutinin or concanavalin A as compared to PBL incubated only with phytohemagglutinin or concanavalin A. In this study we investigated which subpopulations(s) of PBL is involved in this synergistic increase in deoxyribonucleic acid synthesis. Separation of PBL into sheep erythrocyte rosette-forming cells (T cells) and non-rosette-forming cells (B cells) showed that the T cells were responsible for the increased uptake of radiolabel. PBL and T cells had similar dose-response profiles and kinetic patterns. Paralleling this augmented tritiated thymidine uptake was an increase in the number of cells undergoing blast transformation. Delayed-addition experiments showed that the two mitogens must be added within 12 h of each other for maximal augmentation to occur. Finally, preincubation of T cells with lipopolysaccharide had no demonstrable effect on the amount of concanavalin A uptake by these cells. This model may provide unique information about the activation of human peripheral blood T cells compared to activation of these cells by one mitogen.

Cimetidine-induced augmentation of human lymphocyte blastogenesis by mitogen, bacterial antigen, and alloantigen.

The effect of Cimetidine, a histamine-type 2 receptor antagonist, was evaluated on the in vitro proliferative response of normal human peripheral blood lymphocytes (PBLs). Cimetidine (10(-3) to 10(-8) M) increased mitogen-induced blastogenesis by 22% (phytohemagglutinin (PHA) and by 27% (pokeweed) over nondrug-treated control values (P less than 005 for PHA and pokeweed). Preincubation of PBLs with Cimetidine further augmented blastogenesis as much as 2- to 3-fold (P less than 0.005 for both mitogens). Multiple testing of the same normal subject demonstrated consistent reproducibility of increased proliferation by Cimetidine. Similar statistically significant amplifications of the proliferative res-ponse were observed when bacterial antigen (streptokinase-streptodornase) or alloantigen was used to induce blastogenesis. Optimally effective concentrations of Cimetidine ranged from 10(-5) to 10(-7) M, which corresponds to expected clinical serum levels. These observations suggest that a histamine-type 2 receptor antagonist is capable of modulating the proliferative response of PBLs in the absence of exogenously added histamine. The immunoregulatory implication of this Cimetidine-induced proliferative augmentation is discussed in relation to clinical transplantation and cancer immunotherapy.

Differential susceptibility of human peripheral blood lymphocyte subpopulations to mitogenic activation. Influence of methylprednisolone.

The inhibition of the mitogenic activation of human peripheral blood lymphocyte (PBL) subpopulations by methylprednisolone (MP) was dependent on the mitogen used. Purified human T cells were more sensitive to the effects of MP than were B cells or PBLs, especially when these cells were activated by pokeweed mitogen (PWM). MP did not function by inhibiting binding of mitogen to the cell surface. After being mitogenically activated, human lymphocytes were resistant to the effects of MP. These effects of MP were shown to be reversible. Monocytes did not provide a significant degree of protection to mitogenically activated human T cells incubated with MP. These data suggest that MP-induced inhibition of the mitogenic activation of human PBLs may be a reflection of lymphocyte heterogeneity, and that the differential sensitivity of PBLs to MP may be used to isolate functionally different subpopulations of these cells.