Efficacy and tolerability of once-daily telithromycin compared with high-dose amoxicillin for treatment of community-acquired pneumonia.

BACKGROUND: This randomized, double-blind study compared the efficacy and tolerability of the new ketolide antimicrobial telithromycin with that of high-dose amoxicillin in the treatment of community-acquired pneumonia (CAP). PATIENTS AND METHODS: Adult patients (n = 404), with signs and symptoms of CAP and radiologic confirmation were randomized to receive telithromycin 800 mg once daily (n = 199) or amoxicillin 1,000 mg three times a day (n = 205) for 10 days. Clinical and bacteriologic outcomes were assessed at post-therapy test-of-cure (days 17-24) and late post therapy (days 31-36). RESULTS: The clinical cure rate for telithromycin-treated patients (per protocol) pst therapy (days 17-24) was 141/149 (94.6%) and compared well with that for amoxicillin (137/152 (90.1%)). Subset analysis of patients (per protocol) showed high clinical cure rates for patients aged >/= 65 years (telithromycin 21/24, 87.5%; amoxicillin 22/29, 75.9%); those with documented pneumococcal bacteremia (telithromycin 10/10, 100%; amoxicillin 7/9, 77.8%); and patients with a Fine score >/= III (telithromycin 31/34, 91.2%; amoxicillin 38/47, 80.9%). Bacterial eradication rates were comparable between treatments (telithromycin 42/48, 87.5%; amoxicillin 39/45, 86.7%), with 22/23 vs 18/21 Streptococcus pneumoniae strains 9/12 vs 11/13 Haemophilus influenzae strains and all Moraxella catarrhalis isolates (five and three patients, respectively) eradicated at the test-of-cure visit. Both treatments were generally well tolerated. CONCLUSION: Telithromycin 800 mg once daily is a convenient, optimal-spectrum, first-line treatment for CAP in adults, at least as effective and well tolerated as high-dose amoxicillin.

In vitro and in Vivo inhibition of immunoglobulin secretion by the immunosuppressive compound HR325 is reversed by exogenous uridine.

The objective was to demonstrate that the immunosuppressive agent HR325 (an inhibitor of dihydroorotate dehydrogenase, DHODH) inhibits immunoglobulin (Ig) secretion both in vitro and in vivo and that this effect can be reversed with exogenous uridine. In vitro, Ig secretion from mouse splenocytes was induced by lipopolysaccharide (LPS) for 5 days. HR325 inhibited the secretion of IgM and IgG with IC50 values of 2.5 and 2 microm, respectively. Adding uridine (50 microm) increased these values to 70 and 60 microm, respectively. Similarly, the IC50 values of another DHODH inhibitor, brequinar sodium, were also attenuated by uridine from 0.04 to 1 microm for IgM, and 0.012 to 10 microm for IgG. HR325 (and a structural analogue A771726) inhibited LPS-induced kappa light-chain cell surface expression on 70Z/3 cells, a property also reversed by uridine. In vivo, the secondary anti-sheep red blood cell (SRBC) antibody response (unaffected by uridine alone) was inhibited by HR325 and brequinar with respective ID50 values of 38 and 0.6 mg/kg per oral (p.o.). Immunosuppression with HR325 (50 mg/kg) and brequinar (1 mg/kg) was abrogated by uridine. Uridine had no effect on cyclophosphamide-induced (10 mg/kg p.o.) immunosuppression. These data are consistent with the immunosuppressive mechanism of HR325 being the result of pyrimidine depletion in vitro and in vivo.

Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor.

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.

Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide.

Leflunomide is currently in phase-III clinical trials for the treatment of rheumatoid arthritis. In this study, we have focused our efforts on the study of the mechanism of action of the active metabolite of leflunomide, A77 1726, in cells and tissue of human origin. The human high-affinity binding protein for radiolabelled A77 1726 was purified from solubilized U937 membranes by following the binding activity through the purification process and was characterized as the mitochondrial enzyme dihydro-orotate dehydrogenase (DHO-DH). The human and murine enzyme displayed identical pI and molecular mass values on SDS/PAGE (43 kDa), which contrasts notably with previous reports suggesting a molecular mass of 50 kDa for the human enzyme. DHO-DH activity was inhibited by A77 1726 and its analogue HR325 with similar potency in U937 and human spleen membrane preparations. HR325 was found to be anti-proliferative for phytohaemagglutinin-stimulated human peripheral blood mononuclear cells, at the same concentrations that caused accumulation of DHO and depletion of uridine. Supplementation of the cultures with exogenous uridine led to partial abrogation of the anti-proliferative effect. This is in line with our recent demonstration that the anti-proliferative effect in vitro of A77 1726 on lipopolysaccharide-stimulated mouse spleen cells was mediated by DHO-DH inhibition [Williamson, Yea, Robson, Curnock, Gadher, Hambleton, Woodward, Bruneau, Hambleton, Moss et al., (1995) J. Biol. Chem. 270, 22467-22472]. Thus, DHO-DH inhibition by A77 1726 and its analogues is responsible for the anti-proliferative effects in vitro of the compounds on human cells and is likely to be responsible for some of its effects in vivo.

Malononitrilamides synergistically prevent acute and treat ongoing skin allograft rejection with cyclosporine.

The low molecular weight malononitrilamides (MNAs), a new class of immunosuppressive agents, belong to the derivatives of leflunomide's active metabolite, A771726. They have been shown to bind specifically to dehydroorotate dehydrogenase and inhibit de novo pyrimidine biosynthesis, thereby blocking T- and B-cell proliferation and strongly suppressing IgM and IgG antibody production. Here we evaluated their efficacy together with cyclosporine (CyA) in rat skin allotransplantation models, using different strain combinations. Monotherapy of transplanted animals in these models with the MNAs HMR 1279 and HMR 1715 resulted in a significant and dose-dependent prolongation of the graft survival time. Even a short-term application showed efficacy in the prevention of acute rejection. The MNAs were also effective when treatment was started at the time of expected rejection crisis, demonstrating strong therapeutic activity to reverse ongoing acute rejection, whereas CyA was ineffective for the treatment of ongoing allograft rejection episodes. Combination therapy of MNAs with CyA proved to be very effective for the prevention of acute skin graft rejection. Interestingly, whereas CyA alone was unable to treat ongoing acute rejection episodes, comedication of MNAs and CyA, even after a short-term application, was synergistically effective and significantly suppressed ongoing allogeneic skin graft rejection. These results demonstrate that MNAs are potent and well tolerated immunosuppressants with a potential comparable to that of CyA, but they are superior to CyA in their ability to reverse acute rejection episodes. They represent powerful rescue drugs and demonstrate synergistic activity with CyA to prevent acute and treat ongoing skin allograft rejection.

Regulation of alloreactivity in the popliteal lymph node assay by the new immunosuppressants: malononitrilamides.

Malononitrilamides (MNAs) represent a new class of low molecular weight immunosuppressants and have been shown to prevent and reverse ongoing acute allograft rejection and effectively prolong xenograft survival in rodents. MNAs were also found to be potent inhibitors of B and T cell-mediated autoimmune processes and mediate their effects by binding specifically to dihydro-orotate dehydrogenase (DHODH), inhibiting de novo pyrimidine biosynthesis, thereby blocking T and B cell proliferation and strongly suppressing the IgM and IgG antibody production. Here we evaluated the effects of the MNAs (HMR 1279 and HMR 1715) on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local graft-versus-host reaction in Lewis x Brown Norway F1 hybrid rats by measuring the enlargement of the popliteal lymph nodes (PLN) draining the site of allogeneic cell injection. Oral administration of the MNAs dose-dependently prevented the localized lymphoproliferative response in the PLN assay and suppressed the lymph node hyperplasia. The MNAs even acted therapeutically when they were given during an ongoing alloreactivity as late as days 4 or 5 after challenge. Consistent with the mode of action, a complete reversal of the immunosuppression on the lymphoproliferation in vivo was attempted in this protocol by the addition of exogenous uridine during days 0-5. These data suggest the HMR 1279 and HMR 1715 mediate their antiproliferative and immunosuppressive effects in the PLN assay in vivo by decreasing the activity of DHODH in the lymph node cells and thereby inhibiting pyrimidine biosynthesis.

Potencies of leflunomide and HR325 as inhibitors of prostaglandin endoperoxide H synthase-1 and -2: comparison with nonsteroidal anti-inflammatory drugs.

The relative anti-inflammatory activities of the immunomodulators HR325 and leflunomide, or its active metabolite A77 1726, were examined by determining potencies in vitro on prostaglandin endoperoxide H synthase (PGHS) and in vivo in rat air pouch inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) were used as comparators. HR325 was more potent than A77 1726 as an inhibitor of PGHS in guinea pig polymorphonuclear leukocytes (IC50 = 415 and 4400 nM, respectively) and on isolated ovine PGHS-1 (IC50 = 64 and 742 microM) and PGHS-2 (IC50 = 100 and 2766 microM). In vivo, in rat carrageenan air pouch inflammation, HR325 but not leflunomide at 25 mg/kg inhibited accumulation of leukocytes (48%) and PGE2 (61%). HR325 was also more potent than A77 1726 against human peripheral blood mononuclear cell PGHS-1 [IC50 = 1.6 and 25.6 microM (thromboxane B2 production) or 1.1 and 8 microM (PGE2 production)] and lipopolysaccharide-induced PGHS-2 in human adherent peripheral blood mononuclear cells (IC50 = 435 nM and 9.5 microM) and peripheral blood polymorphonuclear leukocytes (IC50 = 91 nM and 3.2 microM). HR325 had low PGHS-2 selectivity in the human (2.5-12-fold) and was a more potent PGHS-2 inhibitor than naproxen, ibuprofen and piroxicam (28-fold). Assays using endogenous arachidonic acid as substrate yielded IC50 values for NSAIDs that were in general markedly lower than those published for assays using 10 microM substrate. With this approach, piroxicam had reasonable activity on human PGHS-2 (IC50 = 260-290 nM). Only NS398 and flufenamic acid were PGHS-2 selective in the human (90-330-fold and 37-60-fold, respectively); the other NSAIDs were either PGHS-1-selective (naproxen, ibuprofen, flurbiprofen and indomethacin) or nonselective (piroxicam and diclofenac). Inclusion of 10% human plasma reduced HR325 potency against PGHS-1 in human peripheral blood mononuclear cells approximately 32-fold (IC50 = 36 microM). Plasma protein binding further reduced HR325 potency (IC50 = 164 microM) and minimized the difference between HR325 and A77 1726 (IC50 = 292 microM) in a whole blood PGHS assay. Whether the greater activity against human PGHS-2 would allow HR325 to exhibit NSAID-like therapeutic effects in humans remains unclear.

Treatment with neutralizing antibodies specific for IL-1beta prevents cyclophosphamide-induced diabetes in nonobese diabetic mice.

Interleukin-1 (IL-1) has been shown to be involved in the pathogenesis of IDDM, but it is not clear which form, IL-1alpha or IL-1beta, is predominantly implicated. In this study, we have evaluated the contribution of IL-1beta by treating diabetes-prone nonobese diabetic (NOD) mice with specific neutralizing antibodies. First, we assessed the neutralizing potential of these antibodies in C57BL/6 mice under acute septic shock by measuring IL-1beta in sera 4 h after lipopolysaccharide injection. One milligram and 0.1 mg of anti-IL-1beta antibodies (Abs) were capable of neutralizing the IL-1beta produced, and the effect persisted for at least 5 days. Second, we evaluated the role of IL-1beta in the cyclophosphamide (CY)-accelerated model of diabetes. Nondiabetic male NOD mice were injected with 200 mg/kg CY and treated twice weekly with anti-IL-1beta Ab. The incidence of diabetes reached 76 and 100% in the control groups treated with 0.25 and 0.1 mg rabbit IgG, respectively. In contrast, only 34% of mice treated with 0.25 mg of anti-IL-1beta Ab became diabetic. In the group treated with 0.1 mg of anti-IL-1beta Ab, 89% of the mice became diabetic in the same period of time, demonstrating that the protective effect was dose dependent. Our results show that IL-1beta is a critical effector molecule in this model of IDDM and that its specific inhibition could be an attractive target for therapeutic intervention.

The alloreactivity in the popliteal lymph node (PLN) assay is regulated by malononitrilamides (MNAs).

Malononitrilamides (MNA 279 and MNA 715) represent a new class of low molecular weight immunosuppressants and belong to the derivatives of the primary metabolite of leflunomide A7771726. They have been shown to prevent and reverse established acute allograft rejection and effectively prolong xenograft survival, and have also been found to be potent inhibitors of B- and T-cell mediated autoimmune processes. The MNAs mediate their effects by binding specifically to dehydro orotate-dehydrogenase (DHODH) and inhibiting de novo pyrimidine biosynthesis, thereby blocking T- and B-cell proliferation and strongly suppressing the IgM and IgG antibody production. In this study we evaluated the effects of MNA 279 and MNA 715 on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local graft-versus-host (GvH) reaction in Lewis x Brown-Norway (LBN) F1-hybrid rats by measuring the enlargement of the PLN draining the site of allogeneic cell injection. Oral administration of one of the two MNAs (7.5 to 50 mg/kg) on day 0, dose-dependently prevented the localized lymphoproliferative response and suppressed the lymph node hyperplasia. The MNAs even acted therapeutically when they were given during an ongoing alloreactivity as late as day 4 or 5 after challenge. Consistent with the mode of action that MNAs inhibit de novo pyrimidine biosynthesis, a complete reversal of the immunosuppression on the lymphoproliferation in vivo was attempted in this protocol by addition of exogenous uridine during days 0 to 5. These data suggest that MNA 279 and MNA 715 mediate their antiproliferative and immunosuppressive effects in the PLN-assay in vivo by decreasing the activity of DHODH in the lymph node cells and thereby inhibiting pyrimidine biosynthesis.

Dihydroorotate dehydrogenase is a high affinity binding protein for A77 1726 and mediator of a range of biological effects of the immunomodulatory compound.

A protein with high affinity (Kd 12 nM) for the immunomodulatory compound A77 1726 has been isolated from mouse spleen and identified as the mitochondrial enzyme dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a pI 9.6-9.8 and a subunit Mr of 43,000. Peptides derived from the mouse protein displayed high microsequence similarity to human and rat dihydroorotate dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate dehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative effects of A77 1726 are mediated by enzyme inhibition and can be overcome by addition of exogenous uridine. The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to counteract inhibition of the murine acute graft versus host response.

Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes.

Mycoplasma arginini TUH-14 partially purified membrane lipoproteins (TUH-14-pp) directly induce secretion of the cytokines involved in the inflammatory response, namely, interleukin 1 (IL-1), tumor necrosis factor alpha, and IL-6, by human monocytes cultured in the absence of serum. The biological activity of each cytokine correlates with its immunoreactivity. Upon stimulation with either TUH-14-pp or lipopolysaccharide, most tumor necrosis factor alpha and IL-6 is secreted in the extracellular compartment, whereas a significant amount of IL-1 remains cell associated. Finally, polymyxin B does not affect secretion of cytokines induced by TUH-14-pp, indicating that mycoplasma lipopolysaccharide does not account for their effects on monocytes. Altogether, our data show that direct interaction of mycoplasma membrane components with human blood monocytes induces secretion of high levels of cytokines known to trigger inflammatory responses. This new concept of membrane-bound active components of mycoplasma may explain its ability to efficiently initiate inflammatory reactions.

Interactions between mycoplasmas and the immune system.

Mycoplasmas are a heterogenous group of prokaryotic organisms causing a wide variety of diseases, including autoimmune disorders. Thus, it is not surprising that various mycoplasmas strains, including Mycoplasma arginini, M. arthritidis, M. neurolyticum and M. pulmonis, are able to regulate the immune response. Though some of the studies of the immunomodulatory action of mycoplasmas have been done in vivo, the majority of the investigations have been conducted in vitro. This has led to the recognition that mycoplasmas are polyclonal activators of both B and T cells from several species, acting through MHC-restricted or -unrestricted pathways. Mycoplasma activation not only induces T-cell proliferation but also leads but to the formation of cytotoxic T cells. We, as well as others, have shown that mycoplasma-mediated B-cell activation induces proliferation as well as Ig secretion, and also that mycoplasma stimulation of lymphocytes may result in the production of cytokines. We communicate here our investigations into the effects of an M. arginini strain on the growth and maturation of preactivated B cells. After an initial biological characterization of the M. arginini effects in vitro, we established the protein nature of the growth-supporting activity and proceeded further on to isolate and identify the responsible proteins. The use of lipid- and lipoglycan-free extracts has allowed us to further extend our studies on the biological activities of the proteins from M. arginini and to compare these results with the effects obtained using live organisms. Furthermore, the study was extended to include a characterization of the in vivo-induced effects of live M. arginini. Altogether, the results from these experiments allow us to conclude that M. arginini is a T-cell independent polyclonal B-cell mitogen, mediated by five identified proteins, inducing growth and Ig secretion of both resting and preactivated B cells.

Functional bioassays for B cell growth factors using polyclonally activated murine spleen B cells.

In this study, we have set up and optimized three distinct T cell-independent polyclonal B cell activation assay systems using highly T cell-depleted murine spleen B cells which were either preactivated in vitro with lipopolysaccharide (LPS) or costimulated with anti-IgM antibodies (anti-mu) or dextran sulfate (DxS). Using these assays, we have investigated the effects of recombinant human or murine interleukins, as well as those of a partially purified T cell-derived factor, designated BSF-LPS. Our results show that none of the interleukins, alone or in combination, is able to maintain growth of the LPS-preactivated B cell blasts, even at the highest concentrations tested, whereas the addition of our BSF-LPS preparation to the cultures significantly increases DNA synthesis. As expected, recombinant murine IL-4 (r mu IL-4) causes a substantial proliferation of anti-mu costimulated B cells. Such anti-mu costimulated B cells also respond, to a lower degree, to recombinant human IL-1 alpha (r hu IL-1 alpha), and do not significantly proliferate upon addition of r mu IL-2, r mu IL-5 or BSF-LPS. On the other hand, r mu IL-5 stimulates very efficiently DxS-costimulated B cells proliferation, whereas r hu IL-1 alpha only exerts a marginal effect; r mu IL-2, r mu IL-4 or BSF-LPS did not maintain growth of DxS-costimulated B cells. We believe that such a thorough investigation of the particular behaviour of activated B cell subpopulations towards various lymphokines provides the background for setting up a valuable bioassay method to differentiate the various interleukins acting on B cells through parallel use of the three distinct T cell-independent polyclonal B cell activation assay systems.

Biochemical characterization of B cell stimulatory factors for lipopolysaccharide-preactivated B cell blasts: distinction from other known lymphokines.

We have performed a biochemical characterization of B cell stimulatory factors (BSF) produced by Con A-stimulated mouse spleen cells that stimulate growth of lipopolysaccharide (LPS)-preactivated B cells (designated BSF-LPS). Two biochemically distinct forms of BSF-LPS were identified in preparative isoelectric focusing, one acidic form having a pI of 3.9-4.4 and a more basic form with a pI 5.2-5.9. The biochemical heterogeneity of the BSF-LPS activity from Con A-stimulated spleen cells was further demonstrated by ion exchange chromatographies using a fast protein liquid chromatography (FPLC) system. The acidic and the basic forms of BSF-LPS could be totally separated from each other and both are distinct from interleukin-2 (IL-2). Moreover, extending the characterization of the BSF-LPS, together with the use of various murine assay systems for BSF, we could formally exclude that IL-4 or IL-5 accounted for the BSF-LPS activities. In summary, our data provide evidence for the existence of heterogeneous BSF-LPS which maintain growth of LPS-preactivated B cell blasts, and show that these factors can be distinguished from the other lymphokines which have been involved in the control of the cell growth of murine B lymphocytes.

Growth-supporting activity of fragment Ba of the human alternative complement pathway for activated murine B lymphocytes.

We have investigated the effects of cleavage of factor B by its activating enzyme, factor D, as well as its activation fragments Bb and Ba, on the growth of mouse spleen B lymphocytes preactivated by LPS. Neither factor B nor factor D show any growth-supporting activity when tested alone. The coaddition of factor B and factor D to serum-free cultures of LPS-preactivated B cell blasts increased the proliferation of the responding cells up to the level obtained by restimulation with LPS. Such growth-supporting activity was shown to be mediated by Ba, whereas Bb did not show any significant effect. Furthermore, this effect was not restricted to the LPS-preactivated B cell blasts; in fact, Ba also supported the growth of in vivo, activated B cell blasts of unprimed mice of the LPS-nonresponder C3H/HeJ strain. In contrast, Ba did not maintain growth of Con A-activated T cells or TCGF-dependent CTL cells. Taken together, these results describe the first biological activity of human Ba as a B cell stimulatory factor.

Enhancement of immunoglobulin secretion by the lymphokine-like activity of a Mycoplasma arginini strain.

B lymphocytes, preactivated by lipopolysaccharide (LPS), could be triggered to growth by a strain of Mycoplasma arginini, while the level of immunoglobulin (Ig) secretion, quantitated as the number of plaque-forming cells (PFC), was low. The PFC response could be increased by the addition of conditioned media (CM) from lectin-activated spleen cells or T cell tumour EL-4 to the culture of preactivated B cells. CM did not by itself induce a significant amount of PFC in the cultures of LPS-preactivated B cells. The maturation enhancing activity was distinct from IL-2 and B cell growth factor as judged by gel exclusion chromatography.