Efficacy and safety of tigecycline compared with vancomycin or linezolid for treatment of serious infections with methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci: a Phase 3, multicentre, double-blind, randomized study.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are causing serious nosocomial infections. Tigecycline was evaluated in hospitalized patients with MRSA or VRE infection. PATIENTS AND METHODS: A randomized (3:1), double-blind, multicentre, Phase 3 study compared the safety and efficacy of tigecycline with vancomycin or linezolid in hospitalized patients with MRSA or VRE infection, respectively. Patients were treated for 7-28 days and the test-of-cure (TOC) assessment was made 12-37 days after the last dose. The primary efficacy endpoint was the clinical response (cure, failure and indeterminate) in the co-primary, microbiologically evaluable (ME) and microbiologically modified intent-to-treat (m-mITT) populations at the TOC assessment. RESULTS: For MRSA infection, clinical cure rates in the ME population (n = 117) were 81.4% (70 of 86 patients) with tigecycline and 83.9% (26 of 31 patients) with vancomycin. In the m-mITT population (n = 133), clinical cure occurred in 75 of 100 tigecycline-treated patients (75.0%) and in 27 of 33 vancomycin-treated patients (81.8%). In patients with complicated skin and skin structure infections caused by MRSA, cure rates were similar with tigecycline or vancomycin (86.4% versus 86.9% in ME population; and 78.6% versus 87.0% in m-mITT population). In patients with MRSA infection, nausea or vomiting occurred more frequently with tigecycline than with vancomycin (41.0% versus 17.9%); most cases were mild, with only three patients discontinuing treatment. In patients with VRE (total enrollment, 15), 3 of 3 and 3 of 8 patients in the ME and m-mITT populations, respectively, were cured by tigecycline, compared with 2 of 3 patients in the ME and m-mITT populations treated with linezolid. CONCLUSIONS: Tigecycline is safe and effective in hospitalized patients with serious infection caused by MRSA. There were too few cases of VRE to draw any conclusions.

Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

Granzyme B independently of perforin mediates noncytolytic intracellular inactivation of vesicular stomatitis virus.

Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe an in vitro model to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host beta-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.

Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene.

We have investigated a possible delivery system for the rat preproinsulin II gene (rI2) utilising a recombinant adeno-associated virus (rAAV) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. The rAAV vector was chosen because it is a safe and nonpathogenic method for gene transfer. The plasmid pBC12BI (ATCC) was purified and digested with restriction enzymes SepI and StuI to release a fragment containing the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene (rI2). Subsequently, the RSV-rI2 gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI2 recombinant plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted terminal repeats for integration and replication and the herpes virus thymidine kinase promoter-driven gene for neomycin resistance (neoR). The cell line 293 (ATCC) was then cotransfected with pLP-1 and helper plasmid pAAV/AD, which is required for viral replication. The rAAV genome, now containing rI2, was rescued using adenovirus and packaged into mature AAV virions termed vLP-1. Finally, human pancreatic adenocarcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 resistance, and screened for insulin production. Successful rescue was confirmed by Southern blot analysis using the rI2 gene probe derived from the original plasmid. The final titer of 1.25 x 10(9) particles/ ml was determined by DNA slot blots using pLP-1 as the standard, HPAC cells were infected with vLP-1 (termed HPAC/rI2). Integration of the rI2 genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI2 gene by PCR. Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat preproinsulin II gene. Successful transduction and stable integration of rI2 into HPAC was achieved. Production of insulin by HPAC/rI2 was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy.

Fas involvement in cytotoxicity mediated by human NK cells.

Lysis of target cells (TC) by cytolytic lymphocytes involves the secretion of cytoplasmic granules containing perforin and serine esterases by the effector cell (EC). Recently, a granule-independent cytolytic mechanism involving the interaction of the apoptosis-triggering Fas antigen (CD95) with Fas ligand (FasL) has been revealed in T cells. However, whether the Fas lytic pathway also functions in NK cells has not been established. We purified human peripheral NK cells (> 98% CD56+) and found that PMA and ionomycin treatment upregulated FasL message and stimulated the NK cells to lyse a Fas+ TC. This lysis was partially inhibited by the anti-Fas-blocking antibody M3 or by Fas.Fc fusion protein. We also found that FasL is constitutively expressed on the human NK-like leukemia cell line YT-INDY and that YT-INDY utilizes a Ca(2+)-independent Fas lytic pathway, as well as the granule pathway. We have previously shown that CD28/B7 interactions are involved in TC recognition by YT-INDY. K562 cotransfected with Fas and B7-1 (K562/Fas/B7) was lysed by YT-INDY at a higher level than a vector-transfected K562 line, whereas K562 transfected with Fas alone was not. Lysis of K562/Fas/B7 cotransfectants was partially Fas-mediated, as indicated by the presence of Ca(2+)-independent, M3-inhibitable lysis. Ca(2+)-independent, Fas-mediated lysis of several TC by YT-INDY was inhibited by anti-CD28 antibody. Anti-LFA-1 also inhibited Fas-mediated cytotoxicity in YT-INDY. Thus, fresh human NK cells and the human NK-like cell line YT-INDY are capable of using the Fas lytic pathway. In YT-INDY, CD28/B7 and LFA-1/ICAM interactions appear to influence the Fas lytic pathway.

Fas-mediated cytotoxicity remains intact in perforin and granzyme B antisense transfectants of a human NK-like cell line.

Cell-mediated cytotoxicity (CMC) has traditionally been thought to involve the release of granule components, including perforin and granzymes, from the effector cell (EC) onto the target cell (TC) membrane. Recently, a granule-independent cytolytic mechanism involving the interaction of Fas antigen (CD95) with Fas ligand has been described. We have generated antisense perforin (YT-xP1) and granzyme B (YT-xGrB) transfectants of the human NK-like cell line YT-INDY. These transfectants have greatly reduced cytolytic ability when compared to the vector-transfected control cell line (YT-neo). In this study, however, we demonstrate that the antisense transfectants retain the ability to lyse Fas+ TC. Fas-mediated lysis is Ca(2+)-independent and is inhibited by a monoclonal anti-Fas blocking Ab, M3. By RT-PCR, we detect message for FasL in unstimulated YT-xP1 and YT-xGrB transfectants, as well as in unstimulated YT-neo. By flow cytometry, we show that YT-neo, YT-xGrB, and YT-xP1 constitutively express surface FasL. These data indicate that in a human NK-like cell line, similar to the murine system, the granule and Fas pathways of cytotoxicity function independently of one another. At least with the TC tested, our data also indicate that the granule and Fas pathways together account for nearly 100% of the cytolytic ability of YT-INDY.

Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability.

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by > 95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by > 95%. Western analysis revealed a 30% decrease in the level of perforin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.

Synergistic inhibition of human cell-mediated cytotoxicity by complement component antisera indicates that target cell lysis may result from an enzymatic cascade involving granzymes and perforin.

A widely accepted theory of lymphocyte-mediated cytotoxicity (CMC) proposes that upon effector cell (EC) and target cell (TC) interaction, release of perforin, serine proteases and other lytic moieties contained within cytoplasmic granules results in TC lysis. Complement activation and the activation of the various enzymatic activities associated with cytotoxic granules have strikingly similar modes of action and both lead to pore formation in their respective targets. We report here that by using antisera to early and late complement components we were able to inhibit CTL, NK and ADCC cytotoxicity up to 100%, even though binding of EC to TC was unaffected. Furthermore, we showed that addition of C1q or C1s (two serine proteases) antisera to C9 antisera, at titers too low to inhibit separately, resulted in synergistic inhibition of CMC. Anti-C1s together with anti-C1q (or anti-C8 with anti-C9) did not result in synergy. This finding supports a cascade model of activation for lytic molecules released from EC. In addition, we demonstrated that anti-C1q and anti-C1s bind to proteins in the 30-kD region and anti-C9 binds to proteins in the 70-kD region, coinciding with published molecular weights of granzymes and perforin, respectively. Finally, lytic ability of purified granules was also inhibited by complement antisera, further suggesting that activation occurs outside of TC. Taken as a whole, these data indicate that TC lysis may be the result of a cascade of events involving granzymes and perforin, analogous to that seen with the complement system.

The lack of NK cytotoxicity associated with fresh HUCB may be due to the presence of soluble HLA in the serum.

Human bone marrow transplantation is becoming more common in the treatment of certain forms of cancer despite the scarcity of HLA matched donors. Because human umbilical cord blood (HUCB) has been used as a source for stem cells in bone marrow transplantation, and because NK cells appear to be important in graft versus leukemia response, we investigated the lytic activity of freshly isolated HUCB NK cells (HUCB-NK) against tumor targets and their ability to differentiate into LAK cells following stimulation with various cytokines. Although cytotoxicity mediated by fresh HUCB-NK was low compared to that of adult peripheral blood lymphocyte-derived NK cells (PBL-NK), the ability of HUCB-NK to bind to K562 target cells (TC) was similar to PBL-NK. In addition, the PBL-NK cytotoxicity of postpartum mothers was also low compared to that of normal adult PBL-NK. When we incubated HUCB for 18 hr in either IL-2 or IL-12, we boosted the level of HUCB-NK cytotoxicity to approximately the level observed in PBL-NK and increased the level of perforin, granzyme A, and granzyme B mRNA expression. In addition, when we incubated HUCB in IL-2, IL-4, IL-7, IL-12, TNF-alpha, IFN-alpha, IFN-gamma, or TGF-beta for 5 days, we observed that HUCB was capable of generating LAK cells only when incubated with either IL-2 or IL-12. In contrast, IL-2, IL-7, IL-12, TNF-alpha, and IFN-gamma all generated LAK cells from adult PBL. When we added to the medium low-dose IL-2 and irradiated K562 as feeder cells (mini-LAK), we were unable to generate LAK activity from HUCB-NK, whereas we could generate it with PBL-NK cells under the same conditions. Addition of serum derived from HUCB in a 4-hr 51Cr release assay with PBL-NK as the effector cells (EC) and K562 as the TC resulted in a 42% decrease in PBL-NK-mediated cytotoxicity. Although we detected no TGF-beta in HUCB serum, we did detect high concentrations of soluble class I MHC (sHLA). To our knowledge, sHLA has not previously been shown to inhibit NK cytotoxicity, although the expression of class I HLA on the surface of TC has been shown to inhibit NK cytotoxicity. To study further the effect of sHLA on cell-mediated cytotoxicity, we added various concentrations of sHLA to EC mediating NK, ADCC, and CTL activities. All were inhibited in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Increase in tumor necrosis factor-alpha mRNA but not perforin mRNA expression in response to two newly characterized anti-LFA-1 monoclonal antibodies.

We have generated two monoclonal antibodies (mAb), designated anti-1B11 and anti-4F9, directed to the human lymphocyte-function-associated antigen-1 (LFA-1). Indirect immunofluorescence with both mAb showed a bimodal distribution of antigen on the surface of T, natural killer (NK), and lymphokine-activated killer (LAK) cells. Neither mAb reacted with the epitopes recognized by TA1 and Mo-1 mAb on the alpha-chain of the heterodimer. Anti-1B11 and anti-4F9 immunoprecipitated polypeptide chains with molecular weights of 177 and 95 kD. Both mAb inhibited cytolytic T lymphocytes (CTL), NK, and LAK cell-mediated cytotoxicity without affecting antibody-dependent cellular cytotoxicity (ADCC). The proliferative responses of T cells to allogeneic cells were inhibited by anti-1B11 and anti-4F9, whereas the responses to phytohemagglutinin P and concanavalin A were not affected. Anti-1B11 and anti-4F9 blocked effector cell (EC)-target cell (TC) conjugate formation by 50%. Only anti-4F9 cross-reacted with LFA-1 on porcine peripheral blood lymphocytes and inhibited porcine NK, LAK, and ADCC activities. Because LFA-1 also functions at the level of signal transduction during T cell activation and we previously showed that CTL rapidly degraded perforin and tumor necrosis factor-alpha (TNF alpha) mRNA after interaction with sensitive TC, we examined the effects of the mAb on the messages for perforin and TNF alpha. Treatment of CTL with anti-1B11 and anti-4F9 induced TNF alpha message and protein levels of TNF alpha, but did not alter perforin mRNA levels.

Human granzyme B is essential for DNA fragmentation of susceptible target cells.

We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YT-INDY and NK3.3, two human cytotoxic cells, were also lysed. EGTA reduced lysis by only 50%, suggesting that a perforin-independent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and 125I-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.

Target cell-directed degradation of perforin mRNA in CTL: lack of correlation with loss of protein and lytic ability.

We have previously shown that CTL and NK cells rapidly down regulate perforin mRNA and become functionally inactive within 4-6 hr after exposure to sensitive target cells (TC). We report here for the first time that CTL also down regulate perforin mRNA upon exposure to resistant, but binding, TC. When three separate human MHC-restricted CTL lines were exposed to resistant TC, perforin mRNA was rapidly degraded. Removal of both extracellular Ca++ and Mg++ prevented perforin message down regulation, whereas removal of Ca++ alone did not, indicating that CTL:TC binding was required. Unlike the response of CTL exposed to sensitive TC, resistant TC did not trigger serine esterase (SE) release, suggesting distinct signalling pathways for perforin mRNA down regulation and granule exocytosis. Moreover, using western analysis, we showed that there was limited (< 10%) perforin protein release after CTL:TC interaction, suggesting that CTL loss of lytic activity after exposure to sensitive TC is not due to massive depletion of perforin. Treatment of CTL with mAb to CD2, CD3, CD2 + CD3, CD8, Class I and LFA-1 did not induce perforin mRNA down regulation. Furthermore, mAb to CD2, CD3, CD8, Class I, Class II, CD54 and LFA-1 did not block TC-mediated perforin mRNA down regulation although lysis of TC was inhibited.

Target cell-directed rapid degradation of TNF-alpha messenger RNA in human cytotoxic T cells.

Previously we have shown that upon exposure to sensitive target cells (TC), human cytotoxic T cells (CTL) and natural killer cells (NK) undergo functional inactivation and specifically degrade mRNA-encoding lytic proteins stored in cytoplasmic granules. These lytic proteins include perforin (PFP) and the serine proteases (granzymes). In this study we show that tumor necrosis factor-alpha (TNF-alpha) mRNA undergoes rapid down-regulation after interaction with TC, in a manner identical to PFP and serine protease mRNA. Both PFP and TNF-alpha messages are down-regulated and maintained at low baseline levels while effector cells (EC) are in contact with TC. Moreover, treatment of CTL with antibodies against CD2, CD3, CD8, and class I could not reproduce TC-directed mRNA down-regulation of TNF-alpha and PFP even though lytic ability was inhibited. Anti-CD2 and anti-CD3, however, markedly induced the expression of TNF-alpha mRNA within 15 min. The increase of TNF-alpha mRNA by anti-CD2 and anti-CD3 was offset by the presence of TC, suggesting that TC supply a negative signal to the CTL resulting in degradation of the TNF-alpha mRNA. Furthermore, treatment of CTL with cycloheximide (CHX) did not prevent PFP message loss and did not allow its recovery, suggesting that de novo protein synthesis is not required for mRNA degradation. In contrast, whereas CHX did not prevent TNF-alpha message loss, CHX allowed recovery of TNF-alpha mRNA within 120 min after TC-directed degradation. Taken as a whole, these data suggest that TC provides a negative regulatory signal that triggers the degradation of TNF-alpha message.