Immunosuppression and regulation: cast in new light?
Explore the source record for details and available documents.
Antilymphocyte Serum↗
Biomedical subjects
David M Rothstein
Publications and source records attributed to David M Rothstein.
Efficacy of novel rifamycin derivatives against rifamycin-sensitive and -resistant Staphylococcus aureus isolates in murine models of infection.
Novel rifamycins (new chemical entities [NCEs]) having MICs of 0.002 to 0.03 microg/ml against Staphylococcus aureus and retaining some activity against rifampin-resistant mutants were tested for in vivo efficacy against susceptible and rifampin-resistant strains of S. aureus. Rifalazil and rifampin had a 50% effective dose (ED50) of 0.06 mg/kg of body weight when administered as a single intravenous (i.v.) dose in a murine septicemia model against a susceptible strain of S. aureus. The majority of NCEs showed efficacy at a lower i.v. dose (0.003 to 0.06 mg/kg). In addition, half of the NCEs tested for oral efficacy had ED50s in the range of 0.015 to 0.13 mg/kg, i.e., lower or equivalent to the oral ED50s of rifampin and rifalazil. NCEs were also tested in the septicemia model against a rifampin-resistant strain of S. aureus. Twenty-four of 169 NCEs were efficacious when administered as a single oral dose of 80 mg/kg. These NCEs were examined in the murine thigh infection model against a susceptible strain of S. aureus. Several NCEs dosed by intraperitoneal injection at 0.06 mg/kg caused a significant difference in bacterial titer compared with placebo-treated animals. No NCEs showed efficacy in the thigh model against a highly rifampin-resistant strain. However, several NCEs showed an effect when tested against a partially rifampin-resistant strain. The NCEs having a 25-hydroxyl moiety were more effective as a group than their 25-O-acetyl counterparts. These model systems defined candidate NCEs as components of potential combination therapies to treat systemic infections or as monotherapeutic agents for topical applications.
Administration, Oral↗
Antigen exposure during enhanced CTLA-4 expression promotes allograft tolerance in vivo.
The role of CTLA-4 in tolerance is primarily inferred from knockout and blocking studies. Anti-CD45RB mediates allograft tolerance in mice by inducing CTLA-4 expression on CD4 cells, providing a novel opportunity to determine how therapeutic enhancement of CTLA-4 promotes tolerance. We now show that induced CTLA-4 expression normally resolves by day 17. Although thymectomy prolongs enhanced CTLA-4 expression, long-term engraftment is unaffected. To address the temporal relationship between increased CTLA-4 expression and engraftment, transplantation was delayed for various times after anti-CD45RB treatment. Delaying transplantation for 7 days (when CTLA-4 expression had peaked but treatment mAb was no longer detectable), resulted in long-term engraftment comparable to transplantation with no delay (day 0). Delaying transplantation from 10 to 18 days led to a progressively poorer outcome as CTLA-4 expression returned to baseline. This suggested that Ag exposure while CTLA-4 expression is enhanced is sufficient to induce long-term engraftment. To substantiate this, on day 0, anti-CD45RB-treated mice received BALB/c vs unrelated alloantigen, followed by transplantation of BALB/c islets 10 days later. Whereas recipients exposed to unrelated Ag experienced acute rejection, recipients exposed to donor Ag achieved long-term engraftment. Anti-CD45RB-treated mice exposed to alloantigen exhibited anergic CD4(+)CD25(-) effector cells and regulatory CD4(+)CD25(+) cells. Moreover, CD25 depletion in the peritransplant period prevented anti-CD45RB-mediated engraftment. Thus, exposure of CD4 cells expressing CTLA-4 to donor Ag is necessary and sufficient to induce long-term engraftment which appears to be mediated by both regulation and anergy.
Animals↗
In vitro time-kill activities of rifalazil, alone and in combination with vancomycin, against logarithmic and stationary cultures of Staphylococcus aureus.
Rifalazil is a novel rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the beta subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that (1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, (2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log10 over either drug used alone and delayed the appearance of rifamycin-resistant mutants, (3) rifalazil and vancomycin in combination killed stationary phase cultures
Anti-Bacterial Agents↗
Enhanced activity of rifalazil in combination with levofloxacin, linezolid, or mupirocin against Staphylococcus aureus in vitro.
Rifalazil is a potent second-generation ansamycin that kills bacterial cells by inhibiting the beta subunit of RNA polymerase. Rifalazil has several improved properties compared with rifampicin, but retains rifampicin's propensity to develop resistant mutants at high frequency. To explore strategies to overcome resistance development, we studied the effects of rifalazil in combination with several different antibiotics in an in vitro time-kill model, against both log phase and stationary phase Staphylococcus aureus cells. Experiments were carried out at high initial cell density so that the frequency and proliferation of resistant mutants could be monitored. We found that each combination was advantageous in terms of enhanced killing and the suppression of mutants, compared with each drug used alone. None of the three combinations was effective against stationary phase cells.
Acetamides↗
Rifalazil pretreatment of mammalian cell cultures prevents subsequent Chlamydia infection.
Chlamydia species are widely disseminated obligate intracellular pathogens that primarily cause urogenital, ocular, and respiratory infections. In these studies, we show that exposing mammalian cells to antibacterial agents prior to Chlamydia inoculation protects the host cells against subsequent challenge by chlamydiae (the protective effect [PE]). Rifalazil exhibited a considerably stronger PE than did azithromycin, rifampin, doxycycline, and ofloxacin. Specifically, 0.002 microg/ml rifalazil incubated for 1 day with a monolayer of McCoy cells was sufficient to protect against a challenge 2 days later with Chlamydia trachomatis serovar D (UW-3). The PE was observed with five different mammalian cell lines and with a variety of C. trachomatis and Chlamydia pneumoniae isolates. The duration of the PE was 6 to 12 days for rifalazil (depending on the cell line), a maximum of 3 days for azithromycin, and less than a day for the other drugs tested. For rifalazil, the PE was shown to be mediated by inhibition of the chlamydial RNA polymerase since mutants with altered RNA polymerases had correspondingly altered PEs. These results suggest that rifalazil may be unique in its ability to prevent infection with obligate intracellular pathogens for a considerable time after treatment. This characteristic may be of particular public health value in reducing reinfection with chlamydiae.
Anti-Bacterial Agents↗
In vitro activity of novel rifamycins against rifamycin-resistant Staphylococcus aureus.
We describe novel rifamycin derivatives (new chemical entities [NCEs]) that retain significant activity against a comprehensive collection of Staphylococcus aureus strains that are resistant to rifamycins. This collection of resistant strains contains 21 of the 26 known single-amino-acid alterations in RpoB, the target of rifamycins. Some NCEs also demonstrated a lower frequency of resistance development than rifampin and rifalazil in S. aureus as measured in a resistance emergence test. When assayed for activity against the strongest rifamycin-resistant mutants, several NCEs had MICs of 2 microg/ml, in contrast to MICs of rifampin and rifalazil, which were 512 microg/ml for the same strains. The properties of these NCEs therefore demonstrate a significant improvement over those of earlier rifamycins, which have been limited primarily to combination therapy due to resistance development, and suggest a potential use of these NCEs for monotherapy in several clinical indications.
Drug Resistance, Bacterial↗
Activity of novel benzoxazinorifamycins against rifamycin-resistant Streptococcus pyogenes.
Explore the source record for details and available documents.
Amino Acid Substitution↗
Development potential of rifalazil and other benzoxazinorifamycins.
Rifalazil and other benzoxazinorifamycins (new chemical entities [NCEs]) are rifamycins that contain a distinct planar benzoxazine ring. Rifalazil has excellent antibacterial activity, high intracellular levels and high tissue penetration, which are attributes that favour its use in treating diseases caused by the obligate intracellular pathogens of the genus Chlamydia. Recent studies have shown that rifalazil has efficacy in the treatment of human sexually transmitted disease caused by Chlamydia trachomatis. The extraordinary potency of rifalazil and other NCEs, such as ABI-0043, extends to the related microorganism, C. pneumoniae, a respiratory pathogen that can disseminate and persist chronically in the vasculature, resulting in increased plaque formation in animal studies. A pivotal clinical trial with rifalazil has been initiated for the treatment of peripheral arterial disease. Other opportunities include gastric ulcer disease caused by Helicobacter pylori and antibiotic-associated colitis caused by infection with Clostridium difficile in the colon. The NCEs could prove to be valuable as follow-on compounds in these indications, as rifampin replacements in antibacterial combination therapy or as stand-alone topical antibacterials (e.g., to treat acne). Neither rifalazil nor NCEs appear to induce the cytochrome P450 3A4, an attribute of rifampin that can result in adverse events due to drug-drug interactions.
Animals↗
Requirements for induction and maintenance of peripheral tolerance in stringent allograft models.
Peripheral tolerance can be achieved in many but not all murine allograft models. The requirements for controlling more aggressive immune responsiveness and generating peripheral tolerance in stringent allograft models are unknown. Understanding these requirements will provide insight toward ultimately achieving tolerance in humans, which are also resistant. We now demonstrate that the combination of donor-specific transfusion, anti-CD45RB, and anti-CD154 uniformly achieves >90-d survival of BALB/c skin allografts on C57BL/6 recipients. Recipients exhibit marked hyporesponsiveness to alloantigen in vitro. In distinct contrast to less rigorous models, engraftment remains absolutely dependent on cytotoxic T lymphocyte antigen 4 signaling, even after grafts are healed, suggesting that prolonged engraftment cannot simply be attributed to more effective depletion of alloreactive T cells but is actively maintained by regulation. Concordantly, we show that both CD4 and CD8 regulatory cells are required and can transfer donor-specific tolerance to naïve recipients. Nonetheless, most recipients ultimately develop gradual graft loss (median survival time = 140 d), suggesting that alloreactive cells emerging from the thymus eventually overwhelm regulatory capacity. In agreement, adding thymectomy to the regimen results in permanent engraftment (>250 d) and donor-specific tolerance not observed previously in this model. These results highlight the potency of both CD4 and CD8 regulatory cells but also suggest that in stringent settings, regulatory T cell longevity and capacity for infectious tolerance compete with prolonged graft immunogenicity and thymic output. These results provide insight into the mechanisms of tolerance in stringent models and provide a rational basis for innovative tolerogenic strategies in humans.
Animals↗
Testicular immune privilege promotes transplantation tolerance by altering the balance between memory and regulatory T cells.
Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8(+) T cells but induces more Ag-specific CD4(+)CD25(+) regulatory T cells than in a conventional site. These CD4(+)CD25(+) cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8(+) T cells or deletion of CD25(+) T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.
Animals↗
Impaired recall of CD8 memory T cells in immunologically privileged tissue.
Foreign Ags that enter immunologically privileged sites such as the eye, brain, and testis persist for an extended period of time, whereas the same Ags are rapidly eliminated at conventional sites. Immune privilege, therefore, provides unwanted refuge for pathogens and tumor cells but is beneficial for the survival of allogeneic grafts. In this study, we asked whether memory T cells can eliminate foreign Ags deposited at an immunologically privileged site by studying CD8 memory T cell-mediated rejection of pancreatic islet allografts placed either in the testis (a privileged organ) or under the kidney capsule (a nonprivileged site) of diabetic mice. We found that CD8 memory T cells reject intratesticular grafts at a significantly slower rate than the rejection of intrarenal grafts. Delayed graft rejection in the testis was not due to reduced homing or proliferation of memory T cells but due to their increased apoptosis at that site. Apoptosis was mediated by the combined actions of two TNFR family members that are up-regulated on activated memory T cells, Fas, and CD30. Therefore, memory T cells survey immunologically privileged tissues but are subject to the immunosuppressive mechanisms present at these sites.
Animals↗
Rifampin-resistant RNA polymerase mutants of Chlamydia trachomatis remain susceptible to the ansamycin rifalazil.
Stable, homotypic mutants of Chlamydia trachomatis for which MICs of rifampin and rifalazil are elevated were isolated by serial passage at sub-MIC concentrations of these compounds. An alternative approach, in which Chlamydia cells were incubated and subsequently passaged three times, all in the presence of the selective agent at concentrations above the MIC, appeared to be a more effective means of selecting for mutants. In every instance where an elevation in the MIC occurred, one or more mutations in the rpoB gene, encoding the rifampin binding site, were detected. With one exception, all rpoB mutants that contained a single mutation conferred lower levels of resistance than mutants containing multiple mutations. Some rpoB mutations conferred very high levels of resistance to rifampin, up to 512 mug/ml. In all cases, mutants remained susceptible to concentrations of rifalazil at or below 0.064 microg/ml. Thus, rifalazil, a compound that is extremely potent against Chlamydia wild-type cells (MIC of 0.00025 microg/ml), may also protect against the selection of mutants at physiologically achievable concentrations.
Chlamydia trachomatis↗
Activities of rifamycin derivatives against wild-type and rpoB mutants of Chlamydia trachomatis.
Rifalazil, a semisynthetic rifamycin, was shown previously to have exceptional potency against Chlamydia trachomatis (MIC of 0.00025 microg/ml). We therefore tested 250 additional rifamycin derivatives and identified 12 with activities that are eightfold more potent than that of rifalazil. These compounds also showed exceptional activities against rifampin-resistant strains that carry missense mutations in the rpoB gene. The antimicrobial potency and intracellular penetration of these agents suggest their potential in treatment of chlamydial infections.
Anti-Bacterial Agents↗
Activation of HTLV-I gene transcription by protein tyrosine phosphatase inhibitors.
Human T-cell leukemia virus type I (HTLV-I) transcription generally depends on the ability of the viral Tax protein to bind the CREB transcription factor and form an active complex by recruiting CBP/p300 coactivators to the long terminal repeat (LTR). Studies have demonstrated that T-cell activating agents that stimulate CREB are potent inducers of HTLV-I transcription. Herein, we demonstrate that bpV[pic], a protein tyrosine phosphatase (PTP) inhibitor activates the HTLV-I LTR in the presence and absence of Tax expression. Optimal activation occurred at 8 h and was synergistic with forskolin or PGE(2). Infected cell lines and cells transfected with HTLV-I proviral DNA were equally responsive to the synergistic effect of bpV and forskolin on HTLV-I gene expression. Activation of the LTR by bpV[pic] was T-cell receptor-independent, but required ZAP70, calcineurin activity and functional calcium entry. Inhibition of the SHP-1 PTP was suggested to be important. Transfection experiments with a CREB dominant-negative mutant and with isolated TRE1- or CREB-responsive reporter constructs and treatment with the MDL-12,330A adenylate cyclase inhibitor all supported the involvement of a CREB/ATF family member in this bpV-dependent activation of the HTLV-I LTR, although CREB itself did not seem to be involved. Analysis of HTLV-I reporter constructs containing mutated CREB-binding sites also implied the involvement of another element in this activation. These results demonstrate for the first time a powerful effect of PTP inhibitors on HTLV-I LTR activity and suggest participation of both CREB-dependent and -independent pathways in this activation.
Cell Line↗
Novel Duchenne muscular dystrophy treatment through myoblast transplantation tolerance with anti-CD45RB, anti-CD154 and mixed chimerism.
Duchenne muscular dystrophy (DMD) is a fatal disease caused by a defect in the skeletal muscle protein, dystrophin. One potential therapy for DMD involves transplantation of myoblasts from normal individuals. Unfortunately, myoblast allografts are particularly immunogenic and transplant tolerance in dystrophic (mdx/mdx) mice has not yet been achieved despite using strategies successful in other allograft models. Here, we attempted to induce 'central tolerance' using either haplo- or fully allogeneic bone marrow after conditioning with low-dose (3 Gy) whole body irradiation and anti-CD154 or anti-CD45RB mAbs. With one exception, these mice lacked persistent chimerism, long-term survival of myoblast allografts, or tolerance. In contrast, the addition of anti-CD45RB to anti-CD154 uniformly resulted in long-lived high-level mixed chimerism, long-term (>100 days) engraftment of allogeneic myoblasts and deletion of donor-reactive cells. Moreover, all recipients exhibited tolerance to second myoblast allografts or donor-specific tolerance to skin transplants performed >80 days after the initial graft. Thus, we now report that anti-CD45RB synergizes with anti-CD40L to promote stable mixed chimerism and robust tolerance to myoblast allografts for the first time. This novel protocol may be applicable to future clinical trials in myoblast transplantation for treatment of DMD and for transplantation of other immunogenic allografts.
Animals↗
Cutting edge: transplantation tolerance through enhanced CTLA-4 expression.
Knockout and blocking studies have shown a critical role for CTLA-4 in peripheral tolerance, however, it is unknown whether augmenting CTLA-4 expression actually promotes tolerance. Here we demonstrate a specific and requisite role for CTLA-4 and its up-regulation in tolerance through anti-CD45RB. First, long-term murine islet allograft survival induced by anti-CD45RB is prevented by CTLA4-Ig, which interferes with B7:CTLA-4 interactions. Second, anti-CD45RB is ineffective in recipients lacking CTLA-4, B7-1, and B7-2. In contrast, CTLA4-Ig, which targets B7 on allogeneic cells, promotes long-term engraftment in these mice. Moreover, anti-CD45RB was effective in B7-deficient controls expressing CTLA-4. Finally, in wild-type mice, CTLA-4 expression returned to baseline 17 days after receiving anti-CD45RB, and was refractory to further increase. Transplantation and anti-CD45RB therapy at this time could neither augment CTLA-4 nor prolong engraftment. These data demonstrate a specific role for CTLA-4 in anti-CD45RB-mediated tolerance and indicate that CTLA-4 up-regulation can directly promote allograft survival.
Abatacept↗
CD45RB-targeting strategies for promoting long-term allograft survival and preventingchronic allograft vasculopathy.
BACKGROUND: CD45RB is a potent immunomodulatory target to achieve long-term allograft survival. We evaluated the in vivo effect of anti-CD45RB monoclonal antibody (mAb) treatment in combination with conventional immunosuppression or costimulatory blockade strategies as a therapeutic modality for future clinical application. METHODS: A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD45RB mAb and conventional immunosuppressive drugs or costimulatory blockade of the CD40/CD154 or B7/CD28 pathway. Chronic rejection was examined histologically for development of chronic allograft vasculopathy. RESULTS: Cyclosporine significantly abrogated the effect of anti-CD45RB therapy. In contrast, rapamycin acted synergistically with anti-CD45RB mAb in promoting long-term allograft survival. CD154 blockade further enhanced the tolerogenic efficacy of anti-CD45RB mAb. These synergistic effects of combination treatments also prevented the development of chronic allograft vasculopathy. CONCLUSION: CD45RB-targeting strategy in combination with the use of rapamycin or costimulatory blockade promotes allograft tolerance and prevents chronic rejection.
Abatacept↗