Robert V. Bundick

Tumour necrosis factor-α blockade suppresses murine allergic airways inflammation

Asthma is a heterogeneous disease that has been increasing in incidence throughout western societies and cytokines, including proinflammatory tumour necrosis factor alpha (TNF‐α), have been implicated in the pathogenesis of asthma. Anti‐TNF‐α therapies have been established successfully in the clinic for diseases such as rheumatoid arthritis and Crohns disease. TNF‐α‐blocking strategies are now being trialled in asthma; however, their mode of action is poorly understood. Based on the observation that TNF‐α induces lymph node hypertrophy we have attempted to investigate this as a mechanism of action of TNF‐α in airway inflammation by employing two models of murine airway inflammation, that we have termed short and long models, representing severe and mild/moderate asthma, respectively. The models differ by their immunization schedules. In the short model, characterized by eosinophilic and neutrophilic airway inflammation the effect of TNF‐α blockade was a reduction in draining lymph node (DLN) hypertrophy, eosinophilia, interleukin (IL)‐5 production and immunoglobulin E (IgE) production. In the long model, characterized by eosinophilic inflammation, TNF‐α blockade produced a reduction in DLN hypertrophy and IL‐5 production but had limited effects on eosinophilia and IgE production. These results indicate that anti‐TNF‐α can suppress DLN hypertrophy and decrease airway inflammation. Further investigations showed that anti‐TNF‐α‐induced inhibition of DLN hypertrophy cannot be explained by preventing l‐selectin‐dependent capture of lymphocytes into the DLN. Given that overall TNF blockade was able to suppress the short model (severe) more effectively than the long model (mild/moderate), the results suggest that TNF‐α blocking therapies may be more effective in the treatment of severe asthma.

The specific monocarboxylate transporter (MCT1) inhibitor, AR-C117977, a novel immunosuppressant, prolongs allograft survival in the mouse.

Novel small molecular weight compounds that act by inhibiting the monocarboxylate transporter (MCT1) receptor have been found to cause profound inhibition of T-cell responses to alloantigen in vitro. Here, we have investigated the ability of one compound in this series, AR-C117977, a potent MCT1 inhibitor, to prevent the acute and chronic rejection of vascularized and nonvascularized allografts in the mouse. Treatment with AR-C117977 or cyclosporin A (CsA) administered at a dose of 30 mg/kg subcutaneously for 15 days to adult CBA. Ca (H2k) mice, commencing either 3 days or 1 day before transplantation, was found to prolong the survival of an allogeneic (C57BL/10 H2b; NZW H2z; or BALB/c H2d) heart, aorta, or skin allograft significantly compared with treatment with vehicle alone (median survival time [MST] AR-C117977 treated 15; 19 and 18 days [skin] and 73; 66 and 67 days ([heart] vs. vehicle treated 8, 8 and 9 days [skin] and 9, 8, 10 days [heart] for B10, NZW and BALB grafts, respectively). AR-C117977 also inhibited the development of transplant arteriosclerosis in aortic allografts partially, but was unable to inhibit alloantibody production after transplantation. The specific MCT1 inhibitor AR-C117977 has potent immunosuppressive properties in vivo effectively preventing acute but not chronic allograft rejection in the mouse.

Dissecting the contribution of innate and antigen-specific pathways to the breach of self-tolerance observed in a murine model of arthritis

Background: The relative roles of innate immunity and antigen-specific T cells in rheumatoid arthritis remain controversial. Previous studies demonstrated that T-helper type 1 cells of irrelevant antigen specificity (ovalbumin) induced a transient arthritis in BALB/c mice, which recapitulates many of the pre-articular and articular features of human disease and is associated with the emergence of autoreactive T and B-cell responses to joint-specific antigens. However, the mechanisms underlying this phenomenon were unclear. Objectives: The aim of this study was to dissect the relative contribution of innate and heterologous antigen-specific pathways to the breach of self-tolerance and pathology observed in this model and how this may result from modified T and B-cell interactions. Methods: To address this issue, experimental arthritis was elicited either by a non-specific inflammatory stimulus alone, by activation of T cells of an irrelevant specificity or a combination of both. Results: The non-specific inflammatory response generated by lipopolysaccharide led to articular inflammation and cartilage erosion, but did not break tolerance to joint-specific antigens. In contrast, local activation of T cells of an irrelevant specificity produced a similar pathological picture but, in addition, induced T-cell responses to unrelated joint-specific antigens with associated activation of autoreactive B cells. These effects could be further potentiated by the addition of lipopolysaccharide. Conclusion: These data demonstrate that non-specific inflammation alone is insufficient to breach self-tolerance. In contrast, T cells of an irrelevant specificity, when triggered locally in an antigen-specific manner, can breach self-tolerance leading to arthritis and autoantibody production, which can then be amplified in a non-specific manner.

The specific monocarboxylate transporter-1 (MCT-1) inhibitor, AR-C117977, induces donor-specific suppression, reducing acute and chronic allograft rejection in the rat

Background. In a search for immunosuppressive drugs having novel mechanisms, monocarboxylate transporter (MCT-1) inhibitors were identified that markedly inhibited immune responses. Here, we report the effects of AR-C117977, a potent MCT-1 inhibitor, on alloimmune responses in the rat. Methods. In vitro activity was determined in a rat mixed lymphocyte response (MLR). In vivo activity was tested in a graft versus host response (GVHR) and in both high (DA to PVG) and low (PVG to DA) responder cardiac allograft models. To assess induction of donor-specific suppression recipients of allogeneic hearts surviving longer than 100 days received a second transplant either of the same donor strain or a third-party donor strain. Effects on chronic graft rejection were assessed histologically by evaluating vasculopathy in long-term surviving grafts and in an obliterative bronchiolitis (OB) model. Results. AR-C117977 inhibited the rat MLR and was more potent than cyclosporin A (CsA). In the rat GVHR model, AR-C117977 gave a dose-related inhibition. In the high responder cardiac allograft model, graft survival in excess of 100 days was achieved with AR-C117977 compared with 20 days with CsA and all the long-term survivors exhibited donor-specific suppression on retransplantation. In the low responder model, both AR-C117977 and CsA induced survival in excess of 100 days. Histology of the long-term surviving grafts suggested reduced vasculopathy associated with chronic rejection. Furthermore, AR-C117977 inhibited the occlusion of transplanted trachea in a OB model. Conclusion. This report describes a MCT-1 specific inhibitor having immunosuppressive activity on alloimmune responses and inducing donor-specific suppression.

Immunosuppressive properties of a series of novel inhibitors of the monocarboxylate transporter MCT-1

We have recently described the immunosuppressive properties of AR‐C117977 and AR‐C122982, representatives of a group of compounds identified as inhibitors of lactate transporters (monocarboxylate transporters; MCTs). These compounds demonstrate the potential therapeutic usefulness of inhibiting MCT‐1, but their physical and metabolic properties made them unsuitable for further development. We have therefore tried to find analogues with similar immunosuppressive efficacy and a more suitable profile for oral administration. Five analogues of AR‐C117977 were synthesised and screened for binding to the transporter, for inhibition of proliferation of both human and rat lymphocytes, for in vivo activity in a model of graft‐versus‐host (GvH) response in the rat, and in high‐ and low‐responder cardiac transplant models in the rat. There was a good correlation between levels of binding of the five analogues to MCT and their inhibition of lymphocyte proliferation in human and rat cells. Furthermore, activity in both the GvH response and the cardiac transplant models correlated well with the determined concentrations of test compound in plasma. These findings on new analogues of MCT‐1 inhibitors have taken us further towards defining the pharmacokinetic properties that may help to identify future drug candidates among inhibitors of MCT‐1.

An investigation of the impact of the location and timing of antigen-specific T cell division on airways inflammation

It is widely accepted that allergic asthma is orchestrated by T helper type 2 lymphocytes specific for inhaled allergen. However, it remains unclear where and when T cell activation and division occurs after allergen challenge, and whether these factors have a significant impact on airways inflammation. We therefore employed a CD4‐T cell receptor transgenic adoptive transfer model in conjunction with laser scanning cytometry to characterize the location and timing of T cell division in asthma in vivo. Thus, for the first time we have directly assessed the division of antigen‐specific T cells in situ. We found that accumulation of divided antigen‐specific T cells in the lungs appeared to occur in two waves. The first very early wave was apparent before dividing T cells could be detected in the lymph node (LN) and coincided with neutrophil influx. The second wave of divided T cells accumulating in lung followed the appearance of these cells in LN and coincided with peak eosinophilia. Furthermore, accumulation of antigen‐specific T cells in the draining LN and lung tissue, together with accompanying pathology, was reduced by intervention with the sphingosine 1‐phosphate receptor agonist FTY720 2 days after challenge. These findings provide greater insight into the timing and location of antigen‐specific T cell division in airways inflammation, indicate that distinct phases and locations of antigen presentation may be associated with different aspects of pathology and that therapeutics targeted against leukocyte migration may be useful in these conditions.

Operational tolerance in nonvascularized transplant models induced by AR-C117977, a monocarboxylate transporter inhibitor.

AR-C117977, a monocarboxylate transporter inhibitor, reduces immune responses both in vitro and in vivo, maintains long-term graft survival, and induces operational tolerance. To evaluate the immunosuppressive limitations of AR-C117977, this study was performed in nonvascularized transplant models noted for their refractive response to standard immunosuppressive agents. Rat skin was transplanted from DA(RT1avl) into PVG(RT1c) and the reverse. Mouse islet allotransplantation was performed with BALB/c H2d donors and C57Bl/6J H2b recipients. In the skin graft model, AR-C117977 monotherapy was associated with long-term skin graft survival in one rat strain combination. AR-C117977 and cyclosporine A (CsA) in combination resulted in significant prolongation of graft survival in both rat strains. CsA monotherapy did not prevent acute rejection in either strain. Islet allograft survival was moderately prolonged with CsA or AR-C117977. AR-C117977 is an efficient immunosuppressive drug in stringent rodent transplant models and further studies are warranted.

The effect of Monocarboxylate Transporter (MCT1) inhibitor, AR-C117977 on accelerated rejection of cardiac grafts in pre-sensitised rats and concordant xenotransplantation

Previous studies have demonstrated the immunosuppressive properties of the monocarboxylate transporter MCT1 inhibitor, AR-C117977. The objective of this study was to evaluate the possible limitations in immunosuppressive efficacy of AR-C117977 in models of hyper acute rejection of cardiac transplants in pre-sensitised rats and in concordant cardiac xenotransplantation. Methods: A primary graft was given without treatment in order to induce sensitisation. At 10 days, a second cardiac transplant was performed, and doses of the test compound were given and compared to or combined with cyclosporin (CsA). Serum samples were retrieved for flow cytometry and measurements of antibody from the presensitised recipients before and after the second transplantation. Results: Survival was significantly prolonged with AR-C117977 in combination with CsA (median 12.5 days) compared with single treatment. AR-C117977 had a better effect on antibody formation and binding to B-cells than CsA, but a similar effect on T-cells. Acute rejection could not be prevented in any of the treatment groups in the xenotransplant model. Conclusion: A short course of the MCT1 inhibitor AR-C117977, in combination with CsA, prevented accelerated acute rejection in presensitised rats, but no longterm graft survival was achieved. Rejection in xenotransplantation was not prevented. Correspondence to: Clara Paul, Agneta Montgomery, Department of Surgery, Institution of Surgical Science, Malmö, Lund University, Sweden, Email: helenclarapaul@gmail.com