Maintaining physiological pH is required for survival, and exposure to alkaline chemicals such as ammonia (smelling salts) elicits severe pain and inflammation through unknown mechanisms. TRPV1, the capsaicin receptor, is an integrator of noxious stimuli including heat and extracellular acidic pH. Here, we report that ammonia activates TRPV1, TRPA1 (another polymodal nocisensor), and other unknown receptor(s) expressed in sensory neurons. Ammonia and intracellular alkalization activate TRPV1 through a mechanism that involves a cytoplasmic histidine residue, not used by other TRPV1 agonists such as heat, capsaicin or low pH. Our studies show that TRPV1 detects both acidic and basic deviations from homeostatic pH.

Summary Heightened nociceptor function caused by inflammatory mediators such as bradykinin contributes to increased pain perception (hyperalgesia) to noxious mechanical and thermal stimuli. While sensitization of the heat transducer TRPV1 largely subserves thermal hyperalgesia, cellular mechanisms underlying mechanical hyperalgesia have been elusive. The role of the mechanically-activated (MA) channel piezo2 (known as FAM38B) present in mammalian sensory neurons is unknown. We test the hypothesis that piezo2 activity is enhanced by bradykinin, an algogenic peptide that induces mechanical hyperalgesia within minutes. Piezo2 current amplitude is increased and inactivation slowed by bradykinin 2 receptor (BDKRB2) activation in heterologous expression systems. Protein Kinase A (PKA) and Protein Kinase C (PKC) agonists enhance piezo2 activity. BDKRB2-mediated effects are abolished by PKA and PKC inhibitors. Finally, piezo2-dependent MA currents in a class of native sensory neurons are enhanced 8-fold by bradykinin via PKA and PKC. Thus, piezo2 sensitization may contribute to PKA- and PKC-mediated mechanical hyperalgesia.

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The prevention of graft rejection in the setting of nonmyeloablative transplant approaches might be mediated by chemotherapy-induced host immunoablation and by the graft-promoting effects of graft-versus-host disease (GVHD). To evaluate whether host immunoablation alone might allow for alloengraftment, we developed an F1-into-parent murine marrow rejection model using host preparative regimens of lethal total body irradiation (TBI; 950 cGy), sublethal irradiation (600 cGy), or combinations of fludarabine (Flu) and cyclophosphamide (Cy). A preparative regimen selectivity index (SI) was calculated to determine whether host lymphocytes were preferentially depleted relative to myeloid cells (SI = number of host myeloid/number host T lymphoid cells remaining after preparative regimen administration). Saline-treated recipients were assigned an SI value of 1.0. Recipients of lethal TBI had reduced myeloid cells relative to T cells (SI = 0.6). In contrast, all Flu/Cy regimens preferentially depleted host T cells: recipients of Flu (100 mg/kg per day)/Cy (50 mg/kg per day) for 10 days (SI = 28.1); recipients of Flu (100 mg/kg per day)/Cy (100 mg/kg per day) for 10 days (SI = 64.1); and recipients of Flu (100 mg/kg per day)/Cy (50 mg/kg per day) for 19 or 27 days (SI = 74.6). The 10-day regimen of Flu/Cy (50 mg/kg per day) did not severely reduce host T cell numbers, nor did it prevent F1 marrow rejection (<1% chimerism, n = 14). In contrast, the 10-day regimen of Flu/Cy (100 mg/kg per day) reduced T-cell numbers below that of lethal TBI recipients and prevented F1 marrow rejection (11.4% chimerism, n = 15); donor chimerism was predominant in lymphoid cells and was stable through day 240 post-BMT. Additionally, the 19- or 27-day regimen of Flu/Cy, which most selectively depleted host T cells, also prevented F1 marrow rejection (6.3% chimerism, n = 15). These results therefore demonstrate that optimized Flu-containing, immunoablative preparative regimens can prevent fully MHC-disparate marrow rejection independent of GVHD.

Summary. Given the known role of the fas cytolytic pathway in B-cell regulation, we evaluated whether fas±fasL interactions might induce chronic lymphocytic leukaemia (CLL) cell death. De novo CLL cells expressed a low level of surface fas, and were not lysed by fasL-bearing cells. CLL cells cultured in media containing the type I cytokines interleukin (IL)-12 or interferon (IFN)-a had increased fas expression, and were readily lysed by fasL-bearing cells. In contrast, the type II cytokine IL-4 did not increase CLL cell fas, and abrogated type I cytokine-induced fas up-regulation. With prolonged culture, IL-4 exposed CLL cells expressed an intermediate level of fas; however, such CLL cells were resistant to fas-mediated lysis. These results indicate that IL-4 inhibits fas-mediated killing of CLL cells at the level of both fas receptor expression and post-receptor events. Additionally, we have de®ned in vitro culture conditions which generate fasL-bearing T cells from CLL patients; such T cells ef®ciently mediated fas-based lysis of autologous fas-positive CLL cells. We therefore conclude that type I and type II cytokines differentially regulate the fas pathway in CLL cells, and that a combination of type I cytokines and fasL-expressing T cells may represent a new approach to the immunotherapy of CLL.