Improving Ambulation and Minimizing Disability with Therapeutic Plasma Exchange in a Stiff-person Syndrome Patient with Recurrent Falls.

Stiff-person syndrome (SPS) is a rare, autoimmune, neuromuscular disorder that manifests with axial and proximal muscle stiffness, rigidity, and painful muscle spasms, often causing progressive disability due to limited movement. First-line therapies comprise symptomatic management with γ-aminobutyric acid-modulating drugs such as benzodiazepines and baclofen. Patients resistant to these treatments are often given intravenous immunoglobulin (IVIg). Severe disease refractory to first-line therapy and IVIg may be treated with therapeutic plasma exchange (TPE) or immunomodulatory agents such as rituximab. Current evidence derived from case reports and case series has shown that roughly half of SPS patients treated with TPE report benefits. Here, we report the case of a 68-year-old man with a 20-year history of severe SPS and recurrent falls who was admitted to the emergency department for a traumatic hip fracture. He had significant rigidity in the axial and extremity muscles with persistent spasms of the quadriceps femoris muscle. Postoperatively, he was unable to participate in physical therapy (PT) due to these symptoms. He previously failed treatment with diazepam, baclofen, and monthly IVIg. Under our care, he underwent seven TPE treatments. By the end of treatment, he reported significant improvement in mobility with a resolution of muscle spasms and was able to be discharged to inpatient rehabilitation. This suggests that TPE may offer an effective, safe treatment modality for patients with severe refractory SPS that may significantly improve mobility and disability associated with the disease.

IL-1beta directly excites isolated rat supraoptic neurons via upregulation of the osmosensory cation current.

Previous studies have shown that IL-1beta can excite the magnocellular neurosecretory cells (MNCs) of the hypothalamus. However, it is not known whether IL-1beta can have direct IL-1 receptor type 1 (IL-1R1)-mediated effects on MNCs, and little is known about the cellular mechanisms by which IL-1beta influences electrical activity in these cells. Here, we used patch-clamp recordings to examine the effects of IL-1beta on acutely isolated rat MNCs. We found that IL-1beta directly excites MNCs in a dose-dependent manner and that this response can be blocked by an inhibitor of the IL-1R1. Voltage-clamp analysis of the current evoked by IL-1beta revealed a linear current-voltage relationship between -90 and -20 mV, and a reversal potential near -35 mV. This value was not affected by reducing the concentration of chloride ions in the external solution, indicating the involvement of a nonselective cation conductance. The effects of IL-1beta were inhibited by Na-salicylate, an inhibitor of cyclooxygenase. Moreover, the effects of IL-1beta were mimicked and occluded by PGE2, and were inhibited by AH-23848, an antagonist of the PGE2 type 4 (i.e., EP4) receptor. The current evoked by IL-1beta was also abolished by 100 microM gadolinium (Gd3+), but was significantly larger when examined in cells preshrunk by negative pressure applied via the recording pipette. IL-1beta alone did not cause changes in cell volume nor in the mechanosensitivity of MNCs. We conclude that IL-1beta directly excites MNCs via an IL-1R1-mediated induction of PGE2 synthesis and EP4 receptor-dependent autocrine upregulation of the nonselective cation conductance that underlies osmoreception.

Selective knock-down of P2X7 ATP receptor function by dominant-negative subunits.

Among the family of P2X ATP-gated cation channels, the P2X7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type characteristics in both agonist affinity and ionic selectivity, strongly suggesting that the heteromeric channels are functionally impaired. The mutant subunits also suppressed the P2X7-dependent pore formation as assessed by uptake of the propidium dye YO-PRO-1 (Molecular Probes, Eugene, OR) in response to 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) in transfected human embryonic kidney 293 cells. Native responses to BzATP as well as ATP-induced ethidium dye uptake were significantly knocked down (31 +/- 9% and 25 +/- 7% of control, respectively) in mouse macrophage cell line RAW264.7 transfected with the mutant subunits. Therefore, these dominant-negative subunits provide selective genetic tools to investigate the functional roles of native P2X7 receptors.

Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels.

Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the functional interaction between native P2X2 and 5-HT3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.

Stretch-inactivated cation channels: cellular targets for modulation of osmosensitivity in supraoptic neurons.

Rat magnocellular neurosecretory cells (MNCs) show an intrinsic sensitivity to acute changes in fluid osmolality. Experiments in acutely isolated supraoptic MNCs have shown that these responses are due to in part to the cell volume-dependent modulation of gadolinium-sensitive 33 pS stretch-inactivated cation (SIC) channels. Previous studies in vivo have shown that the slope (i.e. gain) of the 'osmosensory' relation between VP release and plasma osmolality can be increased or decreased under various physiological and pathological conditions. Here, we review recent work that shows how changes in external [Na] and excitatory neuropeptides such as angiotensin II (Ang II), cholecystokinin (CCK) and neurotensin (NT), may influence osmosensory gain in acutely isolated MNCs. Whole-cell and single-channel recording experiments have revealed that changes in external Na cause proportional changes in osmosensory gain as a result of modified SIC channel permeability and not by affecting mechanotransduction. In contrast, Ang II, CCK, or NT appear to convergently, and directly, stimulate the osmosensory cation conductance in MNCs. Preliminary analysis in current clamp further suggests that osmosensory gain may be increased upon exposure to these excitatory peptides. Whether such mechanisms contribute to the modulation of osmosensory gain in vivo remains to be established.

ADP and AMP induce interleukin-1beta release from microglial cells through activation of ATP-primed P2X7 receptor channels.

P2X(7) is a subtype of ATP-gated channels that is highly expressed in astrocytes, microglia, and other immune cells. Activation of P2X(7) purinoceptors by ATP or 3'-O-(4-benzoyl)-benzoyl ATP (BzATP) induces the formation of cytolytic pores and provokes release of interleukin-1beta from immune cells. We investigated the actions of other endogenous nucleotides on recombinant and microglial P2X(7) receptors using electrophysiology, fluorescence imaging, and interleukin-1beta release measurement. We found that initial application of ADP or AMP to Xenopus oocytes expressing P2X(7) receptors was ineffective. However, when ADP and AMP, but not UTP or adenosine, were applied after a brief exposure to ATP or BzATP, they activated P2X(7) receptors in a dose-dependent manner. Moreover, responses to ADP and AMP were also elicited after exposure to low concentrations of ATP and were recorded several minutes after removal of ATP from the extracellular medium. Whole-cell recordings from mouse microglial cells showed that significant responses to ADP and AMP were elicited only after ATP application. YO-PRO-1 dye uptake imaging revealed that, unlike ATP, prolonged application of ADP or AMP did not cause an opening of large cytolytic pores in mouse microglial cells. Finally, ADP and AMP stimulated the release of interleukin-1beta from ATP-primed mouse and human microglial cells. We conclude that selective sensitization of P2X(7) receptors to ADP and AMP requires priming with ATP. This novel property of P2X(7) leads to activation by ATP metabolites and proinflammatory cytokine release from microglia without cytotoxicity.