Ruksana Huda

Experimental autoimmune myasthenia gravis in the mouse

Myasthenia gravis (MG) is a T cell–dependent antibody‐mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody‐mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques. Curr. Protoc. Immunol. 100:15.8.1‐15.8.26.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis

Objective:To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design:Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting:Investigational intensive care unit at a university hospital. Subjects:Eighteen sheep (37.2 ± 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2–5 × 1011 colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 &mgr;g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringers lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results:In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in Pao2/Fio2 ratio (control group values were 521 ± 22 at baseline [BL], 72 ± 5 at 12 hrs, and 74 ± 7 at 24 hrs, vs. rhAPC group values of 541 ± 12 at BL, 151 ± 29 at 12 hours [p < .05 vs. control], and 118 ± 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 ± 0.02, and at 24 hrs, 0.65 ± 0.08; rhAPC group at BL, 0.24 ± 0.04, and at 24 hrs, 0.45 ± 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 ± 1, and at 24 hrs, 36 ± 4; rhAPC group at BL, 21 ± 1, and at 24 hrs, 28 ± 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 ± 2; control, 17 ± 1; rhAPC, 12 ± 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions:Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Inhaled nitric oxide attenuates reperfusion inflammatory responses in humans.

Background:Reduced bioavailability of endothelium-derived nitric oxide associated with reperfusion could potentially exacerbate the inflammatory response during reperfusion. Evidence suggests the pharmacologic effects of inhaled nitric oxide may extend beyond the pulmonary vasculature, and this is attributed to nitric oxide–derived complexes in blood that ultimately orchestrate antiinflammatory effects. In this study, the authors evaluated the potential for inhaled nitric oxide (80 ppm) to attenuate inflammation instigated by ischemia–reperfusion in a human model using patients undergoing knee surgery where a tourniquet was used to produce a bloodless surgical field. Methods:Inhaled nitric oxide (80 ppm) was administered before tourniquet application and continued throughout reperfusion until the completion of surgery. Venous blood samples were collected before and after reperfusion, for the measurements of nitrate and nitrite, CD11b/CD18, soluble P-selectin, and lipid hydroperoxide. Muscle biopsies were obtained from the quadriceps muscle before skin closure and analyzed for myeloperoxide, conjugated dienes, and nuclear factor-&kgr;B translocation. Results:Administration of inhaled nitric oxide (80 ppm) significantly attenuated the inflammatory response characterized by reduced expression of CD11b/CD18, P-selectin, and nuclear factor &kgr;B compared with the control group. This was accompanied by increased plasma levels of nitrate and nitrite and reduced oxidative stress. Conclusions:Administration of inhaled nitric oxide at 80 ppm significantly reduces inflammation in lower extremity ischemia–reperfusion in humans. This observation supports the concept that during diseases characterized by dysfunction in nitric oxide metabolism, inhaled nitric oxide may be an effective therapy to replenish systemic nitric oxide, thus retarding inflammatory-mediated injury.

Complement and cytokine based therapeutic strategies in myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.

Inflammatory and redox responses to ischaemia/reperfusion in human skeletal muscle

The objective of this study was to identify cellular and plasma marker(s) of post-I/R (ischaemia/reperfusion) in patients undergoing elective knee surgery where a tourniquet was used to facilitate a bloodless surgical field. We evaluated the inflammatory and redox response by measuring the mRNA levels of ICAM-1 (intercellular cell-adhesion molecule-1), MnSOD (manganese superoxide dismutase), GST-mu (glutathione transferase-mu) and Cu/ZnSOD (copper/zinc superoxide dismutase) in the operated muscle and blood cells pre-operatively (pre-tourniquet) and at various times after reperfusion (tourniquet release). We also measured plasma concentrations of IL (interleukin)-6, IL-8, sICAM-1 (soluble ICAM-1), IL-1beta and TNF-alpha (tumour necrosis factor-alpha) using ELISA. Our results show a strong induction of MnSOD and GST-mu in granulocytes (but not in mononuclear cells or muscle) after reperfusion (2 and 4 h). There was no change in the mRNA level of Cu/ZnSOD after reperfusion. An up-regulation of membrane ICAM-1 in muscle and a decrease in sICAM-1 in plasma were detected after reperfusion. Plasma IL-6 and IL-8 levels (but not TNF-alpha or IL-1beta) increased significantly over baseline at 2 and 4 h after reperfusion. Elevated expression of ICAM-1 in muscle, MnSOD and GST-mu in granulocytes and increased levels of plasma IL-6 and IL-8 may be considered as phase- and cell-specific markers of post-I/R of skeletal muscle in humans.

Complement C2 siRNA mediated therapy of myasthenia gravis in mice

Activation of complement components is crucial in the progression and severity of myasthenia gravis and experimental autoimmune myasthenia gravis (EAMG). Mice deficient in complement component C4 or treated with monoclonal antibody to C1q are resistant to EAMG. In this study, we show that inhibition of complement cascade activation by suppressing the expression of a critical low-abundant protein, C2, in the classical complement pathway, significantly improved clinical and immunopathological manifestations of EAMG. Two weeks after a second booster immunization with acetylcholine receptor, when mice exhibit muscle weakness, i.p. injection of C2 siRNA significantly suppressed C2 mRNA in the blood cells and liver of EAMG mice. Treatment of EAMG mice with C2 siRNA, once a week for 5 weeks, significantly improved muscle strength, which was further evidenced by functional AChR preservation in muscle, reduction in number of C3 and membrane-attack complexes at neuro-muscular junctions in forelimb muscle sections, and a transient decrease in serum IgG2b levels. Our study shows for the first time that siRNA-mediated suppression of C2, a component of the classical complement system, following established disease, can effectively contribute to the remission of EAMG. Therefore, C2 siRNA mediated therapy can be applied in all complement mediated autoimmune diseases.

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30μg of recombinant human MuSK in incomplete or complete Freunds adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10μg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30μg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.

Targeting complement system to treat myasthenia gravis

Abstract While the complement system is desired for protective immunity, antibody- and complement-mediated neuromuscular junction (NMJ) destruction, a hallmark of myasthenia gravis (MG) or experimental autoimmune MG (EAMG), is a significant concern. Evidence suggests that the binding of complement factors to the pathogenic anti-acetylcholine receptor (AChR) autoantibody induces the formation of membrane attack complexes (MAC), which ultimately lead to NMJ destruction and muscle weakness. Studies corroborating the evidence show that the complement (C3–C6)-deficient or complement inhibitor (anti-C1q, soluble CR1, anti-C6, and C5 inhibiting peptide)-treated animals are highly resistant to EAMG induction, whereas the deficiency of the naturally occurring complement inhibitors, such as the decay-accelerating factor (DAF), increases EAMG susceptibility. Notably, the complement-inhibited animals do not exhibit significant immunosuppression but only a marginal reduction in the production of certain cytokines and immunoglobulin isotypes. A preliminary clinical trial using antibody-based C5 inhibitor eculizumab has been shown to be of potential use for MG treatment. The inhibition of the classic complement pathway (CCP) alone appears to be enough to suppress EAMG, suggesting that the complement inhibitors targeting specifically the classic pathway could effectively treat MG without causing immunosuppressive and other side effects. For instance, a recent non-antibody-based therapeutic approach selectively targeting the CCP component C2 by small interfering RNA (siRNA) has proven useful in EAMG treatment. The treatment strategies developed for MG might also be beneficial for other complement-mediated autoimmune diseases.

Selective activation of protein kinase C delta in human neutrophils following ischemia reperfusion of skeletal muscle.

Circulatory neutrophils are known to be critical mediators of inflammation and oxidative stress during ischemia reperfusion (I/R) injury. Recent studies have shown an important role for protein kinase C (PKC) in neutrophil survival and function. Activation of specific isotypes of PKC are known to be involved in membrane alteration and motility, oxidative phosphorylation, and apoptosis modulation of neutrophils. However, the role of PKC in neutrophil responses to I/R in the clinical setting has not been studied. In this study, we examined the neutrophil activation of PKC induced by tourniquet-controlled I/R of skeletal muscle in humans. We found that I/R rapidly activates and translocates PKCδ, but not any of the classical forms of PKC (α or β) from cytosol to the particulate fraction of neutrophils. Particulate translocation of PKCδ is sustained up to 4 h after reperfusion and is associated with kinase activity. Postreperfusion activation of PKCδ in neutrophils signals proapoptosis, but does not cause immediate cell death (as revealed by neutrophil morphology study and DNA-laddering assay). This study indicates that calcium-independent novel PKCδ (nPKCδ) might be predominantly involved in regulating membrane functions and survival of neutrophils associated with post-I/R-induced inflammatory oxidative stress.

IgG1 deficiency exacerbates experimental autoimmune myasthenia gravis in BALB/c mice

Myasthenia gravis is an autoimmune disease characterized by muscle weakness due to neuromuscular junction (NMJ) damage by anti-acetylcholine receptor (AChR) auto-antibodies and complement. In experimental autoimmune myasthenia gravis (EAMG), which is induced by immunization with Torpedo AChR in CFA, anti-AChR IgG2b and IgG1 are the predominant isotypes in the circulation. Complement activation by isotypes such as IgG2b plays a crucial role in EAMG pathogenesis; this suggested the possibility that IgG1, which does not activate complement through the classical pathway, may suppress EAMG. In this study, we show that AChR-immunized BALB/c mice genetically deficient for IgG1 produce higher levels of complement-activating isotypes of anti-AChR, especially IgG3 and IgG2a, and develop increased IgG3/IgG2a deposits at the NMJ, as compared to wild type (WT) BALB/c mice. Consistent with this, AChR-immunized IgG1(-/-) BALB/c mice lose muscle strength and muscle AChR to a greater extent than AChR-immunized WT mice. These observations demonstrate that IgG1 deficiency leads to increased severity of EAMG associated with an increase in complement activating IgG isotypes. Further studies are needed to dissect the specific role or mechanism of IgG1 in limiting EAMG and that of EAMG exacerbating role of complement activating IgG3 and IgG2a in IgG1 deficiency.