Depression and recovery of reflex amplitude during electrical stimulation after spinal cord injury.

OBJECTIVE: The aim of this study was to quantify, for the first time, H-reflexes evoked during prolonged trains of wide-pulse neuromuscular electrical stimulation (WP-NMES) in individuals with chronic spinal cord injury (SCI). We hypothesised that after the first H-reflex, reflex amplitudes would be depressed (due to post-activation depression), but would recover and this recovery would be enhanced after a "burst" of 100 Hz WP-NMES. METHODS: Soleus M-waves and H-reflexes evoked during WP-NMES (1 ms pulse width) of the tibial nerve were quantified in nine individuals with SCI. WP-NMES was delivered in two patterns: "constant-frequency" (15 or 20 Hz for 12 s) and "burst-like" (15-100-15 Hz or 20-100-20 Hz; 4 s each phase) at an intensity that evoked an M-wave between 10% and 15% of the maximal M-wave (M(max)). RESULTS: During constant frequency stimulation, after the initial depression from the first to the second H-reflex (1st: 57% M(max); 2nd: 25% M(max)), H-reflexes did not recover significantly and were 37% M(max) at the end of the stimulus train. During the burst-like pattern, after the initial depression (1st: 62% M(max); 2nd: 30%), reflexes recovered completely by the end of the stimulation (to 55% M(max)) as they were not significantly different from the first H-reflex. M-waves were initially depressed (1st: 12% M(max); 2nd: 7% M(max)) then did not change throughout the stimulation and were not significantly different between stimulation patterns. An analysis of covariance indicated that the depression in M-wave amplitude did not account for the depression in H-reflex amplitude. CONCLUSIONS: Relatively large H-reflexes were recorded during both patterns of NMES. The brief burst of 100 Hz stimulation restored H-reflexes to their initial amplitudes, effectively reversing the effects of post-activation depression. SIGNIFICANCE: For individuals with chronic SCI, generating contractions through central pathways may help reduce muscle atrophy and produce contractions that are more fatigue-resistant for rehabilitation, exercise programs, or to perform activities of daily living.

The effects of wide pulse neuromuscular electrical stimulation on elbow flexion torque in individuals with chronic hemiparetic stroke.

OBJECTIVE: Neuromuscular electrical stimulation that incorporates wide pulse widths (1ms) and high frequencies (100Hz; wide pulse-NMES (WP-NMES)) augments contractions through an increased reflexive recruitment of motoneurons in individuals without neurological impairments and those with spinal cord injury. The current study was designed to investigate whether WP-NMES also augments contractions after stroke. We hypothesized that WP-NMES would generate larger contractions in the paretic arm compared to the non-paretic arm due to increased reflex excitability for paretic muscles after stroke. METHODS: The biceps brachii muscles were stimulated bilaterally in 10 individuals with chronic hemiparetic stroke. Four stimulation patterns were delivered to explore the effects of pulse width and frequency on contraction amplitude: 20-100-20Hz (4s each phase, 1ms pulse width); 20-100-20Hz (4s each phase, 0.1ms); 20Hz for 12s (1ms); and 100Hz for 12s (1ms). Elbow flexion torque and electromyography were recorded. RESULTS: Stimulation that incorporated 1ms pulses evoked more torque in the paretic arm than the non-paretic arm. When 0.1ms pulses were used there was no difference in torque between arms. For both arms, torque declined significantly during the constant frequency 100Hz stimulation and did not change during the constant frequency 20Hz stimulation. CONCLUSIONS: The larger contractions generated by WP-NMES are likely due to increased reflexive recruitment of motoneurons, resulting from increased reflex excitability on the paretic side. SIGNIFICANCE: NMES that elicits larger contractions may allow for development of more effective stroke rehabilitation paradigms and functional neural prostheses.

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC-2.

BACKGROUND: The adapter proteins SLP-76 and LAT have been shown to play critical roles in the activation of PLCgamma2 in platelets downstream of GPVI/FcRgamma and the C-type lectin receptor CLEC-2. SLP-76 is constitutively associated with the adapter Gads in platelets, which also binds to tyrosine phosphorylated LAT, thereby providing a potential pathway of regulation of SLP-76. OBJECTIVE: In the present study, we have compared the role of Gads alongside that of LAT following activation of the major platelet glycoprotein receptors using mice deficient in the two adapter proteins. RESULTS: Gads was found to be required for the efficient onset of aggregation and secretion in response to submaximal stimulation of GPVI and CLEC-2, but to be dispensable for activation following stronger stimulation of the two receptors. Gads was also dispensable for spreading induced through integrin alpha(IIb)beta(3) or the GPIb-IX-V complex. Further, Gads plays a negligible role in aggregate formation on collagen at an arteriolar rate of shear. In stark contrast, platelets deficient in the adapter LAT exhibit a marked decrease in aggregation and secretion following activation of GPVI and CLEC-2, and are unable to form stable aggregates on collagen at arteriolar shear. CONCLUSIONS: The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP-76 in response to weak activation of GPVI and CLEC-2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads-independent pathway of platelet activation downstream of LAT.

Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines.

BACKGROUND: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRgamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). OBJECTIVE: To determine why GPVI-FcRgamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. METHODS AND RESULTS: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcRgamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. CONCLUSION: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcRgamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

BACKGROUND: MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cell's contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation. OBJECTIVE: To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin). RESULTS: Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model. CONCLUSIONS: MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

A major role for Scar/WAVE-1 downstream of GPVI in platelets.

BACKGROUND: The small GTPase Rac1 plays a critical role in lamellipodia assembly in platelets on matrix proteins in the absence or presence of G protein-coupled receptor (GPCR) agonists. Rac mediates actin assembly via Scar/WAVE, a family of scaffolding proteins that direct actin reorganization by relaying signals from Rac to the Arp2/3 complex. OBJECTIVE: To evaluate the role of Scar/WAVE-1 in mediating platelet activation and cytoskeletal reorganization. METHODS AND RESULTS: Using specific antibodies, we demonstrate that murine platelets, like human platelets, express Scar/WAVE-1 and Scar/WAVE-2. Lamellipodia formation in Scar/WAVE-1(-/-) platelets is markedly inhibited on immobilized collagen-related peptide (CRP) and on laminin, both of which signal through the collagen receptor GPVI. In contrast, lamellipodia formation on collagen, which requires release of the GPCR agonists ADP and thromboxane A(2), is not altered. Immobilized fibrinogen supports limited formation of lamellipodia in murine platelets, which is not altered in Scar/WAVE-1(-/-) platelets. As with Rac1(-/-) platelets, Scar/WAVE-1(-/-) platelets exhibit a marked inhibition of aggregation in response to CRP, whereas the response to the GPCR agonist thrombin is not altered. Platelet aggregation on immobilized collagen under shear, which is dependent on signaling by matrix and GPCR agonists, was unaltered in the absence of Scar/WAVE-1. CONCLUSION: This study demonstrates a major role for Scar/WAVE-1 in mediating platelet cytoskeletal reorganization and aggregate formation downstream of activation by GPVI but not by GPCR agonists.

GPVI and integrin alphaIIb beta3 signaling in platelets.

This review summarizes recent developments in our understanding of the molecular basis of platelet activation by two distinct types of surface receptor, the immunoglobulin GPVI, and the integrin alphaIIb beta3 (also known as GPIIbIIIa). These two classes of receptor signal through similar yet distinct tyrosine kinase-based signaling cascades leading to activation of phospholipase C gamma2. The significance of these signaling cascades in platelet adhesion and platelet aggregation at arterial rates of shear is discussed.

c-Cbl negatively regulates platelet activation by glycoprotein VI.

BACKGROUND: The adapter protein c-Cbl has emerged as having a potential role in negative regulation of immune receptor signaling. The major platelet-signaling receptor for collagen, glycoprotein VI (GpVI), is associated with the Fc receptor (FcR) gamma-chain, and signals through a similar pathway to immune receptors. c-Cbl is tyrosine-phosphorylated in response to stimulation of GpVI, whereas phosphorylation of c-Cbl in thrombin-activated platelets is dependent on fibrinogen binding to the integrin GpIIb/IIIa. OBJECTIVE: To investigate the role of c-Cbl in platelet signaling. METHODS: Murine platelets lacking functional c-Cbl or Src family kinases were analyzed. RESULTS: Phosphorylation of c-Cbl through GpVI is reduced in murine platelets deficient in the Src-family kinases Fyn and Lyn, demonstrating that they lie upstream of c-Cbl phosphorylation. Phosphorylation of several proteins of the GpVI-signaling pathway, including the FcR gamma-chain, Syk and phospholipase Cgamma2 (PLCgamma2), is increased in the absence of c-Cbl. In line with this, aggregation is potentiated in response to the GpVI-specific collagen-related peptide (CRP) after a slight delay. A delay in potentiation is also seen in response to stimulation by thrombin. CONCLUSIONS: These observations demonstrate that c-Cbl negatively regulates platelet responses to GpVI agonists and to thrombin, with the latter effect possibly being mediated downstream of GpIIb/IIIa. c-Cbl may play a physiological role in helping to prevent unwanted platelet activation in vivo.

Synthesis and antibacterial activity of HMR 3647 a new ketolide highly potent against erythromycin-resistant and susceptible pathogens.

In the search for new ketolides with improved activities against erythromycin-resistant S. pneumoniae and H. influenzae we synthesized a new 11,12 carbamate ketolide substituted by an imidazo-pyridyl side chain: HMR 3647. This compound demonstrated a potent activity against erythromycin susceptible and resistant pathogens, including penicillin G/erythromycin A-resistant S. pneumoniae and H. influenzae. In vivo, HMR 3647 displayed good pharmacokinetic parameters (Cmax = 2.9 microg/ml, bioavailability=49%, AUC0.8 = 17.2 microg.h/l, t1/2=1h) and was shown to have a high therapeutic efficacy in mice infected by various respiratory pathogens, including multi-resistant S. pneumoniae and Gram negative bacteria such as H. influenzae. HMR 3647 appears to be a very promising agent for the treatment of respiratory infections and is currently in clinical trials.

Synthesis and antibacterial activity of ketolides (6-O-methyl-3-oxoerythromycin derivatives): a new class of antibacterials highly potent against macrolide-resistant and -susceptible respiratory pathogens.

In the search for new antibiotics active against macrolide-resistant pneumococci and Haemophilus influenzae, we synthesized a new class of 3-oxo-6-O-methylerythromycin derivatives, so-called "ketolides". A keto function was introduced in position 3 after removal of L-cladinose, a sugar which has long been thought essential. Further modifications of the macrolactone backbone allowed us to obtain three different series of 9-oxime, 11,12-carbamate, and 11, 12-hydrazonocarbamate ketolides. These compounds were found to be very active against penicillin/erythromycin-resistant pneumococci and noninducers of MLSB resistance. The 11,12-substituted ketolide 61 (HMR 3004) demonstrated a potent activity against multiresistant pneumococci associated with a well-balanced activity against all bacteria involved in respiratory infections including H. influenzae, Mycoplasma catarrhalis, group A streptococci, and atypical bacteria. In addition HMR 3004 displayed high therapeutic activity in animals infected by all major strains, irrespective of their resistance phenotype.

Combined hiatal and pleuroperitoneal hernia in a shar-pei.

This article presents an unusual combination of a type IV hiatal hernia and a pleuroperitoneal hernia in a young shar-pei. Pathogenesis, diagnosis, and treatment of both conditions are discussed. At surgery, close examination and palpation of the whole diaphragm are recommended to allow perioperative diagnosis of unexpected defects.