Development and feasibility study of a sensory-enhanced robot-aided motor training in stroke rehabilitation.

Functional impairment of the upper limb is a major challenge faced by many stroke survivors. The present study aimed at developing a novel sensory-enhanced robot-aided motor training program and testing its feasibility in stroke rehabilitation. A specially designed robot handle was developed as an attachment to the Inmotion2 robotic system. This handle provided sensory stimulation through pins connected to small servo motors inside the handle. Vibration of the pins was activated during motor training once pressure on the handle reached a certain threshold indicating an active motion of the study subject. Nine chronic stroke survivors were randomly assigned to either a sensory-enhanced robot-aided motor training group (SERMT) or robot-aided motor training only group (RMT). All participants underwent a 6-week motor training program, performing target reaching movements with the specialized handle with or without vibration stimulation during training. Motor Status (MS) scores were measured for functional outcome prior to and after training. The results showed significant improvement in the total MS scores after training in both experimental groups. However, MS sub-scores for the shoulder/elbow and the wrist/hand increased significantly only in the SERMT group (p<0.05). Future studies are required to confirm these preliminary findings.

Isolation, cloning and functional characterization of porcine mannose-binding lectin.

Binding of mannose-binding lectin (MBL), a C-type lectin, and its associated serine proteases, MASP-1 and MASP-2, to cell surface carbohydrates activates the lectin complement pathway. As MBL plays an important role in innate immunity, it has been cloned and characterized in several species. While the pig may be used as a source of organs/tissues for xenotransplantation, little is known about its MBL, thus, we report the isolation of three monomeric forms of MBL from porcine serum. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie staining of reduced porcine MBL revealed the presence of three monomeric forms with approximate molecular masses of 30 000, 32 000 and 34 000. Protein sequencing identified these monomeric forms as one single protein, suggesting post-translational modification. Western blot analysis demonstrated the cross-reactivity of anti-human MBL polyclonal antibody with porcine MBL. A full-length porcine liver MBL cDNA was isolated and the predicted amino acid sequence exhibited 64.9% identity with human MBL and 50.2% and 56.7% identity with rat A and C MBL, respectively. Furthermore, Northern blot analysis demonstrated the presence of a single ( approximately 1.4-1.6 kilobase pair) transcript in porcine liver. Addition of purified porcine MBL to MBL-deficient human sera augmented N-acetylglucosamine inhibitable C3 deposition to mannan-coated plates in a dose-dependent manner. Taken together, these data demonstrate that porcine and human MBL are highly conserved, sharing structural and functional characteristics.

Isolation, characterization, and cloning of porcine complement component C7.

Activation of the complement system through the classical, alternative, or lectin pathway results in the formation of the terminal complement complex. C7 plays an integral role in the assembly of this complex with target cell membranes. To date, only human C7 has been cloned and characterized; thus, in this study, we characterized the porcine complement component C7. Porcine C7 was isolated by affinity chromatography as a single glycoprotein with an approximate molecular mass of 90 kDa and 100 kDa under reducing and nonreducing conditions, respectively. The full-length porcine C7 cDNA was isolated, and the predicted amino acid sequence exhibited 80% identity with human C7 with conservation of the cysteine backbone and two putative N-linked glycosylation sites. Porcine C7 mRNA expression was detected in all tissues investigated, except polymorphonuclear and mononuclear leukocytes. Addition of purified porcine C7 restored the hemolytic activity of C7-depleted human sera in a dose-dependent manner. A functionally inhibitory mAb against porcine C7 attenuated the hemolytic activity of human, rabbit, or rat sera, suggesting an important conserved C7 epitope among species. These data demonstrate that porcine and human C7 are highly conserved, sharing structural and functional characteristics.

Complement activation after oxidative stress: role of the lectin complement pathway.

The complement system plays an important role in mediating tissue injury after oxidative stress. The role of mannose-binding lectin (MBL) and the lectin complement pathway (LCP) in mediating complement activation after endothelial oxidative stress was investigated. iC3b deposition on hypoxic (24 hours; 1% O(2))/reoxygenated (3 hours; 21% O(2)) human endothelial cells was attenuated by N-acetyl-D-glucosamine or D-mannose, but not L-mannose, in a dose-dependent manner. Endothelial iC3b deposition after oxidative stress was also attenuated in MBL-deficient serum. Novel, functionally inhibitory, anti-human MBL monoclonal antibodies attenuated MBL-dependent C3 deposition on mannan-coated plates in a dose-dependent manner. Treatment of human serum with anti-MBL monoclonal antibodies inhibited MBL and C3 deposition after endothelial oxidative stress. Consistent with our in vitro findings, C3 and MBL immunostaining throughout the ischemic area at risk increased during rat myocardial reperfusion in vivo. These data suggest that the LCP mediates complement activation after tissue oxidative stress. Inhibition of MBL may represent a novel therapeutic strategy for ischemia/reperfusion injury and other complement-mediated disease states.

Mesenteric dysfunction after cardiopulmonary bypass: role of complement C5a.

BACKGROUND: We investigated the effects of cardiopulmonary bypass (CPB) on ileal homeostasis, and the influence of functional inhibition of complement C5a on CPB-induced mesenteric injury. METHODS: Pigs were perfused on CPB for 1 hour and then perfused off CPB for an additional 2 hours. Antiporcine C5a monoclonal antibody (C5a MAb) was administered 20 minutes before onset of CPB to 6 pigs; 6 controls received saline vehicle. Total complement activity, ileal myeloperoxidase, and indices of ileal integrity were examined. RESULTS: Treatment with C5a MAb ameliorated CPB-induced abnormalities in endothelium-dependent relaxation to ADP and substance P, and the hypercontractile response to phenylephrine of ileal microvessels (88 to 168 microm). Ileal myeloperoxidase activity [units/g protein] was 41 +/- 11 in the C5a MAb group, compared to 83 +/- 13 in the saline group (19 +/- 10 base line). Total hemolytic complement activity was similar in the C5a MAb and saline groups (0.6 +/- 0.2 and 0.7 +/- 0.2 CH50 units). During CPB, ileal mucosal blood flow and mucosal pH, edema formation, and epithelial permeability deteriorated similarly in saline and C5a MAb groups. Inducible nitric oxide synthase (iNOS) mRNA expression was similar before and after CPB. CONCLUSIONS: CPB is associated with significant physiologic alterations in mucosal perfusion, epithelial permeability, edema formation, and blood flow regulation. Inhibition of C5a limits neutrophil-mediated impairment of ileal microvascular regulation after bypass, but does not improve extravascular mesenteric dysfunction after CPB.

Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues.

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Attenuation of endothelium-dependent dilation of pig pulmonary arterioles after cardiopulmonary bypass is prevented by monoclonal antibody to complement C5a.

UNLABELLED: We examined whether pulmonary endothelial dysfunction associated with cardiopulmonary bypass (CPB) may be mediated by complement C5a in pigs. Pigs were placed on normothermic CPB for 1 h with or without a previous administration of 1.6 mg/kg anti-C5a monoclonal antibody (MAb), then reperfused for 2 h. Pulmonary tissue myeloperoxidase activity was measured. Expression of nitric oxide synthase (NOS) was measured by reverse transcriptase polymerase chain reaction and Western blotting. Pulmonary arterioles approximately 100 microm in diameter were preconstricted with the thromboxane analog U46619 1 microM, and relaxation responses to adenosine diphosphate 10(-9)-10(-4) M, substance P 10(-12)-10(-6) M, and sodium nitroprusside 10(-9)-10(-4) M were examined in vitro by videomicroscopy. Relaxation to the endothelium-dependent dilators adenosine diphosphate and substance P was attenuated after CPB; this attenuation was prevented by the previous administration of MAb. Relaxation to sodium nitroprusside was not affected by CPB. Neutrophil sequestration, as measured by MPO activity, increased after CPB, either with or without MAb. Transcription of NOS was unchanged by CPB, but translation of constitutive NOS was decreased after CPB, and this decrease was prevented by a previous administration of MAb. We conclude that pig pulmonary endothelial dysfunction associated with CPB may be mediated by C5a. The mechanism may involve changes in NOS translation. IMPLICATIONS: In pigs, pulmonary endothelial dysfunction may occur after cardiopulmonary bypass due to product(s) of complement activation.

Critical role of cAMP response element binding protein expression in hypoxia-elicited induction of epithelial tumor necrosis factor-alpha.

Tissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 +/- 6.1-, 7 +/- 0.8-, and 9 +/- 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-kappaB binding domain, we investigated the influence of hypoxia on NF-kappaB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-kappaB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-kappaB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-alpha, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 +/- 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-alpha and protein release (9 +/- 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-alpha release (86 +/- 3.2% inhibition with 3 mM 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-alpha defining a causal relationship between hypoxia-elicited CREB reduction and TNF-alpha induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators.

Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells.

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 +/- 0. 6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-alpha also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

Complement activation following oxidative stress.

It is clear that complement plays an important role in the inflammatory process following oxidative stress in cellular and animal models. Clinical trials underway with novel complement inhibitors will establish the potential therapeutic benefit of complement inhibition in human disease. For as much as we understand about the role of complement in disease states, many questions remain. How is complement activated on endothelial cells following oxidative stress? What is the ligand for MBL on endothelial cells following oxidative stress? Will inhibition of MBL provide tissue protection to the extent observed with other complement inhibitors such as sCR1 or anti-C5 mAbs? These questions and more will undoubtedly be answered in the next millennium.

Anti-C5a monoclonal antibody reduces cardiopulmonary bypass and cardioplegia-induced coronary endothelial dysfunction.

OBJECTIVE: Because C5a induces tissue injury by activating polymorphonuclear leukocytes, the hypothesis was that inhibition of C5a activity would reduce cardioplegia-related injury. METHODS: Pigs were placed on cardiopulmonary bypass. The hearts were arrested for 1 hour with hyperkalemic cardioplegia. Pigs were then separated from bypass, and the hearts were reperfused for 2 hours. Anti-porcine C5a monoclonal antibody (1.6 mg/kg, intravenously; n = 6) was administered 20 minutes before the onset of cardiopulmonary bypass. Six pigs received saline solution vehicle. Reactivity of coronary arterioles was studied in vitro with videomicroscopy. Microvessels from uninstrumented pigs served as controls for vascular studies. RESULTS: Endothelium-dependent relaxation to adenosine diphosphate (percent relaxation of precontraction) was reduced after cardioplegic reperfusion (63% +/- 14% vs 77% +/- 10% in control at 10 micromol/L; P =.01). This impairment in endothelium-dependent relaxation was improved with anti-porcine C5a monoclonal antibody (80% +/- 22%; P =.01 vs saline solution), as was the impaired endothelium-dependent relaxation to clonidine (64% +/- 12% control; 26% +/- 17% saline solution; 55% +/- 24% anti-porcine C5a monoclonal antibody at 10 micromol/L; P =.01 saline solution vs control or anti-porcine C5a monoclonal antibody). Myeloperoxidase activity was significantly decreased (0.2 +/- 0.2 units/g protein; P =.04) in the anti-porcine C5a monoclonal antibody group compared with 5.2 +/- 2.7 in the saline solution group. CH50 2 hours after bypass was not statistically different (0.57 +/- 0.41 unit and 0.65 +/- 0.41 unit, respectively) between the anti-porcine C5a monoclonal antibody and saline solution groups. Despite less myocardial polymorphonuclear leukocyte infiltration after C5a inhibition, maximum rate of rise of left ventricular pressure, percent segmental shortening, and blood flow through the left anterior descending coronary artery were similar in the anti-porcine C5a monoclonal antibody and saline solution groups. CONCLUSIONS: Inhibition of C5a limits neutrophil-mediated impairment of endothelium-dependent relaxation after cardiopulmonary bypass and cardioplegic reperfusion, but it has no effect on short-term myocardial functional preservation.

Complement activation following reoxygenation of hypoxic human endothelial cells: role of intracellular reactive oxygen species, NF-kappaB and new protein synthesis.

Complement plays an important role in ischemia-reperfusion injury. We recently demonstrated that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) activated the classical complement pathway and augmented iC3b deposition. In the present study, we investigated the potential role of oxygen-derived free radicals, NF-kappaB and new protein synthesis in this model. HUVECs subjected to 12 or 24 h hypoxic stress (1% O2) and then reoxygenated (0.5, 1, 2 or 3 h; 21% O2) in 30% human serum activated complement and deposited iC3b. Addition of hydrogen peroxide (H2O2; 1-100 micromol/l) to normoxic HUVECs increased iC3b deposition in a concentration-dependent manner. H2O2 (10 micromol/l), a concentration that did not significantly increase iC3b deposition on normoxic HUVECs, augmented iC3b deposition on hypoxic/reoxygenated HUVECs. We observed a significant increase in intracellular H2O2 and hydroxyl radical (OH.) production in hypoxic/reoxygenated HUVECs using dihydrorhodamine 123. Further, treatment of HUVECs with dimethylthiourea (DMTU, 1-100 micromol/l), deferoxamine (DEF, 1-100 micromol/l), or oxypurinol (10 micromol/l), but not superoxide dismutase (SOD, 500 U/ml), catalase (300 U/ml) or iron-loaded DEF, attenuated iC3b deposition following hypoxia/reoxygenation in a concentration-dependent manner. Western analysis demonstrated hypoxia-induced nuclear NF-kappaB translocation that increased with reoxygenation. Inhibition of new protein synthesis (i.e. cycloheximide) or inhibition of NF-kappaB (ALLN or SN-50) also significantly decreased iC3b deposition on hypoxic/reoxygenated HUVECs. We conclude that (1) hypoxic/reoxygenated HUVECs generate H2O2 and OH.; (2) treatment of HUVECs with cell permeable reactive oxygen species inhibitors/scavengers (i.e. DEF, DMTU, oxypurinol) but not large molecular weight inhibitors (i.e. catalase or SOD) significantly reduces iC3b deposition and (3) inhibition of new protein synthesis or NF-kappaB activation attenuates iC3b deposition. These data suggest that iC3b deposition on the vascular endothelium may be regulated by intracellular oxygen-derived free radical-induced activation of NF-kappaB, new protein synthesis and activation of the classical complement pathway during ischemia/reperfusion.

Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-C5 therapy.

BACKGROUND: Myocardial ischemia and reperfusion (MI/R)-induced tissue injury involves necrosis and apoptosis. However, the precise contribution of apoptosis to cell death, as well as the mechanism of apoptosis induction, has not been delineated. In this study, we sought to define the contribution of the activated terminal complement components to apoptosis and necrosis in a rat model of MI/R injury. METHODS AND RESULTS: Monoclonal antibodies (mAbs; 18A and 16C) raised against the rat C5 complement component bound to purified rat C5 (ELISA). 18A effectively blocked C5b-9-mediated cell lysis and C5a-induced chemotaxis of rat polymorphonuclear leukocytes (PMNs), whereas 16C had no complement inhibitor activity. A single dose (20 mg/kg i.v.) of 18A blocked >80% of serum hemolytic activity for >4 hours. Administration of 18A before myocardial ischemia (30 minutes) and reperfusion (4 hours) significantly reduced (91%) left ventricular free wall PMN infiltration compared with 16C treatment. Treatment with 18A 1 hour before ischemia or 5 minutes before reperfusion significantly reduced infarct size compared with 16C treatment. A significant reduction in infarct size (42%) was also observed in 18A-treated rats after 30 minutes of ischemia and 7 days of reperfusion. DNA ladders and DNA labeling (eg, TUNEL assay) demonstrated a dramatic reduction in MI/R-induced apoptosis in 18A-treated compared with 16C-treated rats. CONCLUSIONS: Anti-C5 therapy in the setting of MI/R significantly inhibits cell apoptosis, necrosis, and PMN infiltration in the rat despite C3 deposition. We conclude that the terminal complement components C5a and C5b-9 are key mediators of tissue injury in MI/R.

The implications of childbearing in postpubertal girls in Sokoto, Nigeria.

A retrospective analysis of 337 young postpubertal delivered mothers was compared with other parturient women in Sokoto University Teaching Hospital (S.U.T.H.) during a 1-year period. In this analysis, late booking was identified as the most important factor that directly affects the perinatal outcome in young postpubertal pregnant mothers. The problems of postpubertal pregnancy were highlighted in order to motivate individuals towards family planning.

PIP: This investigation was to evaluated the characteristics and the outcome of pregnancy in young postpubertal girls and in other women of childbearing age. A retrospective analysis of 337 young postpubertal delivered mothers was compared with other parturient women in Sokoto University Teaching Hospital, Nigeria, during a 1-year period. Late booking was identified as the most important factor that directly affects the perinatal outcome in young postpubertal pregnant mothers. The problems of postpubertal pregnancy were highlighted in order to motivate individuals towards family planning. The striking features of the young postpubertal mothers in this study as in other reports were relatively low level of education, low socioeconomic status, and social and psychological immaturity. This analysis revealed a relatively low birth weight and low parity in the young adolescent mothers; this agrees with other studies. The high incidence of maternal and fetal complications contradicts some other reports that indicate that adolescent obstetrics present no greater challenge than obstetrics in general. Anemia and prematurity were common in the young mothers. The cesarean section rate was high and the main indication was cephalopelvic disproportion, with the greatest risk for women under 16, due to bone immaturity. For most developing countries of the world, especially where there is inadequate medical care, pregnancy and delivery in young postpubertal girls appear unsafe and must be discouraged through appropriate reproductive health care in the community.