A phase I randomized, double-blind, controlled trial of 2009 influenza A (H1N1) inactivated monovalent vaccines with different adjuvant systems

Methods We conducted a phase I, multicenter, randomized, double-blind, placebo-controlled, multi-arm (10) parallel study involving healthy adults to evaluate the safety and immunogenicity of influenza A (H1N1) 2009 non-adjuvanted and adjuvanted candidate vaccines. Subjects received two intramuscular injections of one of the candidate vaccines administered 21 days apart. Antibody responses were measured by means of hemagglutination-inhibition assay before and 21 days after each vaccination. The three co-primary immunogenicity end points were the proportion of seroprotection >70%, seroconversion >40%, and the factor increase in the geometric mean titer >2.5. Results A total of 266 participants were enrolled into the study. No deaths or serious adverse events were reported. The most commonly solicited local and systemic adverse events were injection-site pain and headache, respectively. Only three subjects (1.1%) reported severe injection-site pain. Four 2009 influenza A (H1N1) inactivated monovalent candidate vaccines that met the three requirements to evaluate influenza protection, after a single dose, were identified: 15 μg of hemagglutinin antigen without adjuvant; 7.5 μg of hemagglutinin antigen with aluminum hydroxide, MPL and squalene; 3.75 μg of hemagglutinin antigen with aluminum hydroxide and MPL; and 3.75 μg of hemagglutinin antigen with aluminum hydroxide and squalene. Conclusions Adjuvant systems can be safely used in influenza vaccines, including the adjuvant monophosphoryl lipid A (MPL) derived from Bordetella pertussis with squalene and aluminum hydroxide, MPL with aluminum hydroxide, and squalene and aluminum hydroxide.

Efeitos da Prostaglandina E1 na gênese de capilares sanguíneos em músculo esquelético isquêmico de ratos: estudo histológico e ultra-estrutural

Objetivo: o objetivo deste trabalho foi estudar os aspectos morfológicos e ultra-estruturais na gênese de capilares sanguíneos em músculo esquelético do membro caudal de ratos submetidos à isquemia sob a ação da Prostaglandina E1 (PGE1), administrada por via intramuscular ou endovenosa. Métodos: foram utilizados 60 ratos (Rattus norvegicus albinus), linhagem Wistar-UEM, distribuídos aleatoriamente em três grupos de 20, redistribuídos igualmente em dois subgrupos, observados no 7o e 14o dias, sendo um grupo controle onde apenas foi provocada a isquemia no membro, outro com a isquemia e a injeção da PGE1 via intramuscular (IM), e outro com a isquemia e a injeção da PGE1 endovenosa (EV). Para análise dos resultados, foram realizadas a coloração com hematoxilina & eosina (HE), a imuno-histoquímica e a microscopia eletrônica de transmissão (MET). Resultados: constatou-se um aumento estatisticamente significante no número de capilares nos subgrupos com o uso da PGE1 IM e EV, através da contagem nos cortes corados com HE. Houve marcação de capilares e vasos de maior calibre nestes mesmos subgrupos, porém, esta reação não foi eficiente para a quantificação dos capilares. Na MET encontraram-se evidências de formação de novos capilares. Conclusões: a PGE1, administrada por via IM ou EV, promoveu, após 14 dias de observação, um aumento no número de capilares no músculo esquelético de ratos submetido à isquemia, identificáveis histologicamente com a coloração em HE. Na análise ultra-estrutural encontraram-se alterações que sugerem, nos animais sob a ação da PGE1, que a neoformação vascular possa ter ocorrido por angiogênese e vasculogênese. A imuno-coloração, apesar da marcação de capilares e vasos maiores, não permitiu estabelecer uma correlação com o aumento de vasos encontrados na coloração com HE.

Ensayos de inmunización en ganado caprino con tiol-proteinasas procedentes de productos de excreción-secreción (E/S) de vermes adultos de "Teladorsagia circumcincta"

Programa de doctorado: Sanidad animal

Botulinum Toxin Type A for the Treatment of Equinus Deformity in to Patients With Mucopolysaccharidosis Type II

Mucopolysaccharidoses are lysosomal storage disorders that are caused by a deficiency in the enzymes that degrade glycosaminoglycans. The accumulation of glycosaminoglycans affects multiple systems, resulting in coarse facial features, short stature, organomegaly, and variable neurological changes from normal intelligence to severe mental retardation and spasticity. Effects on the musculoskeletal system include dysostosis multiplex, joint stiffness, and muscle shortening. This article reports 2 patients with mucopolysaccharidosis type II (Hunter syndrome) who showed progressive equinus deformity of the feet. Both patients were treated with intramuscular botulinum toxin type A injections in the gastrocnemius and the soleus muscles, followed by serial casting. In both patients, passive range of motion, muscle tone, and gait performance were significantly improved. Botulinum toxin type A injections followed by serial casting are a therapeutic option for contractures in patients with mucopolysaccharidosis. However, the long-term effects and the effect of application in other muscles remain unknown.

Ketoprofen in piglets: enantioselective pharmacokinetics, pharmacodynamics and PK/PD modelling

The chiral pharmacokinetics and pharmacodynamics of ketoprofen were investigated in a placebo-controlled study in piglets after intramuscular administration of 6 mg/kg racemic ketoprofen. The absorption half-lives of both enantiomers were short, and S-ketoprofen predominated over R-ketoprofen in plasma. A kaolin-induced inflammation model was used to evaluate the anti-inflammatory, antipyretic and analgesic effects of ketoprofen. Skin temperatures increased after the kaolin injection, but the effect of ketoprofen was small. No significant antipyretic effects could be detected, but body temperatures tended to be lower in the ketoprofen-treated piglets. Mechanical nociceptive threshold testing was used to evaluate the analgesic effects. The piglets in the ketoprofen-treated group had significantly higher mechanical nociceptive thresholds compared to the piglets in the placebo group for 12-24 h following the treatment. Pharmacokinetic/pharmacodynamic modelling of the results from the mechanical nociceptive threshold testing gave a median IC(50) for S-ketoprofen of 26.7 mug/mL and an IC(50) for R-ketoprofen of 1.6 mug/mL. This indicates that R-ketoprofen is a more potent analgesic than S-ketoprofen in piglets. Estimated ED(50) for racemic ketoprofen was 2.5 mg/kg.

Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows

In most mammals, prolactin (PRL) is essential for maintaining lactation, and yet the short-term suppression of PRL during established lactation by bromocriptine has produced inconsistent effects on milk yield in cows and goats. To assess the effect of the long-term inhibition of PRL release in lactating dairy cows, 5 Holstein cows in early lactation received daily intramuscular injections of 1mg of the PRL-release inhibitor quinagolide for 9 wk. Four control cows received the vehicle (water) only. During the last week of the treatments, one udder half was milked once a day (1x) and the other twice a day (2x). Blood samples were harvested at milking in wk -1, 1, 4, and 8. The daily injections of quinagolide reduced milking-induced PRL release but not the basal PRL concentration. Quinagolide induced a faster decline in milk production, which was about 5.3 kg/d lower in the quinagolide-treated cows during the last 4 wk of treatment. During wk 9, the inhibition of milk production by quinagolide was maintained in the udder half that was milked 2x but not in the half milked 1x. Milk production was significantly correlated with the quantity of PRL released at milking. Quinagolide did not affect the release of oxytocin at milking. Serum concentration of insulin-like growth factor-1 was not affected by treatment or correlated with milk production. Serum concentrations of leptin and the calciotropic hormone stanniocalcin were not affected by the treatment. In conclusion, the chronic administration of the PRL-release inhibitor quinagolide decreases milk production in dairy cows. The effect is likely the result of the reduced release of milking-induced PRL and is modulated at the level of the gland by milking frequency.

Distribution of muscarinic receptor subtypes and interstitial cells of Cajal in the gastrointestinal tract of healthy dairy cows

OBJECTIVE: To describe the distribution of muscarinic receptor subtypes M(1) to M(5) and interstitial cells of Cajal (ICCs) in the gastrointestinal tract of healthy dairy cows. SAMPLE POPULATION: Full-thickness samples were collected from the fundus, corpus, and pyloric part of the abomasum and from the duodenum, ileum, cecum, proximal loop of the ascending colon, and both external loops of the spiral colon of 5 healthy dairy cows after slaughter. PROCEDURES: Samples were fixed in paraformaldehyde and embedded in paraffin. Muscarinic receptor subtypes and ICCs were identified by immunohistochemical analysis. RESULTS: Staining for M(1) receptors was found in the submucosal plexus and myenteric plexus. Antibodies against M(2) receptors stained nuclei of smooth muscle cells only. Evidence of M(3) receptors was found in the lamina propria, in intramuscular neuronal terminals, on intermuscular nerve fibers, and on myocytes of microvessels. There was no staining for M(4) receptors. Staining for M(5) receptors was evident in the myocytes of microvessels and in smooth muscle cells. The ICCs were detected in the myenteric plexus and within smooth muscle layers. Distribution among locations of the bovine gastrointestinal tract did not differ for muscarinic receptor subtypes or ICCs. CONCLUSIONS AND CLINICAL RELEVANCE: The broad distribution of M(1), M(3), M(5), and ICCs in the bovine gastrointestinal tract indicated that these components are likely to play an important role in the regulation of gastrointestinal tract motility in healthy dairy cows. Muscarinic receptors and ICCs may be implicated in the pathogenesis of motility disorders, such as abomasal displacement and cecal dilatation-dislocation.

Electroporation-mediated interleukin-10 overexpression in skeletal muscle reduces acute rejection in rat cardiac allografts

OBJECTIVES: Human interleukin 10 (hIL-10) may reduce acute rejection after organ transplantation. Our previous data shows that electroporation-mediated transfer of plasmid DNA to peripheral muscle enhances gene transduction dramatically. This study was designed to investigate the effect of electroporation-mediated overexpression of hIL-10 on acute rejection of cardiac allografts in the rat. METHODS: The study was designed to evaluate the effect of hIL-10 gene transfer on (a) early rejection pattern and (b) graft survival. Gene transfer was achieved by intramuscular (i.m.) injection into the tibialis anterior muscle of Fischer (F344) male recipients followed by electroporation 24 h prior to transplantation. Heterotopic cardiac transplantation was performed from male Brown Norway rat to F344. Four groups were studied (n = 6). Treated animals in groups B1 and B2 received 2.5 microg of pCIK hIL-10 and control animals in groups A1 and A2 distilled water. Graft function was assessed by daily palpation. Animals from group A1 were sacrificed at the cessation of the heart beat of the graft and those in group B1 were sacrificed at day 7; blood was taken for ELISA measurement of hIL-10 and tissue for myeloperoxidase (MPO) measurement and histological assessment. To evaluate graft survival, groups A2 and B2 were sacrificed at cessation of the heart beat of the graft. RESULTS: Histological examination revealed severe rejection (IIIB-IV) in group A1 in contrast to low to moderate rejection (IA-IIIA) in group B1 (p = 0.02). MPO activity was significantly lower in group B1 compared to group A1 (18 +/- 7 vs. 32 +/- 14 mU/mg protein, p = 0.05). Serum hIL-10 levels were 46 +/- 13 pg/ml in group B1 vs. 0 pg/ml in group A1. At day 7 all heart allografts in the treated groups B1 and B2 were beating, whereas they stopped beating at 5 +/- 2 days in groups A1 and A2 vs. 14 +/- 2 days in group B2 (p = 0.0012). CONCLUSIONS: Electroporation-mediated intramuscular overexpression of hIL-10 reduces acute rejection and improves survival of heterotopic heart allografts in rats. This study demonstrates that peripheral overexpression of specific genes in skeletal muscle may reduce acute rejection after whole organ transplantation.

The effect of enamel matrix proteins and deproteinized bovine bone mineral on heterotopic bone formation

To evaluate the osteoinductive potential of deproteinized bovine bone mineral (DBBM) and an enamel matrix derivative (EMD) in the muscle of rats. Sixteen rats were used in this study. The animals were divided in three groups. Group A: a pouch was created in one of the pectoralis profundis muscles of the thorax of the rats and DBBM particles (Bio-Oss) were placed into the pouch. Healing: 60 days. Group B: a small pouch was created on both pectoralis profundis muscles at each side of the thorax midline. In one side, a mixture of EMD (Emdogain) mixed with DBBM was placed into one of the pouches, whereas in the contralateral side of the thorax the pouch was implanted with DBBM mixed with the propylene glycol alginate (PGA--carrier for enamel matrix proteins of EMD). Healing: 60 days. Group C: the same procedure as group B, but with a healing period of 120 days. Qualitative histological analysis of the results was performed. At 60 days, the histological appearance of the DBBM particles implanted alone was similar to that of the particles implanted together with EMD or PGA at both 60 and 120 days. The DBBM particles were encapsulated into a connective tissue stroma and an inflammatory infiltrate. At 120 days, the DBBM particles implanted together with EMD or PGA exhibited the presence of resorption lacunae in some cases. Intramuscular bone formation was not encountered in any group. The implantation of DBBM particles alone, combined with EMD or its carrier (PGA) failed to exhibit extraskeletal, bone-inductive properties.

Iatrogenic sciatic nerve injury in eighteen dogs and nine cats (1997-2006)

OBJECTIVE: To report clinical features associated with iatrogenic peripheral nerve injury in dogs and cats admitted (1997-2006) to a referral teaching hospital. STUDY DESIGN: Retrospective study. ANIMALS: Dogs (n=18), 9 cats. METHODS: Patients had acute signs of monoparesis attributable to sciatic nerve dysfunction that developed after treatment. Neurologic examination and electrodiagnostic testing were performed. Surgical therapy was used for nerve entrapment and delayed reconstructive surgery used in other cases. RESULTS: Of 27 nerve injuries, 25 resulted from surgery (18 with treatment of pelvic injuries). Iliosacral luxation repair resulted in tibial (4 cats) and peroneal (3 dogs) nerve dysfunction. Other causes were intramedullary pinning of femoral fractures (3), other orthopedic surgery (cemented hip prosthesis [2] and tibial plateau-leveling osteotomy [1]), and perineal herniorrhaphy [1]. Nerve injury occurred after intramuscular injection (1 cat, 1 dog). Immediate surgical treatment was removal of intramedullary nails, extruded cement, or entrapping suture. Delayed nerve transplantation was performed in 2 dogs. Within 1 year, 13 patients recovered completely, clinical improvement occurred in 7, and there was no improvement in 7. Five of the 7 dogs that did not recover had acetabular or ilium fracture. CONCLUSION: Iatrogenic sciatic nerve injury occurred most commonly during treatment of pelvic orthopedic diseases and had a poor prognosis. Clinical variation in sciatic nerve dysfunction in dogs and cats can be explained by species anatomic differences. CLINICAL RELEVANCE: Iatrogenic sciatic nerve injury leads to severely debilitating locomotor dysfunction with an uncertain prognosis for full-functional recovery.

Effects of dexamethasone on mRNA abundance of nuclear receptors and hepatic nuclear receptor target genes in neonatal calves

Hepatic nuclear receptors (NR), particularly constitutive androstane receptor (CAR) and pregnane X receptor (PXR), are involved in the coordinated transcriptional control of genes that encode proteins involved in the metabolism and detoxification of xeno- and endobiotics. A broad spectrum of metabolic processes are mediated by NR acting in concert with ligands such as glucocorticoids. This study examined the role of dexamethasone on hepatic mRNA expression of CAR, PXR and several NR target genes. Twenty-eight male calves were allotted to one of four treatment groups in a 2 x 2 arrangement of treatments: feed source (colostrum or milk-based formula) and glucocorticoid administration (twice daily intramuscular dexamethasone). Liver biopsies were obtained at 5 days of age. Real-time reverse transcription polymerase chain reaction was used to quantify mRNA abundances. No effects of feed source on mRNA abundances were observed. For the NR examined, mRNA abundance of both CAR and PXR in dexamethasone-treated calves was lower (p < 0.05) by 39% and 40%, respectively, than in control calves. Abundance of NR target genes exhibited a mixed response. SULT1A1 mRNA abundance was 39% higher (p < 0.05) in dexamethasone-treated calves compared with control calves. mRNA abundance of CYP2C8 tended also to be higher (+44%; p = 0.053) after dexamethasone treatment. No significant treatment effects (p > 0.10) were observed for mRNA abundances of CYP3A4, CYP2E1, SULT2A1, UGT1A1 or cytochrome P450 reductase (CPR). In conclusion, an enhanced glucocorticoid status, induced by pharmacological amounts of dexamethasone, had differential and in part unexpected effects on NR and NR target systems in 5-day-old calves. Part of the unexpected responses may be due the immaturity of NR and NR receptor target systems.

Role of calcium-independent phospholipase A2 in cortex striatum thalamus cortex circuitry-enzyme inhibition causes vacuous chewing movements in rats

RATIONALE: High levels of calcium independent phospholipase A2 (iPLA2) are present in certain regions of the brain, including the cerebral cortex, striatum, and cerebellum (Ong et al. 2005). OBJECTIVES: The present study was carried out to elucidate a possible role of the enzyme in the motor system. METHODS: The selective iPLA2 inhibitor bromoenol lactone (BEL), the nonselective PLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP), and an antisense oligonucleotide were used to interfere with iPLA2 activity in various components of the motor system. Control animals received injections of carrier (phosphate buffered saline, PBS) at the same locations. The number of vacuous chewing movements (VCM) was counted from 1 to 14 days after injection. RESULTS: Rats that received BEL and high-dose MAFP injections in the striatum, thalamus, and motor cortex, but not the cerebellum, showed significant increase in VCM, compared to those injected with PBS at these locations. BEL-induced VCM were blocked by intramuscular injections of the anticholinergic drug, benztropine. Increased VCM was also observed after intrastriatal injection of antisense oligonucleotide to iPLA2. The latter caused a decrease in striatal iPLA2 levels, confirming a role of decreased enzyme activity in the appearance of VCM. CONCLUSIONS: These results suggest an important role for iPLA2 in the cortex-striatum-thalamus-cortex circuitry. It is postulated that VCM induced by iPLA2 inhibition may be a model of human parkinsonian tremor.

Managing chronic whiplash associated pain with a combination of low-dose opioid (remifentanil) and NMDA-antagonist (ketamine)

The aim was to investigate the efficacy of a combination of low-dose remifentanil (REMI) and ketamine (KET) compared to the single drugs and placebo (P) on whiplash associated pain (WAD) in a double-blind, randomized, placebo-controlled, cross-over study. Twenty patients with chronic (>1 year) WAD were included. Four different drug combinations were tested in four sessions: placebo/placebo (P/P), placebo/remifentanil (P/REMI), ketamine/placebo (KET/P) and ketamine/remifentanil (KET/REMI). Target concentrations were 1 and 2ng/ml (stepwise) for remifentanil and 100ng/ml for ketamine. Habitual pain intensity was assessed on a visual analogue scale (VAS). Experimental pain was assessed with electrical stimulation (single and repeated) of tibialis anterior (TA) muscle, pressure pain algometry applied over infraspinatus (IS) and TA muscles and VAS scores after intramuscular hypertonic saline infusion in TA. KET/REMI significantly reduced habitual pain. KET/REMI infused at low REMI target concentration (1ng/ml) significantly elevated electrical intramuscular pain thresholds (single and repeated). Pain thresholds to electrical stimulation were similarly increased by both P/REMI and KET/REMI at 2ng/ml target concentration. Pressure pain thresholds were increased by both KET/REMI and P/REMI. VAS-scores after intramuscular saline were also similarly decreased by both REMI combinations. Seven out of 20 subjects were non-responders (<50% pain relief). No correlation was found between effects on spontaneous pain and experimental pain. KET/REMI showed an analgesic effect on habitual pain. Experimental pain was attenuated by both combinations containing the opioid, however, KET seemed to enhance the effect of REMI on electrical pain thresholds when a low REMI target concentration was used.

[Chronic anterior tibial syndrome]

In two young athletic men we made the diagnosis of chronic tibialis anterior syndrome. Pain during strenuous exercise was localized in the anterolateral aspect of both calves. After stopping exercise the complaints disappeared within 15-20 minutes and not in 2-3 minutes as it would be typical for patients with atherosclerotic peripheral arterial occlusive disease. Diagnosis is based on patient history, normal clinical examination (systolic ankle pressure determined by Doppler-Sonography, electronic segmental oscillography) and increased intramuscular pressure at rest (> 10 mmHg) and after exercise (42 and 35 mmHg). Bilateral fasciotomy was performed in both patients. They are free of pain after 3 respectively 6 months postoperatively.

New method for quantification and statistical analysis of nociceptive reflex receptive fields in humans

A method for quantifying nociceptive withdrawal reflex receptive fields in human volunteers and patients is described. The reflex receptive field (RRF) for a specific muscle denotes the cutaneous area from which a muscle contraction can be evoked by a nociceptive stimulus. The method is based on random stimulations presented in a blinded sequence to 10 stimulation sites. The sensitivity map is derived by interpolating the reflex responses evoked from the 10 sites. A set of features describing the size and location of the RRF is presented based on statistical analysis of the sensitivity map within every subject. The features include RRF area, volume, peak location and center of gravity. The method was applied to 30 healthy volunteers. Electrical stimuli were applied to the sole of the foot evoking reflexes in the ankle flexor tibialis anterior. The RRF area covered a fraction of 0.57+/-0.06 (S.E.M.) of the foot and was located on the medial, distal part of the sole of the foot. An intramuscular injection into flexor digitorum brevis of capsaicin was performed in one spinal cord injured subject to attempt modulation of the reflex receptive field. The RRF area, RRF volume and location of the peak reflex response appear to be the most sensitive measures for detecting modulation of spinal nociceptive processing. This new method has important potential applications for exploring aspects of central plasticity in volunteers and patients. It may be utilized as a new diagnostic tool for central hypersensitivity and quantification of therapeutic interventions.