Fourth Injection Research Articles

Phase II study of subcutaneous alemtuzumab without dose escalation in patients with advanced‐stage, relapsed chronic lymphocytic leukaemia

This phase II study (n = 20) aimed to evaluate type, severity and duration of side-effects and efficacy following subcutaneous (SC) alemtuzumab, without dose-escalation, in advanced-stage relapsed chronic lymphocytic leukaemia (CLL) patients. Alemtuzumab 30 and 3 mg was administered SC simultaneously day 1, followed by 30 mg three times per week. Injection-site-reactions were recorded every 6-24 h until resolved using National Cancer Institute criteria and a new skin toxicity subscale. The first doses of 30 mg and 3 mg produced injection-site-reactions (all but one were grade 1/2) in 13/20 and 9/20 patients, respectively. The second dose (on day 3) resulted in skin-reactions in 10/20 patients and the third, fourth, fifth and sixth injections produced reactions in 6/20, 1/20, 2/20 and 0/20 patients, respectively. Mild "flu-like" symptoms occurred during week 1 in 10/20 patients. All side-effects had subsided by the sixth dose. 15/20 patients (75%) responded (12 partial responses, three complete responses) with a median time-to-treatment-failure of 20 months. Symptomatic cytomegalovirus-reactivation occurred in 6/20 patients. Two deaths occurred: one bacterial pneumonia and one adenovirus-infection. The present study showed how to assess cutaneous-toxicity in detail and that 30 mg alemtuzumab SC administered upfront was well tolerated. Optimized alemtuzumab therapy in properly selected patients may result in high efficacy even in advanced CLL. Our results need to be confirmed in extended studies.

Production of recombinant antigens, polyclonal antibodies for development of an ELISA assay to detect Salmonella spp. in food

Salmonella is one of the most important food-borne pathogens. Early detection of Salmonella is necessary for food safety and hygiene. We have developed an ELISA assay, using polyclonal antibodies which can detect most Salmonella serotypes. To produce the antibodies, the recombinant flagellar antigens or H antigens of S. Enteritidis (H:g,m) and S. Typhimurium (H:i), were utilized to inject into rabbit, due to the fact that they are immunodominant antigenic surface structures of Salmonella and represent for two groups of the most common H antigen types, α cluster and G complex (McQuiston, J. R. et al., 2004). The genes coding for antigens (fliCgm and fliCi) were cloned into vector pGEX-5X-1 and expressed in the strain E. coli BL21(DE3). The recombinant E. coli was cultured in 200ml LB and the expression of fliCgm and fliCi was induced with 0.4mM IPTG. 2.5mg of each GST-fused protein (GST-H:g,m and GST-H:i) was harvested after purifying by GSTrap FF column. The protein was confirmed by SDS-PAGE analysis and Western Blot with the specific anti-H antigen antibody. 100μg of protein in complete Freund adjuvant was injected to rabbit. Blood collection and booster injection were done every 3 weeks. Terminal blood collection was at the fourth booster injection. 100ml of antiserum of each H antigen was obtained and was run through protein A column to purify polyclonal antibody. Polyclonal antibody was successfully conjugated to HRP. The sandwich ELISA procedure utilized the mixture of antibodies. After optimization, the concentration of the mixture of antibodies in the sandwich ELISA method was 250ng of capture antibodies and 100ng of antibody-HRP conjugate. The optimized sandwich ELISA protocol was applied to test 60 Salmonella strains isolated from food samples and check the cross-reactivity with some other bacteria in food. The result showed that 99% of samples was positive of Salmonella presence (OD492>0.3), and only 1% was negative (OD492<0.3). Sandwich ELISA of some other bacteria in food, including E. coli, Shigella spp., Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, resulted in very low values of OD492 (<0.2). In conclusion, the ELISA assay was confirmed the specificity to Salmonella sp. and a very low cross-reactivity to other bacteria presenting in food.

A Case of Polymicrobial Keratitis Violating an Intact Lens Capsule

To report a case of polymicrobial keratitis resistant to topical and intraocular antibiotics with recurrence after penetrating keratoplasty. Case report. We report a case of polymicrobial keratitis caused by Phialophora verrucosa, Candida tropicalis, and Propionibacterium acnes. Initial treatment included topical vancomycin, tobramycin, amphotericin B, voriconazole, and oral fluconazole, as well as subconjunctival amphotericin B. Penetrating keratoplasty was performed, and the infection seemed to have resolved until 4 weeks after keratoplasty. A second penetrating keratoplasty was performed, followed by 4 weekly intracameral injections of voriconazole. Two weeks after the fourth intracameral injection, the infection manifested as an active anterior-chamber reaction. The patient's eye was subsequently enucleated. Histopathologic evaluation showed penetration of the crystalline lens by fungus at the site of synechiae between the intact lens capsule and iris. To our knowledge, this is the first documented case of a polymicrobial keratitis caused by a bacterium, yeast, and a fungus. It is the first histopathologic demonstration of fungal penetration of intact lens capsule from infected iris.

Human alpha 1-antitrypsin therapy induces fatal anaphylaxis in non-obese diabetic mice

Previous studies have shown that human alpha-1 antitrypsin (hAAT) gene delivery prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Furthermore, hAAT protein administration prolongs acceptance of islet allografts. Therefore, we evaluated the use of purified hAAT protein therapy to prevent T1D in NOD mice. Female NOD, non-obese resistant (NOR), Balb/c and C57BL/6 mice were injected intraperitoneally with vehicle alone or vehicle containing hAAT, human albumin or mouse albumin (or mg/injection/mouse; 2x/week). Preparations of clinical-grade hAAT included API(R), Aralast, Prolastin and Zemaira. Surprisingly, hAAT administration was associated with a high rate of fatal anaphylaxis. In studies seeking T1D prevention at 4 weeks of age, 100% mice died after six injections of hAAT. When administrated at 8-10 weeks of age, most (80-100%) NOD mice died following the fourth injection of hAAT, while 0% of Balb/c and C57BL/6 mice and 10% of NOR mice died. Interestingly, repeated injections of human albumin, but not mouse albumin, also induced sudden death in NOD mice. Antibodies to hAAT were induced 2-3 weeks after hAAT administration and death was prevented by treatment with anti-platelet-activating factor along with anti-histamine. In studies of disease reversal in NOD mice, using the four pharmaceutical grade formulations of hAAT, anaphylactic deaths were observed with all hAAT preparations. The propensity for fatal anaphylaxis following antigenic administration appears to be NOD- but not hAAT-specific. The susceptibility of NOD mice to hypersensitivity provides a significant limitation for testing of hAAT. Development of strategies to avoid this unwanted response is required to use this promising therapeutic agent for T1D.

Neonatal PCP Is More Potent than Ketamine at Modifying Preweaning Behaviors of Sprague-Dawley Rats

Treatment with N-methyl-D-aspartate (NMDA) receptor antagonists, such as ketamine (KET) or phencyclidine (PCP), can trigger apoptotic neurodegeneration in neonatal rodents; however, little is known about the behavioral alterations resulting from such treatment. Here, rats were sc treated with saline; 10 mg/kg PCP on postnatal days (PNDs) 7, 9, and 11; 20 mg/kg KET (six injections every 2 h on PND 7); or a regimen of ketamine and 250 mg/kg L-carnitine (KLC) both administered on PND 7 with additional 250 mg/kg doses of L-carnitine given on PNDs 8-11. Postinjection, the home cage behavior of each pup was categorized on PNDs 7-11. Slant board and forelimb hang behaviors were examined on PNDs 8-11 and 12-16, respectively. The initial KET or KLC injections on PND 7 elevated abnormal home cage activity (i.e., paresis and paddling); however, KLC pup behavior returned to normal by the fourth injection, indicating the protective effects of L-carnitine against NMDA antagonist toxicity. PCP treatment caused substantial abnormal home cage activity on each injection day (PNDs 7, 9, and 11). Latencies to turn on the slant board were significantly longer on PND 8 for KET- and PCP-treated pups and PND 10 for PCP-treated pups. On PND 12, the forelimb hang time of PCP-treated pups was significantly shorter. Body weight was decreased on PNDs 8-18 in PCP-treated pups and PNDs 8-10 in KET-treated pups. These data indicate that developmental NMDA antagonist treatment causes short-term behavioral alterations which appear related to motor coordination and may be cerebellar in nature. Furthermore, single PCP injections appear more potent at altering behavior than multiple injections of KET.

Role for des-acyl ghrelin in the responsiveness of plasma hormones and metabolites to ghrelin in Holstein steers

Gastric-derived peptide hormone ghrelin is known for its potent growth hormone (GH) stimulatory effects. The acyl-modification on N-terminal Ser 3 residue is reported to be important to stimulate the ghrelin receptor, GH secretagogue-receptor type1a (GHS-R1a). However, major portion of circulating ghrelin lacks in acylation, and some biological properties of des-acyl ghrelin have been reported in monogastric animals. In the present study, the responsiveness of plasma hormones and metabolites to ghrelin in steers was characterized, and role for des-acyl ghrelin in these changes was investigated. The repeated intravenous administrations of bovine ghrelin (1.0 μg/kg BW) every 2 h for 8 h to Holstein steers significantly increased the plasma acylated ghrelin, total ghrelin, GH, insulin and NEFA levels. The GH responses in peak values and area under the curves (AUCs) were attenuated by repeated injections of ghrelin, however, the responses of plasma total ghrelin were similar. Plasma insulin AUC decreased after fourth injection of ghrelin while plasma NEFA AUCs gradually increased by repeated injections of ghrelin. Pretreatment of des-acyl ghrelin (10.0 μg/kg BW) 5 min prior to the single injection of ghrelin (1.0 μg/kg BW) did not affect the ghrelin-induced hormonal changes. Moreover, the responses of plasma GH to bovine and porcine ghrelin, which differ in C-terminal amino acid residues, were similar in calves. These data show that (1) GH release was attenuated by repeated administration of ghrelin, (2) ghrelin regulates glucose and fatty acid metabolism probably via different pathway, and (3) des-acyl ghrelin is unlikely the antagonist for ghrelin to induce endocrine effects in Holstein steers.

Multiple intracerebral hemorrhages after cervical epidural injections

Epidural steroid injections are commonly used to treat chronic pain secondary to radiculopathy. Complications of epidural injections include unintentional dural puncture, vasovagal syncope, apnea, and hypotension. Cervical epidural injections are associated with higher rates of complications than lumbar or caudal epidural injections.1 Recent American Academy of Neurology guidelines do not recommend epidural injections for radicular pain.2 We report a rare complication of low CSF pressure headache and multiple simultaneous intraparenchymal hemorrhages, occurring 7 days after cervical epidural injection. ### Case report. A 36-year-old left-handed woman with a history of a motor vehicle accident 1 year prior to admission presented with left-sided weakness and paresthesias of her right hand. She had chronic cervical neck pain since her motor vehicle accident, and was treated with a series of four cervical epidural anesthetic injections. She had no complications following the first three injections. The patient then had a fourth cervical epidural injection with no indication of dural puncture. However, approximately 4 days after this epidural injection she developed a positional, holoacranial, throbbing headache. The headache became severe when she was in the upright or sitting position and dramatically decreased with …

Reactivation of hepatitis B in a patient with Crohn’s disease treated using infliximab

We encountered a case of reactivation of hepatitis B virus after administration of infliximab for Crohn's disease. The use of infliximab was considered because the patient displayed abdominal symptoms and perianal lesions. Transaminases were normal, and hepatitis B virus (HBV) DNA was undetectable before treatment, so no antiviral treatment was used, and infliximab and low-dose 6-mercaptopurine were administered. This treatment was effective, but liver dysfunction and reactivation of HBV were observed after the fourth injection of infliximab. This is the first report of Crohn's disease for which infliximab use was continued even after reactivation of HBV was observed. However, liver dysfunction was not improved by lamivudine. Antiviral treatment should be considered before administration of infliximab for patients with HBV.

Lack of Toll‐like receptor 4 decreases lipopolysaccharide‐induced bone resorption in C3H/HeJ mice in vivo

Few in vivo studies have demonstrated whether Toll-like receptor 4 (TLR4) is indispensable for lipopolysaccharide (LPS)-induced bone resorption and little is known about the receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) expression induced by LPS under conditions of lack of TLR4. We compared bone resorption histomorphometrically in C3H/HeN and C3H/HeJ mice that were repeatedly injected with Actinobacillus actionmycetemcomitans LPS into their gingiva every 48 h. RANKL-, interleukin-1beta- and OPG-positive cells in the connective tissue were also compared immunohistochemically. Bone resorption in C3H/HeJ mice in the fourth, seventh, and tenth injection groups was significantly less than that C3H/HeN mice (P < 0.05). The number of RANKL-positive cells in C3H/HeJ mice in the 10th injection group was significantly smaller than that in C3H/HeN mice (P < 0.05). The numbers of interleukin-1beta-positive cells in C3H/HeJ mice in the seventh and tenth injection groups were significantly decreased compared with those in C3H/HeN mice (P < 0.05). The numbers of OPG-positive cells in C3H/HeN and C3H/HeJ mice gradually increased, but there was no significant difference between the two strains of mice. TLR4 is indispensable for LPS-induced bone resorption in vivo.

Targeting Lewis Y (Ley) in Small Cell Lung Cancer with a Humanized Monoclonal Antibody, hu3S193: A Pilot Trial Testing Two Dose Levels

Lewis Y (Le(y)) is a blood group antigen with robust expression on the surface of epithelial tumors, including small cell lung cancer (SCLC), making it a potential target for antibody-based immunotherapy. 3S193, an immunoglobulin G3 monoclonal antibody, has demonstrated superior specificity, affinity, and cytotoxicity over other anti-Le(y) antibodies. A phase I trial of humanized 3S193 (hu3S193) with dosing up to 40 mg/m2 demonstrated tumor targeting without serious toxicities or the development of human anti-human antibodies. We tested the targeting and pharmacokinetics of hu3S193 in patients with SCLC. Eligibility required progressive SCLC treated with up to three previous chemotherapy regimens, measurable disease not previously irradiated, and tumor samples positive for 3S193 by immunohistochemistry. Patients received four weekly injections of hu3S193, five patients at 10 mg/m2 and five patients at 20 mg/m2. The first and fourth injections were radiolabeled with indium-111 for gamma camera imaging. Of 40 patients screened, 25 of 34 (74%) assessable SCLC tumor samples were 3S193 positive by immunohistochemistry. Ten patients were treated with hu3S193; nine completed all four injections. All fluorodeoxyglucose (FDG)-avid lesions >2 cm were visualized on antibody single-photon emission computed tomography. Some lesions overlying vascular structures could not be visualized. No difference was noted in imaging or pharmacokinetics between the first and fourth injections. Toxicities included grade 2 urticaria (n = 1), grade 1 vomiting (n = 2), and grade 2 hypertension (n = 1) transiently after infusion at the higher dose. Given the strong tumor targeting, particularly at the higher dose, the favorable toxicity profile, and the potential for immunomodulatory effects, hu3S193 warrants further investigation in SCLC.

Accumulation of Neurotoxic Thioether Metabolites of 3,4-(±)-Methylenedioxymethamphetamine in Rat Brain

The serotonergic neurotoxicity of 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) appears dependent upon systemic metabolism because direct injection of MDMA into the brain fails to reproduce the neurotoxicity. MDMA is demethylenated to the catechol metabolite N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA). Thioether (glutathione and N-acetylcysteine) metabolites of N-Me-alpha-MeDA are neurotoxic and are present in rat brain following s.c. injection of MDMA. Because multidose administration of MDMA is typical of drug intake during rave parties, the present study was designed to determine the effects of multiple doses of MDMA on the concentration of neurotoxic thioether metabolites in rat brain. Administration of MDMA (20 mg/kg s.c.) at 12-h intervals for a total of four injections led to a significant accumulation of the N-Me-alpha-MeDA thioether metabolites in striatal dialysate. The area under the curve (AUC)(0-300 min) for 5-(glutathion-S-yl)-N-Me-alpha-MeDA increased 33% between the first and fourth injections and essentially doubled for 2,5-bis-(glutathion-S-yl)-N-Me-alpha-MeDA. Likewise, accumulation of the mercapturic acid metabolites was reflected by increases in the AUC(0-300 min) for both 5-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA (35%) and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA (85%), probably because processes for their elimination become saturated. Indeed, the elimination half-life of 5-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA increased by 53 and 28%, respectively, between the first and third doses. Finally, although the C(max) values for the monothioether conjugates were essentially unchanged after each injection, the values increased by 38 and approximately 50% for 2,5-bis-(glutathion-S-yl)-N-Me-alpha-MeDA and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA, respectively, between the first and fourth injections. The data indicate that neurotoxic metabolites of MDMA may accumulate in brain after multiple dosing.

Translation Elongation Factor eEF1A2 Is Essential for Post-weaning Survival in Mice

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.

Acute retinal pigment epithelial tear in the untreated fellow eye following repeated bevacizumab (Avastin™) injections

Acute retinal pigment epithelial tear in the untreated fellow eye following repeated bevacizumab (Avastin™) injections

Food intake suppression induced by fourth ventricular CCK and MTII but not oxytocin and bombesin is partially mediated by activation of the ERK intracellular signaling cascade.

Our previous work suggests that the caudal brainstem mechanisms of food satiation may involve signaling through the mitogen-activated protein kinase ERK1/2 cascade in a population of nucleus of the solitary tract (NTS) neurons. Peripheral administration of CCK and fourth ventricular injection of the stable MC4-receptor agonist MTII led to rapid phosphorylation of ERK1/2 in specific neurons, and CCK as well as MTII-induced suppression of food intake in fasted rats was significantly attenuated by fourth ventricular administration of U0126, a blocker of ERK-phosphorylation. Here, we tested the ability of U0126 to attenuate the satiating effects of oxytocin and bombesin/GRP two other natural ligands that may suppress food intake in the dorsal vagal complex through their hypothalamus-NTS projections. Both, oxytocin (2ig in 4 V) and bombesin (25 ng in 4 V) suppressed food intake similarly in vehicle and U0126-pretreated rats (30 min intake oxytocin: −2.1 and −2.9 g; bombesin: −2.05 and −2.9 g, respectively; all decreases significant, p

Pegaptanib 1-Year Systemic Safety Results from a Safety–Pharmacokinetic Trial in Patients with Neovascular Age-Related Macular Degeneration

To characterize the safety, tolerability, and pharmacokinetics of the pegylated anti-vascular endothelial growth factor (VEGF) aptamer pegaptanib sodium in subfoveal choroidal neovascularization secondary to age-related macular degeneration (AMD). Prospective 2-cohort study: (1) open-label cohort and (2) randomized, double-masked, uncontrolled multicenter trial. In the combined cohorts, 147 subjects with any angiographic subtype of subfoveal choroidal neovascularization secondary to AMD and best-corrected visual acuities (VAs) in the study eye of 20/40 to 20/320 and in the fellow eye of 20/800 or better received pegaptanib sodium. Subjects were randomized to receive intravitreous pegaptanib sodium (1 mg or 3 mg [3- and 10-fold higher than the 0.3-mg approved dose]) every 6 weeks for 54 weeks. Safety assessments included blood chemistries, urinalyses, vital signs, electrocardiograms, serum antipegaptanib antibody assays, adverse events, VAs, and intraocular pressures. After the first, fourth, and eighth injections, serial blood samples were obtained for quantification of pegaptanib plasma concentrations. No antipegaptanib immunoglobulin G (IgG) or IgM antibodies were detected. Few systemic adverse events were noted. Mild or moderate ocular adverse events related to the injection procedure were reported in most patients. Pegaptanib did not accumulate in plasma after multiple doses; systemic exposures were similar after the first, fourth, and eighth doses. The mean apparent terminal half-life was 10 days. Evaluation of blood pressure (BP) and urine protein, both of which are known to be affected by systemic VEGF inhibition, indicated no evidence of a pegaptanib treatment effect on these parameters. Mean BP at the end of year 1 remained below 140 mmHg (systolic) and 90 mmHg (diastolic), levels considered hypertension by the American College of Cardiology. At doses up to 10-fold higher than the 0.3-mg dose approved for the treatment of AMD, pegaptanib sodium was well tolerated, with no detectable clinical evidence of systemic VEGF inhibition (i.e., no clinically meaningful changes in proteinuria or mean BP) and no clinically relevant ocular inflammation. Most ocular adverse events were related to the injection procedure itself and were mild or moderate in severity.

Botulinum Toxin A Injection in ISDN Ointment-Resistant Chronic Anal Fissures

Background: In the treatment of chronic anal fissures (CAFs), surgical sphincterotomy is more commonly being replaced by chemical sphincterotomy. After the good results of our pilot study including 32 patients, we now describe the effect of botulinum toxin A (BT-A) in a consecutive series of 100 patients with isosorbide dinitrate (ISDN) ointment-resistant CAFs. Methods: From October 2002 until August 2005, 100 patients (52 males, 48 females) with a median age of 45 (20–79) years were treated with an injection of 40–100 IU BT-A (Dysport®, Ipsen, The Netherlands) in the internal anal sphincter. Results: After a median follow-up of 10 (4–38) months, 77 of the 100 CAFs (77%) were cured. 20 patients were given a second injection, 1 a third and 1 a fourth injection. In 11 patients a fissure recurred (14%). In 1 patient (1%) there was temporary incontinence due to flatus. Conclusion: With an early response rate of 77% and an overall success rate of 66%, BT-A injections appear to be effective in patients with ISDN ointment-resistant CAFs if initial non-responders are retreated. These results are in concordance with the results of our pilot study. It is a simple technique with little or no side effects, that does not compromise future treatments. Its place in the first-line treatment of CAFs should be investigated further.

Thermogenesis activated by central melanocortin signaling is dependent on neurons in the rostral raphe pallidus (rRPa) area

The central melanocortin system plays a critical role in regulation of energy balance, including thermogenesis in brown adipose tissue (BAT). Activation of the hypothalamic melanocortin signaling stimulates sympathetically-mediated interscapular BAT (IBAT) thermogenesis. The rostral raphe pallidus (rRPa) and adjacent area have been proposed as the location of sympathetic premotor neurons for the central nervous system (CNS) control of IBAT thermogenesis. To determine if neuronal activity in rRPa area is required for the central melanocortin-induced thermogenesis, we studied the effects of inhibition of the activity of neurons in the rRPa area on the sympathetic nerve activity (SNA) to IBAT evoked by lateral ventricular injection of the melanocortin 3/4 receptor (MC3/4R) agonist, MTII, in urethane-chloralose-anesthetized rats and the effects on O 2 consumption induced by third or fourth ventricular injection of MTII in conscious freely moving mice. Icv injection of MTII (1 nmol) significantly increased rat IBAT SNA (+ 741% of control). Both third and fourth ventricular injections of MTII (1 nmol) significantly increased O 2 consumption in conscious C57BL/6J mice (45% higher than that of saline control for third ventricular injection and 44% higher than that of saline control for fourth ventricular injection). The increases in IBAT SNA and in O 2 consumption were reversed by inhibition of neurons in the rRPa and adjacent area with microinjections of glycine or muscimol into rRPa. These results suggest that the neurons in the RPa and its immediate vicinity play an essential role in mediating the increase in IBAT thermogenesis induced by activation of central melanocortin signaling.

Safety and immunogenicity of subcutaneous or intramuscular administration of a monovalent inactivated vaccine against Leptospira interrogans serogroup Icterohaemorrhagiae in healthy volunteers

The safety and immunogenicity of a monovalent inactivated vaccine against Leptospira interrogans serogroup Icterohaemorrhagiae was evaluated in 84 volunteers according to the route of administration, i.e., subcutaneous (SC) or intramuscular (IM), in a double-blind randomised trial. The volunteers were randomised into four groups: SC vaccine; IM vaccine; SC placebo; and IM placebo. Primary vaccination comprised two injections on day 0 and day 14, with a booster after 6 months. A second booster was given 30 months after primary vaccination. Local reactions within 1 h of injections were rare, with no difference between vaccine groups. Local reactions within 3 h were more frequent after the second, third and fourth SC injections than after IM injections. Systemic reactions never occurred within 1 h of vaccination and were rare within 3 days; the rates were comparable for the different vaccine groups. Evolution of the antibody responses, as assessed by microscopic agglutination tests and specific IgG and IgM ELISAs, were similar for both injection routes. IgG seroconversion rates after the first booster were 97% (95% CI 80-100%) for the SC vaccine group, and 96% (95% CI 80-100%) for the IM vaccine group, and both reached 100% for IgG after the second booster. The safety and immunogenicity of the anti-leptospiral vaccine were both good. Monitoring of antibody levels established that a booster dose triggered a strong antibody response in fully vaccinated subjects at 30 months after primary vaccination.

A Novel, Non-immunogenic Fyn SH3-derived Binding Protein with Tumor Vascular Targeting Properties

The generation of novel binding molecules based on protein frameworks ("scaffolds") represents an emerging field in protein engineering, with the potential to replace antibodies for many research and clinical applications. Here, we describe the design, construction, characterization, and use of a novel human Fyn SH3 phage library, containing 1.2 x 10(9) individual clone members. We also present the isolation and in vitro characterization of Fyn SH3-derived proteins binding to the extra-domain B of fibronectin, a marker of angiogenesis. One specific binding clone, named D3, was further evaluated and showed a remarkable ability to stain vascular structures in tumor sections. Furthermore, quantitative biodistribution studies in tumor-bearing mice revealed the ability of D3 to selectively accumulate in the tumor. In contrast to human scFv antibody fragments administered to mice, neither Fyn SH3 WT nor the D3 mutant was immunogenic in mice after four intravenous injections. The extra-domain B binding D3 protein opens new biomedical opportunities for the in vivo imaging of solid tumors and for the delivery of toxic agents to the tumoral vasculature.

Hypothalamic and hindbrain NPY, AGRP and NE increase consummatory feeding responses

Feeding behavior is comprised of both appetitive and consummatory responses to food. Appetitive responses include the motivated acquisition of food. Consummatory responses, including swallowing, are those that move the food from the mouth to the stomach. Intraoral delivery of liquid food bypasses the requirement for appetitive responses and has been used to examine consummatory responses directly in intact rats. In the present study, we administered neuropeptide Y (NPY), agouti-related protein (AGRP) and norepinephrine (NE), into the paraventricular nucleus of the hypothalamus (PVN) or into the fourth cerebral ventricle to examine their effects on the consummatory component of feeding behavior in the rat. To measure consummatory responses, milk (40% lactose free cow's milk diluted with water) was infused intraorally through a chronic cheek fistula (1 ml/min), using an alternating 5 min on − 1 min off schedule, until rejection occurred. We found that both hypothalamic and fourth ventricle injections of NPY, AGRP and NE significantly increased consumption of the intraorally-delivered milk. Our results indicate that the circuitry for modulation of consummatory ingestive responses includes NE, NPY and AGRP receptors operating in both hypothalamic and hindbrain sites.