L-Allylglycine dissociates the neural substrates of fear in the periaqueductal gray of rats

The dorsal (dPAG) and ventral (vPAG) regions of the periaqueductal gray are well known to contain the neural substrates of fear and anxiety. Chemical or electrical stimulation of the dPAG induces freezing, followed by a robust behavioral reaction that has been considered an animal model of panic attack. In contrast, the vPAG is part of a neural system, in which immobility is the usual response to its stimulation. The defense reaction induced by the stimulation of either region is accompanied by anti nociception. Although GABAergic mechanisms are known to exert tonic inhibitory control on the neural substrates of fear in the dPAG, the role of these mechanisms in the vPAG is still unclear. The present study examined defensive behaviors and antinociception induced by microinjections of an inhibitor of gamma-aminobutyric acid synthesis, L-allylglycine (L-AG; 1, 3, and 5 mu g/0.2 mu l), into either the dPAG or vPAG of rats subjected to the open field and tail-flick tests. Passive or tense immobility was the predominant behavior after L-AG (1 or 3 mu g) microinjection into the vPAG and dPAG, respectively, which was replaced with intense hyperactivity, including jumps or rearings, after injections of a higher dose (5 mu g/0.2 mu l) into the dPAG or vPAG. Moreover, whereas intra-dPAG injection of 3 mu g L-AG produced intense antinociception, only weak antinociception was induced by intra-vPAG injections of 5 mu g L-AG. These findings suggest that GABA mechanisms are involved in the mediation of antinociception and behavioral inhibition to aversive stimulation of the vPAG and exert powerful control over the neural substrates of fear in the dPAG to prevent a full-blown defense reaction possibly associated with panic disorder. (C) 2009 Elsevier Inc. All rights reserved.

Effect of estradiol benzoate microinjection into the median raphe nucleus on contextual conditioning

Estrogen deficiency has been associated with stress, anxiety and depression. Estrogen receptors have been identified in the median raphe nucleus (MRN). This structure is the main source of serotonergic projections to the hippocampus, a forebrain area implicated in the regulation of defensive responses and in the resistance to chronic stress. There is reported evidence indicating that estrogen modulates 5-HT(1A) receptor function. In the MRN, somatodendritic 5-HT(1A) receptors control the activity of serotonergic neurones by negative feedback. The present study has evaluated the effect of intra-MRN injection of estradiol benzoate (EB, 600 or 1200 ng/0.2 mu l) on the performance of ovariectormized rats submitted to contextual conditioning. Additionally, the same treatment was given after intra-MRN injection of Way 100635 (100 ng/0.2 mu l). a 5-HT(1A) receptor antagonist. Both doses of EB decreased freezing and increased rearing, indicating an anxiolytic effect. Pretreatment with Way 100635 antagonized the anxiolytic effect of estradiol. On the basis of these results, it may be suggested that estrogens modulate anxiety by acting on 5-HT(1A) receptors localized in the MRN. (C) 2009 Elsevier B.V. All rights reserved.

Anxiety-like symptoms induced by morphine withdrawal may be due to the sensitization of the dorsal periaqueductal grey

Withdrawal from morphine leads to the appearance of extreme anxiety accompanied of several physical disturbances, most of them linked to the activation of brainstem regions such as the locus coeruleus, ventral tegmental area, hypothalamic nuclei and periaqueductal grey (PAG). As anxiety remains one of the main components of morphine withdrawal the present study aimed to evaluating the influence of the dorsal aspects of the PAG on the production of this state, since this structure is well-known to be involved in defensive behaviour elicited by anxiety-evoking stimuli. Different groups of animals were submitted to 10 days of i.p. morphine injections, challenged 2 h after with an i.p. injection of naloxone (0.1 mg/kg), and submitted to the plus-maze, open-field and light-dark transition tests. The effects of morphine withdrawal on anxiety-induced Fos immunolabelling were evaluated in four animals that passed by the light-dark transition test randomly chosen for Fos-protein analysis. Besides the PAG, Fos neural expression was conducted in other brain regions involved in the expression of anxiety-related behaviours. Our results showed that morphine withdrawn rats presented enhanced anxiety accompanied of few somatic symptoms. Increased Fos immunolabelling was noted in brain regions well-known to modulate these states as the prelimbic cortex, nucleus accumbens, amygdala and paraventricular hypothalamus. Increased Fos labelling was also observed in the ventral and dorsal aspects of the PAG, a region involved in anxiety-related processes suggesting that this region could be a common neural substrate enlisted during anxiety evoked by dangerous stimuli as well as those elicited by opiate withdrawal. (c) 2008 Elsevier Inc. All rights reserved,

Serotonin-1A receptors in the dorsal periaqueductal gray matter mediate the panicolytic-like effect of pindolol and paroxetine combination in the elevated T-maze

The beta-adrenergic blocker and 5-HT(1A) receptor antagonist pindolol has been combined with selective serotonin reuptake inhibitors (SSRIs) in patients with depressive and anxiety disorders to shorten the onset of the clinical action and/or increase the proportion of responders. The results of a previous study have shown that pindolol potentiates the panicolytic effect of paroxetine in rats submitted to the elevated T-maze (ETM). Since reported evidence has implicated the 5-HT(1A) receptors of the dorsal periaqueductal gray matter (DPAG) in the panicolytic effect of antidepressants, rats treated with pindolol (5.0 mg/kg, i.p.) and paroxetine (1.5 mg/kg, i.p.) received a previous intra-DPAG injection of the selective 5-HT(1A) antagonist, WAY-100635 (0.4 mu g) and were submitted to the ETM. Pretreatment with WAY-100635 reversed the increase in escape latency, a panicolytic effect, determined by the pindolol-paroxetine combination. These results implicate the 5-HT(1A) receptors of the DPAG in the panicolytic effect of the pindolol-paroxetine combination administered systemically. They also give further preclinical support for the use of this drug combination in the treatment of panic disorder. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

5-HT1A receptors of the lateral septum regulate inhibitory avoidance but not escape behavior in rats

Serotonin in the lateral septum (LS) has been implicated in the modulation of defensive behaviors and in anxiety. However, it is currently unknown whether changes in 5-HT mechanisms in this brain area may selectively affect defensive responses associated with specific subtypes of anxiety disorders recognized in clinical settings. To address this question, we evaluated the effect of the intra-LS injection of the 5-HT1A/7 receptor agonist 8-CH-DPAT (0.6, 3.0, 15.0 nmol) in male Wistar rats exposed to the elevated T-maze animal model of anxiety. This test allows the measurement of two behavioral defensive responses in the same rat: inhibitory avoidance and escape behavior. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. The effects of 8-OH-DPAT were compared to those caused by a standard anxiolytic compound, the benzodiazepine receptor agonist midazolam (MDZ, 20 nmol). We also investigated whether the intra-LS injection of the 5-HT1A receptor antagonist WAY-100635 (0.37 nmol) was able to block the effects of 8-OH-DPAT. All animals were also tested in an open field for locomotor activity assessments. Results showed that whereas intra-LS administration of MDZ decreased avoidance latencies, suggesting an anxiolytic action, 8-OH-DPAT caused the opposite effect. Neither drug affected the escape performance. Intra-LS administration of WAY-100635 blocked the anxiogenic effect caused by 8-OH-DPAT. No changes to locomotion were detected in the open field. The data suggests that LS 5-HT1A receptors are involved in the control of inhibitory avoidance behavior and that a failure in this regulatory mechanism may be of importance to the physiopathology of generalized anxiety disorder. (c) 2008 Elsevier Inc. All rights reserved.

Central substance P NK(1) receptors are involved in fever induced by LPS but not by IL-1 beta and CCL3/MIP-1 alpha in rats

Substance P (SP) is a neuropeptide that can modulate inflammatory mediator release through activation of NK(1) receptors (NK(1)R). Some studies have also suggested the involvement of SP in lipopolysaccharide (LPS)-induced fever. However, the precise contribution of this neuropeptide to the pathways activated during fever is unknown. In this study we investigated the effect of a selective NK(1)R antagonist, SR140333B, on the febrile response induced by LPS and cytokines. Our results show that the systemic injection of SR140333B did not modify the fever induced by LPS at a dose that is able to reduce protein extravasation induced by SP in the skin. On the other hand, intracerebroventricular administration of 5R140333B significantly reduced the fever induced by peripheral injection of LPS. These data emphasize an important role for SP in the central nervous system during the febrile response to LPS, and are reinforced by the fact that intracerebroventricular injection of SP also induced fever in a dose-dependent manner in captopril-treated rats. Considering that the febrile response can result from the generation of several endogenous pyrogens, among them interleukin (IL)-1 beta and macrophage inflammatory protein-1 alpha (CCL3/MIP-1 alpha), we also examined the effect of SR140333B on the fever induced by these cytokines which act through prostaglandin-dependent and independent mechanisms, respectively. Surprisingly, SR140333B did not modify the febrile response to IL-1 beta or CCL3/MIP-1 alpha. Altogether these data suggest that the central action of SP is essential for LPS-, but not for IL-1 beta- or CCL3/MIP-1 alpha-induced fever. (C) 2011 Elsevier B.V. All rights reserved.

Amphetamine modulates cellular recruitment and airway reactivity in a rat model of allergic lung inflammation

Asthma is characterized by pulmonary cellular infiltration, vascular exudation and airway hyperresponsiveness. Several drugs that modify central nervous system (CNS) activity can modulate the course of asthma. Amphetamine (AMPH) is a highly abused drug that presents potent stimulating effects on the CNS and has been shown to induce behavioral, biochemical and immunological effects. The purpose of this study was to investigate the effects of AMPH on pulmonary cellular influx, vascular permeability and airway reactivity. AMPH effects on adhesion molecule expression, IL-10 and IL-4 release and mast cell degranulation were also studied. Male Wistar rats were sensitized with ovalbumin (OVA) plus alum via subcutaneous injection. One week later, the rats received another injection of OVA-alum (booster). Two weeks after this booster, the rats were subjected to AMPH treatment 12 h prior to the OVA airway challenge. In rats treated with AMPH, the OVA challenge reduced cell recruitment into the lung, the vascular permeability and the cellular expression of ICAM-1 and Mac-1. Additionally, elevated levels of IL-10 and IL-4 were found in samples of lung explants from allergic rats. AMPH treatment, in comparison, increased IL-10 levels but reduced those of IL-4 in the lung explants. Moreover, the tracheal responsiveness to methacholine (MCh), as well as to an in vitro OVA challenge, was reduced by AMPH treatment, and levels of PCA titers were not modified by the drug. Our findings suggest that single AMPH treatment down-regulates several parameters of lung inflammation, such as cellular migration, vascular permeability and tracheal responsiveness. These results also indicate that AMPH actions on allergic lung inflammation include endothelium-leukocyte interaction mechanisms, cytokine release and mast cell degranulation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection

Because CD4(+) T cells play a key role in aiding cellular immune responses, we wanted to assess whether increasing numbers of gene-engineered antigen-restricted CD4(+) T cells could enhance an antitumor response mediated by similarly gene-engineered CD8(+) T cells. In this study, we have used retroviral transduction to generate erbB2-reactive mouse T-cell populations composed of various proportions of CD4(+) and CD8(+) cells and then determined the antitumor reactivity of these mixtures. Gene-modified CD4(+) and CD8(+) T cells were shown to specifically secrete Tc1 (T cytotoxic-1) or Tc2 cytokines, proliferate, and lyse erbB2(+) tumor targets following antigen ligation in vitro. In adoptive transfer experiments using severe combined immunodeficient (scid) mice, we demonstrated that injection of equivalent numbers of antigen-specific engineered CD8(+) and CD4(+) T cells led to significant improvement in survival of mice bearing established lung metastases compared with transfer of unfractionated (largely CD8(+)) engineered T cells. Transferred CD4(+) T cells had to be antigen-specific (not just activated) and secrete interferon gamma (IFN-gamma) to potentiate the antitumor effect. Importantly, antitumor responses in these mice correlated with localization and persistence of gene-engineered T cells at the tumor site. Strikingly, mice that survived primary tumor challenge could reject a subsequent re-challenge. Overall, this study has highlighted the therapeutic potential of using combined transfer of antigen-specific gene-modified CD8(+) and CD4(+) T cells to significantly enhance T-cell adoptive transfer strategies for cancer therapy.

Three-monthly intravitreal bevacizumab injections for neovascular age-related macular degeneration: short-term visual acuity results

PURPOSE. To evaluate the change in vision after 3 monthly consecutive intravitreal injections of 1.25 mg of bevacizumab for neovascular age-related macular degeneration (AMD). METHODS. A retrospective analysis of 35 eyes was performed. Visual acuity (VA) at initial visit and at each follow-up visit was compared. The injection of bevacizumab was performed at 30-day intervals and patients were observed for 5 months after the last injection. RESULTS. Of the 35 eyes, 9 had received previous treatment with photodynamic therapy with or without 4 mg of intravitreal triamcinolone. VA was measured in Snellen table and transformed into logMAR for statistical purposes. Mean age was 76.66 years (range, 49-90 years). There were 24(69%) women and 11(31%) men. Mean VA at the initial visit was 0.92 +/- 0.50. At month 1, mean VA was 0.84 +/- 0.51 and at month 2 was 0.74 +/- 0.51. At month 3, mean VA remained 0.74 +/- 0.49. Six and 8 months after the initial visit, VA was 0.79 +/- 0.49 and 0.77 +/- 0.50, respectively. The improvement in VA was statistically significant at month 2 and at the end of the follow-up (8 months) compared with the baseline VA. CONCLUSIONS. Three consecutive monthly injections of intravitreal bevacizumab to treat neovascular AMD is effective in improving VA in the short term. Longer prospective studies should be performed to confirm VA stability after the third injection. (Eur J Ophthalmol 2010; 20: 740-4)

Targeting Antigen-loaded Liposomes to Myeloid Antigen Presenting Cells

Objective: To target antigen-loaded liposomes to myeloid APC in vivo for immunotherapy and to manipulate immune function through liposome composition. Method: Liposomes were loaded with ovalbumin, the lipophilic red fluorescent marker, DiI, with or without QuilA adjuvant then injected either i.v. or s.c. to naı¨ ve C57Bl/6 mice. Spleen, liver and draining LN were stained with MHC class II and various myeloid markers to determine the uptake of liposomes. Frozen sections of spleen and draining LN were stained with FITC-labeled mAb to determine which cells take up the liposomes. To determine the effect on OVA-specific T cell responses, liposomes were administered to Balb/c mice which received DO11.10 OVAspecific TCR transgenic T cells labelled with CFSE. Results: The DiI fluorescence was visualized in MHC class II+ macrophages and DC in draining lymph nodes after s.c. injection and in spleen and liver after i.v injection. Immunofluorescence microscopy shows liposome uptake in marginal zone macrophages and some DC in the T cell areas of the spleen after i.v. injection. Administration of ova-liposomes with or without QuilA stimulated a specific T cell response as measured by CFSE dilution. Conclusion: APC of liver, spleen and LN, and subsequent antigen presentation to T cells can be targeted for immunotherapy by the administration of liposomes encapsulating antigen and adjuvant. Varying the composition and routes of liposome administration is expected to alter the function of the targeted APC and the T cell response.

Charcot`s arthropathy secondary to herpetic encephalitis sequelae: an unusual presentation

Neuropathic arthropathy (Charcot`s arthropathy) is a progressive articular disease associated with a reduced sensorial and protector proprioceptive reflex. Its etiology includes many different conditions such as syringomyelia, traumatic lesion causing medullary deformity, spina bifida, diabetic neuropathy, leprosy neuropathy, neurofibromatosis, amyloid neuropathy, alcohol, and repetitive injection of hydrocortisone into joints, among others. However, the relationship between Charcot`s arthropathy and herpetic encephalitis has not yet been described. Herpes encephalitis causes acute and chronic diseases of the peripheral or central nervous system. It can manifest as subacute encephalitis, recurrent meningitis, or myelitis. It can also resemble psychiatric syndromes, diplopia, sensory changes in the face and limbs, personality changes, frontal dysexecutive syndrome, stiff neck, subclinical alterations of the vestibular function, intracranial hypertension, convulsion, hemiparesis, and generally includes motor components, among others. On the other hand, pure peripheral sensory disturbance has not been described. In this article, we report the clinical case of a patient with Charcot`s arthropathy secondary to pure peripheral sensory polyneuropathy as a consequence of progressive herpetic encephalitis sequelae. In this article, the authors report the first case of Charcot`s arthropathy secondary to herpetic encephalitis.

Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial

Background Homozygous familial hypercholesterolaemia is a rare genetic disorder in which both LDL-receptor alleles are defective, resulting in very high concentrations of LDL cholesterol in plasma and premature coronary artery disease. This study investigated whether an antisense inhibitor of apolipoprotein B synthesis, mipomersen, is effective and safe as an adjunctive agent to lower LDL cholesterol concentrations in patients with this disease. Methods This randomised, double-blind, placebo-controlled, phase 3 study was undertaken in nine lipid clinics in seven countries. Patients aged 12 years and older with clinical diagnosis or genetic confirmation of homozygous familial hypercholesterolaemia, who were already receiving the maximum tolerated dose of a lipid-lowering drug, were randomly assigned to mipomersen 200 mg subcutaneously every week or placebo for 26 weeks. Randomisation was computer generated and stratified by weight (<50 kg vs >= 50 kg) in a centralised blocked randomisation, implemented with a computerised interactive voice response system. All clinical, medical, and pharmacy personnel, and patients were masked to treatment allocation. The primary endpoint was percentage change in LDL cholesterol concentration from baseline. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00607373. Findings 34 patients were assigned to mipomersen and 17 to placebo; data for all patients were analysed. 45 patients completed the 26-week treatment period (28 mipomersen, 17 placebo). Mean concentrations of LDL cholesterol at baseline were 11.4 mmol/L (SD 3.6) in the mipomersen group and 10.4 mmol/L (3.7) in the placebo group. The mean percentage change in LDL cholesterol concentration was significantly greater with mipomersen (-24.7%, 95% CI 31.6 to 17.7) than with placebo (-3.3%, 12.1 to 5.5; p=0.0003). The most common adverse events were injection-site reactions (26 [76%] patients in mipomersen group vs four [24%] in placebo group). Four (12%) patients in the mipomersen group but none in the placebo group had increases in concentrations of alanine aminotransferase of three times or more the upper limit of normal. Interpretation Inhibition of apolipoprotein B synthesis by mipomersen represents a novel, effective therapy to reduce LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia who are already receiving lipid-lowering drugs, including high-dose statins.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Combined Laser and Intravitreal Triamcinolone for Proliferative Diabetic Retinopathy and Macular Edema: One-year Results of a Randomized Clinical Trial

PURPOSE: To evaluate laser combined with intravitreal triamcinolone acetonide (IVTA) for the management of patients with proliferative diabetic retinopathy (PDR) and clinically significant macular edema (CSME). DESIGN: Randomized clinical trial. METHODS: SETTINGS: Single center. STUDY POPULATION: Twenty-two patients with bilateral treatment,naive moderate PDR and CSME. INTERVENTION: Laser (panretinal and macular) photocoagulation was performed in each eye, followed by IVTA in one randomly assigned eye. Best,corrected visual acuity (BCVA), fundus photography, and optical coherence tomography were performed at baseline and at months 1, 3, 6, 9, and 12. MAIN OUTCOME MEASURES:. Changes in BCVA, central macular thickness (CMT), and total macular volume (TMV). RESULTS: The mean logarithm of the minimal angle of resolution (logMAR) BCVA improved significantly, and mean CMT and TMV were significantly reduced in the IVTA group compared with the laser,only group (controls) at all study follow-up visits (P < .001). The mean logMAR BCVA (Snellen equivalent) was 0.44 (20/50(-2)) for the IVTA group and 0.38 (20/50(+1)) for the controls at baseline, and 0.12 (20/25(-1)) for the IVTA group and 0.32 (20/40(-1)) for the controls at 12 months (P < .001.). The mean CMT and TMV were, respectively, 360 mu m and 8.59 mm(3) for the IVTA group and 331 mu m and 8.44 mm(3) for the controls at baseline, and 236 mu m and 7.32 mm(3) for the IVTA group and 266 mu m and 7.78 mm(3) for the controls at 12 months (P < .001). CONCLUSIONS: The combination of laser photocoagulation with IVTA was associated with improved BCVA and decreased CMT and TMV when compared with laser photocoagulation alone for the treatment of moderate PDR with CSME. (Am J Ophthalmol 2009;147:291-297. (C) 2009 by Elsevier Inc. All rights reserved.)

Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

Considering that mycobacterial heat-shock protein 65 (hsp65) gene transfer can elicit a profound antitumoral effect, this study aimed to establish the safety, maximum-tolerated dose (MTD) and preliminary efficacy of DNA-hsp65 immunotherapy in patients with advanced head and neck squamous cell carcinoma (HNSCC). For this purpose, 21 patients with unresectable and recurrent HNSCC were studied. Each patient received three ultrasound-guided injections at 21-day intervals of: 150, 600 or 400 mu g of DNA-hsp65. Toxicity was graded according to CTCAE directions. Tumor volume was measured before and after treatment using computed tomography scan. The evaluation included tumor mass variation, delayed-type hypersensitivity response and spontaneous peripheral blood mononuclear cell proliferation before and after treatment. The MTD was 400 mg per dose. DNA-hsp65 immunotherapy was well tolerated with moderate pain, edema and infections as the most frequent adverse effects. None of the patients showed clinical or laboratory alterations compatible with autoimmune reactions. Partial response was observed in 4 out of 14 patients who completed treatment, 2 of which are still alive more than 3 years after the completion of the trial. Therefore, DNA-hsp65 immunotherapy is a feasible and safe approach at the dose of 400 mg per injection in patients with HNSCC refractory to standard treatment. Further studies in a larger number of patients are needed to confirm the efficacy of this novel strategy.