Electrolytic lesion of the nucleus incertus retards extinction of auditory conditioned fear.

Fear memory circuits in the brain function to allow animals and humans to recognize putative sources of danger and adopt an appropriate behavioral response; and research on animal models of fear have helped reveal the anatomical and neurochemical nature of these circuits. The nucleus (n.) incertus in the dorsal pontine tegmentum provides a strong GABAergic projection to forebrain 'fear centers' and is strongly activated by neurogenic stressors. In this study in adult male rats, we examined the effect of electrolytic lesions of n. incertus on different stages of the fear conditioning-extinction process and correlated the outcomes with anatomical data on the distribution of n. incertus-derived nerve fibers in areas implicated in fear circuits. In a contextual auditory fear conditioning paradigm, we compared freezing behavior in control (naïve) rats (n=23) and rats with sham- or electrolytic lesions of n. incertus (n=13/group). The effectiveness and extent of the lesions was assessed post-mortem using immunohistochemical markers for n. incertus neurons-calretinin and relaxin-3. There were no differences between the three experimental groups in the habituation, acquisition, or context conditioning phases; but n. incertus lesioned rats displayed a markedly slower, 'delayed' extinction of conditioned freezing responses compared to sham-lesion and control rats, but no differences in retrieval of extinguished fear. These and earlier findings suggest that n. incertus-related circuits normally promote extinction through inhibitory projections to the amygdala, which is involved in acquisition of extinction memories.

Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis.

Leishmune, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p=0.0253), suggesting that the combination of additional chemotherapy with Leishmune-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p=0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune in the control and cure of CVL.

Immunogenicity assay of the Leishmune vaccine against canine visceral leishmaniasis in Brazil.

Leishmune is the industrialized version of the FML-saponin vaccine which has been shown to develop 92-95% protection in vaccinated dogs and 76-80% vaccine efficacy against field canine visceral leishmaniasis (CVL) in Brazil. Leishmune has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. In the present investigation, 550 healthy seronegative dogs of endemic and epidemic areas of Brazil were monitored for Leishmune-induced immunogenicity during a 2-year trial. Another group of 588 untreated exposed dogs was also studied in parallel. Both groups were seronegative on day 0. The strong immunogenicity induced by Leishmune vaccine was demonstrated by the 98% of FML-seroconversion, increase in absorbencies, the 82.7% DTH positive reactions and increase in skin test size diameters, the average increase in CD8+ total lymphocytes population in blood (27.1%), expected for QS21 saponin-containing vaccine, the sustained proportions of CD4+ T cells, and the average increased proportions of CD21+ B lymphocytes (42.3%). The Leishmune-induced protection against CVL is demonstrated by the results: 98.8% asymptomatic dogs (at the end of first year) and 99% healthy survivors (at the end of the second year) among vaccinated dogs, compared to the 79.4% asymptomatic and 61% survivor dogs (p<0.001) monitored in the untreated exposed cohort. In spite of the low vaccine coverage, it was possible to detect a 66.1% (p<0.005) reduction in Belo Horizonte and an 80.2% (p<0.005) reduction in Araçatuba of the incidence of CVL among vaccinated dogs, when compared to the global incidence of CVL of each town, respectively. Our preliminary results support the potential use of Leishmune to prevent CVL epidemics.

Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune vaccine.

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune vaccine, formulated with an increased adjuvant concentration (1mg of saponin rather than 0.5mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p<0.0001), a higher and stable IgG2 and a decreasing IgG1 response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune immunotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p<0.0001), the parasitological evidence (p=0.038) and a decrease in Leishmania-specific CD4+ lymphocyte proportions (p=0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune vaccine was subjected to a safety analysis and found to be well tolerated and safe.

Psychological aspects of presymptomatic diagnosis of spinocerebellar ataxia type 2 in Cuba.

OBJECTIVE: In this paper, we present a descriptive study of the first year of implementation of the predictive genetic testing program for spinocerebellar ataxia type 2 (SCA2) in Cuba, which has been structured along the international guidelines for Huntington disease. SUBJECTS AND METHODS: Medical records were retrospectively reviewed from 100 individuals who requested presymptomatic diagnosis. They had been seen between February 2001 and May 2002 at the Ataxia Clinic in the province of Holguín and had completed the protocol. The participants had psychological evaluations before testing and 1 week after being informed of the result. RESULTS: The average age of the participants in the presymptomatic testing for SCA2 was 39.3 years, 62% were female. The main reasons for taking the test were family planning and risk assessment in their children. Family participation in the process was high and there were several additional sources of social support. Before the test, the subjects did not present high levels of anxiety or depression. On informing the patients of the test results indicators for anxiety and depression significantly decreased, and there were no significant differences according to whether the test result had been positive or negative. Catastrophic events were rare during the first week after the test. CONCLUSIONS: The results indicate a strong interest in presymptomatic diagnosis among individuals at risk for SCA2 in Holguín and a lack of significant psychological drawbacks up to 1 week after information of results.

Psychological Aspects of Presymptomatic Diagnosis of Spinocerebellar Ataxia Type 2 in Cuba

In this paper, we present a descriptive study of significant psychological drawbacks up to 1 week after infor-the first year of implementation of the predictive genetic mation of results. Copyright © 2007 S. Karger AG, Baseltesting program for spinocerebellar ataxia type 2 (SCA2) inCuba, which has been structured along the internationalguidelines for Huntington disease. Subjects and Methods:Medical records were retrospectively reviewed from 100 in-dividuals who requested presymptomatic diagnosis. Theyhad been seen between February 2001 and May 2002 at theAtaxia Clinic in the province of Holguín and had completedthe protocol. The participants had psychological evaluationsbefore testing and 1 week after being informed of the result.Results: The average age of the participants in the presymp-tomatic testing for SCA2 was 39.3 years, 62% were female.The main reasons for taking the test were family planningand risk assessment in their children. Family participation inthe process was high and there were several additionalsources of social support. Before the test, the subjects didnot present high levels of anxiety or depression. On inform-ing the patients of the test results indicators for anxiety and...(AU)

Safety trial using the Leishmune vaccine against canine visceral leishmaniasis in Brazil.

A group of 600 healthy and asymptomatic dogs from Brazilian canine visceral leishmaniasis endemic areas was vaccinated with three sc doses of Leishmune which is the industrialized formulation of the FML-saponin, recently licensed for commercialization in Brazil, which previously showed 76-80% vaccine efficacy against canine visceral leishmaniasis. Safety evaluation was performed for 14 days after each vaccine injection and disclosed transient reactions of local pain (40.87%), anorexia (20.48%), apathy (24.17%), local swelling reactions (15.90%), vomit (2.4%) and diarrhoea (1.5%). All effects showed significantly correlating declines, from the first to the third dose (p<0.0001). Most of the noticed reactions of pain (73%), anorexia (79%) and local swelling (84.7%) were mild. No significant differences between puppies and adults dogs were found in the number of adverse reactions. Adult dogs developed however, 94.5% of the small swelling reactions (<3 cm), and indicating that they are more resistant to the inflammatory response promoted by the saponins. No dead by anaphylaxis occurred, and only two dogs (0.1%) showed allergic reactions (facial oedema and itching) after the third dose. Transient alopecia on injection site occurred in only five poodles (0.28%) with total recovery and no need of treatment. All the mild adverse events in response to Leishmune injection were transient and disappeared before the injection of the following vaccine dose, confirming the tolerability of the vaccine. The Leishmune preparation was less haemolytic (HD(50)=180 microg/ml) than expected for a QS21 saponin-containing vaccine, indicating that its formulation with the FML antigen diminished the potential in vitro toxicity.

Assessment of the monoterpene, glycidic and triterpene-moieties' contributions to the adjuvant function of the CP05 saponin of Calliandra pulcherrima Benth during vaccination against experimental visceral leishmaniasis.

The CP05 saponin from Calliandra pulcherrima Benth, shows remarkable similarities to the QS21 saponin of Quillaja saponaria Molina. Both shared a monoterpene hydrophobic moiety, a glycidic chain attached to the triterpene C28, and three sugars attached to C3. Different from QS21, the CP05 does not show the aldehyde group in triterpene C4 involved in TH1 response. Balb/c mice were immunized either intact saponin (CP05), the monoterpene-deprived (BS), the C28 carbohydrate-deprived (HS) or the sapogenin fraction, in formulation with the FML antigen of Leishmania donovani and challenged with 2 x 10(8) amastigotes of L. chagasi. While the CP05 induced 90% survival and 92.1% parasite reduction, a 100% survival and 94.1% protection were detected after the BS-vaccine treatment, indicating that the monoterpene acylated moiety, absent in the BS vaccine, is not necessary for the induction of a protective global TH1 response. Only the DTH response of BS vaccines was mildly lower than that of CP05 vaccinees. Maximal anti-FML antibody, CD4(+) and CD8(+) Leishmania specific lymphocytes, IFN-gamma splenocyte secretion, reduction in parasite load and survival was also detected for the BS vaccine. The HSFML vaccine showed diminished responses in all tested variables, except for IFN-gamma secretion, indicating that the integrity of the carbohydrate moiety attached to C28 is mandatory for the these functions. No protection was induced by the sapogenin-FML indicating that the CP05 triterpene which lacks the C4 aldehyde group, is not a immunostimulating compound. No contribution to protection was detected in the CP05 saponin treated control group supporting the specificity of the FML antigenic preparation.

Immunotherapy against visceral leishmaniasis with the nucleoside hydrolase-DNA vaccine of Leishmania donovani.

The nucleoside hydrolase (NH36) of Leishmania (L.) donovani is a vital enzyme which releases purines or pyrimidines of foreign DNA to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine, the VR1012-NH36 was immunoprotective against visceral and cutaneous murine leishmaniasis. In this work we tested the immunotherapy against Leishmania (L.) chagasi infection, using two doses of 100 or 20 microg VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous garlic extract (8 mg/kg/day by the i.p. route), which was effective in immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was significantly reduced following treatment with 100 microg (91%) and 20 microg (77%) of the DNA vaccine, and by 20 microg DNA vaccine and garlic extract (76%) (p=0.023). Survival was 33% for saline controls, 100% for the 100 microg vaccine, and 83 and 67% for the 20 microg vaccine with and without garlic extract addition, respectively. Garlic treatment alone did not reduce parasite load (p>0.05), but increased survival (100%). The NH36-DNA vaccine was highly effective as a new tool for the therapy and control of visceral leishmaniasis, while the mild protective effect of garlic might be related to an unspecific enhancement of IFN-gamma secretion.

Acylated and deacylated saponins of Quillaja saponaria mixture as adjuvants for the FML-vaccine against visceral leishmaniasis.

The adjuvant of the FML-vaccine against murine and canine visceral leishmaniasis, the Riedel de Haen saponin mixture, was fractionated by ion exchange chromatography on DEAE-cellulose to afford one TLC homogeneous Quillaja saponaria Molina QS21 saponin fraction (18.0%), a mixture of two deacylsaponins (19.4%), sucrose (39.9%), sucrose and glucose (19.7%), rutin (0.8%) and quercetin (2.2%), that were identified by comparison of 1H and 13C NMR spectroscopy. The QS21 shows the typical aldehyde group in C-23 (65% equatorial) and a normonoterpene moiety acylated in C-28. The deacylsaponins show the aldehyde group but do not have the normonoterpene moiety. Balb/c mice were vaccinated with 150 microg of FML antigen of Leishmania donovani and 100 microg of each obtained fraction and further challenged by infection with 10(8) amastigotes of Leishmania chagasi. The safety analysis and the effect on humoral and cellular immune responses and in clinical signs showed that the QS21 saponin and the deacylsaponins are the most active adjuvant compounds of the Riedel the Haen saponin mixture. Both induced the highest and non-significantly different increases in DTH, CD4+ T lymphocytes in spleen, IFN-gamma in vitro, body weight gain and the most pronounced reduction of parasite burden in liver (95% for QS21 and 86% for deacylsaponins; p>0.05). While the QS21 showed mild toxicity, significant adjuvant effect on the anti-FML humoral response before and after infection, and decrease in liver relative weight, the deacylsaponins showed no toxicity, less haemolysis and antibody and DTH responses increased mainly after infection, still inducing a stronger Leishmania-specific in vitro splenocyte proliferation. Our results confirm in the Riedel de Haen saponin extract the presence of deacylsaponins normonoterpene-deprivated which are non-toxic and capable of inducing a specific and strong immunoprotective response in vaccination against murine visceral leishmaniasis.

Leishmune vaccine blocks the transmission of canine visceral leishmaniasis: absence of Leishmania parasites in blood, skin and lymph nodes of vaccinated exposed dogs.

Leishmune vaccine is the first licensed vaccine against canine visceral leishmaniasis. It contains the Fucose-Mannose-ligand (FML) antigen of Leishmania donovani. The potential Leishmune vaccine effect on the interruption of the transmission of the disease, was assayed by monitoring, in untreated (n=40) and vaccinated dogs (n=32) of a Brazilian epidemic area: the kala-azar clinical signs, the FML-seropositivity and the Leishmania parasite evidence by immunohistochemistry of skin and PCR for Leishmanial DNA of lymph node and blood samples. On month 11 after vaccination, untreated controls showed: 25% of symptomatic cases, 50% of FML-seropositivity, 56.7% of lymph node PCR, 15.7% of blood PCR and 25% of immunohistochemical positive reactions. The Leishmune-vaccinated dogs showed 100% of seropositivity to FML and a complete absence of clinical signs and of parasites (0%) in skin, lymph node and blood PCR samples (p<0.01). The positivity in FML-ELISA in untreated dogs significantly correlates with the PCR in lymph node samples (p<0.001) and with the increase in number of symptoms (p=0.006) being strong markers of infectiousness. The absence of symptoms and of evidence of Leishmania DNA and parasites in Leishmune-vaccinated animals indicates the non-infectious condition of the Leishmune-vaccinated dogs.

Intestinal barrier function and secretion in methotrexate-induced rat intestinal mucositis.

Chemotherapy-induced mucositis is an important dose-limiting and costly side effect for which there is no definitive prophylaxis or treatment. This is due in part to the lack of understanding of its pathophysiology and impact on intestinal function. The objectives of this study were to investigate the small intestine barrier function and electrolyte and water transport in an experimental model of methotrexate-induced mucositis, and to correlate these alterations with histological damage. Wistar rats were treated with methotrexate (1.5-3.5 mg/kg) for 3 days to induce mucositis. Intestinal permeability was measured by the urinary excretion rate of lactulose and mannitol following administration by gavage. Intestinal perfusion was performed in vivo for evaluation of water and electrolyte transports. Methotrexate-treated rats lost a significant amount of weight and presented a marked reduction in food intake. Methotrexate induced significant and dose-dependent villous atrophy and elongation of crypts in duodenum, jejunum, and ileum. Methotrexate also induced an increase in sodium and potassium secretion and an important reduction of the mucosa absorptive surface area, shown by the decrease in the mannitol excretion ratio. In conclusion, methotrexate caused major changes in small bowel function by disrupting intestinal permeability and inducing electrolyte secretion in parallel with substantial histological damage.

[Epidemiology of Cuban hereditary ataxia]. / Epidemiología de la ataxia hereditaria cubana.

INTRODUCTION: The hereditary ataxias in Cuba make up the highest concentration of these patients in the world. The spinocerebellar ataxia type 2 (SCA2) molecular form is predominant. OBJECTIVES: To determine the prevalence and incidence of hereditary ataxias. PATIENTS AND METHODS: We made a descriptive study of 440 patients and 1,633 members of families at risk from this disorder in the province of Holguín. We calculated the prevalence rate and incidence. RESULTS: The rate of prevalence of patients in the province is 43 cases per 100,000 inhabitants; the highest rate was 503 cases per 100,000 inhabitants in part of the municipality of Baguanos. The age group that was most affected was that of 30-39 years, with a prevalence of 63.97 cases per 100,000 inhabitants. The rural population showed the highest incidence (62.04 cases per 100,000 inhabitants). The risk of members of affected families showing the disorder was 159.33 cases per 100,000 inhabitants in this province. The highest incidence was 18.08 cases per 100,000 inhabitants in Cacocum, where the incidence in the province was 4.39. CONCLUSIONS: On average the disorder passes from one state to the next every year, which suggests that the extent of the disorder worsens with time. The prevalence and incidence are the highest in the world. This together with the dominant pattern of inheritance, the effect of anticipation and inexorably progressive course of the disorder shows the serious health problem that affects the Eastern region of Cuba.

[Hereditary ataxias in Cuba. Historical, epidemiological, clinical, electrophysiological and quantitative neurological features]. / Las ataxias hereditarias en Cuba. Aspectos históricos, epidemiológicos, clínicos, electrofisiológicos y de neurología cuantitativa.

OBJECTIVE: This review has been designed to describe the main clinical, epidemiological, electrophysiological, molecular and quantitative neurological characteristics in SCA2. DEVELOPMENT: The prevalence rate of patients with ataxia in Holguin province is 43 per 100,000 inhabitants. The prevalence of family members at risk of having this disorder is 159.33 per 100 thousand in this province. The main neurophysiological abnormality observed was reduction in the amplitudes of sensory potentials. These alterations are the expression of a predominantly axonal peripheral lesion with signs of myelin damage. Techniques of quantitative neurology were developed for evaluation of the main disorders of coordination such as asymmetry and adiadochokinesis. In Cuba 125 families have hereditary ataxia, 772 patients and 8 to 10,000 family members are at risk of developing this condition. Seventy percent of the patients with ataxia are concentrated in Holguin province. The most severely affected towns are Báguanos (a rate of 129.20 per 100,000 inhabitants), Holguin (71.85 per 100,000) and Cacocúm (69.83 per 100,000). These are the highest rates in the world. CONCLUSIONS: The commonest molecular form in Cuba is the SCA2, observed in 120 families. Clinically it is characterized by a cerebellar syndrome associated with disorders of eye movements and osteotendinous reflexes.

[Type 2 spinocerebellar ataxia: acceptance of prenatal diagnosis in descendents at risk]. / Ataxia espinocerebelosa tipo 2: aceptación del diagnóstico prenatal en descendientes en riesgo.

INTRODUCTION: Type 2 spinocerebellar ataxia is a hereditary degenerative disorder of the nervous system. Advances in molecular genetics have made it possible to carry out presymptomatic and prenatal studies. A programme to define the strategies and principles for doing this has been devised. OBJECTIVE: To find the level of acceptance of prenatal diagnosis in couples at risk, and to determine the effect of different variables on this acceptance. PATIENTS AND METHODS: We made a descriptive type study of a series of cases. The study group included 226 persons. Forty of these were couples of descendents and the remainder were asymptomatic descendents. We applied a questionnaire, after prior information as to the aims of the study and obtaining the consent of the participants. RESULTS: Regarding acceptance of prenatal diagnosis by couples of descendents, we found that most (77.5%) accepted this and only 2.5% did not. Prenatal diagnosis was accepted by 67.74% of the descendents themselves. CONCLUSIONS: In general there was a high level of acceptance. 159 of the 226 questioned claimed that they would like to have more children and 98.7% of these accepted the test, whilst only 0.01% refused it. The main reasons given were the hope of having healthy children and that the disease would not occur in future generations.

[Type 2 spinocerebellar ataxia: an experience in psychological rehabilitation]. / Ataxia espinocerebelosa tipo 2: una experiencia en la rehabilitación psicológica.

INTRODUCTION: Psychological rehabilitation in patients with neurodegenerative disorders helps to improve their quality of life and provide the most suitable approach to their disease. There are no records of this type of treatment being used in type 11 spinocerebellar ataxia. OBJECTIVES: To evaluate the efficacy of group therapy within the framework of psychological rehabilitation and determine the most favored psychological function markers. PATIENTS AND METHODS: This study is a quasi experimental study of 24 patients with type 2 spinocerebellar ataxia rehabilitated in the CIRAH (Cuba). The procedure involved psychological assessment before and after the strategy for intervention, which consisted of 15 sessions of group therapy. RESULTS: The pathological levels of anxiety were reduced in 50.1% of the cases, and 31.4% of the patients with depression improved. The self assessment markers of a taking step forward were happiness and worry level. The disorder affected all aspects of the life of the patients studied, particularly their interests, family, self esteem and work. CONCLUSIONS: It is possible to improve the attitude of the patient to his disease and his psychological function by means of group therapy during the process of psychological rehabilitation of patients with type 2 spinocerebellar ataxia.