Laboratory of Caribbean brain research organization in the decade of the brain midpoint

CARIBRO was founded in response to the United Nations declaration that the 1990s be designated the Decade of the Brain. The Program of Action is:

1. Annual meetings;
2. Training courses of the Caribbean School of Neurosciences;
3. Network scientific programs;
4. Fellowship programs; and
5. Dissemination of information on neuroscience.

In the same program, a CARIBRO Laboratory was created in one of the Medical Faculties of Havana with the aim to teach students from the Caribbean in neuroscience research. As part of this program, we have been working in lateralized motor functions. Preliminary results in rats show that reaching acquisition allows classification of the animals as right-handed (40%), left-handed (40%), and ambidextrous (20%). Electrolytic lesion of caudate nucleus or amygdala impairs lateralized response. Contralateral lesions increase reaching attempts. Ipsilateral lesions to the preferred forepaw do not affect the reaction. The results remain the same 10, 20, and 90 d after the interference. Pharmacological experiments showed that trihexiphenidil (0.1 mg/kg ip) induced handedness reversion in 50% of the animals, whereas haloperidol (1 mg/kg ip) produced immobility, tremor, and autonomic symptoms. This effect remained the same in young as well as in old animals. We are also working on mathematical modelation. In this sense, preliminary reports about a model for synaptic modification in the framework of the Fukushima hypothesis is discussed.     

Primary cranial vault lymphoma mimicking meningioma

Primary lymphomas of the cranial vault are rare; only six patients have been described in the literature. We report a 75-year-old woman who was admitted to our hospital after a focal seizure. CT showed a homogeneous mass which, on contrast enhancement, was similar to a meningioma. The tumour was excised and found to be a centroblastic, centrocytic non-Hodgkin's lymphoma. Treatment was completed with radiotherapy and chemotherapy.     

A new C6-C3 compound from Cassia grandis

A new compound trans-3-methoxy-4,5-methylene-dioxycinnamaldehyde (1) has been isolated from the aerial parts of Cassia grandis (Caesalpiniaceae) along with the known compounds aloe emodin, centaureidin, catechin, myristicin, 2,4-dihydroxybenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 2,4,6-trimethoxybenzaldehyde, and beta-sitosterol. The structure of 1 was determined by spectral methods.     

Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs. This work was supported by Hungarian National Research Foundation (OTKA I/352 and OTKA II/2622).     

The effect of long term and high dose interferon treatment in Chronic hepatitis C

The results of 43 interferon treatments of 35 patients (23 male, 12 female) are reported. The duration of the treatment was 6–18 months, the dose of interferon was 3x3-5 MU weekly. Complete response (HCV RNA became negative) was found in 11, relapse was observed in 3 patients. Partial response (transaminase levels became normal, or less than twice normal value, but patients remained HCV RNA positive) occurred in 23 cases, relapse was obeserved in 16. The therapy had no effect in 9 cases. The higher dose and longer term interferon therapy resulted in a higher rate of response to the treatment and a reduction in the number of relapses. This work was supported by the Hungarian Ministry of Welfare (No. T-10 064/93).     

Significance of wet autoclave pretreatment in immunohistochemistry

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen-and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regionsassociated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.     

Proliferative (mib1, mdm2) versus anti-proliferative (p53) markers in head and neck cancer. An Immunohistochemical study

Formalin fixed and paraffin embedded samples from 36 squamous cell carcinomas of the larynx and the oral cavity (pT₂N₀M₀, R₀) surrounded by non-tumorous mucosa were studied immunohistochemically using a panel of four different anti-p53 antibodies (CM1, PAbl801, D07, PAb240), a monoclonal anti-mdm2 antibody and MIB1, following wet autoclave antigen retrieval. P53 immunoreactivity was detected in 11/14 laryngeal and in 9/22 oral carcinomas. All p53 positive oral, and all but one laryngeal tumors revealed mdm2 positivity as well, whereas in p53 negative tumors 4/12 and 1/3 mdm2 immunopositive cases were demonstrated, respectively. MIB1 labeling indices of the tumors ranged between 18% – 64% in p53 positive cases, and 10% – 53% in p53 negative ones. The difference was not statistically significant. Close spatial coexpression of p53, mdm2 and MIB1 immunoreactivity was observed at the invasive front of the carcinomas and in the basal and suprabasal layers of the non-tumorous epithelium in all p53 positive cases. However, the MIB1 expression was similarly increased at the invasive margins in carcinomas lacking immunohistochemically detectable p53 alterations. Our results strongly suggest that p53 overexpression does not necessarily correspond to increased rate of proliferation, but rather to mdm2 overexpression and is largely dependent on the anatomical site in case of small and localized squamous cell carcinomas of the head and neck region.     

Polymorphism analysis of the VNTR locus D17S5 in Central Spain

The fragment length polymorphism YNZ22 (D17S5) was analysed for a sample of 207 unrelated individuals living in Madrid (Spanish Caucasians) using PCR-methodology and high resolution separation. Hardy-Weinberg expectations (HWE) were calculated after pooling alleles into four groups. No deviations from HWE were detectable using the conventional ²-test. The power of discrimination was estimated as 0.96 and the mean paternity exclusion chance as 0.7587. A comparison of the allele frequency distribution with those of other Caucasian groups revealed no major differences.     

Involvement of GABA(B) receptors in the motor inhibition produced by agonists of brain cannabinoid receptors

The present study was designed to investigate the possibility of activation of GABA(B) receptors during the motor inhibition caused by cannabimimetics. Adult male rats were injected with an acute dose of arachidonylethanolamide (AEA), Delta(9)- tetrahydrocannabinol (THC), beclofen or vehicle, after pretreatment with CGP 35348, a specific antagonist for GABA(B) receptors, or vehicle, and the behavioral response produced by these compounds tested in an open field. As expected, the administration of either AEA or THC produced a very pronounced motor inhibition, reflected by decreased ambulation and increased time spent in inactivity. The administration of baclofen also produced a marked motor deficit, with similar changes to those observed with both cannabimimetics. Pretreatment with the GABA(B) antagonist, CGP 35348, prevented the motor inhibition induced by baclofen and also attenuated the motor deficit caused by both cannabimimetics, suggesting a role for this receptor. In summary, a GABAergic influence, acting through GABA(B) receptors, seems to be involved in mediating motor effects of cannabimimetics, since the blockade of these receptors attenuates cannabimimetic-induced signs of motor inhibition.