Variable effects of cyclophosphamide in rodent models of experimental allergic encephalomyelitis.

In this study, we have evaluated the effects of cyclophosphamide on the development of experimental allergic encephalomyelitis (EAE) in four EAE rodent models: monophasic EAE in Lewis rats, protracted relapsing (PR)-EAE in DA rats, myelin oligodendrocyte protein (MOG)-induced EAE in C57Bl/6 mice and proteolipid protein (PLP)-induced EAE in Swiss/Jackson Laboratory (SJL) mice. Cyclophosphamide, administered either prophylactically or therapeutically, suppressed most strongly the clinical symptoms of PR-EAE in DA rats. Treated rats in this group also exhibited the lowest degree of inflammatory infiltration of the spinal cord, as well as the lowest levels of nuclear factor kappa B, interleukin-12 and interferon-gamma. Cyclophosphamide prophylactically, but not therapeutically, also delayed significantly the onset of EAE in Lewis rats. In contrast, regardless of the treatment regimen used, was unable to influence the clinical course of EAE in either MOG-induced EAE in C57Bl/6 mice or PLP-induced EAE in SJL mice. This heterogeneous pharmacological response to cyclophosphamide suggests that significant immunopathogenic differences exist among these EAE rodent models that must be considered when designing preclinical studies. In addition, the effectiveness of cyclophosphamide in dark Agouti (DA) rats with PR-EAE suggests that this may be a particularly useful model for studying novel therapeutic approaches for refractory and rapidly worsening multiple sclerosis in human patients.

Comparative study of the immunogenicity and immunoenhancing effects of two hepatitis B core antigen variants in mice by nasal administration.

The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens; however, the mucosal immunogenicity of HBcAg has been poorly studied. In this study, we explored the immunogenicity and the immunoenhancing effect elicited by two different variants of the recombinant complete nucleocapside of HBV in mice by intranasal route. For this purpose, we used as co-administered antigen, the HBV surface protein (HBsAg) and the antibody response in sera was evaluated after each dose. To analyze the specificity of the generated antibody response, the recognition of lineal epitopes was evaluated on a cellulose membrane bearing 12 mer peptides covering the HBcAg sequence. The obtained results evidenced that the intranasal immunogenicity of both variants of HBcAg was similar and high, developing early responses of IgG. The immunoenhancing effect on the HBsAg-specific antibody response was also similar for both variants. The results of the recognition of lineal epitopes study evidenced a similar recognition pattern to all sera and vaginal lavages samples generated by the immunization of mice with the two variants of HBcAg, and also similar to a pool of human anti-HBcAg positive sera samples.

Immunological characterization of two hepatitis B core antigen variants and their immunoenhancing effect on co-delivered hepatitis B surface antigen.

The hepatitis B virus (HBV) core and surface antigens are potent immunogens in animal models and humans. They have been used in vaccine studies for prevention or therapy of HBV diseases and also as carrier molecules in new developments. In this study we explored the nasal immunogenicity of two different variants of the recombinant complete nucleocapsid (HBcAg) as well as their adjuvant effect on hepatitis B surface antigen (HBsAg). To characterize the immune response, the serum IgG antibody response was tested during one year against both antigens, and the serum and vaginal secretions were tested for recognition of linear epitopes of HBcAg for both HBcAg variants. The results obtained evidenced that the intranasal immunogenicity of both HBcAg variants was similar and high, developing early and long lasting IgG responses. A similar recognition pattern to all sera and vaginal washes samples was generated by the two variants of HBcAg, also similar to a pool of human anti-HBcAg positive sera. A synergistic effect in the enhancement of the immunogenicity for both antigens was evidenced in the combined formulation after nasal administration. Taken together, these results would be of interest in the design of more potent therapeutic and preventive vaccines complementing systemic and mucosal responses.

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.

Mucosal immunogenicity of the hepatitis B core antigen.

The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens, however, the mucosal immunogenicity of HBcAg or chimeric HBcAg proteins has been poorly studied and only using the truncated variant of the HBcAg. In this study we explored the mucosal immunogenicity in mice of the recombinant complete nucleocapside of HBcAg. The antigen was administered by different mucosal and parenteral routes. The antibody response in sera was evaluated after each immunization and mucosal lavages were tested with the final extraction. To characterize the immune response, the serum IgG antibody response was tested during six months and also the ratio IgG2a to IgG1 was determined. The results obtained evidenced that the mucosal immunogenicity of HBcAg depended on the administration route, being the intranasal (i.n.) route the one that generated the higher IgG responses in sera, similar in intensity and duration to parenteral administrations. The IgA response in mucosal washes was superior for nasally immunized mice compared to the rest of mucosal and parenteral groups. The nasal route also induced the higher IgG2a to IgG1 ratio, evidencing a Th1-like Ab subclass pattern. In addition to the high Ab responses, preliminary results of the cellular response induced by nasal administration evidenced the induction of strong lymphoproliferative responses in spleen cells.

Acetyl-L-carnitine shows neuroprotective and neurotrophic activity in primary culture of rat embryo motoneurons.

We evaluated the role of acetyl-L-carnitine (ALCAR) in protecting primary motoneuron cultures exposed to excitotoxic agents or serum-brain derived neurotrophic factor (BDNF) deprived. To exclude that ALCAR works as a metabolic source, we compared its effects with those of L-carnitine (L-CAR), that seems to have no neurotrophic effect. A concentration of 10 mM ALCAR, but not L-CAR, significantly reduced the toxic effect of 50 microM N-methyl-D-aspartate (NMDA, % viability: NMDA 45.4+/-2.80, NMDA+ALCAR 90.8+/-11.8; P<0.01) and of 5 microM kainate in cultured motoneurons (% viability: kainate 40.66+/-10.73; kainate+ALCAR 63.80+/-13.88; P<0.05). The effect was due to a shift to the right of the dose-response curve for kainate (EC50 for kainate 5.99+/-1.012 microM; kainate+ALCAR 8.62+/-1.13 microM; P<0.05). ALCAR, but not L-CAR, significantly protected against BDNF and serum-deprivation reducing the apoptotic cell death (% viability respect to control: without BDNF/serum 61.8+/-13.3: without BDNF/serum+ALCAR 111.8+/-13.9; P<0.01). Immunocytochemistry showed an increase in choline acethyltransferase and tyrosine kinaseB receptors in motoneurons treated with ALCAR but not with L-CAR. These results suggest that ALCAR treatment improves the motoneurons activity, acting as a neurotrophic factor.

Nitric oxide produced by non-motoneuron cells enhances rat embryonic motoneuron sensitivity to excitotoxins: comparison in mixed neuron/glia or purified cultures.

The present study compares the sensitivity to chronic exposure to glutamate agonists of SMI-32-positive rat-derived embryonic motoneurons under both mixed neuron/glia and purified cultures. We found that in spite of a trophic role of glia on cultured motoneurons, SMI-32-positive cells are more sensitive to excitotoxicity in the presence of glia than in purified culture, very likely through nitric oxide released by non-neuronal cells. The rank order of potency for inducing toxicity after 48 h incubation was AMPA>kainate>NMDA, with EC(50): 0.43, 4.9 and 49 microM, respectively, in mixed neuron/glia culture and 14, 32 and 135 microM in purified cultures. The effect of NMDA was dose-dependently potentiated by glycine, with similar potency in the two culture conditions. The effect of agonists was completely antagonized by the specific antagonists CNQX, BNQX and MK801 in both culture conditions. Motoneurons were similarly immunoreactive to NR1 and GluR2 antibodies under both mixed neuron/glia and purified cultures, thus confirming the presence of the calcium-impermeant AMPA receptor subtypes and of the obligatory subunit for NMDA receptors. The effect of kainate in mixed neuron/glia culture was reduced by the addition of 40 microM N-nitro-L-arginine or L-NAME, which shifted the EC(50) to 9 microM. By contrast, L-NAME did not modify the effect of kainic acid in purified cultures. These results suggest that the release of nitric oxide by non-neuronal cells in culture enhances glutamate excitotoxicity in SMI-32-positive cells, and that direct activation of ionotropic glutamate receptors is not enough to explain the mechanism of chronic motoneuron degeneration occurring in vivo in amyotrophic lateral sclerosis (ALS).

Multivalent DNA-based immunization against hepatitis B virus with plasmids encoding surface and core antigens.

The immune response against hepatitis B surface and core antigens was evaluated by either coinoculation or independent intramuscular administration of pAEC compact DNA immunization vectors carrying their genes. The pAEC vectors bear just the essential elements for mammalian expression and bacterial amplification. Balb/c mice were immunized with 100 microg of each construct, either alone or in combination. In spite of lacking known immunostimulatory sequences (e.g., AACGTT), significant cellular (proliferative) and humoral immune responses were raised against both antigens. Coadministration of both plasmids maintained the immune response against the two antigens, without interference between them. Modulation of the antigen expression and further immune response, by using the Kozak's translation initiation sequence, was also analyzed. No differences due to its presence or absence were observed.

Increased peripheral benzodiazepine binding sites and pentraxin 3 expression in the spinal cord during EAE: relation to inflammatory cytokines and modulation by dexamethasone and rolipram.

We have studied the mRNA expression of pentraxin 3 (PTX3) and the binding of the peripheral-type benzodiazepine receptor (PBR) ligand, [3H]-PK11195, in the spinal cord of Lewis rats where EAE was actively induced. PTX3 was induced during the active phase of EAE (day 10-14), it remained high up to 30 days and disappeared only 60 days later. Similarly, PK11195 binding peaked at day 14-17 during the recovery and it disappeared by day 60. On the other hand, the levels of TNF and IL-6 in the spinal cord were elevated at the peak and at the onset of clinical signs and returned to non-detectable by day 14-17. Dexamethasone abolished all these changes, while treatment with rolipram, delayed the appearance of the disease and then decreased its severity. However the peaks of TNF, IL-6, PBR and PTX3 levels in spinal cord were only delayed, but not reduced, by rolipram treatment. In conclusion, we show two types of inflammatory changes in EAE: acute, short term changes (TNF and IL-6), that correlate with the disease; and effects such as PTX3 expression and PK11195 binding that last longer after recovery from the disease.

Analysis of enterovirus sequences recovered from the cerebrospinal fluid of patients with epidemic neuropathy.

An outbreak of a previously unknown disease, termed epidemic neuropathy (EN), occurred in Cuba between 1991 and 1993. Although nutritional and oxidative stress in the population were rapidly associated with the disease, several findings were not compatible with such stress being the only cause. In the search for biological factors, samples of the cerebrospinal fluid (CSF) from patients with EN were studied and found to have a slowly progressing cytopathic effect (CPE) on VERO cells. Although the results of several studies indicate the presence of enteroviruses, the CPE and other physico-chemical characteristics are not typical of these viruses. Viral sequences have now been amplified from patients' CSF, using oligonucleotide primers homologous to the enterovirus 5' non-coding region. The sequences of the amplified region showed a high degree of variability (7%-69%) when compared with the coxsackievirus (Cox)A9 Griggs used as the reference strain. Furthermore, sequences differing by > 55% (58%-70%) were isolated from a single individual. These results indicate the generation in stressed individuals of enterovirus quasispecies with altered biological properties, and these could have played a major role in the neurological injury of EN.

Biochemical and pharmacological evidence of a functional role of AMPA receptors in motor neuron dysfunction in mnd mice.

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6-nitro-7-sulphamoyl-benzo(F)quinoxaline-2,3-dione], a selective (rs)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with 'normal' gait (from 50% to 90%, P < 0.05). Receptor binding autoradiography of the competitive N-methyl-D-aspartate (NMDA) antagonist, [3H]CGP 39653, of [3H]AMPA and [3H]kainic acid in spinal cord sections, measured after 1 week of drug washout, were not significantly different in control and mnd mice, and were not modified by NBQX. GluR2/3 immunoreactivity, assessed using Western blotting, was significantly enhanced (by 59%, P < 0.01) in the spinal cord but not in the brain of 28-week-old mnd mice compared to age-matched control mice. NBQX treatment increased GluR2/3 immunoreactivity in the spinal cord of control mice and mnd mice by 327 +/- 74% (P < 0.01) and 212 +/- 52% (P < 0.01), respectively. The changes in GluR2/3 subunits may involve adaptive mechanisms of the receptor and play some role in the protective effect of NBQX. These findings suggest that selective antagonism of ionotropic non-NMDA receptors may be of value in the treatment of motor neuron disease.

Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris.

A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.

Expresión del gen de la quimosina en E. coli / Expression of the chymosin gene in E. coli

En este trabajo se describe la clonación y la expresión en E. coli del gen de la quimosina, enzima de importancia industrial para la producción de quesos. Los clones se identificaron analizando una genoteca de ADN complementario al ARN poli (A) proveniente del estómago de ternero, utilizando como sonda dos oligonucléotidos sintéticos. La región del gen, codificante a la proquimosina, fue expresada bajo el control del promotor triptófano. Se alcanzó una expresión equivalente al 10

de la proteína total. La proteína fue detectada insoluble y formando cuerpos de inclusión. La enzima producida demostró tener propiedades semejantes a la natural.

Expresión del gen de la quimosina en E. coli / Expression of the chymosin gene in E. coli

En este trabajo se describe la clonación y la expresión en E. coli del gen de la quimosina, enzima de importancia industrial para la producción de quesos. Los clones se identificaron analizando una genoteca de ADN complementario al ARN poli (A) proveniente del estómago de ternero, utilizando como sonda dos oligonucléotidos sintéticos. La región del gen, codificante a la proquimosina, fue expresada bajo el control del promotor triptófano. Se alcanzó una expresión equivalente al 10 % de la proteína total. La proteína fue detectada insoluble y formando cuerpos de inclusión. La enzima producida demostró tener propiedades semejantes a la natural.