Diagnosis, gB genotype distribution and viral load of symptomatic congenitally infected CMV patients in Cuba.

OBJECTIVE: Cytomegalovirus (CMV) is the leading cause of viral congenital infection. Some viral factors have been proposed to be CMV pathogenicity markers. The objective of this study was to investigate the frequency of congenital CMV infection in symptomatic patients and the possible association with the CMV glycoprotein B (gB) genotype and viral load. STUDY DESIGN: A total of 361 newborns (NB) and 158 pregnant women (PW) with clinically suspected CMV infection were enrolled. Studied samples included urine, saliva, serum, vaginal swabs and amniotic fluid. CMV infection was diagnosed by multiplex nested PCR. CMV gB genotyping was performed on infected samples, followed by viral load determination. RESULTS: Overall, 18.7% of the tested patients were positive for CMV infection, 19.7% of NB were congenitally infected and 16.5% of PW showed active CMV infection. gB-2 was the most prevalent genotype detected (39/97 patients). gB CMV mixed infections were detected in 12 patients. gB-2 was associated with mono-infections (P<0.01). Mixed infections showed higher levels of viral load compared with gB mono-infection (P=0.03). Hepatomegaly, splenomegaly, jaundice, sepsis-like syndrome and malformations were the most prevalent clinical findings. gB-4 was more frequently associated with sepsis-like syndrome than other gB genotypes (P=0.04, odds ratio=4.3, confidence interval: 0.9 to 21.6). The difference in medians of CMV load was statistically significant among patients presenting different clinical signs (P=0.04). CONCLUSIONS: This study showed that CMV is a frequent cause of congenital infection in symptomatic Cuban patients. Despite gB2 being the most frequently detected, gB-4 was the only genotype associated with clinical features (sepsis-like syndrome in NB). No other associations among specific genotypes and clinical characteristics were found. Further studies are needed to clarify the role that viral load and genotype play in the outcome of congenital infection.

Regulation of immunity by Taeniids: lessons from animal models and in vitro studies.

Taeniidae is the largest family of the Cyclophyllidea order of parasites despite being composed of just two genera: Taenia spp and Echinococcus spp. These parasites are flatworms with a terrestrial life cycle, having an immature or larval stage called metacestode, which develops into the mature form within the intestine of the primary host after being consumed in raw or poorly cooked meat. Consumed eggs hatch into oncospheres, penetrate the intestinal walls and are transported via the bloodstream to later develop into metacestodes within the muscles and internal organs of secondary and sometimes primary hosts, thereby initiating the cycle again. Larval stages of both Taenia spp and Echinococcus spp are well known to produce tissue-dwelling, long-lasting infections; in this stage, these parasites can reach centimetres (macroparasites) and both genera may cause life-threatening diseases in humans. Establishing such long-term infections requires an exceptional ability to modulate host immunity for long periods of time. In this review, we analyse the immunoregulatory mechanisms induced by these tapeworms and their products, mainly discussing the importance of taeniid strategies to successfully colonize their hosts, such as antigen-presenting cell phenotype manipulation and the consequent induction of T-cell anergy, among others.

Antiaterogenicidad in vitro de extractos del alga marina Halimeda incrassata: actividad antioxidante y estudios de migración en células de la musculatura lisa / In vitro antiatherogenicity of extracts from Halimeda incrassata seaweed: antioxidant activity and smooth muscle cell migration studies

Objetivos: El objetivo de este trabajo fue evaluar el potencial ateroprotector in vitro del alga Halimeda incrassata en la migración de células de músculo liso de ratón y la oxidación de lipoproteínas en relación con su actividad antioxidante. Material y métodos: La actividad antioxidante fue determinada mediante los métodos de inhibición de radicales DPPH y la Capacidad antioxidante total (ORAC). La actividad inhibitoria de la oxidación de LDL mediada por iones Cu2+ se determinó por la cuantificación de TBARS y dienos conjugados. El efecto del extracto acuoso sobre la migración de las células de músculo liso se evaluó en la línea de células de músculo liso aórtica de ratón MOVAS-1. Resultados: Se demostró el efecto inhibidor del extracto sobre la oxidación de LDL mediada por Cu2+. El extracto del alga causa inhibición dosis-dependiente de la formación de TBARS (IC50 = 0,8 mg/mL) y dienos conjugados. Las algas tuvieron una alta actividad antioxidante en los ensayos realizados y podría estar relacionada con el contenido de compuestos fenólicos. Conclusiones: Los resultados de este trabajo representan un paso más en la caracterización de la acción ateroprotectora de Halimeda incrassata y evidencian sus posibles aplicaciones como nutracéutico y/o fitofármaco (AU)

Aim: The aim of this work was to evaluate the in vitro atheroprotective potential of the seaweed Halimeda incrassata in smooth muscle cell migration and lipoprotein oxidation in relation to its antioxidant activity. Material and methods: Antioxidant activity was determinate by DPPH• radical scavenging assay and ORAC method. The inhibitory effect of the aqueous extract on LDL oxidation mediated by Cu2+ ions was determinate by TBARS and conjugated diene quantification. The effect of the seaweed aqueous extract on smooth muscle cell migration was evaluated in MOVAS-1 mouse aortic smooth muscle cell. Results: The inhibitory effect of the aqueous extract on lipoprotein oxidation mediated by Cu2+ was demonstrated. Seaweed extract caused dose-dependent inhibition of TBARS (IC50 = 0.8 mg/mL) and conjugated dienes formation. The seaweed had a high antioxidant activity in the assays performed. The activity could be related to the phenolic content of Halimeda incrassata. Conclusions: In summary, the results of this study represent a further step in the characterization of the atheroprotective action of Halimeda incrassata and indicate the seaweed could be used for a nutraceutical and/or phytoterapeutic application (AU)

The treatment with an anti-glycosaminoglycan antibody reduces aortic oxidative stress in a rabbit model of atherosclerosis.

Retained low-density lipoproteins (LDL) by arterial glycosaminoglycans (GAG) are more susceptible to reactive oxygen species-mediated oxidation, contributing to oxidative stress and atherosclerosis. Recently, we reported the properties of the chimeric mouse/human monoclonal antibody chP3R99-LALA to bind sulfated GAG, to inhibit LDL-chondroitin sulfate binding, and to avoid LDL oxidation in vitro. Here, we hypothesized that chP3R99-LALA treatment might reduce aortic oxidative stress in a therapeutic setting. Redox biomarkers and serum lipids were determined by spectrophotometric methods. Subcutaneous administration of five doses (100 µg) of chP3R99-LALA, after Lipofundin administration (2 mL/kg/day, i.v.) during 8 days, reduced atherosclerotic lesion development, which was not associated with a serum lipid modulation. In contrast, the treatment with chP3R99-LALA reduced (p < 0.05) malondialdehyde and protein oxidation, induced a restoration of reduced glutathione level, of the superoxide dismutase and catalase activities and of endothelial nitric oxide level. Thus, the antiatherogenic effect of chP3R99-LALA treatment seems to be associated with a reduction of aortic oxidative stress. These results contribute in understanding the molecular mechanisms associated with chP3R99-LALA atheroprotection and support the use of anti-GAG antibody-based immunotherapy as a potential tool to treat the atherosclerosis.

Probiotics and autoimmunity: an evolutionary perspective.

Probiotics are microorganisms that have demonstrated beneficial effects on human health. Probiotics are usually isolated from the commensal microflora that inhabits the skin and mucosas. We propose that probiotics represent the species of microorganisms that have established a symbiotic relationship with humans for the longest time. Cultural practices of ancient human societies used to favor that symbiosis and the transmission of probiotics from generation to generation. New practices, introduced as a result of industrialization, such as childbirth by surgical delivery, ingestion of pasteurized and synthetic compounds-supplemented food, cleaner homes, indiscriminate use of antibiotics and so on, have led in recent years to the replacement of probiotics by other microorganisms that are not as well adapted to the microenvironments of the human body. These newly settled microorganisms lack many of the beneficial effects of probiotics. Our hypothesis is that the sudden change (from an evolutive perspective) in human intestinal microflora may importantly contribute to the rise in the incidence of autoimmune diseases, observed in the last half a century.

Human immunodeficiency virus and other sexually transmitted diseases in Cuban women.

A cross-sectional study was performed in 60 Cuban women of child-bearing age who were seropositive for human immunodeficiency virus (HIV) and 60 controls. Human papillomavirus (HPV) was identified most frequently, with oncogenic HPV serotypes 16, 33 and 58 detected in HIV-positive patients, and serotypes 11, 33 and 51 in the controls (relative risk 4.41; 95% CI 2.21-8.29). Syphilis and hepatitis B and C viruses were detected exclusively in HIV-seropositive women (p<0.05). Sexually transmitted diseases (STDs) appeared to pose a substantial health problem, especially for HIV-positive women. Clinics should consider screening and treatment for STDs as part of their HIV prevention programmes.

Carboxy-terminally truncated Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induced neutralizing antibodies and resistance to Dengue 4 virus challenge in mice.

We have expressed a recombinant Dengue 4 virus envelope glycoprotein (E4rec), truncated at its C-terminus by 53 amino acids, in Pichia pastoris. The presence of E4rec was confirmed by Western-blot using anti-DEN 4 hyper immune mouse ascitic fluid. E4rec migrated during SDS-PAGE as a 64 kDa protein. Treatment with endoglycosidases showed that the E protein was modified by the addition of short mannose chains and the absence of hyperglycosylation. When administered to BALB-C mice, E4rec elicited a DEN 4 neutralizing antibody response haemagglutination inhibition antibodies and specific memory T cell response. Mice immunized were also significantly protected against lethal DEN 4 virus challenge (86.6%, p < 0.001).

[The application of the polymerase chain reaction technic for the detection of human papillomavirus sequences]. / Aplicación de la técnica de reacción en cadena de la polimerasa para la detección de secuencias de Papillomavirus humano.

The polymerase chain reaction technique was applied to detect sequences of human Papillomavirus (HPV) by controls of cellular lines of cervical cancer and of tissues obtained through biopsy with a HPV-positive clinical diagnosis. A set of consensus oligonucleotides, which are complementary to a highly conserved region within the open reading frame E1 of the viral genome of HPV affecting the cervical mucosa, was used. With these primers it was possible to amplify DNA sequences corresponding to HPV 6 and 11, considered in the low risk group, and to HPV 16, 18, 31 and 33, included in the high risk group. The study of the sensitivity of the amplification technique showed a level of detection of 3,5 viral particles per each cellular diploid genome.

Lesion of nigrostriatal neurons by 6-hydroxydopamine induces changes in rat brain glutathione-S-transferase.

Wistar rats were lesioned into the nigrostriatal pathway with 6-OHDA. The D-amphetamine-induced circling behavior test was performed to evaluated lesion efficiency. Animals that showed more than 620 turns/90 min were named totally lesioned animals (TLA). The group of rats that performed less than 620 turns/90 min were named partially lesioned animals (PLA). The contents of DA and its catabolites in the striata of these groups, and in the same tissue of the untreated animals, were measured. Moreover, the striatal glutathione-S-transferase (GST) specific activity for all groups was tested, and the kinetics parameters for GST purified from the whole brain were evaluated from other three similar groups. The striatal DA depletion on TLA was greater than in PLA. Striatal GST activity showed a significantly bilateral increase in PLA, whereas TLA exhibited only and ipsilateral augment. There were also differences between groups about the kinetic parameters of the purified brain enzyme. The possible role of GST on the interindividual lesion response difference was analyzed.

Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping.

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

[Western blot study of sera from patients with epidemic neuropathy]. / Estudio por western blot de sueros de pacientes con neuropatía epidémica.

The immune response of a group of patients with epidemical neuropathy and of controls was studied by the immunoblotting technique against proteins of the Coxsackie virus and the proteins of slow effect isolated in our laboratory. 13 sera of patients with epidemical neuropathy and 9 sera of controls were studied. Of the 13 sera studied, 8 (61.5%) recognized protein VPI and 2 sera (15.3%) protein VP0 of the strain 47.93. Of the 9 controls studied, 4 (44.4%) recognized protein VPI and 3 (33.3%) protein VP0 only. With the antigen prepared from the slow effect strain it was obtained a specific signal in 5 (38.5%) sera of patients and in 2 sera (22.5%) of controls. It should be stressed that in this last case the protein observed had a molecular weight of 41,300 D, and that its size was smaller than that of the preceding protein detected against the strain 47.93 was of 45,000 D.

[Follicular conjunctivitis caused by an adenovirus. The characterization of its strains by restriction endonucleases]. / Conjuntivitis folicular por Adenovirus. Caracterización de cepas por endonucleasas de restricción.

A study of conjunctivitis cases with a possible viral cause was carried out in Havana City during June and August, 1993. Thirty seven cases from "Ramón Pando Ferrer" Ophthalmological Hospital were studied, of whom 13 isolations (35.1%) in cell culture of Hela cells were obtained. Isolations showed a cytopathic effect characteristic of Adenovirus. A direct immunofluorescence with conjugated serum of rabbit specific to Adenovirus was performed and results were positive in 100% of cases. A DNA restriction analysis of each isolated was performed for their classification. Restriction endonucleases SmaI and Hind III were used and 2 isolates were classified as Adenovirus type 3, and 6 as Adenovirus type 7b, 4 out of 13 isolates were not classified because of the degradation of the genetic material.