Dengue virus identification by transmission electron microscopy and molecular methods in fatal dengue hemorrhagic fever.

Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.

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.

Angiosarcomas arising in edematous extremities: immunostaining for factor VIII-related antigen and ultrastructural features.

Immunostaining for Factor VIII-related antigen was seen in deparaffinized sections from 19 of 20 postmastectomy angiosarcomas and from four of four sarcomas that arose in chronically edematous tissue unrelated to breast carcinoma. Staining was also seen in sections from two malignant hemangioendotheliomas, four capillary hemangiomas, and one granulation tissue specimen. Sections from two lymphangiomas were immunonegative for Factor VIII-related antigen in the endothelium of lymphatic channels, whereas staining was observed in the surrounding normal blood vessels. Electron microscopic study of four postmastectomy angiosarcomas disclosed ultrastructural features (fenestrae, intense pinocytotic activity, cell junctions, and Weibel-Palade bodies) supporting the blood vascular endothelial nature of the neoplastic cells. It is concluded that a neoplastic blood vessel component is present in sarcomas that arise in chronically edematous tissues. It is questionable whether a lymphatic component is also present. These tumors, therefore, should be regarded as angiosarcomas rather than lymphangiosarcomas.

Angiogenesis in the central nervous system: a role for vascular endothelial growth factor/vascular permeability factor and tenascin-C. Common molecular effectors in cerebral neoplastic and non-neoplastic "angiogenic diseases".

Human pathological conditions of the central nervous system (CNS) associated with angiogenesis (i.e. neovascularization) include neoplastic, as well as infectious, ischemic, and traumatic processes. Upregulation of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and tenascin-C (TN-C) is spatially and temporally related to neovascularization. Spatially, VEGF/VPF and TN-C are both found at the site of neovascularization, but they are not detected in areas of normal brain or in areas without neovascularization. Temporally, VEGF/VPF and TN-C are found at the peak of angiogenesis and are not detected when angiogenesis had ceased.

Experimental model of graft vs. host disease in non-immunosuppressed F1 (CBA/J x C57BL/6) mice.

The experimental model of graft vs. host disease (GvHD) has a potential use in the evaluation of different manipulation procedures of the immune system applicable to development of vaccines. In the present study an experimental model of GvHD in F1 (CBA/J x C57BL/6) mice by means of the parenteral inoculation of spleen lymphoid cells from parental male CBA/J to 10-day-old animals (experimental group) was developed. Animals inoculated with Medium 199 (n = 42) (Medium 199 group), or with splenic lymphoid cells either from the hybrids (n = 16) (F1 group), or from mice of the inbred strain Balb/c (n = 10) (Balb/c group) were used as controls. In all groups body and spleen weights, relative spleen index (RSI), and spleen index (SI) were determined. Additionally, histopathologic and morphometric studies were done in the spleens of the animals studied. Significant increases in body and spleen weights, RSI, and lymphocytic perimeter and area were associated with distinctive splenic GvHD lesions found in the experimental group. The experimental SI value was higher than twice the SI value of any of the control groups. We conclude that ours is a useful model of GvHD with many potential applications in the field of vaccine production.

Experimental infection by Haemophilus influenzae type b in inbred mice.

The epidemiological situation of bacterial meningitis is increasing dramatically. There is no doubt that the lack of proper animal models has hampered the achievement of effective prophylactic and therapeutic means. We report the characterization of the experimental disease caused by Haemophilus influenzae type b (Hib) in mice, taking into account its importance as an etiological agent of such a type of meningitis. The high resistance of C57BL/6, CBA/ J and BALB/cJ mice to Hib infection was proven. LD50 of Hib using trypsin or iron dextran as virulence enhancement factors (VEF), both being similar and more than 1000 times lower than that without any VEF, were determined. Lesions of CNS compatible with meningitis were found in about one third of specimens. Hair bristling, conjunctivitis, diarrhea, depression and prostration were the most characteristic symptoms. The proportion of animals which die is highest on the first day, lower on the second and almost zero after 48 h of infection. Water and food intake was higher in control than in infected animals; nevertheless, there were no differences in body weight increase among the mice after 5 days post-infection. Microorganisms were isolated from CSF and blood after 6 h of infection and positive results remained according to the size of the inoculum. Despite the acuteness of the experimental disease, antibiotic treatment with internationally recommended drugs was shown to be effective. Similar results were achieved when hyperimmune serum vs. Hib was applied.

Sequential study of an experimental model of graft vs. host disease (GvHD) in non-immunosuppressed F1 (CBA/J x C57BL/6) mice.

In order to determine the optimal day for the evaluation of an experimental model of GvHD in F1 mice and the histopathologic evolution of the lesions in different organs, we studied 10-day-old F1 (CBA/J x C57BL/6) mice inoculated with splenic lymphoid cells of the male parental CBA/J strain (n = 42) that were sacrificed between 1 and 14 days postinoculation. The evolution of the relative spleen index (RSI) and the histopathologic lesions in different organs were also determined. F1 mice inoculated with Medium 199 were used as controls. Significant RSI increases (p < 0.0001) were found in the experimental group between 2 and 14 days postinoculation, with a peak at the eighth day, associated with the most severe histopathologic lesions in the organs studied. We suggest the eighth day as the optimal time for evaluation of this experimental model.

Histopathologic and humoral study of Balb/c mice inoculated with BCG by different routes.

With the aim of determining the distribution and humoral immunogenicity of the bacillus Calmette-Guérin (BCG) administered by the oral (O), intravenous (IV) and subcutaneous (SC) routes, we studied 54 male Balb/c mice weighing 17-22 g that had been inoculated with BCG (10(6) CFU) by the O (n = 18), IV (n = 18) and SC (n = 18) routes. At weekly intervals we determined the distribution of the microorganism using histopathological techniques including Ziehl-Neelsen staining. Serum samples of the same animals were analyzed by ELISA and Western blot to determine the antibody response to the microorganism. In all groups, distinctive histopathologic lesions harboring the microorganism were found. Using the SC route the lesions were located at the inoculation site, whereas there was systemic dissemination with the O and IV routes, being more prominent with the latter. Anti-BCG antibodies were detected by ELISA in all groups; this response was more intense in the IV group, followed by the SC and O groups. In the Western blot analysis, reactivity against multiple bands and the predominant recognition of a 65 kd band in all groups was observed.

Evaluation of the efficacy of human antimeningococcal immunoglobulin G in infant rats experimentally infected with Neisseria meningitidis group B.

Infants rats, a well known model for the experimental reproduction of bacterial meningitis, were used by us to test the protective potential of antibodies developed in humans who had been vaccinated with the Cuban antimeningitis vaccine (VA-MENGOCBC). Newborn rats were inoculated by the intraperitoneal and intranasal routes with suspensions of Neisseria meningitidis group B bacteria. Bacteremia kinetics were evaluated from blood and brain-spinal fluid cultures. Samples of the central nervous system were taken and smears of backbone fluids prepared for histopathologic evaluations. Characterization of bacteremia evolution, as well as the mean lethal dose of germs and histopathologic features, were determined. After standardization of the model, therapeutic schemes were applied using passive immunization pre- and post-infection with N. meningitidis. A significant level of protection was obtained in relation to control animals that received the same challenge doses.

Viral isolation from cases of epidemic neuropathy in Cuba.

OBJECTIVE: To investigate the possibility of a viral agent in the central nervous system of patients with epidemic neuropathy. DESIGN: Virus isolation attempts, in cell cultures and suckling mice, from cerebrospinal fluid (CSF) of neuropathy patients and controls undergoing lumbar puncture for unrelated reasons. Serologic studies in patients, contacts, and controls. SETTING: An epidemic of optic and peripheral neuropathy affected more than 50,000 people in Cuba in 1991 through 1993. Illness was associated with dietary limitations and increased physical demands accompanying the shortages of food and fuel experienced in Cuba since 1989. Most patients responded to parenteral vitamin therapy, and the epidemic began to subside when oral vitamin supplementation was begun for the entire Cuban population. RESULTS: Coxsackievirus A9 (five isolates) and a similar, less cytopathic virus (100 isolates) were recovered from 105 (84%) of 125 CSF specimens from neuropathy patients. The strains with light cytopathic effect were antigenically related to Coxsackieviruses A9 and B4 by cross-neutralization and immunoblotting assays. Virus persisted in CSF of some patients for 1 to 12 months. Cerebrospinal fluid from patients and both types of virus from cell culture produced illness, including complete posterior flaccid paralysis, in newborn mice, and virus was reisolated from the mice. Mouse tissues and sural nerve biopsy specimens from patients were stained by immunoperoxidase and colloidal gold techniques using hyperimmune rabbit antisera against the virus with light cytopathic effect. CONCLUSIONS: Coxsackievirus A9 or an antigenically related agent with a light cytopathic effect was present in CSF of 84% of 125 patients with epidemic neuropathy. The role of these agents, probably in combination with nutritional factors, in the pathophysiology of the disease requires further investigation.

Mechanism of enterovirus involvement in epidemic neuropathy: hypothesis regarding pathophysiology.

During the epidemic of optic and peripheral neuropathy which occurred in Cuba in 1992-1993, viruses antigenically related to the Coxsackie viruses were isolated from cerebrospinal fluid of patients. Concurrently with the virologic studies, epidemiologic, toxicologic, nutritional, immunologic, and histopathologic investigations were also carried out. Although it was demonstrated that the illness was associated with toxic and nutritional risk factors, it has not been possible to identify a specific etiology for the symptoms observed. Taking into consideration the results obtained in all of the various investigations, we have formulated an integral, multifactorial hypothesis which attempts to explain a pathophysiologic mechanism by which the viruses isolated could participate in the pathogenesis of the illness. We propose that the viral agents produce a persistent infection, and the possibility that they may act as mediator of an autoimmune pathogenic process.

Immunocytolocalization of two protection-inducing antigens of Trichinella spiralis during its enteral phase in immune and non-immune mice.

Monoclonal antibodies (mAb) recognizing epitopes on the 48K (beta stichocyte specific) and the 50/55K antigen (alpha stichocyte specific) were used as first ligands for immunocytolocalization on de-paraffinized sections of infected gut tissue of non-immune and immune CFW strain mice. The enteral phase was studied at 6, 14, 23, 30 hr and 7 days after initiation of infection via the oral route, times corresponding in worm development to the first (L1), second (L2), and third (L3) stage larva and adult. No change in the intensity of the immune reaction with either mAb was noted in parasites developing within immune or non-immune mice for any of the time-points studied. The 48K and the 50/55K antigens were present within the stichocytes at 6 hr. Enterocytes adjacent to some worms also stained positive for both epitopes at this time. Throughout worm development, the amount of each antigen within the worm diminished, until almost none was left at 30 hr. At day 7, the 48K antigen was present within a few stichocyte cells, the canalicular tree, and within the lumen of the midgut. The 50/55K antigen at this time point was localized within only a few stichocyte granules and on the lining of the worm's gut. Embryo stages did not possess either the 48K or 50/55K epitopes. A marked increase in cells bearing IgG in the lamina propria was noted in immune mice when compared with their non-immune counterparts.

Trichinella spiralis: vascular endothelial growth factor is up-regulated within the nurse cell during the early phase of its formation.

The L1 larval stage of Trichinella spiralis induces modification in a portion of striated skeletal muscle cell resulting in the formation of the nurse cell. This specialized host cell is completely encased in a capsule composed mainly of collagen type IV and type VI, which, in turn, is surrounded by a unique rete of vessels whose formation begins on around day 12 after intracellular infection. We investigated the possibility that vascular endothelial growth factor (VEGF) may be up-regulated during nurse cell formation by employing immunohistochemistry and in situ hybridization on synchronously infected mouse muscle tissue. Both VEGF mRNA and VEGF peptide were detected in the developing nurse cell cytoplasm from day 7 up to 16 mo after infection. In addition, VEGF was also detected in cells in the area immediately surrounding the nurse cell on days 15 and 17. On the basis of these results, we propose that hypoxia is induced by T. spiralis within the developing nurse cell some time prior to the up-regulation of VEGF, perhaps as early as day 7. We further propose, on the basis of the continued presence of VEGF in nurse cell cytoplasm, that a constant state of hypoxia cell is maintained.

Monoclonal antibody to dengue capsid protein: its application in dengue studies.

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.

Linfadenitis por mycobacterium incluyendo pacientes infectados por el virus de inmunodeficiencia humana / Mycobacterium lymphadenitis including patients infected with human inmunodefi

El género Mycobacterium provoca infecciones pulmonares y extrapulmonares, de estas últimas predomina la infección ganglionar. Mientras Mycobacterium tuberculosis es el agente causal más importante, en las últimas décadas aumenta la incidencia de otras especies micobacterianas que se han hecho prevalentes en los pacientes positivos al virus de la inmunodeficiencia humana (VIH +) tanto en países desarrollados como en vías de desarrollo. Durante el período enero 2007 hasta diciembre 2009 se procesó en nuestro laboratorio 6540 muestras, 210 muestras fueron obtenidas por biopsia ganglionar, precisamente este constituyó nuestro universo de estudio, 190 (90.4%) muestras se obtuvieron por exéresis quirúrgica, 20 (9.5%) por punción espirativa;17 procedían de pacientes VIH– (8.1%) y 193 procedentes de pacientes VIH+(91.9%). En solo 16 muestras (7.6%) el cultivo BAAR fue positivo; 4 procedentes de pacientes VIH– (25%) y 12 VIH+(75%). La clasificación e identificación micobacteriana demostró la presencia de Mycobacterium tuberculosis en 13 de los casos (81.25%), mientras Mycobacterium avium-intracellulare fue aislado en 3 (18.7%). En los pacientes inmunodeprimidos con linfadenopatía incluidos los pacientes VIH/sida, es muy importante la búsqueda activa de la presencia de BAAR como coinfección oportunista, donde Mycobacterium tuberculosis se mantiene como el agente infeccioso más frecuente, sin embargo la posibilidad de que otras especies micobacterianas también estén presentes no se debe descartar. Nuestro objetivo en este estudio como Laboratorio Nacional de Referencia de TB- Micobacterias fue lograr la caracterización etiológica de linfadenopatías en pacientes en que se sospechaba clínicamente la participación del género Mycobacterium.

Mycobacterium tuberculosis is the most important etiological agent producing pulmonary as well as extrapulmonary infection. During these last decades, the increase in the incidence of infection due to other mycobacteria species is evident. Lymphadenopathy is the most frequent extrapulmonary presentation form of Mycobacterium Genera infection among HIV positive patients either in developed or underdeveloped countries. The aim of this work is to analyze the results obtained during January 2007 - December 2009 in our laboratory. Two hundred ten tissue samples were studied; 190 (90.4%) samples were lymph node biopsied tissues and 20 (9.5%) samples were obtained by fine needle aspiration; 17 were from HIV - patients (8.1%) and 193 from HIV + (91.9%). A total of 16 (7.6%) samples produced a positive culture for BAAR, 4 VIH- (25%) and 12 VIH+ (75%). Classification and identification for mycobacteria confirmed Mycobacterium tuberculosis in 13 of the cases (81.25%), and Mycobacterium avium-intracellulare in three patients (18.7%). The present study once again confirms that BAAR culture has more sensitivity and specificity than histopathologhic studies have. Lymphadenopathy in immunosuppressed patients should by studied for the presence of an BAAR coinfection where M. tuberculosis is still the agent most frequently found, nevertheless, other species of Mycobacteria may be causing infection and should be searched for. Our objective as National Reference Laboratory of Tuberculosis and Mycobacterial was to obtain the etiological characterization of Mycobacterium lymphadenopathy in clinically suspect patients.

Clinical aspects of infection with Trichinella spp.

Isolated cases and outbreaks of infection with Trichinella spp. occur frequently throughout the world, sometimes resulting in fatalities. The clinical presentations of signs and symptoms are remarkably constant for most of the species of Trichinella, but in infections with Trichinella nativa and Trichinella britovi, classical symptoms of trichinellosis may be absent. It is important to be able to correlate the clinical presentation of trichinellosis with the life cycle of these helminths in order to make an accurate diagnosis. Knowledge of the epidemiology of the disease enables the physician to identify other potential cases, since most epidemics can be traced back to a common source of raw or undercooked meat. A comprehensive summary relating the most important clinical variables is presented graphically for easy reference to the text. Symptoms and signs are considered in relation to severity of infection. Laboratory findings and diagnostic techniques, including new modalities (e.g., DNA and antigen detection), are discussed. A discussion of treatment and preventive measures concludes our review.

Analysis of dendritic cells in solitary angiofibromas for factor VIII.

We studied dendritic cells in the dermis of six angiofibromas by the peroxidase-antiperoxidase method in order to determine whether they are endothelial cells. The primary antiserum used was directed to factor VIII, an endothelial cell marker. The dendritic cells were not immunoreactive for factor VIII, suggesting that they are not endothelial cells.

Trichinella spiralis: secreted antigen of the infective L1 larva localizes to the cytoplasm and nucleoplasm of infected host cells.

Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 45-50K. These antigens are stage specific for the infective first stage larva of Trichinella spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance.