Development of Anxiolytic and Depression-like Behavior in Mice Infected with Mycobacterium lepraemurium.

Murine leprosy is a systemic infectious disease of mice caused by Mycobacterium lepraemurium (MLM) in which the central nervous system (CNS) is not infected; nevertheless, diseased animals show measurable cognitive alterations. For this reason, in this study, we explored the neurobehavioral changes in mice chronically infected with MLM. BALB/c mice were infected with MLM, and 120 days later, the alterations in mice were evaluated based on immunologic, histologic, endocrine, neurochemical, and behavioral traits. We found increases in the levels of IL-4 and IL-10 associated with high bacillary loads. We also found increase in the serum levels of corticosterone, epinephrine, and norepinephrine in the adrenal gland, suggesting neuroendocrine deregulation. Mice exhibited depression-like behavior in the tail suspension and forced swimming tests and anxiolytic behavior in the open field and elevated plus maze tests. The neurobehavioral alterations of mice were correlated with the histologic damage in the prefrontal cortex, ventral hippocampus, and amygdala, as well as with a blood-brain barrier disruption in the hippocampus. These results reveal an interrelated response of the neuroimmune--endocrinological axis in unresolved chronic infections that result in neurocognitive deterioration.

The yin/yang of inflammatory status: Blood-brain barrier regulation during sleep.

Sleep loss induces a low-grade inflammatory status characterized by a subtle but sustained increase of pro-inflammatory mediators, which are key regulators of blood-brain barrier function. To investigate the influence of inflammatory status on blood-brain barrier dysfunction induced by sleep restriction we performed an experiment using two strains of mice with different immunological backgrounds, C57BL/6 mice that have a predominant pro-inflammatory response and BALB/c mice that have a predominant anti-inflammatory response. Mice were sleep-restricted during 10 days using the flowerpot technique during 20 h per day with 4 h of daily sleep opportunity. The systemic inflammatory status, blood-brain barrier permeability, and the hippocampal expression of neuroinflammatory markers were characterized at the 10th day. Serum levels of TNF and IFN-γ increased in sleep-restricted C57BL/6 but not in BALB/c mice; no changes in other cytokines were found. Sleep restriction increased blood-brain barrier permeability in C57BL/6 strain but not in BALB/c. The hippocampus of sleep-restricted C57BL/6 mice exhibited an increase in the expression of the neuroinflammatory markers Iba-1, A2A adenosine receptor, and MMP-9; meanwhile in sleep-restricted BALB/c mice the expression of this markers was lesser than the control group. These data suggest that cytokines may be playing a key role in modulating blood-brain barrier function during sleep restriction, and probably the effects are related to Iba-1, MMP-9 and A2A adenosine receptor overexpression.

Blood-Brain Barrier Disruption Induced by Chronic Sleep Loss: Low-Grade Inflammation May Be the Link.

Sleep is a vital phenomenon related to immunomodulation at the central and peripheral level. Sleep deficient in duration and/or quality is a common problem in the modern society and is considered a risk factor to develop neurodegenerative diseases. Sleep loss in rodents induces blood-brain barrier disruption and the underlying mechanism is still unknown. Several reports indicate that sleep loss induces a systemic low-grade inflammation characterized by the release of several molecules, such as cytokines, chemokines, and acute-phase proteins; all of them may promote changes in cellular components of the blood-brain barrier, particularly on brain endothelial cells. In the present review we discuss the role of inflammatory mediators that increase during sleep loss and their association with general disturbances in peripheral endothelium and epithelium and how those inflammatory mediators may alter the blood-brain barrier. Finally, this manuscript proposes a hypothetical mechanism by which sleep loss may induce blood-brain barrier disruption, emphasizing the regulatory effect of inflammatory molecules on tight junction proteins.

Low-dose amphotericin B and murine dialyzable spleen extracts protect against systemic candida infection in mice.

Candida albicans causes opportunistic systemic infections with high mortality (30%-50%). Despite significant nephrotoxicity, amphotericin (AmB) is still used for the treatment of this serious fungal infection. Therefore, alternative treatments are urgently needed. Dialyzable leukocyte extracts have been used successfully to treat patients with mucocutaneous candidiasis, but their effectiveness in systemic candidiasis has not been evaluated. In this study, low-dose AmB (0.1 mg/kg) plus 10 pg of murine dialyzable spleen extracts (mDSE) were tested in a systemic candidiasis mouse model. Survival, tissue fungal burden, kidney damage, kidney cytokines, and serum levels of IL-6 and hepcidin were evaluated. Our results showed that the combined treatment of low-dose AmB plus mDSE improved survival and reduced kidney fungal burden and histopathology; these effects correlated with increased kidney concentration of IFN- γ and TGF- ß 1, decreased levels of TNF- α , IL-6, and IL-10, as well as high levels of systemic IL-6 and hepcidin. Low-dose AmB and mDSE synergized to clear the infectious agent and reduced tissue damage, confirming the efficacy of a low dose of AmB, which might decrease the risk of drug toxicity. Further studies are necessary to explore these findings and its implications in future therapeutic approaches.

Ultrastructural characterization of CD133+ stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications.

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however, this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stem cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic, characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the signal (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133+ stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10(-13) mol iron (9.5 pg) or 7.0 x 10(6) nanoparticles per cell (the measurement was carried out in a volume of 2 muL containing about 6.16 x 10(5) pg iron, equivalent to 4.5 x 10(11) SPIONs).

Treatment with BB-94, a broad spectrum inhibitor of zinc-dependent metalloproteinases, causes deviation of the cytokine profile towards type-2 in experimental pulmonary tuberculosis in Balb/c mice.

BB-94 (batimastat) is a broad- spectrum hydroxamic acid-based zinc metalloproteinase inhibitor that inhibits both the matrix metalloproteinases (MMP) and members of the ADAM family of enzymes such as Tumour Necrosis Factor-alpha Cleaving Enzyme (TACE). These enzymes are involved in the regulation of inflammatory processes in tuberculosis. Balb/c mice infected with M. tuberculosis via the intratracheal route were treated with BB-94 for 1 month, starting on the day of infection. Immunohistochemistry, semiquantitative RT-PCR and ELISA assays for cytokines revealed a deficit in IL-1 and IL-2 expression and a premature bias towards IL-4 expression, accompanied by a delay in granuloma formation and more rapid progression of disease in BB-94-treated animals. This situation corrected itself after the drug was withdrawn at 28 days. In contrast, when BB-94 was administered only after 1 month there were no significant changes apart from the presence of amyloid, and a paradoxically increased expression of IL-1alpha. These results cast light on mechanisms of immunity in tuberculosis and also indicate that in patients treated with similar broad-spectrum MMP inhibitors there may be a risk of inappropriate deviation of some immune responses towards a Type-2 cytokine profile.

Interactions between hormone-mediated and vaccine-mediated immunotherapy for pulmonary tuberculosis in BALB/c mice.

Problems of logistics, compliance and drug resistance point to an urgent need for immunotherapeutic strategies capable of shortening the current 6-month chemotherapy regimens used to treat tuberculosis, or of supplementing ineffective therapy. In this study we sought to define the mechanism of action of two immunotherapies, both of which have previously been shown to prolong survival. Secondly, we wished to identify any clinically useful synergy between these therapies. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance dominated by type 1 cytokines plus tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), followed by a phase of progressive disease. This progressive phase is accompanied by increasing expression of IL-4, and diminished expression of IL-1 and TNF-alpha. Animals in this late progressive phase of the disease (day 60) were treated with two injections (day 60 and day 90) of 0.1 or 1.0 mg of heat-killed Mycobacterium vaccae, or with 3beta, 17beta-androstenediol (AED; 25 microg subcutaneously three times/week), or with both therapies. We show here using four techniques in parallel (morphometry, immunohistochemistry with automated cell counting, semiquantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays of cytokines in lung extracts) that treatment with M. vaccae causes a switch back towards a type 1 cytokine profile, restoration of expression of IL-1alpha and TNF-alpha, and a switch from pneumonia to granuloma. This is very similar to the changes previously seen after treatment with AED. However, there was no evidence for synergy between M. vaccae and AED.

The effects of androstenediol and dehydroepiandrosterone on the course and cytokine profile of tuberculosis in BALB/c mice.

Immunity to Mycobacterium tuberculosis requires a T helper 1 (Th1) cytokine balance accompanied by tumour necrosis factor-alpha (TNF-alpha), and activated macrophages. These facets of the immune response are sensitive to suppression by glucocorticoids (GC), which can reactivate and exacerbate tuberculosis in man and animals. Dehydroepiandrosterone (DHEA) and its derivative, 3beta,17beta androstenediol (AED), are reported to have antiglucocorticoid properties in vivo. We therefore investigated the effects of predetermined optimal doses of these compounds, on the course of pulmonary tuberculosis in an established model in BALB/c mice in which an early phase of Th1-mediated response accompanied by adrenal hyperplasia, is followed by a switch to Th2, progressive loss of TNF-alpha expression and disease progression. Both compounds were protective, particularly AED which caused a fall in bacterial counts and prolonged survival. These effects correlated with the appearance within 3 days of cellular infiltrates rich in cells expressing interleukin-2 (IL-2), IL-1alpha and TNF-alpha, and with partial suppression of the switch to IL-4 producing cells that occurred in controls. AED also caused enhanced development of granulomas at 14 days, and persistence of granuloma formation to 120 days, with a corresponding suppression of areas affected by pneumonia. Much of the therapeutic effect of AED and DHEA was obtained by treating for only the first 3 weeks, which is the phase of adrenal hyperplasia. These results suggest that the ratio of GC to anti-GC steroids may play a role in the pathogenesis of tuberculosis, and further investigation could lead to novel treatment strategies.

Tratamiento de las fracturas subcapitales del fémur / Processing of the subcapital fractures of femur

Presenta un análisis retrospectivo de la casuística de las fracturas subcapitales del fémur, tratadas en el departamento de Ortopedia y Traumatología del Hospital Carlos Andrade Marín de Quito, entre ene. de 1993 hasta oct. 1996. Se incluyen 52 casos, la edad fluctúa entre los 24 años y los 98 años. El uso de prótesis se utilizó en 43 pacientes, en 2 pacientes se requirió de tornillos canulados y los 7 restantes fueron sometidos a tratamiento incruento. La causa de las fracturas se debió a un trauma de baja energía en 50 individuos (caída de propia altura) y en los restantes por trauma de alta energía (atropellamiento).

Osteomielitis en pacientes pediátricos: diagnóstico y tratamiento / Osteomielitis in patients pediatricos: diagnose and processing

Estudia restrospectivamente los ingresos al Hospital Pediátrico Baca Ortiz de Quito, con diagnóstico de osteomielitis. Predominaron los casos en el sexo masculino (57xciento); se determinó que las edades de mayor incidencia fueron 7 años (20xciento), 5 años (16.5xciento), 11 años (13.3xciento), 12 años (10xciento) y una distribución para el resto de casos correspondiente a 2 pacientes por cada grupo. El hueso afectado con mayor frecuencia fue la tibia (66.5xciento, n=20) seguido por el fémur (20xciento) y gentamicina en el 10xciento restante (n=3). Los procedimientos quirúrgicos consistieron en secuestrectomía más curetaje en el 93xciento de casos (n=28), colocación de perlas de gentamicina en 16 sujetos (53xciento) e inmovilización en la totalidad de casos.

Pathogenesis of tuberculosis in mice exposed to low and high doses of an environmental mycobacterial saprophyte before infection.

Mycobacteria are ubiquitous in the environment, but they are not part of the normal human microbial flora. It has been suggested that variable contact with mycobacteria can influence susceptibility to mycobacterial pathogens and the efficacy of subsequent Mycobacterium bovis BCG vaccination. To test this, mice were immunized with high or low doses of an environmental saprophyte, M. vaccae, that is intensely immunogenic as an autoclaved preparation. Two months later, they received an intratracheal challenge with M. tuberculosis H37Rv. Recipients of a low Th1-inducing dose (10(7) organisms) were partially protected and maintained a high ratio of interleukin 2 (IL-2)-positive to IL-4-positive cells in the perivascular, peribronchial, and granulomatous areas of the lung, whereas in unimmunized controls the IL-4-positive cells increased markedly between days 21 and 28. In contrast, recipients of the high dose (10(9) organisms), which primes Th2 as well as Th1 cytokine production, died more rapidly than unimmunized controls and showed massive pneumonia from day 7. The ratio of IL-2-positive to IL-4-positive cells in all compartments of the lung rapidly fell to 1 by day 14 for these animals. These events correlated with cytokine mRNA profiles and with increases in the local toxicity of tumor necrosis factor alpha (TNF-alpha), demonstrable only when a major Th2 component was present. These data indicate that cross-reactive epitopes present in an environmental saprophyte can evoke either protective responses or responses that increase susceptibility to M. tuberculosis. The latter are associated with the presence of a Th2 component and increased sensitivity to TNF-alpha.

Correlation between the kinetics of Th1, Th2 cells and pathology in a murine model of experimental pulmonary tuberculosis.

T-helper 1 (Th1) Th2 kinetics were studied by immunohistochemistry and molecular biology techniques (reverse transcriptase polymerase chain reaction. RT PCR, Southern-blot) during the course of pulmonary tuberculosis induced in BALB/c mice by the intratracheal instillation of the live and virulent strain H-37Rv. The histopathological study clearly showed two phases of the disease. The first one was an acute phase which was characterized by inflammatory infiltrate in the alveolar capillary interstitium, blood vessel and bronchial wall with formation of granulomas. In this acute phase which lasted from 1 to 28 days, a clear predominance of Th1 cells was observed, manifested by a high percentage of interleukin-2 (IL-2) positive cells in the inflammatory infiltrate and granulomas demonstrated by immunohistology, as well as a gradual increment of interferon-gamma (INF-gamma) m-RNA. This was followed by a chronic or advanced phase characterized by pneumonia, focal necrosis and fibrosis, with a Th0 balance due to an equivalent proportion of IL-2 and IL-4 positive cells in the lung lesions, that coincided with the highest level of INF-gamma and IL-4 mRNA. The cytofluorometric analysis of bronchial lavage cells, showed a predominance of CD4 T cells during the acute phase and CD8 T lymphocytes in the chronic phase, gamma-delta T lymphocytes showed two peaks, at the beginning (3 days) and at the end (4 months) of the infection. These results suggest that T-lymphocyte subset kinetics and the pattern of cytokines produced in the lung during tuberculosis infection changed over time and correlate with the type and magnitude of tissue injury.

Evaluation of a pooling method for routine anti-HCV screening of blood donors to lower the cost burden on blood banks in countries under development.

A pooling system was developed for use in anti-HCV screening of voluntary blood donors at the local Central American Red Cross blood banks, in Nicaragua, El Salvador and Honduras. The commercially available second generation anti-HCV screening kit from Abbott Laboratories (North Chicago, IL) was used with a modification in the initial serum dilution procedure. Pools of five sera were selected for routine screening, based on comparative studies of individual samples and of pools with different sample sizes. During the years 1993 and 1994 a total of 89, 148 voluntary blood donors were screened and a positive prevalence rate of 0.35% was established. Of the initially positive samples, 54% confirmed positive, 30% were indeterminate and 16% were negative using the Abbott Matrix test. Significant differences of positive screening prevalence rates were found in the three countries, with average values of 0.50%, 0.23% and 0.08%, respectively, in Nicaragua, El Salvador and Honduras. These initially positive samples also showed a different confirmatory pattern with a positive rate of 64% in Nicaragua, in contrast to 20% in El Salvador. Only a few samples were available for RT-PCR amplification of HCV-RNA; however, this highly sensitive method did not appear to be more helpful than serology in confirming the HCV donor status. Overall, the data obtained indicate a fluctuation of HCV prevalence in voluntary blood donors among the three Central American countries. Further, differences were found in the percentages of initially screened positives and confirmation patterns. This information appears useful for establishing criteria in future screening policies. Thus, we suggest that the use of pooling for anti-HCV screening is beneficial in countries under development, since there are potential cost savings, as well as benefits in establishment of initial prevalence rates.