PPARγ activation ameliorates cognitive impairment and chronic microglial activation in the aftermath of r-mTBI.

Chronic neuroinflammation and microglial activation are key mediators of the secondary injury cascades and cognitive impairment that follow exposure to repetitive mild traumatic brain injury (r-mTBI). Peroxisome proliferator-activated receptor-γ (PPARγ) is expressed on microglia and brain resident myeloid cell types and their signaling plays a major anti-inflammatory role in modulating microglial responses. At chronic timepoints following injury, constitutive PPARγ signaling is thought to be dysregulated, thus releasing the inhibitory brakes on chronically activated microglia. Increasing evidence suggests that thiazolidinediones (TZDs), a class of compounds approved from the treatment of diabetes mellitus, effectively reduce neuroinflammation and chronic microglial activation by activating the peroxisome proliferator-activated receptor-γ (PPARγ). The present study used a closed-head r-mTBI model to investigate the influence of the TZD Pioglitazone on cognitive function and neuroinflammation in the aftermath of r-mTBI exposure. We revealed that Pioglitazone treatment attenuated spatial learning and memory impairments at 6 months post-injury and reduced the expression of reactive microglia and astrocyte markers in the cortex, hippocampus, and corpus callosum. We then examined whether Pioglitazone treatment altered inflammatory signaling mechanisms in isolated microglia and confirmed downregulation of proinflammatory transcription factors and cytokine levels. To further investigate microglial-specific mechanisms underlying PPARγ-mediated neuroprotection, we generated a novel tamoxifen-inducible microglial-specific PPARγ overexpression mouse line and examined its influence on microglial phenotype following injury. Using RNA sequencing, we revealed that PPARγ overexpression ameliorates microglial activation, promotes the activation of pathways associated with wound healing and tissue repair (such as: IL10, IL4 and NGF pathways), and inhibits the adoption of a disease-associated microglia-like (DAM-like) phenotype. This study provides insight into the role of PPARγ as a critical regulator of the neuroinflammatory cascade that follows r-mTBI in mice and demonstrates that the use of PPARγ agonists such as Pioglitazone and newer generation TZDs hold strong therapeutic potential to prevent the chronic neurodegenerative sequelae of r-mTBI.

Overexpression of pathogenic tau in astrocytes causes a reduction in AQP4 and GLT1, an immunosuppressed phenotype and unique transcriptional responses to repetitive mild TBI without appreciable changes in tauopathy.

Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAPP301L mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAPP301L model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.

Deletion of PTEN in microglia ameliorates chronic neuroinflammation following repetitive mTBI.

Traumatic brain injury is a leading cause of morbidity and mortality in adults and children in developed nations. Following the primary injury, microglia, the resident innate immune cells of the CNS, initiate several inflammatory signaling cascades and pathophysiological responses that may persist chronically; chronic neuroinflammation following TBI has been closely linked to the development of neurodegeneration and neurological dysfunction. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been shown to regulate several key mechanisms in the inflammatory response to TBI. Increasing evidence has shown that the modulation of the PI3K/AKT signaling pathway has the potential to influence the cellular response to inflammatory stimuli. However, directly targeting PI3K signaling poses several challenges due to its regulatory role in several cell survival pathways. We have previously identified that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), the major negative regulator of PI3K/AKT signaling, is dysregulated following exposure to repetitive mild traumatic brain injury (r-mTBI). Moreover, this dysregulated PI3K/AKT signaling was correlated with chronic microglial-mediated neuroinflammation. Therefore, we interrogated microglial-specific PTEN as a therapeutic target in TBI by generating a microglial-specific, Tamoxifen inducible conditional PTEN knockout model using a CX3CR1 Cre recombinase mouse line PTENfl/fl/CX3CR1+/CreERT2 (mcg-PTENcKO), and exposed them to our 20-hit r-mTBI paradigm. Animals were treated with tamoxifen at 76 days post-last injury, and the effects of microglia PTEN deletion on immune-inflammatory responses were assessed at 90-days post last injury. We observed that the deletion of microglial PTEN ameliorated the proinflammatory response to repetitive brain trauma, not only reducing chronic microglial activation and proinflammatory cytokine production but also rescuing TBI-induced reactive astrogliosis, demonstrating that these effects extended beyond microglia alone. Additionally, we observed that the pharmacological inhibition of PTEN with BpV(HOpic) ameliorated the LPS-induced activation of microglial NFκB signaling in vitro. Together, these data provide support for the role of PTEN as a regulator of chronic neuroinflammation following repetitive mild TBI.

Dexterity and technique in termite fishing by chimpanzees (Pan troglodytes troglodytes) in the Goualougo Triangle, Republic of Congo.

Although the phenomenon of termite fishing by chimpanzees (Pan troglodytes) has historical and theoretical importance for primatology, we still have a limited understanding of how chimpanzees accomplish this activity, and in particular, about details of skilled actions and the nature of individual variation in fishing techniques. We examined movements, hand positions, grips, and other details from remote video footage of seven adult and subadult female chimpanzees using plant probes to extract Macrotermes muelleri termites from epigeal nests. Six chimpanzees used exclusively one hand (left or right) to grip the probe during termite fishing. All chimpanzees used the same repertoire of actions to insert, adjust, and withdraw the probe but differed in the frequency of use of particular actions. Chimpanzees have been described as eating termites in two ways-directly from the probe or by sweeping them from the probe with one hand. We describe a third technique: sliding the probe between the digits of one stationary hand as the probe is extracted from the nest. The sliding technique requires complementary bimanual coordination (extracting with one hand and grasping lightly with the other, at the same time). We highlight the importance of actions with two hands-one gripping, one assisting-in termite fishing and discuss how probing techniques are correlated with performance. Additional research on digital function and on environmental, organismic, and task constraints will further reveal manual dexterity in termite fishing.

Expression and purification of the surface proteins from Andes virus.

Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 µg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.

Repetitive head trauma and apoE4 induce chronic cerebrovascular alterations that impair tau elimination from the brain.

Repetitive mild traumatic brain injuries (r-mTBI) sustained in the military or contact sports have been associated with the accumulation of extracellular tau in the brain, which may contribute to the pathogenesis of neurodegenerative tauopathies. The expression of the apolipoprotein E4 (apoE4) isoform has been associated with higher levels of tau in the brain, and worse clinical outcomes after r-mTBI, though the influence of apoE genotype on extracellular tau dynamics in the brain is poorly understood. We recently demonstrated that extracellular tau can be eliminated across blood-brain barrier (BBB), which is progressively impaired following r-mTBI. The current studies investigated the influence of repetitive mild TBI (r-mTBI) and apoE genotype on the elimination of extracellular solutes from the brain. Following intracortical injection of biotin-labeled tau into humanized apoE-Tr mice, the levels of exogenous tau residing in the brain of apoE4 mice were elevated compared to other isoforms, indicating reduced tau elimination. Additionally, we found exposure to r-mTBI increased tau residence in apoE2 mice, similar to our observations in E2FAD animals. Each of these findings may be the result of diminished tau efflux via LRP1 at the BBB, as LRP1 inhibition significantly reduced tau uptake in endothelial cells and decreased tau transit across an in vitro model of the BBB (basolateral-to-apical). Notably, we showed that injury and apoE status, (particularly apoE4) resulted in chronic alterations in BBB integrity, pericyte coverage, and AQP4 polarization. These aberrations coincided with an atypical reactive astrocytic gene signature indicative of diminished CSF-ISF exchange. Our work found that CSF movement was reduced in the chronic phase following r-mTBI (>18 months post injury) across all apoE genotypes. In summary, we show that apoE genotype strongly influences cerebrovascular homeostasis, which can lead to age-dependent deficiencies in the elimination of toxic proteins from the brain, like tau, particularly in the aftermath of head trauma.

Atopic Dermatitis and Cardiovascular Risk in Pediatric Patients: A Systematic Review and Meta-Analysis.

Cardiovascular guidelines recommend early screening and preventative treatment for children with chronic inflammatory diseases. Atopic dermatitis (AD) is associated with cardiovascular risk in adults, but data in children are limited. We systematically searched for studies that examined the association between childhood AD and cardiovascular risk factors and outcomes. Data from 10 publications, including 577,148 individuals, revealed an association between AD and ischemic heart disease (n = 3, OR = 1.68, 95% confidence interval [CI] = 1.29-2.19) and diabetes (n = 4, OR = 1.31, 95% CI = 1.12-1.53), but this did not persist among studies that adjusted for potential confounders (n = 2, OR = 0.98, 95% CI = 0.35-2.75). Similarly, there was an association with lipid disorders but not across the entire population distribution (n = 7, OR = 1.24, 95% CI = 1.13-1.36, 95% prediction interval = 0.95-1.61). AD was not associated with hypertension (n = 5, OR = 1.15, 95% CI = 0.98-1.34, 95% prediction interval = 0.81-1.62) or stroke (n = 2, OR = 1.24, 95% CI = 0.94-1.62). Studies lacked detail on AD severity and important confounders such as body mass index, and the certainty of evidence was very low to low on the basis of GRADE (Grading of Recommendations, Assessment, Development and Evaluation) assessments. Currently, data do not support a clinically meaningful increase in cardiovascular risk for children with AD.

Social and Political Trust in a Low Trust Society.

We study the causal relationship between social and political trust in a low trust society, a setting where this topic has received very little attention. We focus on contemporary Chile, a relatively consolidated new democracy lacking many of the conditions that fosters trust such as high socioeconomic equality, weak social divisions, or universal welfare policies. Our empirical analysis is based on a 4-wave panel survey applied to a representative sample of 2000 Chilean adults interviewed face to face each year between 2016 and 2019. Based on statistical models with varying specifications and assumptions, we find support for the institutionalist view that claims that political trust exercises a positive influence over social trust. However, contrary to recent findings for some European democracies (Sønderskov & Dinesen 2016, Seifert 2018), we also find that social trust positively affects political trust. Our results suggest that the positive relationship between both types of trust travels to different political settings, and that there is no minimum threshold required in levels of trust for this relationship to emerge.

TGFß signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment.

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-ß (TGFß) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFß receptor 2 (TGFßR2) in lung epithelium were generated and crossed to cell fate reporter mice that express ß-galactosidase (ß-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFßR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFßR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/ß-gal(+)) fibroblasts. Attenuation of TGFß signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.

Metabolomics in endometriosis: challenges and perspectives for future studies.

Endometriosis is a complex disease characterized by inflammation and the growth of endometrial- like glands and stroma outside the uterine cavity. The pathophysiology of endometriosis is not entirely understood, however, with a prevalence of ~10% of women in their reproductive years, the disease symptoms significantly affect the quality of life of millions of women globally. Metabolomic studies have previously identified specific metabolites that could be a signature of endometriosis. This approach could potentially be used as a non-invasive tool for early diagnosis and provide a better understanding of endometriosis pathophysiology. This review aims to provide insight as to how endometriosis affects the metabolome by reviewing different studies that have used this approach to design follow-up studies. The search query included the term 'endometriosis' in combination with 'metabolomics', 'lipidomics', or 'sphingolipidomics' published between 2012 and 2020. We included studies in humans and animal models. Most studies reported differences in the metabolome of subjects with endometriosis in comparison to healthy controls and used samples taken from serum, endometrial tissue, follicular fluid, urine, peritoneal fluid, or endometrial fluid. Statistically significant metabolites contributed to group separation between patients and healthy controls. Reported metabolites included amino acids, lipids, organic acids, and other organic compounds. Differences in methods, analytical techniques, and the presence of confounding factors can interfere with results and interpretation of data. Metabolomics seems to be a promising tool for identifying significant metabolites in patients with endometriosis. Nonetheless, more investigation is needed in order to understand the significance of the study results. LAY SUMMARY: Endometriosis is a chronic disease affecting the quality of life in one out of every ten women during their reproductive years, causing pain and infertility. It is characterized by inflammation and growth of tissue like the endometrium (uterus lining) outside the uterine cavity. Studies have searched for a predictor of endometriosis-associated changes by observing small molecules necessary for metabolism on a large scale (metabolomics). Metabolomics could serve to resolve one of the biggest challenges that patients with endometriosis face: a delay in diagnosis. In this review, the authors summarize identified potential biomarkers from various bodily fluids and tissues that are characteristic of metabolic processes observed in endometriosis. Biomarkers include cell growth, cell survival, high energy demand, oxidative stress, and fatty acid levels. A metabolomics approach offers promise as a non-invasive tool to identify significant metabolite changes in patients with endometriosis, potentially leading to earlier diagnoses and new opportunities for back-translational strategies.

Effectiveness of an intervention to reduce stigma towards people with a severe mental disorder diagnosis in primary health care personnel: Programme Igual-Mente.

This study assessed the effectiveness of a program (called Igual-Mente, Equal-Mind) designed to reduce stigma in primary health care personnel. A random clinical trial was performed (ISRCTN46464036). There were 316 primary care professionals and technicians who were randomized and assigned to the experimental or control group. The program considered as strategies the education, the contact and the development of skills. There were six sessions with the primary care staff and two sessions with the managers of the health centers. It was executed by two facilitators, a professional psychologist and an expert by experience, i.e., a person diagnosed with a severe mental disorder (SMD). Attitudes, social distance, and humane treatment behaviors toward people with SMD were assessed. The intervention was effective in reducing stigma attitudes y social distance towards people diagnosed with SMD. The magnitude of the changes ranged from moderate to high in all these variables and the effects were maintained for four months after the end of the program. Regarding humane treatment behaviors, the effects were less clear. This study shows good results indicating that well-designed interventions can effectively reduce stigma towards people diagnosed with SMD, which is one of the main challenges of health systems.

Psychometric Properties of the Patient Health Questionnaire-9 in Elderly Chilean Primary Care Users.

Background: This study aimed to assess the measurement properties (reliability, factor structure, and criterion validity) of the Patient Health Questionnaire (PHQ-9) as an instrument for screening major depressive disorder (MDD) in elderly primary care users in Chile. Method: About 582 participants aged between 65 and 80 years were enrolled from primary care centers. They completed the Composite International Diagnostic Interview (CIDI), a survey with sociodemographic characteristics and the PHQ-9. Results: The PHQ-9 revealed an acceptable internal consistency (ω = 0.79 [95% CI: 0.75-0.80] and α = 0.78 [95% CI: 0.75-0.81]); confirmatory factor analysis demonstrated a good fit for both 1- and 2-factor solutions. The chi-square difference test (χ2 = 0.61, gl = 1, p = 0.43) and correlation between the somatic and the cognitive-effective latent factors were very high (r = 0.97, p < 0.001), indicating that the 1 factor model was more parsimonious. Utilizing the CIDI as the gold standard, the area under the curve (AUC) was 0.88 (SE = 0.04, 95% CI: 0.84-0.90). The optimal cut-off score of ≥ 6 yielded good sensitivity and specificity for detecting MDD (0.95 and 0.76, respectively). However, considering the clinical utility index, the cut-off score of ≥9 proved to be a more effective marker for discarding cases of MDD. Conclusion: The PHQ-9 has adequate psychometric properties for elderly primary care users. In clinical settings, it showed its greatest utility in ruling out the presence of an MDD, however, its clinical value for identifying possible cases of MDD is limited. In cases above the cut-off point, it is recommended to perform a more thorough evaluation.

Standardization of a colorimetric technique for determination of enzymatic activity of diamine oxidase (DAO) and its application in patients with clinical diagnosis of histamine intolerance.

BACKGROUND: Diamine Oxidase (DAO) has an essential role for degradation of exogenous histamine in the intestine; thus, histamine intolerance (HI) mainly has been correlated to a low concentration and/or activity of this enzyme. The objective of the study was to standardize a colorimetric technique to measure the enzymatic activity (function) of hDAO to then apply it to a series of 22 patients with a clinical diagnosis of HI. METHODS: For the standardization variables such as volume and type of sample, incubation time, wavelength of maximum absorption, types of substrates, and concentration of oxidized ascorbate were evaluated. Then the activity and concentration of DAO was determined in 22 patients diagnosed with HI and 22 healthy subjects. RESULTS: The mean of serum DAO concentration in the 22 patients was of 9.268 ± 1.124 U/mL. The mean of serum DAO concentration in the 22 controls was of 20.710 ± 2.509 U/mL, being significantly higher (P value 0.0002) the mean of the samples. The mean of serum DAO activity of the patients was of 1.143 ± 0.085 U/L and the controls was 1.533 ± 0.119 U/L, significantly greater than the patients (P value 0.011). In addition, the sensitivity of both techniques was 0.63. In the measuring of DAO concentration the specificity was 0.9, constituting a good diagnostic test, especially to rule out the true negatives. The determination of DAO activity had a specificity of 0.68. CONCLUSIONS: Although we used a small number of patients and controls and the absorbance values were lower than expected, statistically significant differences were found in the levels of concentration and DAO activity between the patients with histamine intolerance and the controls. Therefore, the measuring of DAO concentration and DAO activity is a good diagnostic strategy for study suspect cases of HI. The simultaneous use of both assays allows to reduce positive and negative false results, for example, patients with normal DAO levels that could present a dysfunction in the activity of this enzyme.

Use of TEVI-R to describe passive vocabulary in children belonging to a Mapuche community. / Uso del TEVI-R para describir el vocabulario pasivo en niños de la etnia Mapuche.

PURPOSE: To describe the level of passive vocabulary in boys and girls belonging to the Mapuche ethnic group, using the Vocabulary Test in Images Revised version (TEVI-R). METHODS: A cross-sectional study was carried out. Twenty-six children, both gender, between 4 to 7 years old participated in the study. The performance of passive vocabulary was measured through the application of the TEVI-R, analyzing the variables age and gender, as well as performing an analysis of the items and the number of errors. RESULTS: The performance of these children is not influenced by gender or age. There are potential sources of error in the items of the instruments related to the cultural, geographical relevance and graphic quality of them. CONCLUSION: No association was observed by gender or differences by age in the study population. The possibility of developing new instruments or revising the available ones is discussed, given the characteristics of their native language, obtaining reliable results and respecting the elements that are part of their culture.

OBJETIVO: Describir el nivel de vocabulario pasivo en niños y niñas pertenecientes a la etnia mapuche, utilizando el Test de Vocabulario en Imágenes versión Revisada (TEVI-R). MÉTODO: Se llevó a cabo un estudio de corte transversal. Participaron 26 niños, de ambos géneros cuyas edades fluctuaron entre 4 y 7 años de edad. Se midió el desempeño de vocabulario pasivo mediante la aplicación del TEVI-R, analizando las variables edad y género, además de realizar un análisis de los ítems con mayor cantidad de errores. RESULTADOS: El rendimiento de estos niños no se ve influenciado ni por género, ni por edad. Existen potenciales fuentes de error en los ítems de los instrumentos relacionados a la pertinencia cultural, geográfica y a la calidad gráfica de los mismos. CONCLUSIÓN: No se observó asociación por género ni diferencias por edad en la población estudiada. Se discute la posibilidad de elaborar nuevos instrumentos o revisar los disponibles, dadas las características de su lengua materna, con la finalidad de recabar resultados fiables y respetar los elementos que forman parte de los marcadores propios de su cultura.

An Integrative Program to Reduce Stigma in Primary Healthcare Workers Toward People With Diagnosis of Severe Mental Disorders: A Protocol for a Randomized Controlled Trial.

Background: People with severe mental disorders (SMDs) have higher disease and death rates than the general population. Stigma (negative attitudes and perceptions) contributes to limited access to health services and a lower quality of medical assistance in this population, and it is manifested as negative attitudes, social distance, and discrimination toward this social group. For these reasons, healthcare workers are a priority group for anti-stigma interventions. This study aims to assess the effectiveness of a program specifically designed to decrease negative attitudes and social distance and increase inclusive behaviors in healthcare workers toward people with SMD. Methods: The study will be a randomized clinical trial. A minimum of 210 healthcare workers from 11 primary care centers in the province of Concepción, Chile, will be randomly chosen to receive the program or be part of the control group. There will be a pre-, post-, and 4-months evaluation of social distance, attitudes, and behaviors of participants toward people with SMD using standardized scales such as the social distance scale, which is a scale of clinician attitude toward mental illness adapted from attitudes of clinicians toward mental illness, and self-reports. The intervention program will consist of education strategies, direct, and indirect contact with people diagnosed with SMD, and skill development. There will be six face-to-face sessions directly with the participants and two additional sessions with the directors of each healthcare center. The program will involve a facilitator who will be a healthcare professional and a co-facilitator who will be a person diagnosed with SMD. Discussion: This study will evaluate an intervention program especially designed to reduce stigma in healthcare workers toward people with SMD, a topic on which there is little background information, particularly in low- and middle-income countries. It is important to have interventions with proven effectiveness for this purpose to ensure equity in healthcare services. Trial Registration: This study was registered under ISRCTN.com (ISRCTN46464036).