Building evidence for improving vaccine adoption and uptake of childhood vaccinations in low- and middle-income countries: a systematic review.

Vaccine coverage for children is an important indicator of the performance of national health and immunization systems. Most of the existing literature has targeted mothers' low educational level, living in underserved districts and/or remote rural areas and economic poverty that are correlated with low immunization coverage but the supply- and demand-side constraints to immunization in low- and middle- income countries (LMICs) are not well understood. The reliability of claimed administrative immunization coverage in these contexts is questionable. To address these barriers within the present Expanded Programme on Immunization (EPI), the difficulties related to inadequate vaccination uptake must be addressed in more depth. Building on already produced literature, this study aims to determine the extent of immunization coverage among children in LMICs, as well as to fill in the gaps in awareness about system-level obstacles that currently hinder the effective delivery and uptake of immunization services through EPI. By two reviewers, a literature search using PubMed and Google Scholar along with targeted grey literature was conducted on the 2nd of June 2021 by following PRISMA guidelines. The search techniques for electronic databases used both Medical Subject Headings (Mesh) and free-text words were tailored to each database's specific needs using a controlled vocabulary that was limited to the English language from 2000 and 2020. Of the 689 records, eleven articles were included in this review meeting the inclusion criteria. In total, five articles related to vaccination coverage, four studies on components of the routine immunization system, one article on the implementation of new and under-utilized vaccines and one were on vaccines financing. We evaluated the quality of the included studies and extracted into tables created by one investigator and double-checked by another. Review findings suggest that specific strategies to reduce inequality may be required. Vaccine procurement and pricing strategies, as well as vaccine customization to meet the needs of LMICs, are all critical components in strengthening immunization systems. Our findings could be used to establish practical strategies for countries and development partners to address coverage gaps and improve vaccination system effectiveness. Supplementary Information: The online version contains supplementary material available at 10.1007/s40267-021-00890-7.

Point-of-care ultrasound to confirm endotracheal tube cuff position in relationship to the cricoid in the pediatric population.

BACKGROUND: Anatomically, the subglottic area and the cricoid ring are the narrowest portions of the larynx. To limit the potential for damage related to mucosal pressure injuries from the presence of an endotracheal tube, the cuff should be placed below the cricoid in children. Previously, no clinical or imaging method has been used in real time to determine the exact location of the endotracheal tube cuff after endotracheal intubation. Point-of-care ultrasound may provide an option as a safe and rapid means of visualizing the endotracheal tube cuff and its relationship to the cricoid ring thereby achieving ideal endotracheal tube cuff positioning-below the cricoid. METHODS: In this prospective, nonrandomized trial, point-of-care ultrasound was used following endotracheal intubation in children to evaluate the position of the endotracheal tube cuff in relationship to the cricoid and tracheal rings. After anesthesia was induced and the trachea was intubated, the endotracheal tube cuff and its position in relation to the cricoid and tracheal rings were identified in the longitudinal plane using point-of-care ultrasound. With the patient's neck in a neutral position, the level of the proximal (cephalad) margin of the saline-filled cuff of the endotracheal tube was identified and recorded in relationship to the cricoid and tracheal rings. The ideal position is defined as the cephalad margin of the endotracheal tube cuff below the level of the cricoid. RESULTS: The study cohort included 80 patients, ranging in age from 1 to 78 months. In all patients, the cuff of the ETT, cricoid, and tracheal rings were identified. The cephalad end of the endotracheal tube cuff was found at the level of the cricoid in 16.3% of patients, at the first tracheal ring in 27.5% of patients, at the second tracheal ring in 23.8% of patients, at the third tracheal ring in 17.5% of patients, and at below the fourth tracheal ring in 15% of patients. Initial endotracheal tube cuff position had no significant association with age, height, weight, endotracheal tube size, and endotracheal tube type. CONCLUSION: Point-of-care ultrasound provides a rapid and effective means of identifying the position of the endotracheal tube cuff in relationship to the cricoid ring. The technique may have applications in the perioperative arena, emergency departments, and intensive care units.

Attitudes and practices of postgraduate medical trainees towards research--a snapshot from Faisalabad.

OBJECTIVE: To assess the attitudes and practices of postgraduate medical trainees towards research. METHODS: It was a self-administered questionnaire based cross-sectional survey conducted on 55 conveniently selected trainees in Allied Hospital, Faisalabad. RESULTS: Only 11 trainees read journals monthly, seven had written an article for a journal, 51 regarded reading literature important, 39 intended to engage in future research and 37 said they received inappropriate research training. The major reasons cited for poor research activity in Pakistan were poor research training and awareness. CONCLUSION: Though the attitudes towards research were positive, they were deficient practically in terms of reading and writing literature. There is an immediate need to improve research training in our educational institutes to facilitate the development of the local literature both in terms of research utilization and production

Cardiovascular health--behaviour of medical students in Karachi.

OBJECTIVE: To assess the health behaviour and perceptions of medical students towards cardiovascular disease. METHODS: This descriptive cross-sectional survey was conducted on 203 Pakistani medical students enrolled in a private medical college in Karachi, Pakistan using systematic random sampling. It was based on a self-administered questionnaire. RESULTS: Eight percent smoked, 9% were overweight, 33% had a family history of coronary artery disease, 32% regulated dietary fat intake, 28% exercised regularly, 62.1% knew personal blood pressure and 5.4% personal cholesterol levels. Regarding developing cardiovascular disease in the future, 62% showed concern but only 54% of these adopted preventive practices. About 46% believed medical college life had a harmful effect on their health. Gender, family history and personal health status perception were behavior modifying influences (p<0.05). Reasons reported for their behavior were: no need of prevention at their age (38.3%) and never thinking about these issues (37.0%). CONCLUSIONS: The study shows a high prevalence of coronary artery disease family history, inappropriate dietary intake, physical inactivity; poor screening practices and lack of awareness. The results underscore the urgent need to promote preventive knowledge and practices among medical students, if they are to become prevention oriented physicians and counsel patients on preventive strategies to counter the rapidly increasing burden of cardiovascular diseases effectively.

Down-regulation of beta-catenin TCF signaling is linked to colonic epithelial cell differentiation.

The beta-catenin TCF pathway is implicated in the regulation of colonic epithelial cell proliferation, but its role in the regulation of cell differentiation is unknown. The colon carcinoma cell line, Caco-2, spontaneously undergoes G(0)/G(1) cell cycle arrest and differentiates along the absorptive cell lineage over 21 days in culture. In parallel, we show that beta-catenin-TCF activity and complex formation are significantly down-regulated. The down-regulation of beta-catenin-TCF signaling was independent of APC, which we characterized as having a nonsense mutation in codon 1367 in Caco-2 cells, but was associated with a decrease in TCF-4 protein levels. Total beta-catenin levels increased during Caco-2 cell differentiation, although this was attributable to an increase in the membrane, E-cadherin-associated, fraction of beta-catenin. Importantly, down-regulation of beta-catenin-TCF signaling in undifferentiated Caco-2 cells by three different mechanisms, ectopic expression of E-cadherin, wild-type APC, or dominant negative TCF-4, resulted in an increase in the promoter activities of two genes that are well-established markers of cell differentiation, alkaline phosphatase and intestinal fatty acid binding protein. These studies demonstrate, therefore, that in addition to its established role in the regulation of cell proliferation, down-regulation of the beta-catenin-TCF pathway is associated with the promotion of a more-differentiated phenotype in colonic epithelial cells.

The Sp family of transcription factors in the regulation of the human and mouse MUC2 gene promoters.

Modulation of mucin gene expression is an important component both in the early steps of colon cancer development and in later tumor progression. Previous work from our laboratory and others has suggested that the Sp family of transcription factors may play an important role in the regulation of the human MUC2 gene. To determine whether this was an essential element, we extended our work to the cloning and analysis of 3.5 kb of the 5'-flanking region of the mouse Muc2 (mMuc2) gene. Comparative analysis between the mouse and human MUC2 promoter regions has identified a strong sequence homology between the mouse and human genes, including the presence of GC-rich boxes, the location and composition of which are maintained in the mouse and human genes. We show that these GC boxes are binding sites for Sp-family transcription factors and are functionally important since mithramycin, an inhibitor of Sp1/Sp3 binding, blocks MUC2 gene expression in HT29 cells. Furthermore, by a combination of gel shift analysis and site-directed mutagenesis, we have identified the relative contribution of individual GC boxes, and of the factors they bind, to the regulation of the mouse Muc2 promoter, which appears to be different in the mouse and human genes. Finally, we demonstrate by overexpressing Sp1 and Sp3 that the functional difference between the proximal promoter region of the MUC2 gene in the two species is not attributable to differential ability of this region to bind members of the Sp family of transcription factors, but rather to the different anatomy of the individual GC boxes in the mouse and human proximal promoters.

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

The cell cycle regulator p27kip1 contributes to growth and differentiation of osteoblasts.

The cyclin-dependent kinase (cdk) inhibitors are key regulators of cell cycle progression. p27 and p21 are members of the Cip/Kip family of cdk inhibitors and regulate cell growth by inactivating cell cycle stage-specific CDK-cyclin complexes. Because down-regulation of osteoprogenitor proliferation is a critical step for osteoblast differentiation, we investigated expression of p27 and p21 during development of the osteoblast phenotype in rat calvarial osteoblasts and in proliferating and growth-inhibited osteosarcoma ROS 17/2.8 cells. Expression of these proteins indicates that p21, which predominates in the growth period, is related to proliferation control. p27 levels are maximal postproliferatively, suggesting a role in the transition from cell proliferation to osteoblast differentiation. We directly examined the role of p27 during differentiation of osteoprogenitor cells derived from the bone marrow (BM) of p27-/- mice. BM cells from p27 null mice exhibited increased proliferative activity compared with BM cells from wild-type mice and formed an increased number and larger size of osteoblastic colonies, which further differentiated to the mineralization stage. Although p27-/- adherent marrow cells proliferate faster, they retain competency for differentiation, which may result, in part, from observed higher p21 levels compared with wild type. Histological studies of p27-/- bones also showed an increased cellularity in the marrow cavity compared with the p27+/+. The increased proliferation in bone does not lead to tumorigenesis, in contrast to observed adenomas in the null mice. Taken together, these findings indicate that p27 plays a key role in regulating osteoblast differentiation by controlling proliferation-related events in bone cells.

AP-1 and vitamin D receptor (VDR) signaling pathways converge at the rat osteocalcin VDR element: requirement for the internal activating protein-1 site for vitamin D-mediated trans-activation.

Responsiveness of genes to steroid hormones is a complex process involving synergistic and/or antagonistic interactions between specific receptors and other nonreceptor transcription factors. Thus, DNA recognition elements for steroid hormone receptors are often located among binding sites for other trans-acting factors. The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, stimulates transcription of the tissue-specific osteocalcin (OC) gene in osteoblastic cells. The rat OC vitamin D response element contains an internal acitvating protein-1 (AP-1) site. Here, we report for the first time that this AP-1 site is critical for the transcriptional enhancement of rat osteocalcin gene expression mediated by vitamin D. Precise mutations were introduced either in the steroid half-elements or in the internal AP-1 sequences. One mutation within the internal AP-1 site retained vitamin D receptor/retinoid X receptor binding equivalent to that of the wild-type sequence, but resulted in complete loss of vitamin D inducibility of the OC promoter. These results suggest a functional interaction between the hormone receptor and nuclear oncoproteins at the rat OC vitamin D response element. This cooperation of activities may have important consequences in physiological regulation of osteocalcin transcription during osteoblast differentiation and bone tissue development in vivo.

Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts.

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.

Multiple levels of steroid hormone-dependent control of osteocalcin during osteoblast differentiation: glucocorticoid regulation of basal and vitamin D stimulated gene expression.

We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts.

Species-specific glucocorticoid and 1,25-dihydroxyvitamin D responsiveness in mouse MC3T3-E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression.

The mouse MC3T3-E1 cell line is nontumorigenic and undergoes a typical program of osteoblast differentiation in vitro, producing a bone-like mineralized extracellular matrix. We report responses of these cells to dexamethasone (Dex) and 1,25-(OH)2D3 that are in contrast to findings from other osteoblast culture systems. First, chronic exposure of both early- and late-passaged MC3T3-E1 cells to 10(-7) M Dex, initiated during the proliferation period, blocked osteoblast differentiation, in contrast to the enhanced differentiation observed in cultures of fetal rat calvarial-derived cells. Secondly, 1,25-(OH)2D3 did not up-regulate expression (messenger RNA or protein synthesis) of the endogenous mouse osteocalcin (OC) gene. Several lines of evidence are presented that suggest this response is caused by sequence specific properties of the mouse OC vitamin D response element. We also observed both qualitative and quantitative differences in expression of cell growth (histone H2B) and phenotype-related genes (collagen, OC, osteopontin, glucocorticoid receptor, and 1, 25-(OH)2D3 receptor), between pre- and postmineralization stage osteoblasts, in response to 24 h steroid hormone treatment. Our findings in MC3T3-E1 cells are consistent with current concepts of selective influences of 1,25-(OH)2D3 and glucocorticoids as a function of osteoblast maturation. However, the inhibition of osteoblast differentiation by chronic Dex at 10(-7) M and the down-regulation of OC by 1,25-(OH)2D3 are novel observations relevant to species-specific responsiveness of mouse bone-expressed genes to steroid hormones during osteoblast differentiation.

YY1 regulates vitamin D receptor/retinoid X receptor mediated transactivation of the vitamin D responsive osteocalcin gene.

The responsiveness of genes to steroid hormones is principally mediated by functional interactions between DNA-bound hormone receptors and components of the transcriptional initiation machinery, including TATA-binding protein, TFIIB, or other RNA polymerase II associated factors. This interaction can be physiologically modulated by promoter context-specific transcription factors to facilitate optimal responsiveness of gene expression to hormone stimulation. One postulated regulatory mechanism involves the functional antagonism between hormone receptors and nonreceptor transcription factors interacting at the same hormone response element. Here we demonstrate that the multifunctional regulator YY1 represses 1,25-dihydroxyvitamin D3 (vitamin D)-induced transactivation of the bone tissue-specific osteocalcin gene. We identify YY1 recognition sequences within the vitamin D response element (VDRE) of the osteocalcin gene that are critical for YY1-dependent repression of vitamin D-enhanced promoter activity. We show that YY1 and vitamin D receptor (VDR)/retinoid X receptor heterodimers compete for binding at the osteocalcin VDRE. In addition, we find that YY1 interacts directly with TFIIB, and that one of the two tandemly repeated polypeptide regions of TFIIB spanning the basic domain is responsible for this interaction. TFIIB and VDR can also interact directly, and these factors synergize to mediate transactivation. Our results suggest that YY1 regulates vitamin D enhancement of osteocalcin gene transcription in vivo by interfering with the interactions of the VDR with both the VDRE and TFIIB.

Bone tissue-specific transcription of the osteocalcin gene: role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box.

Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.