Understanding demand for, and feasibility of, centre-based child-care for poor urban households: a mixed methods study in Dhaka, Bangladesh.

BACKGROUND: Centre-based child-care has potential to provide multiple health and development benefits to children, families and societies. With rapid urbanisation, increasing numbers of low-income women work with reduced support from extended family, leaving a child-care vacuum in many low- and middle-income countries. We aimed to understand perceptions of, and demand for, centre-based child-care in Dhaka, Bangladesh among poor, urban households, and test the feasibility of delivering sustainable centre-based child-care. METHODS: We used sequential mixed methods including a household survey (n = 222) and qualitative interviews with care-givers (n = 16), community leaders (n = 5) and policy-makers (n = 5). We co-produced and piloted a centre-based child-care model over ten-months, documenting implementation. A co-design focus group with mothers, parents' meetings, and qualitative interviews with child-care centre users (n = 5), non-users (n = 3), ex-users (n = 3) and staff (2) were used to refine the model and identify implementation issues. RESULTS: We found 24% (95% CI: 16,37%) of care-givers reported turning-down paid work due to lack of child-care and 84% (95% CI:74, 91%) reported wishing to use centre-based child-care and were willing to pay up to 283 Takka (~$3.30) per month. Adjusted odds of reported need for child-care among slum households were 3.8 times those of non-slum households (95% CI: 1.4, 10). Implementation highlighted that poor households needed free child-care with food provided, presenting feasibility challenges. Meta-inference across quantitative and qualitative findings identified the impact of the urban environment on child-care through long working hours, low social capital and fears for child safety. These influences interacted with religious and social norms resulting in caution in using centre-based child-care despite evident need. CONCLUSION: Sustainable provision of centre-based care that focuses on early childhood development requires subsidy and careful design sensitive to the working lives of poor families, particularly women and must respond to the dynamics of the urban environment and community values. We recommend increased research and policy focus on the evaluation and scale-up of quality centre-based child-care, emphasising early-childhood development, to support low-income working families in urban areas.

5-Fluorouracil incorporation into DNA of CF-1 mouse bone marrow cells as a possible mechanism of toxicity.

Isolated CF-1 mouse bone marrow cells were exposed for 1 hr to 5-fluorouracil (FUra) at concentrations from 1.8 to 50 microM and then washed and suspended in a soft agar growth medium to assess the effect on toxicity (measured as reduction in colony growth compared to control). These data were used to determine specific toxic concentrations ranging from 25 to 90% lethal doses. Subsequent studies examined in parallel the effect of these toxic concentrations of FUra on the possible sites of toxicity including: (a) inhibition of thymidylate synthetase activity using a modified 3H release assay; (b) incorporation of FUra into RNA (FUra-RNA); and (c) incorporation of FUra into DNA (FUra-DNA). Thymidylate synthetase activity was slightly decreased (75% of control) after 1-hr exposure to a 50% lethal dose and was not significantly further reduced as the FUra concentration was increased to an 85% lethal dose. Furthermore, subsequent exposure of FUra-treated cells to a nontoxic thymidine dose (5 microM) failed to reverse toxicity. FUra-RNA increased during 1-hr exposure to increasing concentrations of FUra (25 to 90% lethal doses). Although initially suggesting a relationship between the level of FUra-RNA and toxicity, subsequent studies in cells exposed to FUra in the presence of uridine demonstrated a significantly decreased toxicity while, at the same time, a minimal decrease of FUra in RNA. In contrast, FUra-DNA was significantly decreased in the presence of uridine and correlated with decreased toxicity. In additional subsequent studies, an apparent decrease in subsequent DNA synthesis was observed (measured by 32P or [3H]thymidine incorporation into DNA) as the level of FUra-DNA increased. In conclusion, FUra is demonstrated to be incorporated into DNA of isolated CF-1 mouse bone marrow cells, and the level of FUra-DNA appears to be closely associated with toxicity and inhibition of further DNA synthesis. The parallel studies of thymidylate synthetase activity and FUra-RNA suggest that FUra-DNA may be an unrecognized mechanism of FUra toxicity in these cells.

Dihydrofluorouracil, a fluorouracil catabolite with antitumor activity in murine and human cells.

Dihydrofluorouracil (FUH2), the initial catabolite of 5-fluorouracil (FUra), was examined to determine whether this derivative had antitumor activity or host cell (bone marrow) toxicity. Studies were undertaken with Ehrlich ascites tumor and bone marrow cells isolated from CF-1 mice. Cells were exposed for 1 h either to no drug (control) or to varying concentrations, ranging from 1 to 250 microM, of either FUra, FUH2, or alpha-fluoro-beta-alanine. Cells were then cultured and colony formation was assessed after 10 to 14 days. Ehrlich ascites tumor cells were more sensitive to FUra [50% lethal dose (LD50) = 18 microM] than to FUH2 [LD50 = 50 microM], with no sensitivity to alpha-fluoro-beta-alanine even at 250 microM. Bone marrow cells had a toxicity profile similar to that of FUra (LD50 = 10 microM) but were relatively insensitive to FUH2 (LD50 greater than 250 microM), with no sensitivity to alpha-fluoro-beta-alanine. Subsequent studies examined colony formation of the human breast carcinoma cell line MCF-7 following 1 h exposure to varying concentrations of FUra and FUH2. These cells were less sensitive to both FUra (LD50 approximately 80 microM) and FUH2 (LD50 approximately 350 microM). Initial studies on the mechanism of toxicity of FUH2 demonstrated that this FUra catabolite could produce inhibition of thymidylate synthase activity in Ehrlich ascites tumor cells with a pattern similar to that resulting from exposure to FUra. This is the first study to demonstrate that FUH2 (a quantitatively important catabolite of FUra) is cytotoxic, and it suggests that FUH2 may contribute to the toxicity of FUra in vivo, possibly by being anabolized to FUra.

Alteration of the secondary structure of newly synthesized DNA from murine bone marrow cells by 5-fluorouracil.

The present study evaluates the effects of 5-fluorouracil (FUra) on the structure of newly synthesized DNA purified from bone marrow cells. DNA synthesis was decreased by 30 and 45% of control in the presence of 19 and 100 microM FUra, respectively. Furthermore at these concentrations of FUra, the DNA strand sizes were smaller as determined by alkaline sucrose gradients. Enzymatic digestion of the DNA demonstrated that most of the FUra (greater than 90%) was localized in the internucleotide linkage and not at the chain terminus. As the concentration of FUra was varied, the percentage of FUra at the chain terminus was unchanged, suggesting that the decrease in chain size as well as inhibition of DNA synthesis was not due to chain termination. DNA that had been synthesized in the presence of FUra was shown to fragment after increasing time as demonstrated by alkaline sucrose gradient analysis. This time-dependent fragmentation was associated with an increased number of strand breaks as determined by neutral and alkaline sucrose gradient analysis. A parallel study demonstrated a time-dependent excision of FUra from DNA over this same time period. In summary, these studies demonstrate an association between the excision of FUra from DNA and the changes in secondary structure of newly synthesized DNA.

Potentiation of the activity of 1-beta-D-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: association with enhanced fragmentation of mature DNA.

We have examined the interaction between 1-beta-D-arabinofuranosylcytosine (ara-C) and the macrocyclic lactone protein kinase C activator bryostatin 1 in the human promyelocytic leukemia cell line HL-60. Preexposure of cells to 10 nM bryostatin 1 for 24 h, followed by an additional 24-h incubation with 10 microM ara-C, resulted in greater than additive inhibitory effects toward clonogenic HL-60 cells. In a series of alkaline elution assays, cells preincubated with bryostatin 1 and prelabeled with [3H]thymidine exhibited a significant increase in DNA fragmentation following exposure to ara-C in comparison to cells exposed to ara-C alone. This increase in DNA damage was apparent at both neutral and alkaline pH and was not protein associated. In contrast, studies using cells pulse-labeled with [3H]thymidine immediately before analysis suggested that bryostatin 1 pretreatment did not increase the ability of ara-C to interfere with DNA replicative intermediates. Additional studies demonstrated that the increase in DNA fragmentation induced by bryostatin 1 and ara-C preceded both loss of cell membrane integrity (as determined by trypan blue exclusion) as well as depletion of intracellular ATP and NAD pools. Furthermore, the enhanced inhibitory effects of bryostatin 1 and ara-C toward clonogenic HL-60 cells did not appear to result from the induction of cellular differentiation. Finally, agarose gel electrophoresis of DNA obtained from cells exposed to both bryostatin 1 and ara-C revealed a pattern of integer multiples of 180- to 200-base pair fragments commonly associated with endonucleolytic cleavage; the extent of this fragmentation was considerably greater than that observed in cells exposed to ara-C alone. Taken together, these findings suggest that exposure of HL-60 cells to bryostatin 1 renders them more susceptible to ara-C-related DNA damage and that this phenomenon contributes to the cytotoxic effects of this drug combination. They also raise the possibility that bryostatin 1, perhaps through modulation of intracellular signaling events in leukemic cells, has the capacity to potentiate ara-C-related apoptosis or programmed cell death.

Interpretation of the DermaLab Combo® pigmentation and vascularity measurements in burn scar assessment: an exploratory analysis.

BACKGROUND: The DermaLab Combo® measures pigmentation and vascularity of a burn scar more reliably than the modified Vancouver Scar Scale (mVSS). This study aims to examine how the DermaLab Combo® continuous measurements of pigmentation and vascularity of burns scars relate to the mVSS, a standard clinical scar assessment method; and secondly, to obtain evidence to support the concurrent validity of DermaLab Combo® measurements for pigmentation and vascularity. METHOD: Scar assessments were performed on an index burn scar of 100 subjects using two methods: the mVSS (two raters) and the DermaLab Combo® device (one rater). Using the DermaLab Combo®, measurements of pigmentation and vascularity for the index scar and an adjacent normal skin site were obtained. Indices were generated to represent the scar pigmentation (melanin index, MI%) and scar vascularity (erythema index, EI%) relative to the patient's matched normal skin. Exploratory univariate and bivariate analyses were conducted and the concordance of classification by mVSS score using DermaLab® cut-off values was assessed. RESULTS: For pigmentation, the results suggest a 80% classification concordance for the DermaLab Combo® MI% values into mVSS pigmentation categories (hypopigmentation, normal pigmentation and hyperpigmentation) using two predictors (MI% and EI%) and visually fitted discriminant axis cut-offs. Due to the high degree of overlap of EI% values between the vascularity categories, meaningful classification of EI% values using the mVSS was not possible. CONCLUSION: Quantifying percentage changes in melanin and erythema relative to matched normal skin improved understanding of the DermaLab Combo® pigmentation and vascularity measurements. The DermaLab Combo® pigmentation MI% values were able to be classified into pigmentation categories of the mVSS, and pigmentation classification concordance was further improved with consideration of the scar's DermaLab Combo® vascularity EI% values. The DermaLab Combo® is an objective tool; however, while the measurement provides continuous numerical data that may be useful for identifying change over time in clinical scar monitoring of pigmentation and vascularity, further work will be useful to understand the DermaLab Combo® measurements to optimise the interpretation of these data.

Levels of tumor necrosis factor-alpha (TNF-alpha) and soluble TNF receptors in chronic venous leg ulcers--correlations to healing status.

This study tested the hypothesis that excessive tumor necrosis factor-alpha (TNF-alpha) levels in chronic venous leg ulcers are associated with impaired healing. TNF-alpha was measured by two enzyme-linked immunosorbent assays and a bioassay (KYM-1D4) in paired wound fluid samples collected during the nonhealing and healing phases from 21 human patients with venous leg ulcers. Soluble TNF receptor levels (p55 and p75) were also measured. The levels of immunoreactive TNF-alpha were significantly higher in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers (p < 0.005), whereas the levels of bioactive TNF-alpha were not. Statistical analysis confirmed that TNF-alpha bioactivity relative to the amount of immunoreactive TNF-alpha was downregulated in wound fluid from nonhealing ulcers compared with healing ulcers. The levels of soluble p55 and p75 receptors in wound fluid showed a significant linear correlation (p < 0.001), suggesting a partially coordinated or common regulatory mechanism for the cleavage of transmembrane TNF receptors in chronic venous ulcers in vivo. Although the levels of soluble p75 receptors were significantly higher in nonhealing wound fluid compared with healing wound fluid (p < 0.025), these levels were theoretically inadequate to substantially neutralize the bioactivity of the accompanying TNF-alpha levels on their own. The bioactivity accompanying the elevated levels of immunoreactive TNF-alpha in wound fluid from nonhealing ulcers may have been further down-modulated by an additional mechanism. Because healing was initiated without a significant decline in the level of bioactive TNF-alpha, TNF-alpha-mediated events may not be the key events contributing to the impaired healing seen in chronic venous ulcers.

Combined radiotherapy and chemotherapy in the management of local-regionally advanced vulvar cancer.

PURPOSE: To determine, in a retrospective single institutional study, the role of concurrent radiotherapy and chemotherapy in the treatment of local-regionally advanced vulvar cancer. METHODS AND MATERIALS: From 1984 to 1991, 20 patients with locally extensive primary or recurrent carcinoma of the vulva were treated with initial combined radiotherapy and chemotherapy. Seven patients had Federation Internationale de Gynecologie et d'Obstretrique Stage III disease, 10 had Stage IV disease, and three were treated for recurrent disease. None of these patients were considered candidates for primary radical vulvectomy and groin node dissection. Median radiation doses to regions of microscopic disease and gross tumor were 40 Gy (range 30-54 Gy) and 54 Gy (34-70.4 Gy), respectively. All patients received 2 or 3 cycles of 5-Fluorouracil concurrently with radiotherapy. In addition, five patients received Cis-platinum, and one Mitomycin-C. Median at-risk follow-up interval was 37 months. RESULTS: Ten patients had complete resolution of tumor to initial chemoradiotherapy, and eight of these have remained free of tumor relapse. Eight other patients had partial responses, with tumor bulk reduced by > 50%, while the remaining two patients had local-regionally progressive disease. Six of the patients with partial responses had residual tumor successfully resected, although four subsequently recurred. For the entire group of 20 patients, the actuarial 3- and 5-year local control rates were 48% each, and the corresponding disease-specific survival rates were 59% and 49%. There was a suggestion that better local control was obtained in patients who received gross tumor radiation doses > or = 50 Gy. Skin reaction was the major acute toxicity and responded well to conservative management. Long-term sequalae were limited to skin and subcutaneous atrophy. CONCLUSION: These results indicate that initial combined radiotherapy and chemotherapy is effective in the management of advanced vulvar cancer.

DNA repair following incorporation of 5-fluorouracil into DNA of mouse bone marrow cells.

5-Fluorouracil (FUra) was previously demonstrated to be incorporated into DNA at cytotoxic concentrations in mouse bone marrow cells. Subsequently, we showed that under these conditions FUra exhibited a time-dependent removal from DNA accompanied by a decrease in DNA strand length. In the present study we utilized hydroxyurea to inhibit semiconservative DNA synthesis while monitoring DNA repair by measuring the incorporation of [3H]dThd into double-stranded DNA (DNAds), which can be separated from DNA at the replication fork (DNAss) by benzoylated-naphthoylated-DEAE cellulose. Under these conditions we assessed DNA repair in cells that had previously been exposed for 1 h to varying cytotoxic concentrations of FUra. The results demonstrate an increase in labelling of DNAds with increasing FUra concentrations, with no appreciable increase in incorporation of label into DNAss. In conclusion, this study demonstrates that DNA repair occurs following incorporation of FUra. The failure to repair DNA damage at higher FUra concentrations may have a role in the cytotoxicity of this drug.

Reliability of scar assessments performed with an integrated skin testing device - the DermaLab Combo(®).

BACKGROUND: The DermaLab Combo(®) is a device with potential to make objective measurements of key scar components - pigmentation, vascularity, pliability and thickness. This study assessed the inter-rater and test-retest reliability of these measurements. METHOD: Three raters performed scar assessments on thirty patients with burn scars using the DermaLab Combo(®). Measurements of pigmentation, vascularity, pliability and thickness were made and intra-class correlation coefficients (ICC) were derived for inter-rater and test-retest reliability. RESULTS: Inter-rater reliability was found to be "excellent" in the 'best' and 'worst' areas of the index scar and normal skin for pigmentation (ICC: 0.94-0.98) and thickness (ICC: 0.86-0.96). Test-retest reliability was also "excellent" for pigmentation (ICC: 0.87-0.89) and thickness (ICC: 0.92-0.97) in all areas. Vascularity showed "good" to "excellent" inter-rater reliability (ICC: 0.66-0.84) in all areas however test-retest reliability was "low" (ICC: 0.29-0.42). Test-retest reliability was "excellent" for pliability (ICC: 0.76-0.91). Technical limitations were encountered making measurements in some scars for thickness, and in particular, pliability. CONCLUSION: The DermaLab Combo(®) measured pigmentation, thickness and pliability with "excellent" reliability. If future studies provide protocols to improve test-retest reliability of vascularity measurements and obtain pliability measurements more successfully, the DermaLab Combo(®) will be valuable device for scar assessment.

A modified Vancouver Scar Scale linked with TBSA (mVSS-TBSA): Inter-rater reliability of an innovative burn scar assessment method.

BACKGROUND: Current scar assessment methods do not capture variation in scar outcome across the burn scar surface area. A new method (mVSS-TBSA) using a modified Vancouver Scar Scale (mVSS) linked with %TBSA was devised and inter-rater reliability was assessed. METHOD: Three raters performed scar assessments on thirty patients with burn scars using the mVSS-TBSA. Scoring on pigmentation, vascularity, pliability and height was undertaken for the 'best' and 'worst' areas of each scar. Raters allocated the total body surface area of the scar (%TBSA) to three mVSS categories (<5, 5-10, >10). Intra-class correlation coefficient (ICC) and weighted kappa statistic (kw) were used to assess inter-rater reliability. The data were also analysed for clinically relevant misclassifications between pairs of raters. RESULTS: Total mVSS scores showed 'fair to good' agreement (ICC 0.65-0.73) in the 'best' area of the scar while there was 'excellent' agreement in the 'worst' scar area (ICC 0.85-0.88). The kw of the individual mVSS components ranged from 0.44 to 0.84 and 0.02 to 0.86 for 'best' and 'worst' scar areas, respectively. Determination of scar %TBSA had 'excellent' reliability (ICC 0.91-0.96). Allocation of scar %TBSA to severity category <5 mVSS demonstrated 'good to excellent' reliability (ICC 0.63-0.80) and 'fair to good' reliability (ICC 0.42-0.74) for 5-10 mVSS category. However, misclassifications were observed for the total mVSS score in the 'worst' scar area and the allocation of scar %TBSA in the <5 mVSS category. CONCLUSION: Inter-rater reliability of mVSS scores depends on the severity of the scar area being assessed. The mVSS-TBSA method of allocation of scar %TBSA to two broad mVSS categories, namely <5 and ≥5 mVSS, has 'good to excellent' reliability. The mVSS-TBSA has demonstrated utility for both clinical and research purposes; however, there is potential to misclassify scar outcome in some cases.

Association of TGFß1 and clinical factors with scar outcome following melanoma excision.

All patients with primary cutaneous malignant melanoma undergo surgical excision to remove the tumour, resulting in scar formation. There is marked variation in the aesthetic appearance of scars following surgery but limited knowledge about the genetic factors affecting non-keloid, surgical scar outcomes. This study aimed to investigate the role of known clinical factors and genetic polymorphisms in pigmentation and wound repair genes in non-keloid scar outcome, following melanoma excision. Participants were 202 cases who underwent a standardized scar assessment following surgical melanoma excision and provided a DNA sample. Genetic association analyses between single nucleotide polymorphisms (SNPs) from 24 candidate genes and scar outcome data were performed, controlling for relevant clinical factors. Following adjustment for multiple testing, SNP rs8110090 in TGFß1 was significantly associated with both the primary scar outcome (a combination score reflecting vascularity, height and pliability, p = 0.0002, q = 0.01) and the secondary scar outcome (a combination score reflecting vascularity, height, pliability and pigmentation, p = 0.0002, q = 0.006). The minor allele G was associated with a poorer scar outcome. Younger age, time elapsed since excision, absence of kidney failure and eczema, presence of thyroid problems and infection were also associated with poorer scar outcome and were adjusted for in the final model, along with scar site. Results from this study suggest that genes involved in wound healing may play a role in determining scar outcome. Associations observed between scar outcome and clinical factors reinforce current clinical knowledge regarding factors affecting scarring. Replication studies in larger samples are warranted and will improve our understanding of the underlying mechanisms of scarring, potentially help to identify patients at risk of poor scar outcomes.

Anderson-Fabry disease. Characteristic ultrastructural features in cutaneous blood vessels in a 1-year-old boy.

Electron microscopy of clinically uninvolved skin taken from a 12-month-old male child with biochemically proven angiokeratoma corporis diffusum showed characteristic lamellar lipid deposits within endothelial and perithelial cells of dermal blood vessels. Ultrastructural examination of skin may aid the early identification of males affected with Anderson-Fabry disease long before the development of specific skin lesions.

Leukemia-specific DNA sequences in leukocytes of the leukemic member of identical twins.

The discovery in human leukemic cells of particulate elements encapsulating 70S RNA and RNA-directed DNA polymerase made possible the synthesis of a [(3)H]DNA probe that could detect leukemia-specific sequences in the DNA of normal and leukemic individuals. In an earlier study of a series of unrelated leukemic patients, we established that the nuclear DNA of their leukemic cells contain particle-related sequences that cannot be detected in leukocytes of normal individuals. This result is inconsistent with the virogene concept that demands the inclusion of one complete copy of oncogenic information in the genome of every normal cell. The present study carries this analysis one step further by showing, with two sets of identical twins, that the leukemic member contains particle-related sequences in the DNA of his leukocytes that cannot be detected in the leukocytes of his healthy identical sibling. This finding implies that the additional leukemia-specific information found in the DNA of the leukemic individuals must have been inserted subsequent to fertilization. This outcome argues against the virogene hypothesis or any other etiologic concept that invokes vertical transmission through the germ line of the particle-related information found uniquely in the DNA of leukemic cells.

Effects of bryostatin 1 and rGM-CSF on the metabolism of 1-beta-D-arabinofuranosylcytosine in human leukaemic myeloblasts.

The effects of the protein kinase C activator bryostatin 1, either with or without recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) were examined with respect to the in vitro metabolism of ara-C in leukaemic myeloblasts obtained from 10 patients with acute myelogenous leukaemia (AML). Coincubation of cells with 12.5 x 10(-9) M bryostatin 1 and 10(-5) M ara-C for 4 h resulted in a significant increase in ara-CTP formation (compared to controls) in 6/10 specimens (mean increase 106%; range 38-255%), and no change in the remainder. In contrast, coincubation of cells with 1.25 ng/ml rGM-CSF resulted in a significant increase in only one specimen, and decreases in two. Bryostatin 1 also significantly increased ara-C DNA incorporation in 6/9 evaluable samples, including two which did not display an increase in ara-CTP formation. Coincubation of cells with both bryostatin 1 and rGM-CSF did not lead to a further increase in ara-CTP formation or ara-C DNA incorporation compared to values obtained with either agent alone. Finally, exposure of blasts to bryostatin 1 for 24 h before ara-C led to an increase in ara-CTP formation in 3/8 additional specimens, and a decrease in one sample displaying evidence of bryostatin 1-induced macrophage differentiation. Incubation of cells with both rGM-CSF and bryostatin 1 for this period resulted in ara-CTP levels equivalent to those obtained with bryostatin 1 alone. These studies indicate that while bryostatin 1 exerts a heterogeneous effect on ara-C metabolism in leukaemic myeloblasts, it is capable of potentiating ara-C phosphorylation in a subset of patient samples, including some that do not exhibit an increase in response to rGM-CSF. They also raise the possibility that bryostatin 1-induced potentiation of ara-C metabolism in some leukaemic cells may contribute, at least in part, to the antileukaemic efficacy of this drug combination.