Improving emergency obstetric care and reversing the underutilisation of vacuum extraction: a qualitative study of implementation in Tete Province, Mozambique.

BACKGROUND: Maternal and perinatal mortality in Mozambique were declining at a slow pace, despite progress in coverage of institutional childbirth. Implementation of quality emergency obstetric care including vacuum extraction remained inadequate. In 2015-2017, Tete Province achieved remarkable progress in improving emergency obstetric care and reversing the underutilisation of vacuum extraction, with encouraging results for maternal and perinatal outcomes, despite severe resource constraints. This paper presents the experience of Tete Province, generating a rich, contextualised understanding, which might provide generalizable insights and lessons. METHODS: This qualitative study design is used to present Tete's experience in improving emergency obstetric care and reversing the underutilisation of vacuum extraction, drawing on principles from implementation science and applying a systems thinking approach. Sources include routine data, documents, social media messages, and the lived experience of the authors, all intimately involved in the implementation process during 2014-2017. Iterative learning and analysis, involving all authors, led to the final interpretations. RESULTS: Within a context of severe resource constraints, Tete applied 4 interventions (training, accreditation, audit, monitoring and evaluation with feedback) to improve the implementation of emergency obstetric care. Considerable progress was achieved in vacuum extraction and other signal functions of emergency obstetric care and in the decision-making process for caesarean sections, contributing to important reductions in the provincial institutional maternal mortality and stillbirth rates. Facilitating factors include attributes of the vacuum extraction itself, of the structural and organisational environments in which it was introduced, of the people involved in implementation, and of the process through which the implementation was rolled-out. CONCLUSIONS: The lessons from implementation science and systems thinking can contribute to surprising results in the improvement of emergency obstetric care including the use of vacuum extraction, even in a severely resource-constrained setting. The creation of conditions for real change, with empowerment of the staff and managers at the front-line of day-to-day practice in Tete may inspire others in similar conditions and circumstances. The underutilisation of vacuum extraction in middle- and low-income countries is indeed a missed opportunity. Its reversion is possible and provides a good chance to make considerable difference in maternal and perinatal outcomes.

Effect of 1,25(OH)2D3 on BALB/c mice infected with Leishmania mexicana.

The most active metabolite of vitamin D, 1,25(OH)2D3 is a steroid hormone implicated in a wide range of cell functions such as differentiation, proliferation and apoptosis. Leishmania mexicana causes two kinds of cutaneous leishmaniasis: localized or diffuse. In this work we explored the effect of treatment of 1,25(OH)2D3 on a susceptible leishmaniasis mice model. A significant reduction in the lesion size was found in animals treated with 1,25(OH)2D3. Well preserved tissue and presence of large numbers of eosinophils and fibroblasts was found in the group treated with 1,25(OH)2D3. By contrast, destroyed epidermis was observed with large amount of neutrophils and epithelioid macrophages, on infected groups without 1,25(OH)2D3 treatment. The production of pro-inflammatory cytokines in mice infected and treated with 1,25(OH)2D3 was lower than the animals infected without 1,25(OH)2D3 treatment. Interestingly, there were no differences in the number of parasites in both groups. Finally, the amount of collagen was higher in animals with treatment compare with animals without 1,25(OH)2D3 treatment. In summary, mice treated with 1,25 (OH) 2D3 reflect a healing process without elimination of L. mexicana.

Polymorphisms of innate immunity receptors in infection by parasites.

The innate immune system is the first line of defence against infection by pathogenic bacteria, virus and parasites and is also responsible for initiating an adaptive immune response. In contrast to the receptors of adaptive immunity (TCRs and antibodies) which are generated by gene recombination, receptors of the innate immune system are encoded in the germline and are thus inherited from generation to generation. Although evolutionarily selected, the genes encoding the innate recognition receptors show variations among individuals, and these polymorphisms may have an impact on the ability of an individual to deal with an infection. In recent years, several polymorphisms have been identified in innate recognition receptors, and efforts are being made to determine whether these polymorphisms are associated with a higher or lower susceptibility to infectious diseases. These studies will allow a better understanding of the role of innate receptors in specific diseases and are valuable in the design of preventive or therapeutic interventions to fight the disease. In this review, we summarize studies aimed at determining the influence of polymorphisms in innate recognition receptors on the susceptibility to diseases caused by parasites.

Mast cells are activated by Leishmania mexicana LPG and regulate the disease outcome depending on the genetic background of the host.

The regulatory effect of mast cells on the pathogenesis of leishmaniasis is unclear. We report a comparative analysis of TLR2 membrane expression, TNF-α, IL-10 and MIP-1α production, and granule release of bone marrow-derived mast cells (BMMCs) from susceptible BALB/c and resistant C57BL/6 mice, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG). We studied the kinetics of mast cell degranulation and parasite numbers in lesions of both mouse strains infected with L. mexicana. We found that BMMCs of C57BL/6 mice expressed more TLR2 and produced higher levels of both cytokines and MIP-1α, whereas BALB/c BMMCs significantly augmented their granule release. Lesions of BALB/c mice showed higher levels of degranulated mast cells at 3 h of infection, whereas after 3 days of infection, the number of degranulated mast cells in C57BL/6 was higher than in BALB/c lesions. Throughout infection, BALB/c mice harboured more parasites. The regulatory effect of mast cells seems to depend on the genetic background of the host: mast cells of BALB/c mice facilitate disease progression due to an augmented inflammatory response early in the infection, whereas mast cells of C57BL/6 mice produce cytokines that regulate inflammation and maintain an elevated number of immune cells in the lesions, promoting disease control.

Agrobacterium-mediated transformation of the terpenoid indole alkaloid-producing plant species Tabernaemontana pandacaqui.

Plants of the Apocynaceae family produce a wide range of terpenoid indole alkaloids (TIAs) which have important pharmaceutical applications. Studies of the molecular mechanisms controlling TIA biosynthesis may eventually provide possibilities to improve product yield by genetic modification of plants or cell cultures. However, these studies suffer from the lack of transformation/regeneration protocols for Apocynaceae plants. We chose to study the feasibility of Agrobacterium tumefaciens-mediated transformation of Tabernaemontana pandacaqui, because of the availability of an efficient regeneration procedure for this member of the Apocynaceae family. A procedure to produce transgenic T. pandacaqui plants was established, albeit with low efficiency. Transgenic expression was demonstrated of an intron-containing ß-glucuronidase reporter gene and of a gene coding for the TIA biosynthetic enzyme strictosidine synthase from Catharanthus roseus, another Apocynaceae species.

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding.

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells.

Tau protein seems not to be a useful routine clinical marker of axonal damage in multiple sclerosis.

BACKGROUND: The descriptions of early axonal damage in patients with multiple sclerosis (MS) prompted the search of body fluid markers. However, the studies addressing this issue in MS present conflicting results. AIM: To assess the levels of tau protein in patients with definite MS. PATIENTS AND METHODS: Cerebrospinal fluid (CSF) samples from 50 patients with definite diagnosis of MS (33 F, 17 M; mean age: 33.6 years) and from 19 age-matched individuals without organic neurological diseases (11 F, 8 M), entered this study. With regard to the clinical course, the MS patients were classified as follows: 32 relapsing-remitting (RR); two secondary progressive (SP), and four primary progressive (PP). Twelve patients had clinical isolated syndromes (CIS). The mean duration was 36.1 months (range: 15 days to 20 years). Tau protein was measured in the CSF by double antibody sandwich ELISA. RESULTS: The median tau and the cut-off values of the controls were 104.9 and 175.3 pg/mL, respectively. We found that most MS patients presented normal values. In addition, the clinical features - course, duration, Expanded Disability Status Scale (EDSS) value, Poser index of progression, Multiple Sclerosis Severity Score - did not significantly influence the tau levels in the MS group. CONCLUSION: Our study showed similar CSF tau concentrations in MS patients with different clinical characteristics. This suggests that tau protein does not seem to be a useful routine clinical marker of axonal damage.

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants.

The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 monooxygenases, was purified from a cell suspension culture of the higher plant Catharanthus roseus. Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans-cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identify of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animals species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways.

A promoter region that controls basal and elicitor-inducible expression levels of the NADPH:cytochrome P450 reductase gene (Cpr) from Catharanthus roseus binds nuclear factor GT-1.

NADPH:cytochrome P450 reductase (CPR) is essential for the activation of cytochrome P450 enzymes, which are involved in a wide variety of metabolic pathways in plants, including those related to defence responses. In the subtropical plant Catharanthus roseus several cytochrome P450 enzymes operate in the biosynthesis of defence-related terpenoid indole alkaloids (TIAs). In agreement with the importance of CPR in defence, Cpr mRNA levels in C. roseus were found to be enhanced by fungal elicitor preparations that also induce TIA biosynthesis and P450 gene expression. Here we describe the isolation of a C. roseus genomic DNA clone covering the 5' part of the Cpr gene and 1.6-kb of upstream sequences. Mapping of the transcription start site showed the untranslated leader sequence is approximately 280 bp long. To study the control of gene expression by the Cpr promoter, transcriptional fusions between Cpr promoter fragments and the gusA reporter gene were generated and their expression was analyzed in stably transformed tobacco plants. The Cpr promoter fragment extending from -1510 to -8, with respect to the ATG start codon, conferred basal and elicitor-inducible expression on the gusA reporter gene, strongly indicating that the Cpr gene of C. roseus is indeed controlled by this promoter region. Progressive deletion from the 5' end of the promoter to position -632 had little effect on gusA expression. However, deletion to position -366 resulted in a complete loss of basal activity and largely eliminated elicitor-induced expression, indicating that the region from -632 to -366 contains the main transcription-enhancing cis-regulatory sequences. Electrophoretic mobility shift assays with tobacco nuclear extracts showed that binding sites for nuclear factor GT-1 are redundant in the Cpr promoter, but absent from the downstream part of the leader sequence. The presence of strong GT-1 binding sites in the main enhancer region (-632 to -366), is suggestive of a functional role for this factor in basal expression and elicitor responsiveness of the Cpr promoter.

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.