Fetal and post-natal outcomes in offspring after intrauterine metformin exposure: A systematic review and meta-analysis of animal experiments.

AIMS: The impact of maternal metformin use during pregnancy on fetal, infant, childhood and adolescent growth, development, and health remains unclear. Our objective was to systematically review the available evidence from animal experiments on the effects of intrauterine metformin exposure on offspring's anthropometric, cardiovascular and metabolic outcomes. METHODS: A systematic search was conducted in PUBMED and EMBASE from inception (searched on 12th April 2023). We extracted original, controlled animal studies that investigated the effects of maternal metformin use during pregnancy on offspring anthropometric, cardiovascular and metabolic measurements. Subsequently, risk of bias was assessed and meta-analyses using the standardized mean difference and a random effects model were conducted for all outcomes containing data from 3 or more studies. Subgroup analyses were planned for species, strain, sex and type of model in the case of 10 comparisons or more per subgroup. RESULTS: We included 37 articles (n = 3133 offspring from n = 716 litters, containing n = 51 comparisons) in this review, mostly (95%) on rodent models and 5% pig models. Follow-up of offspring ranged from birth to 2 years of age. Thirty four of the included articles could be included in the meta-analysis. No significant effects in the overall meta-analysis of metformin on any of the anthropometric, cardiovascular and metabolic offspring outcome measures were identified. Between-studies heterogeneity was high, and risk of bias was unclear in most studies as a consequence of poor reporting of essential methodological details. CONCLUSION: This systematic review was unable to establish effects of metformin treatment during pregnancy on anthropometric, cardiovascular and metabolic outcomes in non-human offspring. Heterogeneity between studies was high and reporting of methodological details often limited. This highlights a need for additional high-quality research both in humans and model systems to allow firm conclusions to be established. Future research should include focus on the effects of metformin in older offspring age groups, and on outcomes which have gone uninvestigated to date.

Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA-1), comparison with P. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion.

Apical membrane antigen 1 is a candidate vaccine component for malaria. It is encoded by a single copy gene and has been characterised in a number of malaria species as either an 83-kDa de novo product (Plasmodium falciparum; Pf AMA-1) or a 66-kDa product (all other species). All members of the AMA-1 family are expressed during merozoite formation in maturing schizonts and are initially routed to the rhoptries. Processed forms may subsequently be associated with the merozoite surface. Because of the unique occurrence of the 83-kDa form in P. falciparum we were interested to determine whether the phylogenetically closely related chimpanzee malaria Plasmodium reichenowi shared characteristics with Pf AMA-1. Here we show that the molecular structure, the localisation and processing are similar to that of Pf AMA-1 and that in vitro growth inhibitory mAbs reactive with Pf AMA-1 also inhibit P. reichenowi growth in an in vitro assay. Polymorphism in the 83-kDa AMA-1 family was analysed through comparison of Pr ama-1 with Pf ama-1 alleles, which showed the most significant evidence for selection maintaining polymorphism in Domains I-III of AMA-1 in P. falciparum. The most substantial divergence between Pr AMA-1 and Pf AMA-1 sequences was in the N-terminal region unique to the 83-kDa form of AMA-1. It was confirmed that the specific Pr ama-1-type allele was not present among P. falciparum parasites in an African population, and an allele coding for lysine at amino acid 187 was uniquely associated with field isolates in this population.

High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris: strong immunogenicity in Macaca mulatta immunized with P. vivax AMA-1 and adjuvant SBAS2.

The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Deltaglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Deltaglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Deltaglyc43-487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant, Pichia-expressed PV66Deltaglyc43-487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization.

The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the full-length 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.

Transfection systems for animal models of malaria.

Transfection of malaria parasites is a rapidly emerging technology that offers great promise for the investigation of many aspects of infection. Animal models of malaria have always played an important role in the investigation of the disease. In this article, two of the Dutch groups that have been involved in combining transfection with animal models describe the relevant techniques and recent vector developments for the expression of transgenes, giving examples of their application.

Transfection of the primate malaria parasite Plasmodium knowlesi using entirely heterologous constructs.

The recently developed transfection systems for Plasmodium berghei and Plasmodium falciparum offer important new tools enabling further insight into the biology of malaria parasites. These systems rely upon artificial parasite-host combinations which do not allow investigation into the complex interactions between parasites and their natural hosts. Here we report on stable transfection of Plasmodium knowlesi (a primate malaria parasite that clusters phylogenetically with P. vivax) for which both natural and artificial experimental hosts are available. Transfection of this parasite offers the opportunity to further analyze the biology of antigens not only in a natural host but also in hosts that are closely related to humans. To facilitate future development of integration-dependent transfection in P. knowlesi, completely heterologous plasmids that would reduce homologous recombination at unwanted sites in the genome were constructed. These plasmids contained the pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase (dhfr-ts) from Toxoplasma gondii or P. berghei, under control of either (a) P. berghei or (b) P. falciparum promoters. Plasmids were electroporated into mature P. knowlesi schizonts and these cells were injected into rhesus monkeys (Macaca mulatta). After pyrimethamine treatment of these monkeys, resistant parasites were obtained that contained the plasmids. Promoter regions of both P. berghei and P. falciparum controlling dhfr-ts expression were effective in conferring pyrimethamine resistance in P. knowlesi, indicating that common signals control gene expression in phylogenetically distant Plasmodium species.

Plasmodium vivax: in vitro antiparasitic effect of cyclosporins.

Plasmodium vivax is, next to P. falciparum, the second important human malaria parasite. In view of reports on developing chloroquine resistance, research on new drugs that are active against P. vivax is necessary. Due to a requirement for continuous addition of reticulocytes, long-term in vitro culture of P. vivax is not practicable. Conventional drug assays, i.e., culturing for a time equivalent to one asexual cycle of development in the presence of drugs, and then measuring propagation, are therefore not readily performed. In this report the in vitro susceptibility of P. vivax to cyclosporin A and a new, nonimmunosuppressive derivative, SDZ NIM 811, was investigated using parasite material obtained from an infected Aotus monkey. The assay was initiated with blood containing ring-stage parasites and was ended when parasites were multinucleate, but before merozoite release. The results were compared with the in vitro susceptibility of P. falciparum to these drugs in a conventional and a short assay. As an indicator of parasite propagation [3H]hypoxanthine incorporation was measured. The susceptibility of P. vivax to cyclosporins was found to be intermediate to that of P. falciparum in the conventional and in the much less sensitive short assay, and SDZ NIM 811 proved to be as active as cyclosporin A.

Habitual dietary calcium intake and blood pressure change around the menopause: a longitudinal study.

In a 10-year follow-up study, blood pressure and dietary intake were measured annually in 167 healthy perimenopausal normotensive women. Their initial ages ranged between 49 and 56 years and habitual calcium intake between 560 and 2580 mg/day (mean 1110 mg/day); they lived in the mixed rural/industrial community of Ede, the Netherlands. The longitudinal design provided an opportunity to study the 'natural history' of blood pressure and the effect of dietary calcium during and after the period of ovarian failure. For data analysis, person-time experience was divided into three menopausal periods. Based on years relative to menopause three menopausal cohorts were created starting 2 years before, 2 years after and 6 years after menopause, each was followed for 4 years. Changes in systolic (SBP) and diastolic blood pressure (DBP) during the menopausal periods were adjusted for change in body mass index and other relevant variables in multiple regression analysis. An average decline in SBP of 6 mm Hg was observed in the period of 2 years before menopause to 6 years after menopause, and an increase of almost 5 mm Hg in the period between 6 and 10 years after menopause. A significant change in DBP was not observed. Neither changes in, nor the absolute level of, calcium intake showed any relevant association with blood pressure change. Ovarian failure seems to reverse temporarily the increase in blood pressure due to aging. The results do not suggest that a habitual calcium intake exceeding 800-1000 mg/day (the current Recommended Daily Allowance for adults) is effective in preventing hypertension during the peri- and postmenopausal period.