Cross-sectional study in Madagascar demonstrates efficacy of virtual mentoring and flipped classroom modifications of neonatal resuscitation programme Helping Babies Breathe.

AIM: The Covid-19 pandemic necessitated virtual adaptation of the neonatal resuscitation programme Helping Babies Breathe (HBB). This study assessed one such virtually mentored and flipped classroom modification in Madagascar. METHODS: A cross-sectional study was performed in September 2021 and May 2022. Healthcare providers were identified by local collaborating organisations. United States-based master trainers collaborated with local trainers on virtually mentored trainings followed by independent trainings. Master trainers were available for consultation via Zoom during the virtual training. A flipped classroom modification and traditional didactic method were compared. Primary outcomes were knowledge and skill acquisition, evaluated by written assessments and objective structured clinical examinations. RESULTS: Overall, 97 providers completed the curriculum. Written assessment scores improved in both training models (traditional-74.8% vs 91.5%, p < 0.001; flipped classroom-89.7% vs 93.6%, p < 0.05). There was no significant difference among written assessment scores (92.8% vs 91.5%, p = 0.62) and significantly higher objective structured clinical examination scores (97.3% vs 89.5%, p < 0.001) for the independent training compared to the virtually mentored training. CONCLUSION: The virtually mentored HBB training was followed by a successful independent training as measured by participant knowledge and skill acquisition, supporting the efficacy of virtual dissemination.

Age- and passage-dependent upregulation of fibroblast elastase-type endopeptidase activity. Role of advanced glycation endproducts, inhibition by fucose- and rhamnose-rich oligosaccharides.

It could be shown using the in vitro cell culture aging model, that elastase-type endopeptidase activity is progressively upregulated with successive passages (in vitro aging). Similar results were obtained previously by determining elastase-type activity as a function of age in aorta extracts (human) and skin extracts (mouse). Among the possible mechanisms involved we tested the role of advanced glycation endproducts (AGEs) on this process. AGE-production was shown to increase with age, exemplified by the exponential age-dependent crosslinking of collagen, demonstrated by Fritz Verzár, already in 1963. Several AGEs significantly upregulated elastase-type activity when added to the culture medium of fibroblasts. This effect appears to be mediated by some AGE-receptors as shown previously, and could be inhibited by a 5 kDa rhamnose-rich oligosaccharide (RROP-3) as well as by a fucose-rich oligosaccharide (FROP-3). When present in the culture media, RROP-3 and FROP-3 efficiently inhibited the passage-dependent upregulation of elastase-type activity expressed by human skin fibroblasts. The use of specific inhibitors and zymography suggested that matrix metalloproteinases (MMP)-9 activation and expression are mainly involved. A detailed discussion is proposed for the interpretation of age-dependent modifications of tissues as vascular wall and skin in the light of these and related experiments, highlighting the role of several specific receptors in the mediation of the observed reactions.

Expression of senescence-associated beta-galactosidase (SA-beta-Gal) by human skin fibroblasts, effect of advanced glycation end-products and fucose or rhamnose-rich polysaccharides.

Expression by cells of the SA-beta-Gal was shown to be a reliable indicator of the switch mechanism used by cells to enter the senescent phenotype. We used this method in order to explore the variation of SA-beta-Gal-positive cells with passage number and time spent in culture. Both parameters produced an increase of SA-beta-Gal-positive cells. The addition of a Maillard-product (advanced glycation end-product=AGE) to the fibroblast cultures also increased SA-beta-Gal expression. Fucose- and rhamnose-rich oligo- and polysaccharides (FROPs and RROPs, respectively) provided a significant protection against this AGE-induced increase of SA-beta-Gal-positive cells. It is speculated that these processes might well play an important role in skin aging.

Collagen biosynthesis in cell culture: comparison of corneal keratocytes and skin fibroblasts. Effect of rhamnose-rich oligo- and polysaccharides.

Corneal keratocytes are often confounded with fibroblasts, although their matrix-synthetic phenotype is quite different as shown by the nature and relative amount of the different collagens and proteoglycans-glycosaminoglycans synthesized. In these experiments, we compared the concentration of collagens excreted in the culture medium by human corneal keratocytes and skin fibroblasts at three consecutives passages. Although the keratocytes excreted less collagen at earlier passages, they approached and reached fibroblasts at later passages. This can be taken as an indication of the progressive loss of a specific keratocyte phenotype with increasing passages (in vitro aging). The effect of rhamnose-rich oligo- and polysaccharides on collagen secretion also confirmed the subtle differences between these two cell-types, as well as the difference between saturation density of both cell types at confluence and the proportion of dead cells floating on top of the culture medium. These differences were also attenuated with passage number without disappearing completely. The significance of these findings will be discussed in the light of previous results in our and other laboratories on matrix-secreting phenotypes and aging.

Effect of cellular aging on collagen biosynthesis: II. Collagen synthesis and deposition by a human skin fibroblast strain over 25 passages.

Collagen synthesis and accumulation were studied by serial cultures of human dermal fibroblasts. A freshly seeded strain of cells was compared to cryopreserved cells from the same donor. Up to 25 passages there was no clear sign of reaching phase III, the decline of the culture. This was ascertained by the count of the dead cells floating on top of the culture fluid, the time to reach saturation density and the number of cells at confluence, counted after trypsinization. Although collagen synthesis oscillated to some extent, the average value of collagen deposited by the cells did not show any clear sign of decrease of collagen synthesis, on the contrary, there was an increase between the 15th and 25th passages. A review of the literature revealed that recent experiments on the age-dependent variation of maximal passage number did not confirm previous results showing a progressive decline, which was much delayed in recent studies as compared to previous records. The same appears to happen with collagen synthesis, found by earlier investigators to decline with increasing passage numbers. This is not confirmed, at least up to passage 25 in the present experiments. As skin tissue is progressively lost with age, our results are more in favor of increasing matrix degradation with age as an important factor of the age-dependent loss of skin tissue, more than decrease of matrix synthesis.

Effect of cellular aging on collagen biosynthesis: I. Methodological considerations and pharmacological applications.

The study of the age and passage dependent modifications of collagen biosynthesis requires a simple, rapid and reproducible procedure adaptable to serial cell cultures. To make such a method comparable to other methods of collagen determination, we calibrated a colorimetric procedure both by hydroxyproline (HYP) determinations and in terms of collagen concentration. For collagen types I and IV, widely different slopes were obtained with the colorimetric procedure. To further refine the procedure, we tempted to completely inhibit collagen synthesis by beta-aminopropionitrile (beta APN) added to cultures in order to obtain a negative control. Using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide (MTT-test), it could be shown that relatively high concentrations of beta APN are tolerated by the cells. It appeared, however, that even the highest concentration of beta APN (1mM) still tolerated by the fibroblasts did not completely inhibit collagen synthesis. At low concentrations, beta APN even stimulated cell-proliferation. The colorimetric procedure calibrated in terms of collagen type I concentration, was therefore retained for the serial determination of collagen synthesis and accumulation. We shall here describe the methodological details of its validation as well as its application for the pharmacological study of the effect of aging on collagen biosynthesis. Among the factors involved, the accumulation of advanced glycation end-products (AGEs) might well play an important role. Several of such AGE-products showed a significant inhibition of collagen deposition. On the contrary, retinol, ascorbic acid as well as the rhamnose-rich oligo- and polysaccharides (RROPs) did produce a significant upregulation collagen deposition. Polysaccharide preparations, rich in rhamnose and fucose (the EROB-mixture) could protect against the AGEs-induced inhibition of collagen accumulation.

[Demonstration of the cytotoxic effect of Advanced Glycation Endproducts (AGE-s)]. / Démonstration de l'effet cytotoxique des produits avancés de la glycation (AGE-s).

Advanced Glycation End-products (AGE-s) were shown to exhibit a number of potentially harmful properties in contact with cells and tissues. As their concentrations increases with age, faster even in hyperglycemic individuals, they are considered important for aging- and age-associated pathologies, especially for athero-arteriosclerosis and type II diabetes. We describe here the methods used for the demonstration of a direct cytotoxicity of several AGE-products when added to human skin fibroblast cultures. This cytotoxicity was still demonstrable when cells, previously cultured with AGE-s, were transferred to new medium without AGE-s. This effect, the remanence of cytotoxicity in absence of AGE-s, suggests a certain degree of inheritance, possibly by epigenetic mechanisms, of the cytotoxic effect of AGE-s, mediated by the AGE-receptors (RAGE-s) and inhibited by free radical-scavengers, such as L-Carnosine, Catalase and Rhamnose-rich oligo- and polysaccharides. Such cytotoxicity can occur not only on the skin but also in other tissues. It appears thus that besides the crosslinking of collagen and other macromolecules, the products of the Maillard reaction can exert their harmful cytotoxic effects directly on the cells.

Protection by rhamnose-rich polysaccharides against the cytotoxicity of Maillard reaction products.

Previous experiments have shown that AGE-products added to human skin fibroblast cultures increased the number of dead cells floating on top of the culture fluid and took up vital dye [1]. In these experiments, we tested several rhamnose-rich polysaccharides for protection against the cytotoxic effect of AGE-s. Added at relatively low concentrations (between 10 and 100 microg/ml) to the culture medium, several of the tested rhamnose-rich oligo- and polysaccharides (RROP-s) gave a significant protection against AGE-induced cytotoxicity. Their effect on cell proliferation was also tested. The number of cells at saturation density was also shown to be influenced by AGE-products added to the cultures. This effect was also, at least partially, corrected by the rhamnose-rich oligo- and polysaccharides. These substances might therefore be considered as of potential therapeutical interest against hyperglycemia induced cytotoxic effects as in type II-diabetes.

Effect of advanced glycation end-products on cell proliferation and cell death.

The effect of advanced glycation end products (AGE-s) was studied on the proliferation and cell death of human skin fibroblasts in culture. Several AGE-products were prepared from proteins, a peptide and amino acids, using Glucose or Fructose, with or without Fe2+. The AGE preparations increased cell death at the 7th day, after only 72 hours of incubation. Some of these glycation products modified also proliferation. This effect of AGE-s was even maintained without these products in fresh medium for a second period of incubation up to 10 days from the start of the experiment. In order to explore the role of AGE-receptors, especially of AGE-receptor and of growth factor receptors (fibroblast and epidermal growth factors receptors), antibodies to these receptors were added to cell cultures and their effect on both cell death and proliferation were determined as for the AGE-s. These anti-receptor antibodies imitated to some extent the results obtained with AGE-s, producing increase of cell death and proliferation, followed above a certain concentration of antibodies by a decrease and a new increase or plateau. This might correspond to the internalization of the receptors followed by a re-expression on the cell membrane. The role of receptor-mediated Reactive Oxygen Species-production was also explored using scavengers: N-acetyl-cysteine (NAC), L-Carnosine, superoxide dismutase (SOD) and Catalase. Several of these scavengers decreased cell death, suggesting that Reactive Oxygen Species-production is partially involved in the observed phenomena.