Striae Distensae: In Vitro Study and Assessment of Combined Treatment With Sodium Ascorbate and Platelet-Rich Plasma on Fibroblasts

Introduction: Striae distensae (SD) appear clinically as parallel striae, lying perpendicular to the tension lines of the skin. SD evolve into two clinical phases, an initial inflammatory phase in which they are called “striae rubrae” (SR) and a chronic phase in which they are called striae albae (SA). Fibroblasts seem to play a key role in the pathogenesis of stretch marks. This study was aimed at describing and analyzing stretch marks-derived fibroblasts (SMF), the differences between SR- and SA-derived fibroblasts (SRF, SAF), testing two treatments in vitro (sodium ascorbate and PrP) on SAF. Material and Methods: To characterize the SMF, the expression of alpha smooth muscle actin (alpha SMA) was investigated. Type I collagen expression was measured in SAF, before and after adding different PrP concentrations and sodium ascorbate in the culture medium. Results were processed through statistical analysis models using the Student’s t-test. Results: A significant increase in alpha SMA (P <0.001) was observed in SRF. SAF treated with PrP and sodium ascorbate showed a resumption of their metabolic activity by an increase in collagen type I production and cell proliferation. After 24 h of incubation with PrP 1% and PrP 5% + sodium ascorbate, cell viability was increased by 140% and 151% and by 156 and 178% after 48 h, respectively, compared to the control. Conclusion: Our study shows that a biologically mediated improvement in SMF metabolic activity is possible. Our promising results require further trials to be able to confirm the reproducibility of this combined treatment, particularly in vivo. No Level Assigned: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable

Structural and Functional Insights into the Malaria Parasite Moving Junction Complex

Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics

Role of oxidative stress in physiological albumin glycation: A neglected interaction

Protein glycation is a key mechanism involved in chronic disease development in both diabetic and nondiabetic individuals. About 12–18% of circulating proteins are glycated in vivo in normoglycemic blood, but in vitro studies have hitherto failed to demonstrate glucose-driven glycation below a concentration of 30 mM. Bovine serum albumin (BSA), reduced BSA (mercaptalbumin) (both 40 g/L), and human plasma were incubated with glucose concentrations of 0–30 mM for 4 weeks at 37 °C. All were tested preoxidized for 8 h before glycation with 10 nM H2O2 or continuously exposed to 10 nM H2O2 throughout the incubation period. Fructosamine was measured (nitroblue tetrazolium method) at 2 and 4 weeks. Oxidized BSA (both preoxidized and continuously exposed to H2O2) was more readily glycated than native BSA at all glucose concentrations (p = 0.03). Moreover, only oxidized BSA was glycated at physiological glucose concentration (5 mM) compared to glucose-free control (glycation increased by 35% compared to native albumin, p &#60; 0.05). Both 5 and 10 mM glucose led to higher glycation when mercaptalbumin was oxidized than when unoxidized (p &#60; 0.05). Fructosamine concentration in human plasma was also significantly higher when oxidized and exposed to 5 mM glucose, compared to unoxidized plasma (p = 0.03). The interaction between glucose concentration and oxidation was significant in all protein models (p &#60; 0.05). This study has for the first time demonstrated albumin glycation in vitro, using physiological concentrations of albumin, glucose, and hydrogen peroxide, identifying low-grade oxidative stress as a key element early in the glycation process

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca2+-independent, as inhibition of Ca2+ channels or culture in the absence of Ca2+ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation

High Affinity Antibodies to Plasmodium falciparum Merozoite Antigens Are Associated with Protection from Malaria

Malaria kills almost 1 million people every year, but the mechanisms behind protective immunity against the disease are still largely unknown. In this study, surface plasmon resonance technology was used to evaluate the affinity (measured as k(d)) of naturally acquired antibodies to the Plasmodium falciparum antigens MSP2 and AMA1. Antibodies in serum samples from residents in endemic areas bound with higher affinities to AMA1 than to MSP2, and with higher affinities to the 3D7 allele of MSP2-3D7 than to the FC27 allele. The affinities against AMA1 and MSP2-3D7 increased with age, and were usually within similar range as the affinities for the monoclonal antibodies also examined in this study. The finding of MSP2-3D7 type parasites in the blood was associated with a tendency for higher affinity antibodies to both forms of MSP2 and AMA1, but this was significant only when analyzing antibodies against MSP2-FC27, and individuals infected with both allelic forms of MSP2 at the same time showed the highest affinities. Individuals with the highest antibody affinities for MSP2-3D7 at baseline had a prolonged time to clinical malaria during 40 weeks of follow-up, and among individuals who were parasite positive at baseline higher antibody affinities to all antigens were seen in the individuals that did not experience febrile malaria during follow up. This study contributes important information for understanding how immunity against malaria arises. The findings suggest that antibody affinity plays an important role in protection against disease, and differs between antigens. In light of this information, antibody affinity measurements would be a key assessment in future evaluation of malaria vaccine formulations

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents