Local erythropoietin signaling enhances regeneration in peripheral axons

Erythropoietin (EPO) and its receptor (EPO-R), mediate neuroprotection from axonopathy and apoptosis in the peripheral nervous system (PNS). We examined the impact and potential mechanisms of local EPO signaling on regenerating PNS axons in vivo and in vitro. As a consequence of injury, peripheral nerve axons and DRG neurons have a marked increase in the expression of EPO and EPO-R. Local delivery of EPO via conduit over 2 weeks to rat sciatic nerve following crush injury increased the density and maturity of regenerating myelinated axons growing distally from the crush site. In addition, EPO also rescued retrograde degeneration and atrophy of axons. EPO substantially increased the density and intensity of calcitonin gene-related peptide (CGRP) expression within outgrowing axons. Behavioral improvements in sensorimotor function also occurred in rats exposed to near nerve EPO delivery. EPO delivery led to decreased nuclear factor kappaB (NFkB) activation but increased phosphorylation of Akt and STAT3 within nerve and dorsal root ganglia neurons indicating rescue from an injury phenotype. Spinal cord explant studies also demonstrated a similar dose-dependent effect of EPO upon motor axonal outgrowth. Local EPO signaling enhances regenerating peripheral nervous system axons in addition to its known neuroprotection. Exogenous EPO may have a therapeutic role in a large number of peripheral nerve diseases through its impact on regeneration.     

The patient with an odontogenic infection.

The routine use of antibacterial agents for the management of odontogenic infections has not been shown to be effective and is inappropriate. Most of these bacterial infections can be resolved satisfactorily through an approach that incorporates debridement (primary dental care) in conjunction with local anesthesia. Odontogenic infections are polymicrobial. Facultative anaerobes, particularly viridans streptococci, accompanied by strict anaerobes, appear to predominate in all types of odontogenic infections. When antibacterial chemotherapy is indicated, the drug of choice should be either the most effective drug against the infective pathogens or the least toxic alternative among several available agents. It should also be emphasized that drugs seldom exert their beneficial effects without also causing adverse side effects. Dealing with this certainty, the clinician familiar with the mechanisms of action, principles of disposition, and therapeutic and adverse effects of antibacterial agents, has the advantage.     

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate

Hepatitis C virus (HCV) is a global disease burden, and a preventive vaccine is needed to control or eradicate the virus. Despite the advent of effective antiviral therapy, this treatment is not accessible to many patients and does not prevent reinfection, making chronic hepatitis C an ongoing global health problem. Thus, development of a prophylactic vaccine will represent a significant step toward global eradication of HCV. HCV exhibits high genetic variability, which leads frequently to immune escape. However, a considerable challenge faced in HCV vaccine development is designing an antigen that elicits broadly neutralizing antibodies. Here, we characterized the immunogenicity of a vaccine based on a soluble, secreted form of the E1E2 envelope heterodimer (sE1E2.LZ). Sera from mice immunized with sE1E2.LZ exhibited an anti-E1E2–specific response comparable to mice immunized with membrane-bound E1E2 (mbE1E2) or a soluble E2 ectodomain (sE2). In competition-inhibition ELISA using antigenic domain-specific neutralizing and nonneutralizing antibodies, sera from sE1E2.LZ-immunized mice showed nearly identical or stronger competition toward neutralizing antibodies when compared with mbE1E2. In contrast, sera from mice immunized with sE2, and to a lesser extent mbE1E2, competed more effectively with nonneutralizing antibodies. An assessment of neutralization activity using both HCV pseudoparticles and cell culture–derived infectious HCV showed that immunization with sE1E2.LZ elicited the broadest neutralization activity of the three antigens, and sE1E2.LZ induced neutralization activity against all genotypes. These results indicate that our native-like soluble glycoprotein design, sE1E2.LZ, induces broadly neutralizing antibodies and serves as a promising vaccine candidate for further development.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5), and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct acting antivirals (DAAs), but several issues have blunted their effectiveness in eradicating HCV. In particular, multiple public health and financial barriers (7, 8) restrict access to DAAs in areas with high incidence of infection and DAAs do not prevent reinfection. Moreover, HCV infection is largely asymptomatic and often does not generate sterilizing immunity, thereby contributing to reinfection or continued disease progression (7, 9, 10). Collectively, these issues have resulted in a continued rise in HCV infections.Acute HCV infections can be cleared by host immunity in ∼25% of cases. Among individuals who clear their first infection, the rate of clearance rises to 80% for subsequent infections, indicating an effective immune memory response (11–14). This type of natural protective immunity to HCV requires the induction of broadly neutralizing antibodies to E1E2 ectodomains and T cell responses to the structural and nonstructural proteins (15–17). The above clinical observations suggest that, if a vaccine candidate could induce broadly neutralizing antibody and cell-mediated immune responses equivalent to that seen in spontaneous clearance, such a vaccine would be highly effective at preventing HCV infection. An HCV vaccine therefore remains an essential proactive measure to protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (17, 18).A number of challenges exist that have thus far limited progress toward developing a prophylactic vaccine against HCV. One major challenge in developing a successful vaccine for HCV has been the remarkable genetic diversity of the virus which has six major genotypes (genotypes 1 to 6), in addition to two less-common genotypes (19) (genotypes 7 and 8), and intragenotypic diversity resulting in 90 total subtypes (20). Moreover, shielding of important neutralizing epitopes with glycans (21, 22), and the presence of immunodominant nonneutralizing epitopes (23–26) deflect the immune response from conserved regions that mediate virus neutralization. Multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, but their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited. In particular, immunological assessment in chimpanzees of an E1E2 vaccine produced superior immune responses as compared with E2 administered alone and resulted in sterilizing immunity against homologous virus challenge (27, 28), but with less cross-neutralization capacity against heterologous isolates (29). In addition, an E1E2 formulation tested in humans is well-tolerated (30). However, due to the limited neutralization breadth observed in the human clinical trial (31, 32), using native E1E2 as a vaccine is not likely to provide sufficient protection from HCV infection. Rather, optimization of E1E2 to improve its immunogenicity and capacity to elicit bnAbs through rational design appears to be the preferred path for developing an effective B cell-based vaccine (33).An additional bottleneck contributing to the difficulty in generating protective B cell immune responses required for an effective HCV vaccine is preparation of a homogeneous E1E2 antigen. HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (34–36). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (37) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (36, 38, 39) and interactions between E1 and E2 ectodomains (40–42). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and scavenger receptor type B class I (SR-B1) coreceptors as part of a multistep entry process on the surface of hepatocytes (43–46). Neutralizing antibody (nAb) responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (25, 47–52). A significant impediment to the uniform production of an immunogenic E1E2 heterodimer that could be utilized for vaccine development is the association of the antigen with the membrane via the TMDs (36, 53). Progress has been made in the production and purification of the membrane-bound E1E2 complex via immunoaffinity purification (54, 55) or the use of tags that allow protein A (56) or anti-Flag (57) chromatography. While these methods produce high-quality samples, they all involve harsh elution conditions. How such conditions might influence sample quality at a scale required for vaccine trials is unclear. Furthermore, intracellular expression and membrane extraction limits the ability to produce large quantities of sufficient homogeneity required for both basic research and vaccine production.In contrast, viral glycoproteins of influenza hemagglutinin (58), respiratory syncytial virus (RSV) (59), SARS-CoV-2 (60), and others (61, 62) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. In addition, HIV gp120-gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (63, 64). Recent efforts have made strides toward liberating the E1E2 complex from the membrane in its native form (65, 66). In particular, our previous work (66) showed that a soluble E1E2 (sE1E2) using the Fos/Jun leucine zipper (LZ) coiled-coil as a scaffold (sE1E2.LZ) is antigenically intact, as the protein is recognized by E1E2-specific mAbs AR4A and AR5A (67). Moreover, sE1E2.LZ elicited nAbs in mice immunized with the antigen, making this scaffold a promising potential platform for engineering of additional HCV vaccine candidates.Here, we describe the immunogenicity of our native-like secreted E1E2 construct sE1E2.LZ and compare it with the membrane-bound E1E2 complex (mbE1E2) and a secreted form of the E2 ectodomain (sE2). Immunization of mice with sE1E2.LZ produced sera possessing anti-E1E2 antibodies at levels comparable to mice immunized with mbE1E2 or sE2. Moreover, the antibody response in sE1E2.LZ-immunized mice is skewed more toward nAbs relative to non-nAbs than the other two antigens. Remarkably, sera from sE1E2.LZ-immunized mice exhibited broader neutralization activity than either mbE1E2 or sE2 when assessed using both pseudotyped HCV particles (HCVpp) and cell culture-derived HCV (HCVcc), suggesting that this sE1E2 platform represents a favorable starting point for developing scaffolded E1E2 vaccine candidates.     

Comparative Study on Nutrition and Lifestyle of Information Technology Workers from Romania before and during COVID-19 Pandemic

The study aimed to evaluate lifestyle factors among Information Technology (IT) workers from Romania before and during the pandemic. We used an online applied questionnaire, filled in by 1638 respondents, that assessed nutrition status-Body Mass Index (BMI), weight and diet change, physical activity, alcohol consumption, number of hours working in front of the computer, stress, and sleep. Multivariate logistic regression was used to establish the lifestyle factors that lead to weight gain. Although the level of physical activity (PA) was low before the pandemic, the results of our study showed a further decrease in physical activity. In total, 61.1% of men and 71.1% of women performed PA for less than 30 min per day. Weight gain was reported in 50.5% of men and 45.3% of women (mean weight gain was 5.11 ± 3.52 kg) as a result of increased screen time (with a mean of 3.52 ± 4.29 for females and 3.05 ± 2.09 for males, p > 0.05 h per day) and the reported changes in diet. Despite the popularity of home-cooked foods, the intake of vegetables and fruit remained low. The quality of sleep was poor for 55.7% of the respondents. Public health and corporation policies are required to encourage a healthy lifestyle and avoid chronic diseases.     

Encapsulation of lipopeptides within liposomes: effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C8, C12 and C16. The C12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers.     

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS.     

Malignancy may adversely influence the quality and behaviour of oocytes

A case series of eight cycles of in-vitro fertilization (IVF) in five women diagnosed with malignant disorders is presented. These patients chose to defer definitive treatment for a chance for preservation of potential fertility. The response of these patients to ovarian stimulation, and the outcome, was compared with 17 IVF cycles in 12 age- matched patients with isolated tubal infertility. An apparent adverse influence of malignant disease on the quality and behaviour of oocytes was observed. Despite a comparable total number of oocytes per cycle in the two groups, a significantly reduced percentage of mature oocytes was retrieved per cycle from patients with malignant diseases. The oocytes from patients with malignant disorders were of a poorer quality and exhibited a significantly impaired fertilization rate compared to the controls. We propose that neoplastic processes, irrespective of the site or cell of origin, may have a detrimental impact on the biology of oocytes, an effect akin to that seen on spermatozoa in men with certain malignancies.      

Investigation of the Possible Functions of PACAP in Human Trophoblast Cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.