The positive side of the coin: Sars-Cov-2 pandemic has taught us how much Telemedicine is useful as standard of care procedure in real life

Clinical Rheumatology - Patients and health workers were at high risk of infection during the Sars-Cov-2 pandemic lockdown. For this reason, other medical and clinical approaches such as...     

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

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, but multiple public health and financial barriers (7, 8), along with the possibility of reinfection or continued disease progression (7, 9, 10), have resulted in a continued rise in HCV infections. An HCV vaccine remains essential to proactively protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (11, 12). The challenges posed by HCV sequence diversity (12, 13), glycan shielding (14, 15), immunodominant nonneutralizing epitopes (16–19), and preparation of a homogeneous E1E2 antigen all contribute to the difficulty in generating protective B cell immune responses. Although multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited (20). Optimization of E1E2 to improve its immunogenicity and elicitation of bnAbs through rational design may lead to an effective B cell-based vaccine (21).HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (22–24). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (25) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (24, 26, 27) and interactions between E1 and E2 ectodomains (28–30). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and SR-B1 coreceptors as part of a multistep entry process on the surface of hepatocytes (31–34). Neutralizing antibody responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (18, 35–40). Structural knowledge of bnAb antibody–antigen interactions, which often target E2 epitopes in distinct antigenic domains B, D, or E (18, 41, 42), can inform vaccine design efforts to induce bnAb responses against flexible HCV epitopes (43–45). E1E2 bnAbs, including AR4A, AR5A (46), and others recently identified (38), are not only among the most broadly neutralizing (35) but also represent E1E2 quaternary epitopes unique to antibody recognition of HCV.Although much is known about bnAb responses to E1E2 glycoproteins, induction of B cell-based immunity with an E1E2-based vaccine immunogen (47–49) has remained difficult. The inherent hydrophobicity of E1 and E2 TMDs (24, 50) may impede uniform production of an immunogenic E1E2 heterodimer that could be utilized for both vaccine development and E1E2 structural studies. Although partial E1 and E2 structures have been determined (39, 51–54), many other enveloped viruses have structures of a complete and near-native glycoprotein assembly (55–59), providing a basis for rational vaccine design (60–62). Viral glycoproteins of influenza hemagglutinin (63), respiratory syncytial virus (RSV) (55), severe acute respiratory syndrome coronavirus 2 (64), and others (65, 66) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. 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 (56, 67). Previously described E1E2 glycoprotein designs include covalently linked E1 and E2 ectodomains (68, 69), E1E2 with TMDs intact and an immunoglobulin G (IgG) Fc tag for purification (70), as well as E1 and E2 ectodomains with a cleavage site (68), which presented challenges for purification either due to intracellular expression or to high heterogeneity. Two recently described scaffolded E1E2 designs, while promising, have not been shown to engage monoclonal antibodies (mAbs) that recognize the native E1E2 assembly, though they were engaged by E1-specific and E2-specific mAbs, as well as coreceptors that recognize E2 (71). Therefore, these presentations of E1E2 glycoproteins may not represent a native and immunogenic heterodimeric assembly, and thus their potential as vaccine candidates remains unclear.Here, we describe the design of a secreted E1E2 glycoprotein (sE1E2) that mimics both the antigenicity in vitro and the immunogenicity in vivo of the native heterodimer through the scaffolding of E1E2 ectodomains. In testing our designs, we found that both replacing E1E2 TMDs with a leucine zipper scaffold and inserting a furin cleavage site between E1 and E2 enabled secretion and native-like sE1E2 assembly. We assessed the size, heterogeneity, antigenicity, and immunogenicity of this construct (identified as sE1E2.LZ) in comparison with full-length membrane-bound E1E2 (mbE1E2). sE1E2.LZ binds a broad panel of bnAbs to E2 and E1E2, as well as coreceptor CD81, providing evidence of assembly into a native-like heterodimer. An immunogenicity study indicated that sera of mice injected with sE1E2.LZ neutralize HCV pseudoparticles at levels comparable to sera from mice immunized with mbE1E2. This sE1E2 design is a form of the native E1E2 heterodimer that both improves upon current designs and represents a platform for structural characterization and engineering of additional HCV vaccine candidates.     

Lemon juice has protective activity in a rat urolithiasis model

Background

The use of herbal medicines (medicinal plants or phytotherapy) has recently gained popularity in Europe and the United States. Nevertheless the exact mechanism of the preventive effects of these products is still far to be clearly established, being its knowledge necessary to successfully apply these therapies to avoid stone formation.

Methods

The effect of oral lemon juice administration on calcium oxalate urolithiasis was studied in male Wistar rats. Rats were rendered nephrolithic by providing drinking water containing 0.75% ethylene glycol [v/v] (EG) and 2% ammonium chloride [w/v] (AC) for 10 days. In addition to EG/AC treatment, three groups of rats were also gavage-administered solutions containing 100%, 75% or 50% lemon juice [v/v] (6 μl solution/g body weight). Positive control rats were treated with EG/AC but not lemon juice. Negative control rats were provided with normal drinking water, and were administered normal water by gavage. Each group contained 6 rats. After 10 days, serum samples were collected for analysis, the left kidney was removed and assessed for calcium levels using flame spectroscopy, and the right kidney was sectioned for histopathological analysis using light microscopy.

Results

Analysis showed that the rats treated with EG/AC alone had higher amounts of calcium in the kidneys compared to negative control rats. This EG/AC-induced increase in kidney calcium levels was inhibited by the administration of lemon juice. Histology showed that rats treated with EG/AC alone had large deposits of calcium oxalate crystals in all parts of the kidney, and that such deposits were not present in rats also treated with either 100% or 75% lemon juice.

Conclusion

These data suggest that lemon juice has a protective activity against urolithiasis.     

Effects of atorvastatin on high-density lipoprotein apolipoprotein A-I metabolism in dogs

BACKGROUND: The mechanisms involved in the decline of high-density lipoprotein (HDL) levels at a higher dose of atorvastatin have not yet been elucidated. We investigated the effects of atorvastatin on HDL-apolipoprotein (apo) A-I metabolism in dogs, a species lacking cholesteryl ester transfer protein activity. MATERIALS AND METHODS: Seven ovariectomized normolipidaemic female Beagle dogs underwent a primed constant infusion of [5,5,5-(2)H(3)] leucine to determine HDL-apo A-I kinetics before and after atorvastatin treatment (5 mg kg(-1) d(-1) for 6 weeks). Plasma lipoprotein profiles, activity of HDL-modifying enzymes involved in reverse cholesterol transport and hepatic scavenger receptor class B type I (SR-BI) expression were also studied. RESULTS: Atorvastatin treatment decreased HDL-cholesterol levels (3.56 +/- 0.24 vs. 2.64 +/- 0.15 mmol L(-1), P < 0.05). HDL-triglycerides were not affected. HDL-phospholipids levels were decreased (4.28 +/- 0.13 vs. 3.29 +/- 0.13 mmol L(-1), P < 0.05), as well as phospholipids transfer protein (PLTP) activity (0.83 +/- 0.05 vs. 0.60 +/- 0.05 pmol microL(-1) min(-1), P < 0.05). Activity of lecithin: cholesterol acyl transferase (LCAT), hepatic lipase (HL) and SR-BI expression did not change. HDL-apo A-I absolute production rate (APR) was higher after treatment (twofold, P < 0.05) as well as fractional catabolic rate (FCR) (threefold, P < 0.05). This resulted in lower HDL-apo A-I levels (2.36 +/- 0.03 vs. 1.55 +/- 0.04 g l(-1), P < 0.05). Plasma lipoprotein profiles showed a decrease in large HDL(1) levels, with lower apo A-I and higher apo E levels in this subfraction. CONCLUSIONS: Although a high dose of atorvastatin up-regulated HDL-apo A-I production, this drug also increased HDL-apo A-I FCR in dogs. This effect could be explained by a higher uptake of apo E-enriched HDL(1) by hepatic lipoprotein receptors.     

Human platelet alloantigens (HPA) 1, HPA2, HPA3, HPA4, and HPA5 polymorphisms in sickle cell anemia patients with vaso‐occlusive crisis

Objectives: Vaso‐occlusive crisis (VOC) is a significant cause of morbidity and mortality in sickle cell anemia (SCA) patients. Insofar as polymorphism in human platelet alloantigen (HPA) exhibit a prothrombotic nature, we hypothesized that specific HPA polymorphic variants are associated with VOC. We investigated the distribution of HPA1, HPA2, HPA3, HPA4, and HPA5 alleles genotypes among VOC and non‐VOC control SCA patients. Patients/methods: This was a case–control study. Study subjects comprised SCA patients with (VOC group; n = 127) or without (Steady‐state group; n = 130) VOC events. HPA genotyping was done by PCR‐SSP. Results: Significantly higher frequencies of HPA‐2b, HPA‐3b, and HPA‐5b alleles, and marked enrichment of HPA‐3b/3b, HPA‐5a/5b, and HPA‐5b/5b genotypes, were seen in VOC than in control SCA patients. Taking homozygous wild‐type genotypes as reference, univariate analysis identified HPA‐3a/3b, HPA‐3b/3b, and HPA‐5b/5b to be associated with VOC. Multivariate analysis confirmed the independent association of only HPA‐3a/3b and HPA‐3b/3b genotypes with VOC. HPA‐3 genotypes were significantly correlated with VOC frequency, type, and medication, and requirement for hospitalization. While both HPA 3a/3b (P = 0.002; OR = 2.94; 95% CI = 1.49–5.77) and 3b/3b (P = 0.006; OR = 3.16; 95% CI = 1.40–7.17) genotypes were associated with need for hospitalization, only HPA‐3b/3b was associated with VOC frequency, type (localized vs. generalized), and medication (narcotics vs. NSAIDs). Conclusion: This confirms the association of HPA polymorphisms with SCA VOC, of which HPA‐3 appears to be independent genetic risk factors for SCA VOC.     

Female Circumcision: Toward an Inclusive Practice of Care

Female circumcision is a cultural tradition that includes cutting of female genitals without medical necessity. Over 130 million girls and women have been circumcised globally. This article reports on partial findings from a qualitative study that examined the lives of Somali Muslim women who were circumcised. A reoccurring theme of resentment toward North American health care practitioners who condemn the women for having experienced the practice of circumcision in their birth country was found. Discussion will include the physical and social stigma, the complex legal aspects, and ways to deal with female circumcision in a culturally competent manner.