Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.     

Resisted sprint training with partner towing improves explosive force and sprint performance in young soccer players – a pilot study

The purpose of this study was to examine the effects of non-resisted (NRS) and partner-towing resisted (RS) sprint training on legs explosive force, sprint performance and sprint kinematic parameters. Sixteen young elite soccer players (age 16.6 ± 0.2 years, height 175.6 ± 5.7 cm, and body mass 67.6 ± 8.2 kg) were randomly allocated to two training groups: resisted sprint RS (n = 7) and non-resisted sprint NRS (n = 9). The RS group followed a six-week sprint training programme consisting of two “sprint training sessions” per week in addition to their usual soccer training. The NRS group followed a similar sprint training programme, replicating the distances of sprints but without any added resistance. All players were assessed before and after training: vertical and horizontal jumping (countermovement jump (CMJ), squat jump (SJ), and 5-jump test (5JT)), 30 m sprint performance (5, 10, and 20 m split times), and running kinematics (stride length and frequency). In the RS group significant (p < 0.05) changes were: decreased sprint time for 0–5 m, 0–10 m and 0–30 m (-6.31, -5.73 and -2.00%; effect size (ES) = 0.70, 1.00 and 0.41, respectively); higher peak jumping height (4.23% and 3.59%; ES = 0.35 and 0.37, for SJ and CMJ respectively); and 5JT (3.10%; ES = 0.44); and increased stride frequency (3.96%; ES = 0.76). In the NRS group, significant (p < 0.05) changes were: decreased sprint time at 0–30 m (-1.34%, ES = 0.33) and increased stride length (1.21%; ES = 0.17). RS training (partner towing) for six weeks in young soccer players showed more effective performances in sprint, stride frequency and lower-limb explosive force, while NRS training improved sprint performance at 0–30 m and stride length. Consequently, coaches and physical trainers should consider including RS training as part of their sprint training to ensure optimal sprint performance.     

Antimicrobial activity of chemically and biologically synthesized silver nanoparticles against some fish pathogens

Pathogens isolated from fish appear to possess considerable antimicrobial resistance and represent a problem for the economy and public health. Natural antimicrobial substitutes to traditional antibiotics represent an essential tool in the fight against antibiotic resistance. Nanotechnology has shown considerable potential in different research fields, and the antimicrobial properties of silver nanoparticles are known. Silver has been used for medical purposes since ancient times because of its bactericidal properties, and the highly reactive surfaces of silver nanoparticles (AgNPs) indicate that they might have a function in antimicrobial applications. This work aimed to study the antimicrobial properties of biologically produced AgNPs from Origanum vulgare leaves compared to chemically produced AgNPs. Both types were characterized by UV–vis spectrophotometry, TEM, and dynamic light scattering and tested against three bacterial strains (Streptococcus agalactiae, and Aeromonas hydrophila, both isolated from Nile tilapia and Vibrio alginolyticus, isolated from sea bass) and three fungal strains (Aspergillus flavus, Fusarium moniliforme, and Candida albicans, all isolated from Nile tilapia). Disk diffusion test and evaluation of ultrastructure changes of tested microorganisms treated with AgNPs by transmission electron microscopy were performed. Moreover, the hemolytic properties of AgNPs were studied on chicken and goat red blood cells. The results obtained declare that the green biological production of silver nanoparticles is safer and more effective than the chemical one; moreover, AgNPs have interesting dose-dependent antimicrobial properties, with better results for biologically produced ones; their effectiveness against tested bacterial and fungal strains opens the way to their use to limit fish diseases, increase economy and improve human health.     

Potent universal beta-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel β-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human β-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human β-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.     

Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat

The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca²⁺]_i andCa²⁺ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca²⁺), the resting length and[Ca²⁺]_i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 10⁵ M) caused transient increasein [Ca²⁺]_i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca²⁺]_i transient was notsignificantly different, but the maintained [Ca²⁺]_i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa²⁺-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca²⁺]_i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca²⁺]_i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca²⁺]_i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca²⁺]_i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca²⁺]_i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca²⁺]_i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (10⁸ M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca²⁺]_i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca²⁺]_i and contraction of vascularsmooth muscle triggered by Ca²⁺ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca²⁺]_i due to Ca²⁺release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca²⁺]_i in vascular smoothmuscle of female rats is greater in SHR than WKY rats.

     

Interference free and simplyfied liquid chromatography-based determination of thiopurine S-methyltransferase activity in erythrocytes

The determination of the thiopurine S-methyltransferase activity (TPMT; EC 2.1.1.67) has become an important issue during thiopurine therapy due to its known genetic polymorphism resulting in a wide range of TPMT activity. Therefore, the standard thiopurine drug regimen is associated with increased hematopoetic toxicity in patients with low or absent TPMT activity, whereas patients with high activity may be insufficiently treated. However, presently available methods are labour intensive and time consuming and tend towards too high or too low enzyme activity due to their methodological approach. The use of instable substrate solutions (6-MP or 6-TG), organic solvents like dimethyl sulfoxide and too high substrate and co-substrate saturation concentrations contribute to this phenomenon. We therefore, established an optimized and fast isocratic HPLC linked TPMT assay based on the enzymatic methylation of mercaptopurine or thioguanine in RBC lysates with S-adenosyl-l-methionine as methyl donor. Unspecific non-enzymatic methylation was not detectable. The recovery of 6-methyl-mercaptopurine was 97-102%, the intra- and interday variation between 1.0 and 5.0%, respectively. The assay dispenses with a time consuming extraction procedure with organic solvents, a heating step, and a gradient elution and is therefore, favourable for clinical routine application. The TPMT activity was measured in 62 untreated children with acute lymphoblastic leucemia at the time of diagnosis (activity = 34.0+/-10.6 nmol/g Hb/h, range: 11.5-55.4 nmol/g Hb/h) and in 12 adult healthy volunteers (62.8+/-7.7 nmol/g Hb/h, range: 48-82 nmol/g Hb/h) reflecting the wide measurable TPMT activity found in erythrocytes.     

Approach to diagnosis and pathological examination in bronchial Dieulafoy disease: a case series

Background

We previously observed that allergen-exposed mice exhibit remodeling of large bronchial-associated blood vessels. The aim of the study was to examine whether vascular remodeling occurs also in vessels where a spill-over effect of bronchial remodeling molecules is less likely.

Methods

We used an established mouse model of allergic airway inflammation, where an allergic airway inflammation is triggered by inhalations of OVA. Remodeling of bronchial un-associated vessels was determined histologically by staining for α-smooth muscle actin, procollagen I, Ki67 and von Willebrand-factor. Myofibroblasts were defined as and visualized by double staining for α-smooth muscle actin and procollagen I. For quantification the blood vessels were divided, based on length of basement membrane, into groups; small (≤250 μm) and mid-sized (250–500 μm).

Results

We discovered marked remodeling in solitary small and mid-sized blood vessels. Smooth muscle mass increased significantly as did the number of proliferating smooth muscle and endothelial cells. The changes were similar to those previously seen in large bronchial-associated vessels. Additionally, normally poorly muscularized blood vessels changed phenotype to a more muscularized type and the number of myofibroblasts around the small and mid-sized vessels increased following allergen challenge.

Conclusion

We demonstrate that allergic airway inflammation in mice is accompanied by remodeling of small and mid-sized pulmonary blood vessels some distance away (at least 150 μm) from the allergen-exposed bronchi. The present findings suggest the possibility that allergic airway inflammation may cause such vascular remodeling as previously associated with lung inflammatory conditions involving a risk for development of pulmonary hypertension.     

Expression of apolipoprotein C-III in McA-RH7777 cells enhances VLDL assembly and secretion under lipid-rich conditions

Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion efficiency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL₁. Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL₁ secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identified in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions.     

Regulation of yeast glycogen metabolism and sporulation by Glc7p protein phosphatase.

Glc7p is an essential serine/threonine type 1 protein phosphatase (PP1) from the yeast Saccharomyces cerevisiae, which has a role in many processes including cell cycle progression, sporulation, glycogen accumulation, translation initiation, and glucose repression. Two hallmarks of PP1 enzymes are very high amino acid sequence conservation and association of the catalytic subunit with a variety of noncatalytic, regulatory subunits. We tested the hypothesis that PP1 sequence conservation was the result of each PP1 residue playing a role in multiple intermolecular interactions. Analysis of 24 glc7 mutants, isolated primarily by their glycogen accumulation traits, revealed that every mutated Glc7p residue altered many noncatalytic subunit affinities and conferred unselected sporulation traits to various degrees. Furthermore, quantitative analysis showed that Glc7p affinity for the glycogen-binding noncatalytic subunit Gac1p was not the only parameter that determines the glycogen accumulation by a glc7 mutant. Sds22p is one Glc7p noncatalytic subunit that is essential for mitotic growth. Surprisingly, several mutant Glc7p proteins had undetectable affinity for Sds22p, yet grew apparently normally. The characterization of glc7 diploid sporulation revealed that Glc7p has at least two meiotic roles. Premeiotic DNA synthesis was undetectable in glc7 mutants with the poorest sporulation. In the glc7 diploids examined, expression of the meiotic inducer IME1 was proportional to the glc7 diploid sporulation frequency. Moreover, IME1 hyperexpression could not suppress glc7 sporulation traits. The Glc7p/Gip1p holoenzyme may participate in completion of meiotic divisions or spore packaging because meiotic dyads predominate when some glc7 diploids sporulate.