Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation

Silane-coated silica particle solutions (ISolate(TM) and PureSperm)TM)) and iodixanol (OptiPrep(TM)) were compared to polyvinylpyrrolidone (PVP)-coated silica particles (Percoll(TM)) in their efficacy to recover spermatozoa by gradient centrifugation for use in assisted reproductive procedures. Efficacy was assessed in terms of percentages of sperm recovery, sperm vitality and motility, normal sperm morphology and normal sperm chromatin condensation. No significant difference was found in the recovery of spermatozoa for men with both normal sperm counts and oligozoospermia, between PVP-coated and silane-coated particle solutions. Iodixanol had significantly lower sperm recovery compared to the other products. Sperm vitality, progressive motility, normal morphology and normal chromatin condensation did not differ significantly between any of the sperm isolation products.      

Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-gamma

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus- specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.      

Arrhythmogenic right ventricular dysplasia: polymorphism of clinical manifestations

We observed 15 patients with arrhythmogenic right ventricular dysplasia (ARVD): 9 with definite and 5 with probable ARVD (modified European Criteria, 2010). Eight patients had typical ARVD (frequent right ventricular extrasystoles, nonsustained right ventricular tachycardia without heart failure with or without myocarditis). Five patients had ARVD with progressive heart failure (right- or biventricular with or without myocarditis). Two patients had full scale arrhythmic form (sustained right ventricular tachycardia without or with right ventricular dilation, with or without myocarditis). In 3 cases diagnosis was confirmed morphologically or with DNA-diagnostics. This material allowed us to highlight the following specific points related to diagnostics of ARVD. Detection of fat at MRT is not obligatory for diagnosis, fat can be detected by MSCT; ventricular arrhythmias can move backwards in the picture of the disease; leading clinical manifestation can be unexplained right ventricular insufficiency; ARVD can be combined with other genetic cardiomyopathies as well as with infectious immune myocarditis (up to 50% of patients); elevated titer of anticardiac antibodies is not characteristic for isolated ARVD; myocardial biopsy allows to verify both ARVD and concomitant myocarditis. The paper also contains discussion of the role of myocarditis in various forms of ARVD and possibilities of its diagnosis and treatments.     

Anicteric hepatoxicity: a potential health risk of occupational exposures in Nigerian petroleum oil refining and distribution industry

Background

Literature abounds linking one’s job to certain unpalatable health outcomes. Since exposures to hazardous conditions in industrial environments often results in sundry health effects among workers, we embarked on this study to investigate the hepatic health effects of occupational activities in the petroleum refining and distribution industry.

Method

Biochemical markers of liver functions were assayed in plasma, using Reflotron dry chemistry spectrophotometric system. The study was conducted on randomly selected workers of Port Harcourt Refining Company (PHRC) and Pipelines and Petroleum Product Marketing Company (PPMC) both in Alesa-Eleme near Port Harcourt, Nigeria, as well as non-oil work civil servants serving as control subjects.

Result and conclusion

Results showed that, bilirubin ranged 0.3-1.6 mg/dl with a mean of 0.66±0.20mg/dl among the oil workers as against 0.5-1.00mg/dl with a mean of 0.58±0.13mg/dl in non-oil workers, Alkaline phosphatase ranged 50.00-296.00u/l (mean: 126.21±39.49u/l) in oil workers as against 40.20-111u/l (mean: 66.83±18.54u/l) for non-oil workers, Aspartic transaminases (AST) ranged 5.80-140.20u/l (mean: 21.81±11.49u/l) in oil workers against 18.00-44.00u/l (mean: 26.89±6.99u/l) for non-oil workers, while Alanine transaminases (ALT) ranged 4.90-86.00u/l (mean: 22.14±11.28u/l) in oil workers as against 10.00-86.60u/l (mean: 22.30±10.22u/l) for the non-oil workers. A close study of the results revealed that although the mean values for all the studied parameters were still within the parametric reference ranges, however, relative to the referents, there were significant increases (P<0.05) in plasma bilirubin (though anicteric) and alkaline phosphatase that was not matched with a corresponding increase in the plasma transaminases, suggesting a possibility that toxic anicteric hepatoxicity is part of the potential health effects of sundry exposures in the Nigeria petroleum oil refining and distribution industry. Gender differentiation data showed that though the mean values for the parameters were higher in males than females, the increases were not significant in most cases (P>0.05), whereas data for age and exposure period classifications revealed that irrespective of the age of the worker, the effects are likely to start after the first five years, manifesting fully after the first decade of occupational exposures. Thus, an update of industrial/occupational health measures is necessary for a safer and healthier work environment.     

Analysis of the relationship between age and treatment response in migraine

In migraine, headache severity varies with age. As a consequence, the effectiveness of medication may also depend on a patient's age. The purpose of this study was to assess the combined effect of age and drug treatment on headache characteristics. Using data from clinical trials of sumatriptan in adolescents and adults, we show how the interaction between age and drug exposure can be parameterised as a covariate on a Markov model that describes the decline of headache severity over three clinically defined stages (no relief, relief and pain-free status). The model explains important clinical observations: (i) the rates at which the pain relief and pain-free status were attained were found to be inversely related to age; (ii) in placebo-treated patients, the mean transit time from 'no relief' to 'relief' is 3 h for young adolescents and increases to 6 h for patients aged ≥ 30 years; and (iii) sumatriptan reduces the transit time to 2 h, irrespective of age. These findings indicate that the therapeutic gain over placebo increases with age. Prospective studies of antimigraine drugs should take this relationship into account when extrapolating efficacy data from adults to adolescents.     

Ex-vivo investigations on endoluminal laser therapy of varicosis – An optimization process

ObjectiveThis study was performed to compare the acute effects of different endoluminal heating procedures, by which obstruction of insufficient veins could be induced in an established standardized experimental ex-vivo model.MethodsThe model consists of the subcutaneous foot vein from recently slaughtered cows. Different energy sources (laser and radiofrequency), application devices and energy application parameters were compared in an iterative optimizing process. Following energy application the vein tissue was harvested and prepared for histology.ResultsBy means of the developed ex-vivo model reproducible conditions for the application of endoluminal thermal energy were enabled in each specimen. Radiofrequency-treated veins showed macroscopic shrinkage, contraction of vessels and induration of tissue. Histologically a circumferential and homogenous thickening of the vessel wall could be described. Dependent on the laser treatment protocol, in-situ dissection of the treated veins showed a broad spectrum of effects. These effects ranged from blood deposits in perivascular tissue and perivascular thermal lesions to transmural coagulated vein tissue inducing obstruction of the lumen.ConclusionThis standardized model is applicable to investigate acute effects of endoluminal treatment protocols. Changes in the protocol could be easily performed and investigated in order to develop optimized treatment protocols. These ex-vivo experiments indicate that endovenous laser treatment should be modified and needs treatment standardization to ensure a controlled homogenous circumferential thermal damage.     

A randomized double-blind placebo-controlled crossover study of subcutaneous sumatriptan in general practice

Objective. To evaluate the therapeutic response to sumatriptan in the acute migraine attack. Material and methods. Two hundred and thirty migraineurs diagnosed by their general practitioners in accordance with their usual practice were included in the study. The patients treated two migraine attacks at home by subcutaneous injection of sumatriptan or placebo for the first attack and the alternative medication, i.e. placebo or sumatriptan, for the second attack (crossover). Following treatment, a neurology resident interviewed and examined the patients, Results. When sumatriptan was compared to placebo, significantly more of the 209 evaluable patients reported headache relief at I h (56% vs 8%, p < 0.001) and 2 h (62% vs 15%, p < 0.001) after the first injection. Resolution of nausea, photophobia, and phonophobia was significantly more common in patients on sumatriptan than in those on placebo ( p < 0.001 for all comparisons). The adverse events were usually transient and of mild or moderate severity; however, three patients withdrew due to adverse events. Ninety-five percent of patients evaluated by a neurology resident met the IHS criteria for migraine. Conclusion. In general practice, sumatriptan taken subcutaneously using an autoinjector at home was an effective and well tolerated acute treatment for migraine.     

Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product

To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (K_d ∼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H₅-bisDHBS) (K_d = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)]²⁻ from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)]²⁻ binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)₂]³⁻. The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)]²⁻ (K_d ∼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins.With the rapid rise in bacterial resistance to antibiotics, a better understanding of cooperative behavior in microbial communities is urgently needed for the development of novel approaches to controlling infections caused by resistant bacteria (1, 2). As an essential nutrient, iron is often a growth-limiting factor for beneficial, commensal, and pathogenic bacteria alike, not only due to its low solubility in water under aerobic conditions at and around neutral pH, but also because the host organism and competing microbes actively limit its availability (3, 4). Microorganisms evolved efficient Fe(III) uptake mechanisms to overcome this challenge, a common strategy being the production of siderophores, small Fe(III)-chelating molecules with high affinity and selectivity for Fe(III), with over 500 examples known to date (5). The sharing of siderophores is a recognized example for positive cooperativity that has been linked to bacterial virulence (6). The best-characterized siderophores are hexadentate ligands that form coordinatively saturated, octahedral 1:1 complexes with Fe(III) (3, 5, 7), the most studied being the triscatecholate enterobactin (H₆-ENT) produced by many enteric bacteria (8).In Escherichia coli, enterobactin is synthesized within the cell and secreted through the cell membranes to capture Fe(III) from the environment. The resulting Fe(III)-enterobactin complex [Fe(ENT)]³⁻ is recognized by the outer membrane receptor FepA and actively transported into the periplasm. In the periplasm, [Fe(ENT)]³⁻ is sequestered by the periplasmic binding protein (PBP) FepB, which transfers it to an inner membrane transporter for further transport into the cytoplasm (9). Once there, [Fe(ENT)]³⁻ is hydrolyzed by an intracellular esterase to release Fe(III) for use in the cell (8).Along with the development of structurally diverse siderophores, microorganisms adapted their associated receptor and transport proteins for the uptake of the appropriate Fe(III) complexes (9). To gain a competitive advantage, many bacteria have evolved to poach siderophores produced by other bacteria. Campylobacter jejuni, for example, does not itself produce siderophores yet possesses an uptake system that is able to use siderophores from competing species (10). Initially, it was proposed that in C. jejuni [Fe(ENT)]³⁻ is transported across the outer membrane by the receptors CfrA and CfrB (11). Once in the periplasm, [Fe(ENT)]³⁻ was proposed to bind to the PBP CeuE, the resulting complex enabling the transport of the ferric siderophore into the cytoplasm (12, 13).In addition, increasing numbers of lower-denticity siderophores are being isolated from bacterial cultures and found to coordinate Fe(III) and mediate its uptake (14–18). For example, the trilactone backbone of enterobactin makes it prone to hydrolysis, and although this lability is necessary to allow the intracellular release of Fe(III) from the siderophore, it in addition leads to its slow degradation in aqueous media (7, 19–21). Three hydrolysis products are formed: tris(2,3-dihydroxybenzoyl-l-Ser) (H₇-trisDHBS), bis(2,3-dihydroxybenzoyl-l-Ser) (H₅-bisDHBS), and 2,3-dihydroxybenzoyl-l-Ser (H₃-monoDHBS), with all three found in the growth medium of E. coli (Fig. 1). Although enterobactin, once secreted, is also available to other cells (producers or nonproducers), it can only be used once because Fe(III) release requires its hydrolysis. The enterobactin hydrolysis products, however, could be used again as secondary, lower-denticity siderophores.Open in a separate windowFig. 1.Molecular structure of enterobactin, its hydrolysis products, the siderophore mimic H₆-MECAM, and a selection of tetradentate siderophores.It has been demonstrated that the human pathogens C. jejuni (10, 22, 23) and Vibrio cholerae (24), the causes of food poisoning and cholera, respectively, can use enterobactin hydrolysis products for the uptake of Fe(III) from their environment. Both are known not to produce enterobactin.An alternative Campylobacter Fe(III) acquisition model that relies on these linear hydrolysis products was recently proposed based on the finding that Cee, the sole trilactone esterase of C. jejuni and Campylobacter coli, is located in the periplasm, i.e., these bacteria are unable to degrade enterobactin within the cytoplasm (25). The model suggests that, once the Fe(III) complex of enterobactin enters the periplasm, its ester backbone is cleaved by Cee, which is highly efficient in hydrolyzing both the Fe(III) complex and apo-enterobactin. The resulting hydrolysis products, mainly the tetradentate ligand bisDHBS⁵⁻ and the bidentate ligand monoDHBS³⁻, are then used to mediate the subsequent transport of Fe(III) into the cytoplasm.The identification of the esterase Cee and in particular the observation that bisDHBS⁵⁻ can be used independently of enterobactin (25) raise important questions about the siderophore preference of the PBP CeuE and its role in the iron uptake in C. jejuni. By using siderophore mimics, we previously demonstrated that CeuE can bind the Fe(III) complexes of both hexadentate and tetradentate catecholate ligands (26, 27). Our cocrystal structures revealed that CeuE interacts with the coordinatively saturated Fe(III) complex of the hexadentate mimic MECAM⁶⁻ through electrostatic interactions and hydrogen bonding, whereas it binds the coordinatively unsaturated complex of the tetradentate mimic 4-LICAM⁴⁻ by recruiting the side chains of two amino acid residues (His227 and Tyr288) to complete the coordination sphere of the Fe(III) center. We established that His227 and Tyr288 are conserved among a subset of related PBPs, including VctP from V. cholerae, and suggested that this subset of PBPs undergo similar structural changes to adapt to the binding of lower-denticity sidero-phores (27). The recent report that V. cholerae most efficiently uses trisDHBS⁷⁻ and bisDHBS⁵⁻ for the acquisition of Fe(III) provided a partial confirmation of this prediction (24).Here, we report that CeuE binds [Fe(bisDHBS)]²⁻ with much higher affinity than the Fe(III) complex of enterobactin, reveal the structural basis for this difference in binding strength, and examine key aspects of the relevant Fe(III) coordination chemistry in solution.