Final Dilution Research Articles

Stability of diluted chlorhexidine for skin testing in drug allergy evaluations

BackgroundChlorhexidine gluconate (CHX), a common cause of perioperative anaphylaxis, is frequently used for skin testing in allergy evaluations. While CHX’s maximal non-irritating concentrations are known, the stability of its dilutions for skin testing remains unexplored, particularly when using sterile water for injection (SWFI) or normal saline (NS) as diluents. ObjectiveTo evaluate the stability and precipitation of CHX when diluted with SWFI or NS for drug allergy skin testing. MethodsCHX dilutions (5 mg/mL to 0.002 mg/mL) were prepared using SWFI and NS. High-performance liquid chromatography (HPLC) and UV-Vis spectrophotometry were used to assess stability and precipitation over 48 hours. Turbidity was measured at various time points to monitor precipitation. ResultsHPLC analysis showed no significant differences in peak heights between CHX-SWFI and CHX-NS dilutions. However, visible precipitation and increased turbidity (>100 NTU) were observed in CHX-NS at higher concentrations (5 mg/mL) after 60 minutes. No precipitation occurred in CHX-SWFI at any concentration for 48 hours. ConclusionFor CHX skin testing, SWFI is the preferred diluent at concentrations above 0.02 mg/mL to avoid precipitation. Using NS for the final dilution from 0.02 mg/mL to 0.002 mg/mL is feasible and reduces injection pain. Reagents can be prepared up to 24 hours prior to testing, except for CHX-NS at 5 mg/mL.

Improved method for quantification of intact oxaliplatin by ultra high performance liquid chromatography-inductively coupled plasma mass spectrometry: Applications to clinical and speciation studies

Interest is increasing in the use of different liquid chromatography techniques coupled online to mass spectrometry for the quantification of platinum anticancer drugs in human plasma to inform cancer chemotherapy. We developed, validated and studied the application of a method for quantification of intact oxaliplatin in human plasma using ultra high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (UHPLC-ICP-MS). Plasma samples were processed instantly after collection from patients to preserve oxaliplatin speciation by methanol-deproteinization, and storage of diluted supernatants (plasma:methanol 1:2 v/v) at −80 °C. UHPLC separation of intact oxaliplatin and internal standard (carboplatin) was achieved using a C18 column and linear gradient mobile phase (Mobile phase A: water-methanol (97:3 v/v), 0.075 mM sodium dodecyl sulfate, 9.79 nM thallium adjusted to pH 2.5 with trifluoromethanesulfonic acid; Mobile phase B: 100 % methanol (v/v)) with ICP-MS detection to monitor platinum and thallium at m/z 195 and 205, respectively. The limit of quantification was 50 nM in methanol-deproteinized diluted plasma (1:2 v/v). Linearity was established for calibration standards ranging from 50 to 500 nM made in methanol-deproteinized diluted plasma (1:2 v/v), and for dilution of higher concentration samples in blank matrix containing internal standard (final dilution 1:29 v/v). Intra-day and inter-day accuracy ranged from 96.8 to 103 % of nominal concentration and precision from 0.62 to 2.49 % coefficient of variation. Recovery was complete and a matrix effect confirmed the requirement for matrix-matched standards. Intact oxaliplatin was stable during storage for at least 473 days, and during analysis, in methanol-deproteinized diluted plasma (1:2 v/v). The method was applied to determining the plasma concentrations of intact oxaliplatin in patients undergoing cancer chemotherapy, and studies of oxaliplatin degradation in vitro. This improved method based on UHPLC-ICP-MS will allow more specific, efficient and reliable quantification of intact oxaliplatin in human plasma.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB – TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to −3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias – 4 to – 25. The bias between [VIAB – TMOT] and VLPP ranged from – 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

Optimisation of cryovials and milt dilution ratios to upscale carp sperm cryopreservation for use in hatchery seed production

This study reports the experimental results carried out to optimise containers and miltdilution ratios for carp sperm cryopreservation, in order to make the process simple andcompatible with the needs of commercial carp seed production, which is a small-scale andlow technology sector in Asia. The procedure optimisation for up-scaling was assessedby the ability of cryopreserved sperms to fertilise Cyprinsu carpio eggs and hatchingpercentage, in three different experiments. In the first experiment, we tested the effect ofmilt to different extender dilutions (1:4, 1:6 and 1:8); the second experiment tested theeffect of container capacities (0.5 ml French straw; 2 ml and 5 ml cryovials) with two dilutionratios (1:3 and 1:6) and in the third experiment, the efficacy of pre-mix of extender salts andindividually pre-weighed salts stored over 5 months was compared with fresh preparationof extender. Out of the three dilutions tested, two dilutions viz., 1:4 and 1:6, yielded hatchingabove 50%. We also used the milt cryopreserved at dilution of 1:3 which was post-thawdiluted with extender, to make final dilution of 1:6, to fertilise the eggs. This post-thawdilution in the ratio 1:6, yielded the results comparable to the milt cryopreserved in 1:6dilution. This approach can help small-scale seed producers to store more sperm withinthe limits of the cost of liquid nitrogen. The extender salt composition, pre-weighed andpre-mixed, was also found to give comparable results to the fresh composition after storageover 5 months. This can assist in developing ready to use, cost-effective, working kitsavailable to semi-technical hatchery seed producers. The parameters optimised in the studyhas potential to be transformed as an easy strategy with the use of 5 ml vial, cryopreservedat 1:3 dilution, with further post-thaw dilution at site, thus aiding in fertilisation of largevolume of eggs produced by high fecund carp species within the limited time available formaintaining good gamete quality. Keywords:Cryobanking, Cryovials, Dilution, Salt premix

Antiviral Potential of Specially Selected Bulgarian Propolis Extracts: In Vitro Activity against Structurally Different Viruses.

Propolis is a natural mixture of resins, wax, and pollen from plant buds and flowers, enriched with enzymes and bee saliva. It also contains various essential oils, vitamins, mineral salts, trace elements, hormones, and ferments. It has been found that propolis possesses antimicrobial, antiviral, and anti-inflammatory properties. We have studied the antiviral activity of six extracts of Bulgarian propolis collected from six districts of Bulgaria. The study was conducted against structurally different viruses: human coronavirus strain OC-43 (HCoV OC-43) and human respiratory syncytial virus type 2 (HRSV-2) (enveloped RNA viruses), human herpes simplex virus type 1 (HSV-1) (enveloped DNA virus), human rhinovirus type 14 (HRV-14) (non-enveloped RNA virus) and human adenovirus type 5 (HadV-5) (non-enveloped DNA virus). The influence of the extracts on the internal replicative cycle of viruses was determined using the cytopathic effect (CPE) inhibition test. The virucidal activity, its impact on the stage of viral adsorption to the host cell, and its protective effect on healthy cells were evaluated using the final dilution method, making them the focal points of interest. The change in viral infectivity under the action of propolis extracts was compared with untreated controls, and Δlgs were determined. Most propolis samples administered during the viral replicative cycle demonstrated the strongest activity against HCoV OC-43 replication. The influence of propolis extracts on the viability of extracellular virions was expressed to a different degree in the various viruses studied, and the effect was significantly stronger in those with an envelope. Almost all extracts significantly inhibited the adsorption step of the herpes virus and, to a less extent, of the coronavirus to the host cell, and some of them applied before viral infection demonstrated a protective effect on healthy cells. Our results enlarge the knowledge about the action of propolis and could open new perspectives for its application in viral infection treatment.

P-078 In vitro effects of polycarbophil vaginal moisturizing gel on sperm motility and vitality

Abstract Study question The aim of this study was to evaluate the possible effects of intravaginal polycarbophil gel in sperm quality in an in vitro model. Summary answer Incubation of human sperm samples with a vaginal moisturizer containing polycarbophil gel 10% resulted in important reduction of sperm motility and vitality. What is known already The possible effect of lubricants on sperm quality has been a matter of debate for over 50 years. Despite the publication of a few studies, the real impact of lubricants on male gametes is remains unknown. Some commercial lubricants have been tested but so far there is a lack of studies evaluating the impact of vaginal moisturizers on seminal quality. Vaginal dryness is a recurring and significant complaint affecting couples trying to conceive who may use lubricants, vaginal moisturizers or even natural oils that could advsersely interfere with seminal quality. Study design, size, duration This paired prospective study involved seminal samples obtained fom 14 men undergoing fertility treatment at a private IVF clinic in Brazil from August/2020 to July/2022. The study was approved by the local review board and each participant signed an informed consent. Each sample was divided into two aliquots: 20% polycarbophil gel (final dilution 10%) or medium only. Statistical analysis was performed using D’Agostino/ Pearson, General Linear Model, Wilcoxon, Friedman’s. P values <0.05 were considered significant. Participants/materials, setting, methods Normozoospermic samples of 14 patients (alpha 0.05, power 0.8)were collected by masturbation, 2 to 5 days after the last ejaculation. After liquefaction ,10 μmL were analyzed using a Makler chamber according to World Helath Organization (WHO) guidelines. Samples were mixed 1:1 v/v with 20% policarbophil gel or with culture medium only, then incubated for 24h at 34 °c. Analysis were performed at 0, 15, 30 min and 24h to determine progressive motility and vitality (eosin-nigrosin test) Main results and the role of chance There was a significant decrease of sperm motility according to the WHO classification in both experimental conditions over time, but the reduction was more evident in the samples treated with polycarbophil gel (p < 0.0001 group x time, General LinearModel for Repeated Measure). The proportion of progressively motile sperm reduced immediately upon contact of the seminal sample with polycarbophil gel (time point 0 min) and decreased throughout the experiment, contrasting to incubation with culture medium (control), which only affected substantially sperm motility after 24h. In the validation cohort, the proportion of progressively motile sperm decreased from a median of 52% in fresh samples to 11% after 24h incubation with culture medium and 0% after 24h incubation with polycarbophil gel (p < 0.0001). Sperm vitality at the end of the experiment also differed significantly between treatments, with medians of 62.5% after exposure to culture medium and 47% after exposure to polycarbophil gel (p = 0.020, Wilcoxon’s test) Limitations, reasons for caution Results represent samples from a selected group of men undergoing fertility treatment at a private center in Brazil. The effect of polycarbophil gel in fertile males may be different. Moreover, the in vitro setting used here does not accurately mimic the vaginal environment which could interfere with the results. Wider implications of the findings Polycarbophil-based gels and similar products may harm sperm vitality and motility, therefore woment trying to conceive should avoid the use of such products until more studies prove otherwise. Trial registration number Not applicable

Silver nanoparticles and cellulose microfiber micro-composite from banana (Musa acuminata) waste: green synthesis, antioxidant property and antimicrobial capacity

The green chemistry promotes the synthesis of nanomaterials from plant extracts as a new climate intelligent alternative to the use of conventional protocols based on costly and toxic chemicals. Therefore, this research was undertaken to analyses the efficiency of banana (peels and rachis) waste extracts in the production of a micro-composite composed by silver nanoparticles (AgNPs) and cellulose microfibers (CMF) respectively. Results showed the synthesis of 24 nm diameter spherical particles AgNPs, with a peak of absorbance at 410 nm, in (v/v) water:ethanol extracts of banana peels at a final dilution of 3.10-2. Concomitantly, 50-350 µm in length and 5-10 µm of diameter CMF were obtained via the oxalic acid hydrolysis of the oven-dried banana rachis. The micro-composite (AgNPs-CMF) and AgNPs displayed an active reducing capacity over 60% determined by the DPPH test, and active bacterial activity against E. Coli and S. aureus in Petri dishes. Overall results support the use of banana waste for the synthesis of AgNPs and CMF for industrial purposes.

Perioperative Monitoring with Global Coagulation Test in Severe Hemophilia a without Inhibitor on Emicizumab Prophylaxis Undergoing Orthopedic Surgery

INTRODUCTION Emicizumab is a bispecific antibody used to prevent bleeding episodes in patients with severe hemophilia A (PwHA) without inhibitor. However, using FVIII to manage breakthrough bleeding or invasive procedures in patients without inhibitors remains necessary. Current guidelines recommend using chromogenic FVIII (FVIII:C) levels based on bovine reagents to monitor the hemostatic response to the combination of both drugs. However, the perioperative hemostatic management of PwHA undergoing prophylaxis with emicizumab and FVIII remains challenging. Here we describe the hemostatic management and monitoring using global coagulation test of two PwHA without inhibitor on emicizumab prophylaxis undergoing orthopaedic surgery with FVIII. METHODS Perioperative monitoring for two patients was performed, measuring FVIII:C levels using bovine-derived reagents. Samples for global coagulation test were collected in tubes with Corn Trypsin Inhibitor to prevent activation of the contact pathway. Clotting time (CT) was evaluated by Rotational Thromboelastometry (ROTEM®) using whole blood activated by a low concentration of tissue factor (TF) solution (final dilution 1:50,000 of EXTEM reagent) plus recalcification. Thrombin Peak (TP) was assessed by thrombin generation test (TGT) in plasma, also using a low amount of TF and phospholipids (PPP-LOW, Stago). ROTEM and TGT were performed in samples collected before and after FVIII administration during the perioperative process. Samples obtained before FVIII dosing were also spiked in vitro with equivalent therapeutic amounts of FVIII products. RESULTS Patient 1 is a 55-year-old male with severe HA under EMI prophylaxis (1.5 mg/week). He underwent an ankle arthrodesis with osteosynthesis due to severe hemophilic arthropathy. Plasma-derived FVIII/FVW concentrate (pdFVIII/FVW) was used to provide effective hemostatic coverage during the procedure. He received the first dose before surgery and the last dose on day +7 after surgery. The postoperative course was uneventful, and he was discharged on day +4. Patient 2 is a 52-year-old male with a medical history of HIV infection, lymphoma in complete remission and severe HA on EMI prophylaxis (6 mg every four weeks). He was referred to an orthopaedic surgeon due to cubital tunnel syndrome secondary to hemophilic arthropathy that required surgery. A single dose (50 Ul/kg) of extended half-life recombinant FVIII (rFVIII EHL) concentrate was administered before intervention. He was discharged on day +2 in the absence of bleeding complications. He received an additional dose of rFVIII EHL on day +3 after the surgery. Table 1 shows FVIII:C levels in both patients. Both global coagulation tests showed that treatment with emicizumab was insufficient for complete normalization of hemostatic parameters. Clotting time was longer, and thrombin generation was reduced in emicizumab-treated patients compared to healthy controls before the intervention (Figure 1). We observed an improvement and normalization of CT and TP values after administering both FVIII products (plasma-derived or recombinant with EHL) during the perioperative period. This improvement was similar to that obtained with in vitro spiking of equivalent doses of FVIII (Figure 1). Additionally, a correlation between FVIII:C levels and CT and TP values was also observed. CONCLUSIONS The global coagulation test were used to evaluate the hemostatic response to FVIII treatment in PwHA on emicizumab prophylaxis during invasive procedures. Considering that both tests provide a more comprehensive assessment of the hemostatic state of the interaction of emicizumab and FVIII, these tests may complement FVIII levels in complex cases of bleeding or surgery. Funding: ISCIII-FEDER PI19/00631; Catedra UAM-Roche Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Lupus Anticoagulant Titer Is Associated with Elevated Anti-Phosphatidyl-Serine/Prothrombin Antibody Levels

Background Laboratory diagnosis of the antiphospholipid antibody syndrome (APS) requires evidence of persistently positive lupus anticoagulant (LAC) activity in clot-based assays and/or the presence of medium-high titer anticardiolipin (aCL) and/or anti-beta 2 glycoprotein I (aB2GPI) antibodies. Although aCL and aB2GPI methodologies provide quantitative information about the antibody titer, LAC testing is interpreted in a qualitative fashion (presence versus absence). Objectives In this study, we describe a method for measuring the LAC titer and evaluate the relationship between the LAC titer, other antiphospholipid antibodies and clinical outcomes. Methods Residual plasma from 51 LAC-positive samples (50 unique patients) were included in this study. To estimate LAC-titers, samples were tested undiluted and subjected to serial (dependent) twofold dilutions in pooled normal plasma (PrecisionBiologic) up to a final dilution of 1:128, as appropriate. The dilute Russell's viper venom screening time (DRVVT screen, LA Check, PrecisionBiologic) and activated partial thromboplastin screening time (APTT, SynthasIL, Werfen) were measured for each dilution. A four-parameter logistic model of the dilution factor versus the clotting time was used to interpolate the LAC-titer at the upper limit of normal for both assays (APTT of 37s and DRVVT screen ratio of 1.2). Measurements of aCL, aB2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) were performed using ELISA methodologies (QUANTA Lite, Werfen). Available clinical data were extracted from the electronic medical record. Continuity corrected chi-square test was used as a categorical test of association and quantile regression was used to compare medians for interval/ratio data across subgroups (Stata v17). Results There was a strong positive linear relationship between the APTT and DRVVT titers (Pearson's correlation coefficient, r=0.92, Figure 1A). There was only a moderate relationship between the APTT titer and the APTT screen in the undiluted sample (r=0.50) and almost no relationship between the DRVVT titer and the DRVVT screen ratio or DRVVT screen/confirm ratio in the undiluted sample (r=0.06 and r=0.13, respectively). Data-driven partitioning identified two DRVVT titer groups: low-titer (<10) and high-titer (≥10). Empirical cutpoint estimation (Youden) identified an APTT titer threshold of 5 for predicting the DRVVT titer group. Among patients with available clinical history, 15 of 25 (60%) low-DRVVT-titer and 16 of 20 (80%) high-DRVVT-titer patients had thromboembolic/obstetric events (p=0.26). Autoimmune diagnoses were more prevalent in the low-DRVVT-titer group (19 of 25 patients, 13 with SLE) than the high-DRVVT-titer group (8 of 20 patients, 3 with SLE, p=0.03). Comparing the low and high-DRVVT-titer groups, median aCL IgM (p=0.27), aCL IgG (p=0.99), aB2GPI IgM (p=0.72) and aB2GPI IgG (p=1.00) titers were not different. High-DRVVT-titer samples had a significantly higher median aPS/PT IgM compared to low-DRVVT-titer samples (p <0.01, Figure 1B) but there was no difference observed for IgG aPS/PT (p=0.57, Figure 1B). Conclusions APTT and DRVVT screening times are not strongly correlated with the corresponding APTT or DRVVT LAC-titer and may not indicate LAC potency. High DRVVT LAC-titers were strongly associated with IgM aPS/PT levels, but not with aCL, aB2GPI or IgG aPS/PT levels. Further research is required to evaluate the association between the LAC-titer and recurrent thrombosis in APS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Structural and physical characterization of iron-oxide based inks for inkjet printing

Iron-oxide nanopowders, hematite and magnetite, are basic compounds applied in several steps for the preparation of magnetic inks. These steps included ball milling of the nanopowders in dowanol, mixing with polysiloxane binder dissolved in pure ethanol, and final dilution in hexanol to adjust the rheological parameters of the inks applied in the inkjet printing technology under ambient conditions. Various experimental methods, carried out for the samples in all forms (powder, ink, and layer), have yielded basic data concerning morphology, chemical, and physical properties and allowed to follow their changes during individual processing steps. The phase analysis of both the pristine iron-oxides did not confirm the pure single-phase compositions. The hematite contained also a substantial amount of magnetite and maghemite contributing to its unusual high saturation magnetization, Ms, of 66 Am2kg−1. The magnetite contained about 38 % maghemite and its Ms was 117 Am2kg−1. The magnetic parameters of both the iron-oxides in the nanopowder and ink forms were comparable contrary to those measured in the solidified state after printing. Substantial decrease in the Ms values and changes of the remnant magnetization and coercivity were observed in both cases. The obtained results were verified by measurement of a designed magnetite core printed on the flexible foil and tested as a magnetic sensor. Nevertheless, despite the worse magnetic parameters, the relative permeability, approximately 2, was twice as high as that reported by other authors.

Sensitive Immunochromatographic Assay (ICA) for the Determination of Thiamethoxam in Fruit, Vegetables, and Natural Water

Rapid on-site detection of thiamethoxam, a widely used neonicotinoid insecticide, is required to ensure food safety and environmental health. In this study, we produced specific and high-affinity monoclonal antibodies against thiamethoxam and developed an immunochromatographic assay (ICA) strip. Monoclonal antibodies labeled with colloidal gold particles were used as the probe, nitrocellulose membrane as the solid carrier, thiamethoxam-ovalbumin (OVA) conjugate as the test line, and goat anti-rat immunoglobulin G (IgG) as the control line. The visual limit of detection of the test strip was 0.5 µg/L thiamethoxam and the cutoff value was 2.0 µg/L. The test strip enabled rapid visual detection (within 10 min) of thiamethoxam. Matrix interferences were eliminated by diluting the sample extracts with the optimized working buffer solution (phosphate buffer containing 5% methanol v/v, pH 8.0); the final dilutions used were: 50-fold for cucumber, pear, and bok choy extracts, 5-fold for apple extracts, and 2-fold for pond water. Using the recovery test, we verified that the test strip was suitable for rapid on-site determination of thiamethoxam residues in fruit, vegetable, and water samples. The developed immunochromatographic assay is easy to use, rapid, environment-friendly, and suitable for on-site screening and detection of thiamethoxam residues in food and environmental samples.

White Wine-Induced Endothelium-Dependent Vasorelaxation in Sprague-Dawley Rats.

The vasodilatory activity and polyphenolic content of commercially available white wine is low compared to red wines. This study assessed the vasodilator potential of white wines produced by four different fermentation processes: (1) white wine produced by the standard procedure; (2) grapes left to macerate completely for 30 days; (3) grapes left to macerate up to half of unfermented sugar; and (4) wine produced by cooling the must. All tested wine samples were analyzed for their phenolic content, antioxidant capacity, and ethanol content. Vasodilation was examined in the norepinephrine pre-contracted isolated rat aortas of male Sprague-Dawley rats randomly exposed to cumulative concentrations (0.1‰ to 8‰ final dilutions in organ baths) of each of the tested wine samples with or without quercetin and/or gallic acid supplementation, in the absence/presence of NOS inhibitor L-NAME. Standard procedure and the procedure involving must cooling gives wine with lower phenolic content, antioxidant capacity, and lower vasodilator potential, respectively. L-NAME inhibited vasodilation to all wine samples. Quercetin with or without gallic acid supplementation restored vasodilation. Results show that vasodilation to white wine is NO-dependent and suggest the possibility of increasing the antioxidant capacity and vasodilatory potential of white wine using different production procedures, depending on quercetin content.

Carcinosinum produces phenotypic changes of 4T1 tumor cells in vitro

Considering that there are few published studies that specifically address the exclusive use of Carcinosinum in different potencies and, most of them focused on genotypic and clinical effects, the present study was proposed to identify possible phenotypic changes, including viability, expression of HER-2 and metastatic abilities, using 4T1 cells in vitro as a model. Carcinosinum was tested in different homeopathic potencies (12cH; 30cH; 200cH) mechanically prepared using sterile pure water. The time space between preparing the potencies and using them was 24 hours.The final dilutions were inserted into the culture medium in a volume equal to 10%, at the time of cell seeding. The same succussed vehicle used to prepare the drugs (70% ethanol) diluted 1:100 in sterile pure water was used as control. All treated cells were cultured in 25 mL flasks, with cell density of 5 x 105 cells/mL. After 24 hours of treatment, cells were analyzed for apoptosis index using Annexin V kit and the Countess® system. The morphology of 4T1 cells was monitored by staining cell smears with hematoxylin-eosin and Giemsa methods. HER-2 expression was assessed by immunocytochemistry and metalloproteinase activity was assessed by zymography. The determination of the cytokine profile was performed using Cytometric Bead Array (CBA). The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. Carcinosinum 30cH presented the highest apoptotic index and reduction of MMP-9-Pro expression; Carcinosinum 200cH produced the highest positivity for HER-2 and no specific effect was seen after the treatment with Carcinosinum 12cH. No change in cytokine expression was seen among treatments. We conclude that Carcinosinum 30cH and 200cH can change phenotypic features important to tumor development in vitro. The clinical meaning of these data deserves further investigation.

Effect of glycerol concentration, glycerol removal method, and straw type on the quality and fertility of frozen chicken semen

The long-term semen cryopreservation is increasingly crucial for conservation of endangered livestock and poultry species. Glycerol is the most widely used cryoprotectant for freezing chicken semen. Continuous improvement in details with glycerol may help increase the fertility of post-thawed semen. Two experiments were performed in the present study to investigate the effects of glycerol concentration, removal method, and straw type on the quality of post-thawed sperm. In experiment 1, glycerol concentration (3%, 5%, 7%, 9%, 11%, and 13%) and glycerol removal method (final dilution ratio 1:1, 1:2, 1:4, 1:8, 1:16, and 1:20) combination groups were investigated for post-thawed sperm quality, residual glycerol concentration, and fertility to find the best combinations. Experiment 2 was performed to evaluate the effects of straw type (0.25 and 0.5 mL) and glycerol concentration (3%, 5%, 7%, 9%, 11%, and 13%) on the post-thawed sperm quality. Results showed that post-thawed sperm motility of 6 glycerol concentration groups were different (P < 0.01). Sperm motility of 5%, 7%, 9%, 11% and 13% was higher than that of 3% (P < 0.01). There was no difference among different concentrations of glycerol in VSL, VCL, VAP, ALH, WOB, BCF, LIN, or STR (P > 0.05). As for the glycerol removal method, sperm motility of 1:8 dilution was the highest, followed by 1:1 and 1:2, while the difference among groups was not statistically significant (P = 0.11). Glycerol concentration and removal method had no interaction effect on sperm motion parameters (P > 0.05). The highest fertility (48.70%) was found for the 5% and 1:2 combination. There was no difference for sperm motility between 0.25 and 0.5 mL straws (P > 0.05). Glycerol concentration and straw type had no interaction effect on the sperm motion parameters (P > 0.05). It can be concluded from these observations that the combination of 5% glycerol and 1:2 dilution rendered higher fertility should be suggested in practice, and that both 0.25 and 0.50 mL straws fit the present procedure.

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. For the experiment, semen extender was supplemented with Mito TEMPO (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p<0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p<0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Effects of 200cH medications on mice bone marrow cells and macrophages

Paracelsus once wrote: "All things are poison and nothing is without poison, only the dose permits something not to be poisonous." Latter Hahnemann formulated the law of similars, preparations which cause certain symptoms in healthy individuals if given in diluted form to patients exhibiting similar symptoms will cure it. Highly diluted natural complexes prepared according to Hahnemann’s ancient techniques may represent a new form of immunomodulatory therapy. The lack of scientific research with highly diluted products led us to investigate the in vivo and in vitro actions of commonly used medications. Here we describe the results of experimental studies aimed at verifying the effects of Mercurius solubilis, Atropa Belladonna, Lachesis muta and Bryonia alba. All medications were at 200cH dilution. Animals were maintained for 7 days and were allowed to drink the medications, which were prepared in a way that the final dilution and agitation (200cH) was performed in drinking water. The medication bottle was changed and sucussed every afternoon. Co-culture of non treated mice bone marrow cells and in vitro treated peritoneal macrophages were also performed. After animal treatment the bone marrow cells were immunophenotyped with hematopoietic lineage markers on a flow cytometer. We have determined CD11b levels on bone marrow cells after culture and co-culture with treated macrophages and these macrophages were processed to scanning electron microscopy. We have observed by morphological changes that macrophages were activated after all treatments. Mercurius solubilis treated mice showed an increase in CD3 expression and in CD11b on nonadherent bone marrow cells after co-culture with in vitro treatment. Atropa Belladonna increased CD45R and decreased Ly-6G expression on bone marrow cells after animal treatment. Lachesis muta increased CD3, CD45R and, CD11c expression and decreased CD11b ex vivo and in nonadherent cells from co-culture. Bryonia alba increased Ly-6G, CD11c and CD11b expression ex vivo and when in co-culture CD11b was increased in adherent cells as well as decreased in nonadherent cells. With these results we have demonstrated that highly diluted medications act on immune cells activating macrophages, and changing the expression profile of hematopoietic lineage markers. Highly diluted medications are less toxic and cheaper than other commonly used medications and based on our observations, it is therefore conceivable that this medications which are able to act on bone marrow and immune cells may have a potential therapeutic use in clinical applications in diseases were the immune system is affected and also as regenerative medicine as it may allow proliferation and differentiation of progenitor cells.

Study of the Effects of Homeopathic Medicine Carcinosinum on Mammary Adenocarcinoma (4T1 cells) in vitro.

Background: There are few published researches about the exclusive use of Carsinosinum in several potencies to treat cancer. The name Carcinosinum refers to any homeopathic preparation of epithelial cancerous tissues and is especially indicated when there are any hereditary and familial antecedents of cancer, tuberculosis, diabetes, pernicious anemia or a combination of two or more of these diseases. Homeopathic complexes which include Conium Maculatum, Sabal Serrulata, Thuja Occidentalis and Carcinosinum can reduce in 23% the incidence of prostate cancer in vivo and in 38% the tumor volume, compared to untreated groups. Another in vivo study revealed reduction of symptoms and increase of survival time in mice bearing Ehrlich ascitic carcinoma, after treatment with Carcinosinum 200cH. In vitro, Carcinosinum 200cH can increase the expression of the pro-apoptotic gene p53. However, mice treated with Carcinosinum 6cH had the highest percentage and diversity of symptoms compared to other treatments, which demonstrate the importance of homeopathic potency in pro or anti-carcinogenic action. Considering that the literature on this subject is still rare and focused on genotypic and clinical effects, the present study was proposed, with the aim of identifying the possible phenotypic changes, including viability, HER-2 expression and metastatic skills, using 4T1 cells in vitro as a model, after treatment with Carcinosinum in different homeopathic working dilutions (12cH; 30cH; 200cH), prepared mechanically (Denise Machine, Autic®) in our laboratory using sterile pure water, from a commercial matrix (HN Cristiano, São Paulo, Brazil) stocked in 70% hydro-alcoholic solution. The final dilutions were inserted in the culture medium in a volume equal to 10%, at the time of cell seeding. The same succussioned vehicle used to prepare the medicines (70% hydro-alcoholic solution), from the same batch and diluted 1:100 in sterile pure water, was used as control. All treated cells were cultivated in bottles of 25ml with cell density of 5 x 105 cells / ml and, after 24 hours of treatment, they were analyzed for the apoptosis index using the Annexin V kit and measured by the Countess® system. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. The results obtained up to now show that the treatment with Carcinosinum 12cH produced a different pattern of cell death compared to the other treatments, with significant reduction in apoptosis index (one-way ANOVA, p=0.01) and clear hydropic degeneration phenotypic pattern. The analysis of HER-2 expression and metastatic skill will be the next step of this research.

Platelets Serve as Circulating Mediators of Cardioprotection by Remote Ischemic Conditioning in Healthy Volunteers

Background Brief cycles of occlusion and reperfusion in a tissue/organ remote from the heart protect the myocardium from sustained severe ischemia/reperfusion injury. Such remote ischemic conditioning (RIC) is operative in all species tested so far, including humans. Although unclear in its details, the underlying signal transfer involves humoral factors as evidenced by the transfer of cardioprotection via plasma or plasma-derivatives from one individual to another individual´s heart, even across species. We have recently demonstrated, that the platelet count increases in response to RIC in healthy volunteers within 60 min.1 Aim To study whether or not platelets serve as carriers for RIC's cardioprotective signalling. Methods Venous blood was collected from healthy volunteers (2 females/ 3 males, 32±7 years) before and 60 min after RIC (3 × 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation). Blood (80 mL) was drawn into polypropylene tubes containing sodium citrate (3.2 %), apyrase (0.1 U/ml) and prostaglandin E1 (1 µmol/L)and centrifuged (100 × g) for 15 min at room temperature. The obtained platelet-rich plasma was centrifuged again (1000 × g) for 12 min and the platelet pellet washed twice with buffer (pH 6.5) and finally re-suspended in buffer containing bovine serum albumin (3.5 mg/mL, pH 7.35). The platelet count was adjusted to 2.5 × 103 platelets/µL. This platelet suspension was either used for infusion experiments (washed platelets) or further processed by supplementing CaCl2 (1 mmol/L) followed by centrifugation (14.000 × g) at 4oC for degranulation; the supernatant was then retrieved (releasate). Functionality of washed platelets was assessed by light transmission aggregometry in response to stimulation by protease-activated receptor 1 activating peptide. Rat hearts were isolated and buffer-perfused at constant pressure (70 mmHg). Hearts were infused with washed platelets (25.000/mL × min) or with the releasate (final dilution 1:10) for 8 min followed by 2 min washout before being subjected to 30 min global zero-flow ischemia followed by 120 min reperfusion. Infarct size was demarcated by triphenyl tetrazolium chloride staining and calculated as percent of total ventricular mass. Results Infarct size was reduced by infusion of washed platelets retrieved after RIC in comparison to those retrieved before RIC (Figure A). Similar results were obtained by infusion of platelet-free releasates after RIC (Figure B). Conclusion In response to RIC in healthy volunteers, platelets serve as carriers for cardioprotective factors. The precise transfer of platelet protective factors from the circulation to the myocardium and the nature of the platelet-derived factors remain to be identified. 1H R Lieder et. al., P2589, Europ Heart J, 40 (1), 2019, ehz748.0915.

Peculiarities of the Physicochemical Properties of Hydrated C60 Fullerene Solutions in a Wide Range of Dilutions

Hydrated fullerene C60 (HyFn) is a supramolecular object in which the nanosized fullerene molecule is enclosed in a multilayer shell of water molecules. Despite the fact that fullerene C60 is chemically rather inert, aqueous solutions of HyFn exhibit a wide spectrum of biological activity in particular in low and ultra-low concentrations. Thus, physical and chemical properties of aqueous solutions of HyFn in a wide range of its dilutions are of interest. Here we compared some physical and chemical properties of aqueous systems prepared by successive 100-fold dilutions of HyFn (10–7 M) with deionized water, with their intensive shaking at each stage up to the calculated HyFn concentration of 10–31 M and of the corresponding “dilutions” of deionized water prepared in the same manner (controls). We studied the character of рН changes in dilutions when titrating them with HCl and NaOH. It turned out that HyFn dilutions had significantly higher buffering capacity against acidification with HCl than control water “dilutions.” At the highest acidity reached pH in all HyFn dilutions was almost 0.3 units higher than in the respective controls. Average buffering capacity of HyFn dilutions and water controls when titrated with NaOH did not differ. However, differences in buffering capacity could be seen between consecutive dilutions of HyFn at their titration either with NaOH or with HCl. Most prominent differences were observed between consecutive HyFn dilutions in the range of calculated concentrations 10–17–10–31 M titrated with NaOH while no significant differences in pH between equivalent “dilutions” of control water were observed. Similar though less prominent variations in buffering capacity between consecutive HyFn dilutions titrated with HCl were also noticed. Thus, titration with an acid and especially with an alkali made it possible to reveal differences between individual dilutions of HyFn, as well as differences between HyFn dilutions and corresponding dilutions of water. These features may be due to complexity in the structural properties of aqueous systems, which, supposedly, can arise due to the emergence of heterogenous aqueous regions (“clouds”) in the course of their dilutions with intensive mixing at each stage. In order to find out if such heterogeneity is a characteristic for HyFn dilutions we used the method of drying microsphere-containing droplets, whose aqueous base were either HyFn dilutions in the range of calculated HyFn concentration 10–7–10–31 M or respective water controls. It was found that a significant part of HyFn dilutions is characterized by mesoscopic heterogeneity. It showed up by the tendency of microspheres to concentrate in a specific way resembling ornaments once the droplets had dried. As the degree of HyFn dilution increased, the number of dried droplets with an ornament-like microsphere distribution increased. Same was also observed in water control drops. However, for the dilutions of HyFn equivalent to concentrations 10–19–10–31 M the percentage of complexly structured dried up droplets reached 60–80%, while for dried out drops of respective water controls it did not exceed 15–20%. Thus, the physicochemical properties of high dilutions of hydrated fullerene differ not only from each other dependently on the dilution level, but also from those of high dilutions of water, which can be explained by the structuredness and heterogeneity of these aqueous systems. Therefore, upon dilution process the properties of the solutions change according to complex and non-linear laws so that final dilutions cannot be identical in their properties and features to those of the initial solutions (before dilutions process) and to the untreated water. Dilution process, in view of the aforementioned, should not be underestimated when analyzing properties of the solutions, having shown to be able to affect dramatically properties of the solutions.

A Comparison of Urine Dilution Ability between Adult Dominant Polycystic Kidney Disease, Other Chronic Kidney Diseases, and Healthy Control Subjects: A Case-Control Study.

The final dilution of urine is regulated via aquaporin-2 water channels in the distal part of the nephron. It is unclear whether urine dilution ability in autosomal dominant polycystic kidney disease patients (ADPKD patients) differs from other patients with similar degree of impaired renal function (non-ADPKD patients). The purpose of this case control study was to measure urine dilution ability in ADPKD patients compared to non-ADPKD patients and healthy controls. Methods. Eighteen ADPKD, 16 non-ADPKD patients (both with chronic kidney disease, stage I-IV), and 18 healthy controls received an oral water load of 20 ml/kg body weight. Urine was collected in 7 consecutive periods. We measured free water clearance (CH2O), urine osmolality, urine output, fractional excretion of sodium, urine aquaporin2 (u-AQP2), and urine epithelial sodium channel (u-ENaC). Blood samples were drawn four times (at baseline, 2 h, 4 h, and 6 hours after the water load) for analyses of plasma osmolality, vasopressin, renin, angiotensin II, and aldosterone. Brachial and central blood pressure was measured regularly during the test. Results. The three groups were age and gender matched, and the patient groups had similar renal function. One hour after water load, the ADPKD patients had an increased CH2O compared to non-ADPKD patients (2.97 ± 2.42 ml/min in ADPKD patients vs. 1.31 ± 1.50 ml/min in non-ADPKD patients, p0.029). The reduction in u-AQP2 and u-ENaC occurred earlier in ADPKD than in non-ADPKD patients. Plasma concentrations of vasopressin, renin, angiotensin II, and aldosterone and blood pressure measurements did not show any differences that could explain the deviation in urine dilution capacity between the patient groups. Conclusions. ADPKD patients had a higher CH2O than non-ADPKD patients after an oral water load, and u-AQP2 and u-ENaC were more rapidly reduced than in non-ADPKD patients. Thus, urine-diluting capacity may be better preserved in ADPKD patients than in non-ADPKD patients.