Spectrum of Albumin Auto‐Agglutinins

The properties of four albumin‐agglutinating sera demonstrating a spectrum of reactivities are reported. All cause pan‐ and auto‐agglutination in albumin. Direct and indirect reacting caprylate‐dependent agglutinins are described, as are two sera with undefined specificity not related to the presence of caprylate stabilizer in the albumin. Immunoelectrophoretic evidence was obtained to support the concept of albumin‐immunoglobulin complex formation in the genesis of albumin agglutination. Most albumin agglutinins react only with albumin stabilized by short‐chain fatty acids; however, other specificities may be represented. Albumin agglutinins should be clearly identified in the blood bank to prevent confusion with blood group antibodies.     

Red Cell Recovery and Leukocyte Depletion Following Washing of Frozen‐Thawed Red Cells

Human red blood cells frozen by the high glycerol method and washed in the Haemonetics, IBM, and Elutramatic cell washers showed uniformly low hemolysis and modest differences in the loss of intact cells during washing. Residual leukocytes ranged from an apparent absence of cells to as many as 10³/mm³. Configuration changes in the Haemonetics bowl designed to improve mixing also improved leukocyte removal.     

A Comprehensive In Vivo and In Vitro Assessment of the Drug Interaction Potential of Red Ginseng

?Purpose: Red ginseng is one of the world's most popular herbal medicines; it exhibits a wide range of pharmacologic activities and is often co-ingested with other herbal and conventional medicines. This open-label, randomized, 3-period study investigated the in vivo herb–drug interaction potential for red ginseng extract with cytochrome P-450 (CYP) enzymes and organic anion-transporting polypeptide (OATP) 1B1.?Methods: Fifteen healthy male volunteers (22-28 years; 57.1-80.8 kg) were administered a single dose of cocktail probe substrates (caffeine 100 mg, losartan 50 mg, omeprazole 20 mg, dextromethorphan 30 mg, midazolam 2 mg, and pitavastatin 2 mg) and single or multiple doses of red ginseng extract for 15 days.?Findings: The pharmacokinetic profiles of the probe substrates and metabolites after single- or multiple-dose administration of red ginseng extracts were comparable to the corresponding profiles of the control group. The geometric mean ratio of AUC_0–t and 90% CIs for the probe substrate drugs between the control and multiple doses of red ginseng for 15 days were within 0.8 to 1.25 (CYP2C9, CYP3A4, and OATP1B1 probe substrates) or slightly higher (CYP1A2, CYP2C19, and CYP2D6 probe substrates). Additional assessments of the in vitro drug interaction potential of red ginseng extracts and the ginsenoside Rb1 on drug-metabolizing enzymes and transporters using human liver microsomes, cryopreserved human hepatocytes, and transporter-overexpressed cells were negative.?Implications: Red ginseng poses minimal risks for clinically relevant CYP- or OATP-mediated drug interactions and is well tolerated. Clinical Research Information Service registry no.:     

Demonstrating effectiveness—the concept of numbers‐needed‐to‐treat

When assessing a health-care intervention the main question is—does it work? Often, a more difficult question needs to be answered—how well does it work? Systematic reviews and meta-analyses help to provide answers to both questions. Too often though, the results are expressed in a way that leaves the reader asking, ‘what is the result?’ Numbers-needed-to-treat is a simple method for conveying the answers. It can be applied to any chosen clinical outcome with dichotomous data, and the results can be understood by doctor, patient and the public. This paper discusses the concept of numbers-needed-to-treat and gives worked examples using trials in H. pylori eradication and lowering of serumcholesterol.     

Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Nonhuman primates (NHPs) are critical for biomedical research due to their close genetic and physiologic relationship to humans. In addition, according to the classification criteria proposed by the International Union for the Conservation of Nature, approximately 63% of the 702 species and subspecies of NHP in the world are at some level of risk of extinction.^21,31 Thus, efficient assisted reproductive technologies (ARTs) are required to 1) understand primate reproductive processes, 2) advance NHP biomedical model development and 3) advance species conservation efforts.The rhesus macaque (Macaca mulatta) is one of the most commonly used NHP species for research studies relevant to human health and disease.⁴² Advances in the use of ARTs in this important species include cryopreservation of sperm;⁴¹ artificial insemination;³⁴ in vitro fertilization (IVF);³ and intracytoplasmic sperm injection (ICSI).³⁰ Establishing effective and reproducible rhesus macaque ARTs is critical in allowing the birth of live offspring after blastomere nuclear transfer,²⁸ somatic cell nuclear transfer,¹⁰ mitochondrial replacement in oocytes,⁴³ and autologous grafting of prepubertal rhesus testis, followed by ICSI and embryo development.¹⁶ Moreover, recent advances in gene editing now allow NHP genome manipulation in one cell embryos (zygotes) for the subsequent generation of transgenic models of human disease.¹¹Genetic resource banking or cryobanking, (the low temperature storage of gametes, embryos, and tissues) is crucial to preserving genetic material for future use in ARTs. Cryobanking allows the preservation of material from genetically valuable individuals independent of the long-term goal for its use (biomedical models, domestic species breeding programs, species conservation, etc.).⁶ Moreover, cryobanking makes sperm readily available when needed, without the need to maintain live male sperm donors or personnel who are trained to collect fresh sperm.However, spermatozoa cryopreservation and recovery of viable sperm after thawing is still a major concern in NHP ARTs and would benefit from further improvement.⁵⁰ Although several studies have investigated the optimal conditions for primate sperm preservation,^{12-14,25,33,39-41,53,54} major gaps exist in our understanding of what is critical for promoting the survival of sperm after thawing. For example, IVF success using fresh sperm varied between 50% to 61%^50,51 with 55% of the embryos reaching the blastocyst stage,⁵¹ whereas IVF with cryopreserved sperm resulted in 82 ± 13% of the ova developing into embryos, of which only 39 ± 21% reached the blastocyst stage.⁴¹ One process that needs further optimization in rhesus macaques is sperm vitrification. Vitrification is the process of obtaining a glass-like solid to avoid ice crystal formation; it is achieved either by rapid cooling or using additives.⁵² Although successful vitrification of human sperm has been reported,^19,20 the only study that assessed sperm vitrification in rhesus macaques reported no motility after thawing.²⁹ Therefore, further studies are necessary on how to successfully cryopreserve sperm from rhesus macaques.An important factor to consider with regard to sperm cryopreservation is how to isolate good quality sperm after thawing (that is, progressively motile sperm with an intact plasma membrane and acrosome and low levels of DNA fragmentation). Several studies have compared different sperm preparation methods in various species, including bulls,^2,23 humans,¹⁵ and dogs,²² but no studies to date have systematically compared various methods in rhesus macaques.Therefore, the objectives of the current study were to 1) compare 2 methods for cryopreservation of sperm from rhesus macaques with regard to sperm viability characteristics after thawing; 2) compare 4 different methods of removal of cryopreservation reagents and sperm isolation after thawing, and (3) evaluate the overall efficacy of the 2 best sperm isolation methods for use in IVF.     

The Effect of Long-Term In Vitro Incubation on the Intermediates of Red Cell Metabolism

Whole human blood was incubated for periods of up to 70 hours at 37 C under conditions that would cause minimal modifications in normal metabolic controls. Insofar as possible, the conditions utilized were similar to those found in vivo . Control of pH within the limits of 7.2 to 7.5 was achieved by the addition of isotonic Tris buffer or isotonic TES buffer. Glucose, inorganic phosphate, adenine, and pyruvate were added as needed throughout the incubation period. The ADP/ATP ratio and levels of ATP, total adenine nucleotides, 2,3-diphosphoglycerate and other metabolic intermediates were maintained near those of fresh erythrocytes. The maintenance and control of red blood cell adenine nucleotide levels are discussed, with particular emphasis on factors affecting the rate of incorporation of adenine into adenine nucleotides.     

Preservation of Red Blood Cell 2,3‐Diphosphoglycerate in Stored Blood Containing Dihydroxyacetone

In a search for red blood cell metabolites which would preserve 2,3‐DPG during storage of blood, it was discovered that dihydroxyacetone (DHA) prolonged the maintenance of 2,3‐DPG levels for up to four weeks of storage, compared to about one week for presently used preservatives such as CPD. Four‐week preservation of 2,3‐DPG at normal levels was desired. CPD‐adenine was used as the starting point and a formulation having a pH of 7.0 and a DHA concentration of 20 millimoles per liter of blood was developed. The 2,3‐DPG level at four weeks of storage was proportional to DHA concentration in the 5 to 20 mM range. The osmotic fragility, red blood cell ATP levels, and plasma sodium, potassium, and hemoglobin during four weeks of storage in CPD‐adenine‐DHA were similar to those in blood stored in CPD‐adenine.     

骨髓细胞液氮保存21-25年后的细胞活力体外研究

为探讨适合临床应用的骨髓细胞保存方法,揭示细胞长期低温保存效果,将20人份骨髓加入DMSO-AuP(10%二甲亚砜、10%自体血浆)或DMSO-HES-HuA(5%DMSO、6%羟乙基淀粉、4%人血白蛋白)保护剂,分装2毫升/管,600-800管/人份,自控程序降温仪或-80℃低温冰箱预冻降温、液氮中保存21-25年。取冷冻骨髓细胞于38℃复温,检测细胞相关指标。结果表明:加保护剂的细胞标本在-80℃低温冰箱中为低速降温,-30℃前,与自控程序降温仪设定1℃/min的低降温速率接近。DMSO-HES-HuA与DMSO-AuP比较,红细胞形态畸形率分别为(3.5±1.5)%和(12.6±4.8)%;溶血率分别为(3.3±1.6)%和(23.1±5.1)%(p0.05);前者渗透性脆性不变,后者减低;红细胞、血小板、粒单系造血祖细胞、长期培养起始细胞回收率,前后者对比分别为(96.1±1.8)%、(70.0±9.5)%、(49.2±10.9)%、(54.2±13.8)%vs(76.3±5.6)%、(52.7±8.1)%、(43.5±12.3)%、(47.2±13.6)%。用上述保护剂配合上述降温法保存后,细胞在间充质干细胞培养上,生长特性良好。同一保护剂用上述两种降温法保存后,细胞各种回收效果差异不显著(p0.05)。结论:细胞在液氮中保存21-25年,其细胞形态和回收活力(率)良好;保存如骨髓这种细胞成分并非单一的标本时,用5%DMSO-6%HES-4%HuA为保护剂的-80℃冰箱预冻降温后液氮保存,方法简便、经济、易操作,适合临床应用。     

Circulation of Concentrated One‐day‐old Platelets in Vivo

Platelet concentrates of rat and human origin were stored at 22 C or at 4 C for up to 24 hours without additives. Transfusion of these concentrates into thrombocytopenic recipients demonstrated that: (1) storage of rat or human platelets at 4 C for up to 24 hours did not affect their recovery in vivo ; (2) storage at 22 C resulted in a marked reduction in the viability of rat platelets but only a slight reduction in the viability of human platelets as adjudged by these criteria; (3) at 24 hours posttransfusion, the residual increment of platelets stored at 22 C was significantly higher than that of platelets stored at 4 C. The pH of the concentrates (rat and human) stored at 4 C remained slightly alkaline while the pH of those stored at 22 C., especially rat platelets, was significantly reduced. The deleterious effects of storage of platelets at 4 C are well known. These effects, however, do not preclude their usefulness when a limited objective of arresting or preventing hemorrhage for short periods is pursued. When daily platelet transfusions are feasible, platelets stored for 24 hours at 4 C in the absence of fresh material are adequate for clinical use.     

In Vitro Activity of Ceftazidime-Avibactam Combination in In Vitro Checkerboard Assays

To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae, 19 Escherichia coli, 1 Citrobacter freundii, and 3 Enterobacter cloacae) and 26 strains of Pseudomonas aeruginosa, all with known resistance mechanisms such as extended-spectrum β-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains (n = 69) were analyzed in more detail. For the Enterobacteriaceae strains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC₅₀ and MIC₉₀ being 8 and 16 mg/liter, respectively). For the ceftazidime-resistant P. aeruginosa strains, considerable inhibition of β-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on these in vitro data, avibactam is a promising inhibitor of different β-lactamases, including ESBLs and carbapenemases.     

The Effects of Cyanate In Vitro on Red Blood Cell Metabolism and Function in Sickle Cell Anemia

Cyanate, which is in equilibrium with urea, combines with the α-amino group of the aminoterminal valine of hemoglobin in an irreversible, specific carbamylation reaction. Partial carbamylation (0.72 residues/hemoglobin tetramer) as determined by cyanate-¹⁴C incorporation or hydantoin analysis diminishes the in vitro sickling phenomenon. Since cyanate may react not only with hemoglobin but also with functional groups of other red blood cell proteins, the in vitro effect of cyanate was studied on sickle cells. Cells were incubated with 10 mM KCl (control) or 10 mM KNCO (carbamylated) for 1 hr, washed, and resuspended in autologous plasma. Glycolysis, ATP and 2,3-diphosphoglyceric acid (DPG) stability, autohemolysis, and osmotic fragility were not affected by carbamylation. Potassium loss in carbamylated cells (2.8 mmol/liter) was less than in control cells (9.0 mmol/liter). Pyruvate kinase activity of carbamylated cells was decreased (~25%) but the activities of other glycolytic enzymes were similar to those of control cells. Oxygen affinity of carbamylated sickle, normal, and DPG-depleted normal cells increased, and was a sensitive index of the degree and duration of reaction with cyanate. The reactivity of carbamylated cells to DPG was similar to control cells. DPG-depleted carbamylated cells regenerated DPG and increased the P₅₀ when incubated with pyruvate, inosine, and phosphate. The Bohr effect of normal and of sickle cells was not affected (Δlog P₅₀/Δ pH=-0.48 and -0.53, respectively) after carbamylation. The reserve buffering capacity of plasma offset the slightly diminished (~15%) CO₂ capacity of carbamylated cells so that whole blood CO₂ capacity, pH, and P_CO2 were normal. These studies provide further support for the potential clinical use of cyanate in treating and preventing the anemia and painful crises of sickle cell disease.     

二甲基亚砜联合羟乙基淀粉的非程控降温法冻存脐血造血干细胞研究

为了探索有效低温保存脐血造血干／祖细胞的冷冻保护剂和技术，进行了二种方法试验。第一种方法联合使用二甲基亚砜(DMSO)和羟乙基淀粉(HES)(分子量120000)作为冷冻保护剂并用非程控降温冷冻技术；第二种方法只使用DMSO作为冷冻保护剂并用程控降温仪冷冻技术保存脐血。对二种方法的效果进行比较。结果表明：15份脐血冻存后细胞存活率、CFU—GM回收率，无论在使用非程控降温的第一种方法和使用程控降温仪的第二种方法均无显著性差异。183份脐血质量检测结果及21例临床移植结果也显示两种方法无显著性差异。结论：联合使用DMSO和HES作为冷冻保护剂并用非程控降温冷冻的方法对脐血造血干细胞有良好的保护作用，是一种简单易行、省时省力、适合于脐血造血干细胞库大批量冻存脐血的方法。     

In Vitro Studies of Tobramycin, an Aminoglycoside Antibiotic

Tobramycin is an aminoglycoside antibiotic which has excellent antibacterial activity against Pseudomonas, Staphylococcus aureus, and many members of the Enterobacteriaceae. Most strains of Serratia, Providence, Streptococcus, and Diplococcus pneumoniae were resistant to concentrations of tobramycin which could be achieved in man. Tobramycin was effective against certain Pseudomonas strains resistant to gentamicin. The growth medium used to determine the inhibitory level of tobramycin had a significant effect upon the minimal inhibitory concentration. Calcium and magnesium ions inhibited the bactericidal effect of tobramycin. Tobramycin and carbenicillin acted in a synergistic manner. Ethylenediaminetetraacetic acid did not act in a synergistic manner with tobramycin. Broth-dilution susceptibility tests and disc-diffusion tests in agar (10-μg discs) showed excellent correlation except with Proteus strains.     

Racial Differences in Oxidative Stress and Inflammation: In Vitro and In Vivo

African American race is an independent risk factor for enhanced oxidative stress and inflammation. We sought to examine whether oxidative‐stress and inflammatory markers that are typically measured in humans also differ by race in cell culture. We compared levels between African American and Caucasian young adults and then separately in human umbilical vein endothelial cells (HUVECs) from both races. We found heightened oxidative stress and inflammation in the African Americans both in vitro and in vivo. African American HUVECs showed higher nitric oxide (NO) levels (10.8 ± 0.4 vs. 8.8 ± 0.7 μmol/L/mg, p = 0.03), Interleukin‐6 (IL‐6) levels (61.7 ± 4.2 vs. 23.9 ± 9.0 pg/mg, p = 0.02), and lower superoxide dismutase activity (15.6 ± 3.3 vs. 25.4 ± 2.8 U/mg, p = 0.04), and also higher protein expression (p < 0.05) of NADPH oxidase subunit p47phox, isoforms NOX2 and NOX4, endothelial nitric oxide synthase (NOS), inducible NOS, as well as IL‐6. African American adults had higher plasma protein carbonyls (1.1 ± 0.1 vs. 0.8 ± 0.1 nmol/mg, p = 0.01) and antioxidant capacity (2.3 ± 0.2 vs. 1.1 ± 0.3 mM, p = 0.01). These preliminary translational data demonstrate a racial difference in HUVECs much like that in humans, but should be interpreted with caution given its preliminary nature. It is known that racial differences exist in how humans respond to development and progression of disease, therefore these data suggest that ethnicity of cell model may be important to consider with in vitro clinical research. Clin Trans Sci 2011; Volume 4: 32–37     

Studies of the Metabolic Integrity of Human Red Blood Cells after Cryopreservation

Human red blood cells subjected both to the sub-zero temperatures and processing of a low-glycerol-rapid-freeze method for cryopreservation and to subsequent storage at 4 C, under a variety of conditions, consistently maintained concentrations of ATP comparable to those of their nonfrozen, nonprocess d counterparts. Their potassium content was similarly unaffected. The cell sodium was increased by about 10 per cent as a result of the cryopreservation procedure, and during a subsequent storage period at 4 C it increased to a final concentration of from 10 to 13 per cent greater than that of the nontreated red blood cells. Cryopreserved red blood cells were capable of reinitiating sodium and potassium transport activities with a facility equal to that of nonfrozen cells which indicates that the cryoprocessing is protective of enzyme systems essential for this mechanism.     

甲基纤维素体外分离骨髓移植物中红细胞的效果

“ABO”血型不合的骨髓移植和一部分自体骨髓移植 ,需分离骨髓移植物中的红细胞。本研究观察了用甲基纤维素分离移植物中红细胞的效果。用生理盐水配制的 0 .5 %甲基纤维素 (MC)与采集的骨髓移植物按一定比例混匀 ,静置 ,然后沉降红细胞。对 1 8份骨髓移植物分离的结果显示 ,单个核细胞和CD34+ 细胞回收率分别达(83 .8± 5 .2 ) %和 (90 .3± 7.2 ) % ,CFU GM产率为 (60 .8± 2 2 .4) / 2× 1 0 5个单个核细胞 ;红细胞残留率仅为 (4.3±1 5) %。结论 :此方法可用于骨髓移植     

Bioeffects of Low‐Intensity Ultrasound In Vitro

Objective. The purpose of this study was to evaluate the potential molecular mechanism of low‐intensity ultrasound‐induced apoptosis by analyzing protein profile alteration in response to ultrasound exposure. Methods. Human hepatocarcinoma SMMC‐7721 cells were used in this study. Cell viability was measured by a trypan blue dye exclusion test. Morphologic changes were examined by light microscopy. Apoptosis was assessed by phosphatidylserine externalization and DNA fragmentation. The pattern of the mitochondrial membrane potential decrease was determined by flow cytometry. Protein profile alteration was analyzed by comparative proteomics based on 2‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Results. Low‐intensity ultrasound (3.0 W/cm², 1 minute, cells incubated for 6 hours after ultrasound exposure) induced early apoptosis (mean ± SD, 26.5% ± 6.2%) significantly (P < .05) with minimal lysis in human hepatocarcinoma cells in vitro. On a molecular level, several proteins, eg, cellular tumor antigen protein 53, BH3‐interacting domain death agonist, apoptosis regulator Bcl‐2, and heme oxygenase 1 were identified as responding to ultrasound irradiation, suggesting that mitochondrial dysfunction and oxidative stresses were involved in ultrasound‐induced apoptosis. It was also assumed that mitofilin‐regulated crista remodeling may be a potential channel of mitochondrial membrane permeabilization pore formation involved in low‐intensity ultrasound‐induced apoptosis. Conclusions. This study suggests that 2 potential molecular signaling pathways are involved in ultrasound‐induced apoptosis. It is a first step toward low‐intensity ultrasound‐induced apoptotic cancer therapy via understanding its relevant molecular signaling and key proteins.