scholarly journals Assessment of Inflammatory Response on Pyrogenic Induction by In vitro and In vivo Methods

2021 ◽  
Vol 12 (6) ◽  
pp. 7668-7684
Keyword(s):  
Human Blood ◽  
Safety Concern ◽  
Sandwich Elisa ◽  
Well Being ◽  
Severe Reaction ◽  
High Concentration ◽  
Healthy Human ◽  
Response Level

The incidence of pyrogenic contaminations creates a paramount safety concern in the field of biotherapeutics, as it compromises the normal well-being of an individual. After exposure to pyrogenic agents, the febrile response can create a severe reaction from shock and further complications leading to death. So it is imperative that the medical aids, as well as the parenteral drugs, must be pyrogen-free. The possible reliability of continuous supply of healthy human blood can be overcome by implementing a novel approach by pooling the blood samples for checking the pyrogenicity. In this study, an indigenously developed sandwich ELISA method to quantify pro-inflammatory cytokine (IL-1β) activated after pyrogenic exposure was used. The study unraveled the possibility of using the human pooled blood system to detect the endogenous pyrogen (IL-β) after exposure to endotoxins from Gram-positive and negative bacteria and chemical toxicants such as phytohemagglutinin (PHA) and 2,4,6-trinitrophenol (TNP). The results indicate that upon exposure to the high concentration of endotoxin such as LPS (5EU/ml) and LTA (1µg/ml), the maximum release of IL-1β was observed within 2h of post-stimulation. The stimulation with chemical toxicants PHA and TNP triggered a sudden release of IL-1β within 2h in both cases after 30µg/ml treatment. The study conveys a better platform for providing a continuous supply of healthy human blood, thus minimizing personal variations for the detection of IL-1β for evaluating in vitro pyrogenic response. It also depicts understanding the possible underlying mechanism of the pyrogenic response level of inflammatory cytokines and the possible rise in biochemical parameters leading to complications that affect the normal homeostasis by lipopolysaccharide-induced in rabbits.

Immunological investigations of oligoethylene glycols functionalized with desaminotyrosine and desaminotyrosyltyrosine

2013 ◽  
Vol 1569 ◽  
pp. 9-14 ◽  
Author(s):  
Konstanze K. Julich-Gruner ◽  
Toralf Roch ◽  
Nan Ma ◽  
Axel T. Neffe ◽  
Andreas Lendlein
Keyword(s):  
Human Blood ◽  
High Concentration ◽  
Inflammatory Conditions ◽  
Tnf Α ◽  
High Concentrations ◽  
Pharmaceutical Agents ◽  
Immunological Evaluation ◽  
Necrosis Factor

ABSTRACTBiomaterials require thorough in vitro testing before being applied in vivo. Unwanted contaminations of biomaterials but also their intrinsic properties can cause uncontrolled immune response leading to severe consequences for the patient. Therefore, immunological evaluation of materials for biomedical applications is mandatory before entering clinical application. In order to introduce physical netpoints, the aromatic compounds desaminotyrosine (DAT) and desaminotyrosyl-tyrosine (DATT) were successfully used to functionalize linear and star-shaped oligoethylene glycol (lOEG and sOEG) as previously described. The materials showed properties of surfactants and have potential to be used for solubilization of lipophilic drugs in water. Furthermore, the materials are susceptible for H2O2 degradation as determined by MALDI-ToF MS analyses. This is important for potential in vivo applications, as macrophages can release reactive oxygen species (ROS) under inflammatory conditions. As it is known that surfactant solutions of high concentration can lead to cell lysis, the effects of OEG-DAT(T) solutions on murine RAW macrophages were investigated. Even at highest OEG-DAT(T) concentration of 1000 µg·mL-1 the viability of the RAW cells was not significantly impaired. Additionally, the polymers were incubated with whole human blood and the production of inflammatory cytokines such as the tumor necrosis factor (TNF)-α and interleukin (IL)-6 was determined. Only at high concentrations, the OEG-DAT(T) solution induced low levels of TNF-α and IL-6, indicating that a mild inflammatory reaction could be expected when such high OEG-DAT(T) concentrations are applied in vivo. Similarly, the OEG-DAT(T) solution did not induce ROS in monocytes and neutrophils after incubation with whole human blood. Conclusively, the data presented here demonstrate that OEG-DAT(T) do not lead to a substantial activation of the innate immune mechanisms and could therefore be investigated for solubilizing pharmaceutical agents.

Changes in the Ultrastructure and Number of Platelets Induced by an Adrenochrome Complex

1970 ◽  
Vol 23 (02) ◽  
pp. 237-260 ◽  
Author(s):  
G. J Stewart
Keyword(s):  
The Body ◽  
High Concentration ◽  
Electron Microscopic ◽  
Healthy Human ◽  
Thin Sections ◽  
Microscopic Appearance ◽  
Sequence Of Events ◽  
Shadow Cast

SummaryAdrenochrome semicarbazone injected intramuscularly into apparently healthy human volunteers resulted in a decrease in the number of circulating platelets after 30 min. The decrease ranged from 9 to 44% with an average of 24%. The platelet count returned to near normal by 60 min. A similar concentration had no effect in vitro. The sodium salicylate carrier used to solubilize the adrenochrome complex had no effect on platelet numbers in vivo or in vitro. Blood taken 30 min post injection and prepared by a fixation-shadow cast technique contained many disintegrating platelets and a high concentration of membrane fragments and free organelles. Thin sections of platelets revealed extensive intracellular alteration, but physically intact limiting membranes. Many free vesicles of various sizes and shapes and a few free mitochondria were shown in cross section.Two major metabolites isolated from human urine after adrenochrome semicarbazone injection caused platelet disruption when added to citrated PRP in vitro. The disruption was reflected by a decrease in the number of platelets and by the electron microscopic appearance of fixed-shadow cast preparations.It is suggested that the sequence of events observed in this study may represent, greatly exaggerated, a process which occurs in the body constantly as a small amount of epinephrine seems to be metabolized through an adrenochrome pathway.

scholarly journals Blood Glutathione Disulfide: In Vivo Factor or in Vitro Artifact?

2002 ◽  
Vol 48 (5) ◽  
pp. 742-753 ◽  
Author(s):  
Ranieri Rossi ◽  
Aldo Milzani ◽  
Isabella Dalle-Donne ◽  
Daniela Giustarini ◽  
Lorenzo Lusini ◽  
...  
Keyword(s):  
Human Blood ◽  
Reversed Phase ◽  
Published Data ◽  
Glutathione Disulfide ◽  
Fluorescence Detector ◽  
Healthy Human ◽  
Low Concentrations ◽  
Human Blood Erythrocytes

Abstract Background: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 μmol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. Methods: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. Results: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30–150 μmol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide. Conclusions: GSSG is present in healthy human blood at low concentrations (2–6 μmol/L), and most published data on GSSG may be affected by artifacts.

Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

1964 ◽  
Vol 12 (01) ◽  
pp. 232-261 ◽  
Author(s):  
S Sasaki ◽  
T Takemoto ◽  
S Oka
Keyword(s):  
Molecular Weight ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Molecular Structures ◽  
Severe Reaction ◽  
Clinical Use ◽  
Molecular Weights ◽  
Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

scholarly journals Current Knowledge on Functionality and Potential Therapeutic Uses of Donkey Milk

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1382
Author(s):  
Mina Martini ◽  
Iolanda Altomonte ◽  
Domenico Tricò ◽  
Riccardo Lapenta ◽  
Federica Salari
Keyword(s):  
Animal Models ◽  
Randomized Clinical Trials ◽  
Current Knowledge ◽  
Saturated Fatty Acids ◽  
Cow Milk ◽  
Donkey Milk ◽  
Alpha Linolenic Acid ◽  
High Concentration

The increase of knowledge on the composition of donkey milk has revealed marked similarities to human milk, which led to a growing number of investigations focused on testing the potential effects of donkey milk in vitro and in vivo. This paper examines the scientific evidence regarding the beneficial effects of donkey milk on human health. Most clinical studies report a tolerability of donkey milk in 82.6–98.5% of infants with cow milk protein allergies. The average protein content of donkey milk is about 18 g/L. Caseins, which are main allergenic components of milk, are less represented compared to cow milk (56% of the total protein in donkey vs. 80% in cow milk). Donkey milk is well accepted by children due to its high concentration of lactose (about 60 g/L). Immunomodulatory properties have been reported in one study in humans and in several animal models. Donkey milk also seems to modulate the intestinal microbiota, enhance antioxidant defense mechanisms and detoxifying enzymes activities, reduce hyperglycemia and normalize dyslipidemia. Donkey milk has lower calorie and fat content compared with other milks used in human nutrition (fat ranges from 0.20% to 1.7%) and a more favourable fatty acid profile, being low in saturated fatty acids (3.02 g/L) and high in alpha-linolenic acid (about 7.25 g/100 g of fat). Until now, the beneficial properties of donkey milk have been mostly related to whey proteins, among which β-lactoglobulin is the most represented (6.06 g/L), followed by α-lactalbumin (about 2 g/L) and lysozyme (1.07 g/L). So far, the health functionality of donkey milk has been tested almost exclusively on animal models. Furthermore, in vitro studies have described inhibitory action against bacteria, viruses, and fungi. From the literature review emerges the need for new randomized clinical trials on humans to provide stronger evidence of the potential beneficial health effects of donkey milk, which could lead to new applications as an adjuvant in the treatment of cardiometabolic diseases, malnutrition, and aging.

scholarly journals Development and biopharmaceutical evaluation of extended release formulation of tramadol hydrochloride based on osmotic technology

2009 ◽  
Vol 59 (1) ◽  
pp. 15-30 ◽  
Author(s):  
Pramod Kumar ◽  
Sanjay Singh ◽  
Brahmeshwar Mishra
Keyword(s):  
Drug Release ◽  
Extended Release ◽  
Relative Bioavailability ◽  
Pharmacokinetic Parameters ◽  
Healthy Human ◽  
Tramadol Hydrochloride ◽  
Release Formulation ◽  
Extended Release Formulation

Development and biopharmaceutical evaluation of extended release formulation of tramadol hydrochloride based on osmotic technologyExtended release formulation of tramadol hydrochloride (TRH) based on osmotic technology was developed and evaluated. Target release profile was selected and different variables were optimized to achieve it. Formulation variables such as the level of swellable polymer, plasticizer and the coat thickness of semipermeable membrane (SPM) were found to markedly affect drug release. TRH release was directly proportional to the levels of plasticizer but inversely proportional to the levels of swellable polymer and coat thickness of SPM. Drug release from developed formulations was independent of pH and agitation intensity but dependent on osmotic pressure of the release media.In vivostudy was also performed on six healthy human volunteers and various pharmacokinetic parameters (cmax,tmax,AUC0-24,MRT) and relative bioavailability were calculated. Thein vitroandin vivoresults were compared with the performance of two commercial TRH tablets. The developed formulation provided more prolonged and controlled TRH release compared to the marketed formulation.In vitro-in vivocorrelation (IVIVC) was analyzed according to the Wagner-Nelson method. The optimized formulation (batch IVB) exhibited good IVIV correlation (R= 0.9750). The manufacturing procedure was found to be reproducible and formulations were stable over 6 months of accelerated stability testing.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

Novel Self-Micro Emulsifying Drug Delivery System for safe intra muscular delivery with improved pharmacodynamics and pharmacokinetics

2021 ◽  
Vol 18 ◽  
Author(s):  
Subheet Kumar Jain ◽  
Neha Panchal ◽  
Amrinder Singh ◽  
Shubham Thakur ◽  
Navid Reza Shahtaghi ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Drug Delivery System ◽  
Delivery System ◽  
Inflammatory Diseases ◽  
Diclofenac Sodium ◽  
Physical Stability ◽  
In Vivo Studies ◽  
High Concentration

Background: Diclofenac sodium (DS) injection is widely used in the management of acute or chronic pain and inflammatory diseases. It incorporates 20 % w/v Transcutol-P as a solubilizer to make the stable injectable formulation. However, the use of Transcutol-P in high concentration leads to adverse effects such as severe nephrotoxicity, etc. Some advancements resulted in the formulation of an aqueous based injectable but that too used benzyl alcohol reported to be toxic for human use. Objective: To develop an injectable self-micro emulsifying drug delivery system (SMEDDS) as a novel carrier of DS for prompt release with better safety and efficacy. Methods: A solubility study was performed with different surfactants and co-surfactants. The conventional stirring method was employed for the formulation of SMEDDS. Detailed in vitro characterization was done for different quality control parameters. In vivo studies were performed using Wistar rats for pharmacokinetic evaluation, toxicological analysis, and analgesic activity. Results: The optimized formulation exhibited good physical stability, ideal globule size (156±0.4 nm), quick release, better therapeutics, and safety, increase in LD50 (221.9 mg/kg) to that of the commercial counterpart (109.9 mg/kg). Further, pre-treatment with optimized formulation reduced the carrageenan-induced rat paw oedema by 88±1.2 % after 4 h, compared to 77±1.6 % inhibition with commercial DS formulation. Moreover, optimized formulation significantly (p<0.05) inhibited the pain sensation in the acetic-acid induced writhing test in mice compared to its commercial equivalent with a better pharmacokinetic profile. Conclusion: The above findings confirmed that liquid SMEDDS could be a successful carrier for the safe and effective delivery of DS

scholarly journals A 70-Kilodalton Recombinant Heat Shock Protein ofCandida albicans Is Highly Immunogenic and Enhances Systemic Murine Candidiasis

1998 ◽  
Vol 66 (5) ◽  
pp. 2154-2162 ◽  
Author(s):  
Carla Bromuro ◽  
Roberto La Valle ◽  
Silvia Sandini ◽  
Francesca Urbani ◽  
Clara M. Ausiello ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mononuclear Cells ◽  
Cell Mediated Immunity ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
Ifn Γ

ABSTRACT The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-γ), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-γ was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-γ upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

Sign in / Sign up

Export Citation Format

Share Document