Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2.
- MeSH
- buněčné kultury MeSH
- ELISA MeSH
- endoteliální buňky * MeSH
- lidé MeSH
- myocyty hladké svaloviny MeSH
- trénovaná imunita * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BK virus (BKPyV) is a causative agent of BKPyV-associated nephropathy and graft rejections in kidney transplant patients. It establishes persistent infection in the kidneys, which can lead to reactivation in an immunosuppressed state or transmission to kidney recipients. Complications in the case of donor-derived infections can be caused by differences between the four known BKPyV subtypes, as prior infection with one subtype does not guarantee protection against de novo infection with other subtypes. The recipient and donor pretransplant serotyping is not routinely performed since simple ELISA tests employing antigens derived from the major viral capsid protein 1 (VP1) are hindered by the high cross-reactivity of anti-VP1 antibodies against all subtypes. Identifying subtype-specific epitopes in VP1 could lead to the design of specific antigens and the improvement of serodiagnostics for kidney transplantation. We aimed to study the surface residues responsible for the interactions with the subtype-specific antibodies by focusing on the DE and EF loops of VP1, which have only a small number of distinct amino acid differences between the most common subtypes, BKPyV-I and BKPyV-IV. We designed two mutant virus-like particles (VLPs): we introduced BKPyV-I characteristic amino acid residues (either H139N in the DE loop or D175E and I178V changes in the EF loop) into the base sequence of a BKPyV-IV VP1. This way, we created BKPyV-IV mutant VLPs with the sequence of either the BKPyV-I DE loop or the BKPyV-I EF loop. These mutants were then used as competing antigens in an antigen competition assay with a panel of patient sera, and changes in antibody reactivity were assessed by ELISA. We found that the changes introduced into the BKPyV-IV VP1 EF loop restrict antibody recognition in most samples and that converting the BKPyV-IV DE loop into its BKPyV-I equivalent attracts anti-VP1 BKPyV-I antibodies. Although our results did not lead to the discovery of a subtype-specific epitope on the VP1, they suggested that the arrangement of the EF loop in VP1 might dictate the mode of interaction between virus and anti-VP1 antibodies in general and that the interactions between the antibodies and the viral capsid might be very complex. Consequently, an antigen competition assay as an assay to distinguish between BKPyV serotypes might prove difficult to interpret.
- MeSH
- ELISA MeSH
- ledviny MeSH
- lidé MeSH
- nemoci ledvin * MeSH
- sérotypizace MeSH
- transplantace ledvin * MeSH
- virus BK * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The study was designed to evaluate the medical relevance of total homocysteine (tHcy), total antioxidant capacity (TAC), and malondialdehyde (MDA) before and after chemotherapy for women with breast cancer (BRCA). Blood samples were taken from Oncology Unit in Merjian Teaching hospital in Hilla city (Iraq). Sixty patients suffering from breast cancer (BRCA) were enrolled in this study, and twenty-one apparently healthy subjects were considered as a control group control. We found that significantly increased lipid peroxidation, measured as MDA, was demonstrated in the serum of BRCA patients who were not taken any medication was higher than in the control group (P<0.01) were (25±15) nmol/ml of MDA levels in BRCA patient and (14.5±7.9) nmol/ml of MDA levels in healthy controls. We found that treatment by chemotherapy resulted in a significant increase in MDA levels when compared with MDA levels in patients who were not taken any medication. The tHcy level in BRCA patients before and after treatment were changed. In addition, it is found that the mean serum TAC levels in BRCA patients were significantly less than in the control group. Moreover, a positive correlation was observed between the activity of chemotherapy and MDA levels in the patient and the same correlation between tHcy levels and TAC levels while a negative correlation was observed between TAC levels with MDA levels in the patient group.
The study was designed to evaluate the medical relevance of Malondialdehyde (MDA) (a marker of oxidative stress) and total antioxidant capacity (TAC) in coronavirus disease 2019 (COVID-19) groups, cured groups, and control groups; before and after taking the vaccine. Blood samples were taken from Oncology Unit in Al-Mahaweel hospital in Hilla city. Sixteen patients, sixteen cured patients, thirty control, sixteen subjects were taken one dose of Pfizer vaccine, sixteen subjects were taken two doses of Pfizer vaccine. We found that significantly increased lipid peroxidation, measured as MDA, was demonstrated in the serum of COVID-19 patients and TAC decreased in patients when compared with the control groups. Inversely, we found the mean MDA levels decrease and increase in TAC levels in cured patients when compare with COVID-19 patients. In addition, it is found that subjects were taken one dose or two doses of the Pfizer vaccine have less MDA levels and more TAC levels than the COVID-19 vaccine for that reason the Pfizer vaccines play the important role in the activity of immune systems.
- MeSH
- angiotensin konvertující enzym 2 metabolismus MeSH
- antioxidancia analýza metabolismus MeSH
- COVID-19 * komplikace metabolismus MeSH
- ELISA metody MeSH
- lidé MeSH
- malondialdehyd analýza metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- vakcína BNT162 MeSH
- vakcíny proti COVID-19 škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinické zkoušky kontrolované MeSH
- pozorovací studie MeSH
Background and objectives: Atherosclerosis is the underlying pathology of ischemic heart disease. There are many aggravating metabolic and oxidant parameters which are participating together overwhelming the pathology of vascular stenosis. Adipokines play a positive metabolic effect in healthy individuals and oxidation reaction greatly impacts the lipid metabolism and might negatively impact the condition. In the present study, we aimed to characterize the level of adiponectin, obestatin, and redox parameters in atherosclerotic patients.Methods: Serum was collected from atherosclerotic patients and froze to be ready for analysis.Results: The results indicated that hyperlipidemia significantly reduced adiponectin, obestatin, and antioxidant enzymes (catalase, glutathione, glutathione-S-transferase, and glutathione peroxidase) together with a significant increase in oxidant byproduct (malondialdehyde) and modulated lipid parameters (cholesterol, triglyceride, and high-density lipoprotein).Conclusion: The study concluded that atherosclerosis is associated with reduced antioxidant enzymes, obestatin, and adiponectin levels and increased lipid levels. These parameters play a great role in the patho-logical status of coronary stenosis.
- MeSH
- adiponektin krev metabolismus MeSH
- antioxidancia analýza metabolismus MeSH
- ateroskleróza etiologie patofyziologie MeSH
- ELISA MeSH
- ghrelin krev metabolismus MeSH
- hyperlipidemie * diagnóza etiologie patofyziologie MeSH
- koronární stenóza patofyziologie MeSH
- lidé MeSH
- malondialdehyd krev metabolismus MeSH
- metabolismus lipidů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinická studie MeSH
Cílem sdělení je seznámit s formami očních projevů visceral larva migrans u dětí, jak dokresluje rozsáhlá fotodokumentace. Oční larvální toxokaróza (OLT) má i v dětském věku rozdílné klinické projevy, kdy vliv má věkové zastoupení. Nejčastěji bývá přítomen periferní granulom oka často s trakčním vitreálním pruhem táhnoucím se z periferie sítnice k papile zrakového nervu. Následuje granulom zadního pólu oka zasahující většinou od makulární krajiny do střední periferie sítnice vždy s vitritidou. U dětí se může OLT projevit i postižením optického nervu (cystický granulom hlavy zrakového nervu nebo neuropatie s vitritreální reakcí), fulminativní endoftalmitidou a vzácně i difúzní chorioretinitidou. Diagnostika se opírá o klinický oftalmologický nález a také laboratorní vyšetření hladin protilátek s případnou eosinofilií. Histologické vyšetření může prokázat kulovitou polyploidní osifikaci v cévnatce na zadním pólu oka jako následek fibrotizace a kalcifikace postupující z v okolí vstřebané larvy. Celková kombinovaná léčba anthelmetikem a kortikoidem je obtížná, a ne vždy přináší dobrý efekt ve smyslu uspokojivého zlepšení zrakové ostrosti. Projevy OLT u malých dětí jsou stále spojeny v diferenciální diagnóze s retinoblastomem a klinikou dalších nitroočních chorob.
The aim of this paper is to present an outline of forms of ocular manifestations of visceral larva migrans in children, as illustrated by the extensive photographic documentation. Ocular larval toxocariasis (OLT) has various clinical manifestations even in childhood age, in which age representation has an influence. The most common is presence of peripheral granuloma of the eye, frequently with a tractional vitreal streak leading from the retinal periphery to the optic nerve papilla. This is followed by granuloma of the posterior pole of the eye, usually reaching from the macular landscape to the central retinal periphery, always with vitritis. In children OLT may be manifested also in affliction of the optic nerve (cystic granuloma of the head of the optic nerve or neuropathy with vitreal reaction), fulminant endophthalmitis and in rare cases also diffuse chorioretinitis. The diagnosis rests upon a clinical ophthalmological finding, as well as laboratory examination of the levels of antibodies with potential eosinophilia. Histological examination may demonstrate spherical polypoid ossification in the choroid at the posterior pole of the eye as a consequence of fibrotisation and calcification, proceeding from the surrounding area of the absorbed larva. General combined treatment with antihelminthics and corticosteroids is arduous and does not always produce the desired effect in the sense of a satisfactory improvement of visual acuity. In differential diagnostics, manifestations of OLT in small children are still associated with retinoblastoma and a clinical picture of other intraocular diseases.
- MeSH
- anthelmintika aplikace a dávkování farmakokinetika terapeutické užití MeSH
- chorioretinitida etiologie MeSH
- diferenciální diagnóza MeSH
- dítě MeSH
- ELISA metody MeSH
- endoftalmitida etiologie MeSH
- larva migrans viscerální diagnóza etiologie MeSH
- lidé MeSH
- oči patologie MeSH
- toxokaróza * diagnóza farmakoterapie prevence a kontrola MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- přehledy MeSH
OBJECTIVES: For the proper diagnosis of toxoplasmosis it is essential to determine the stage of the infection, for which the most preferred method is IgG avidity test. The avidity index (AI) should initially be low (AI≤0.3) in the acute phase and increase during the infection. However, persistent low avidity can occur in patients with latent toxoplasmosis, which can complicate the interpretation of the results. The aim of the study is to explain the causes of this phenomenon. METHODOLOGY: A retrospective study was carried out with 717 serum samples collected from 442 patients from the categories of pregnant and non-pregnant women, men, and newborns + infants (age < 0.5 year). The trends of AI kinetics were evaluated in repeatedly examined patients. The frequency of cases with low avidity was compared in individual categories of patients and in groups of people with acute and non-acute toxoplasmosis. RESULTS: The proportion of patients with initially low avidity was 42.1% in the acute toxoplasmosis group while it was 13.0% in the non-acute groups. In uninfected newborns with anti-Toxoplasma antibodies transmitted from the mother, a decrease in IgG avidity levels over time was observed, resulting in 29.2% of samples showing low (improper) avidity. While the dynamics of IgG avidity and the frequency of cases of improperly low avidity were similar in men and pregnant and non-pregnant women, the category of newborns and infants differed substantially for these indicators. CONCLUSIONS: Due to acceptable specificity and negative predictive value, high avidity can rule out acute toxoplasmosis, but moderate sensitivity complicates the possibility of its confirmation. The results of the avidity test must be interpreted in the context of the results of other methods.
- MeSH
- afinita protilátek MeSH
- ELISA metody MeSH
- imunoglobulin G * MeSH
- imunoglobulin M MeSH
- kojenec MeSH
- lidé MeSH
- novorozenec MeSH
- protilátky protozoální MeSH
- retrospektivní studie MeSH
- toxoplazmóza * MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The QuantiFERON®-Monitor (QFM) is an assay that measures interferon-γ production and was developed to provide an objective marker of complex immune response. In this study, we evaluated the use of the QFM test in patients with two forms of multiple sclerosis (MS), relapsing-remitting form treated with fingolimod (fMS) and secondarily progressive form not treated pharmacologically (pMS), and in healthy controls (HC). We hypothesized that IFN-γ levels would be lower in those subjects who are relatively more immunosuppressed and higher in those with normal or activated immune function. METHODS: This single-center observational study was conducted from November 2020 to October 2021 and compared results in three groups of patients: 86 healthy controls, 96 patients with pMS, and 78 fMS. Combination of lyophilized stimulants was added to 1 ml heparinized whole blood within 8 hr of collection. Plasmatic IFN-γ was measured using the ELISA kit for the QFM and data were obtained in IU/ml. RESULTS: The results showed that controls had nearly 2-fold higher levels of IFN-γ (QFM score) in median (q25, q75) 228.00 (112.20, 358.67) than the MS patient groups: pMS 144.80 (31.23, 302.00); fMS 130.50 (39.95, 217.07) which is statistically significant difference P-value: HC vs. pMS = 0.0071; HC vs. fMS = 0.0468. This result was also confirmed by a validation analysis to exclude impact of variable factors, such as disease duration and Expanded Disability Status Scale scores. CONCLUSIONS: Results showed that controls had higher levels of IFN-γ production than the MS patient groups and suggest that MS patients included in this study have a lower ability of immune system activation than HC. Results confirm that fingolimod is able to suppress production of IFN-γ. The fact that the QFM score of MS patients is significantly lower than that of HC may indicate a dysfunctional state of the immune system in baseline conditions.
- MeSH
- biologické markery * analýza MeSH
- chybná diagnóza MeSH
- diagnostické techniky gastrointestinální klasifikace trendy MeSH
- ELISA MeSH
- feces * mikrobiologie MeSH
- gastrointestinální nemoci diagnostické zobrazování klasifikace MeSH
- lidé MeSH
- metody pro přípravu analytických vzorků metody MeSH
- odběr biologického vzorku metody MeSH
- pracovníci laboratoře MeSH
- prediktivní hodnota testů MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
Matrix Gla protein (MGP) je cirkulující protein s nízkou molekulární hmotností, který působí jako přirozený inhibitor kalcifikace. Řadíme ho do skupiny proteinů označených jako vitamin K dependentní proteiny. Jeho hlavní funkcí je prevence ukládání vápníku do měkkých tkání. Pro správné fungování musí být MGP dostatečně karboxylován a fosforylován, což je proces, který vyžaduje vitamin K2 jako kofaktor gamma-glutamylkarboxylasy. Nedostatek vitaminu K se projevuje kardiovaskulárním onemocněním, nedostatečnou mineralizací kostní tkáně a vznikem kalcifikujících deposit v měkkých tkáních. Cílem práce bylo navržení metod pro sledování hladiny MGP a defosforylovaného nedostatečně karboxylovaného MGP (dp-uc MGP) za využití imunochemické detekce a aplikovat je na analýzu reálných pacientských vzorků pro použití v klinických laboratořích, kde by tyto markery mohly sloužit jako potencionální markery závažnosti kloubních onemocnění, kardiovaskulárních onemocnění a také jako markery vypovídající o stavu vitaminu K v organismu.
Matrix Gla protein (MGP) is a circulating protein with a low molecular weight that acts as a natural inhibitor of calcification. It belongs to the group of vitamin K‐dependent proteins. For its proper function, MGP must undergo vitamin K-dependent carboxylation and phosphorylation. Its main function is the prevention of soft-tissue calcification. It requires vitamin K2 as a cofactor for gamma glutamyl carboxylase. Vitamin K insufficiency is manifested by cardiovascular diseases, insufficient mineralization of bone tissue and the formation of calcifying deposits in soft tissues. The aim of this study was to design methods for monitoring the levels of MGP and dp-uc MGP by using immunochemical detection and to apply them to the analysis of real samples for use in clinical laboratories. These markers could serve as potential markers of the severity of joint diseases, cardiovascular diseases and also as markers indicating the status of vitamin K in organism.
