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ABSTRACT Introduction: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Patients with SLE exhibit multiple serum autoantibodies, including anti-neutrophil cytoplasmic antibodies (ANCAs). There are two main techniques to detect ANCAs: indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). In this study, an attempt was made to determine the frequency and clinical associations of ANCAs in patients with SLE. Methods: A cross-sectional study was conducted in a tertiary care hospital in Colombia that included 74 patients with SLE. The presence of ANCAs was assessed using IIF with ethanol-fixed slides, and ELISA was used to detect antibody specificities for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Results: Of the 74 patients with SLE evaluated, 60 (81.1%) of them were ANCA-positive by IIF. By contrast, only one patient showed specificity for PR3-ANCA by ELISA. The relevance of ANCA positivity by IIF and clinical and serological features was significant for renal involvement (p = .0174), and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (p = .0308). Conclusion: ANCAs are common in the serum of patients with SLE, as detected by ethanol-fixed slides with IIF staining. However, detection of specificity to PR3 and/or MPO is rare, thus highlighting the importance of detecting these autoantibodies by different techniques.
RESUMEN Introducción: El lupus eritematoso sistémico (LES) es una enfermedad autoinmune sistémica. Los pacientes con LES muestran múltiples autoanticuerpos séricos, incluyendo los anticuerpos anticitoplasma de neutrófilo (ANCA, por sus siglas en inglés). Existen 2 técnicas principales para la detección de ANCA: inmunofluorescencia indirecta (IFI) y ensayo por inmunoadsorción ligado a enzimas (ELISA). En este estudio nuestro objetivo fue determinar la frecuencia y las asociaciones clínicas de los ANCA en pacientes con LES. Métodos: Realizamos un estudio transversal de 74 pacientes con LES en un hospital de alta complejidad de Colombia. La presencia de ANCA se evaluó por IFI, utilizando láminas con fijación de etanol, y con ELISA para determinar las especificidades para mieloperoxidasa (MPO)-ANCA y proteinasa 3 (PR3)-ANCA. Resultados: Fueron evaluados 74 pacientes con LES, 60 (81,1%) de ellos fueron positivos para ANCA. Por el contrario, solo un paciente mostró especificidad para PR3-ANCA por ELISA. La relación entre la positividad para ANCA por IFI y las características clínicas y serológicas fue estadísticamente significativa para compromiso renal (p = 0,0174) y para el índice de actividad de la enfermedad (Systemic Lupus Erythematosus Disease Activity Index [SLEDAI]) (p = 0,0308). Conclusiones: Los ANCA detectados mediante fijación con etanol por técnicas de IFI, son comunes en pacientes con LES. Sin embargo, la detección de especificidades para PR3 o MPO es rara; se destaca la importancia de la evaluación de estos autoanticuerpos mediante diferentes técnicas.
Subject(s)
Humans , Adult , Immunoproteins , Blood Proteins , Skin and Connective Tissue Diseases , Connective Tissue Diseases , Antibodies, Antineutrophil Cytoplasmic , Amino Acids, Peptides, and Proteins , Lupus Erythematosus, SystemicABSTRACT
Objective To compare different blocking methods for eliminating autofluorescence of mouse liver frozen sections, so as to find the best method to reduce the interference to immunofluorescence positive signals and improve the accuracy of immunofluorescence. Methods Intrasplenic injection-liver colonization nude mice: Hepa1-6-GFP cells were intrasplenically injected into male athymic BALB/c nude mice to create liver colonization models. Liver tissues were frozen and continuously sectioned. Sections were blocked with AB reagent (A reagent: streptavidin reagent, B reagent: biotin reagent), blocking buffer, AB reagent＋blocking buffer, or acetone＋AB reagent＋blocking buffer, and then the autofluorescence of the frozen sections was detected. C57BL/6 mice: the liver tissues of C57BL/6 mice were frozen and continuously sectioned, and then the sections were blocked with AB reagent, blocking buffer, AB reagent＋blocking buffer, or acetone＋AB reagent＋blocking buffer. Liver macrophages (Kupffer cells) were labeled with F4/80, and then the autofluorescence of mouse liver frozen sections was detected. Results In the immunofluorescence staining of liver tissue frozen sections, all the above four blocking methods could reduce the autofluorescence of liver sections, and acetone＋AB reagent＋blocking buffer group had the best effect. Conclusion The combined buffers (acetone＋AB reagent＋blocking buffer) has the best effect in eliminating the autofluorescence of mouse liver frozen section.
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Objective To study the time-dependent expression and distribution of acetylcholinesterase （AChE） during skin incised wound healing in mice, and discuss its effect in wound healing as well as the feasibility of using it as a reference index for wound age estimation. Methods A total of 45 C57BL/KsJ mice were randomly divided into one control group and eight incised groups. The skin incised wound model was established in the incised groups with samples of skin wounds taken at 6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 10 d and 14 d post-injury respectively, while the uninjured skin tissue was extracted in the control group. Expression and distribution of AChE in skin samples were detected by immunohistochemistry, double immunofluorescence and Western blotting. Results Immunohistochemistry results indicated that AChE was mainly detected in infiltrating polymorphonuclear cells （PMNs） 6 to 12 h post-injury. A large number of AChE-positive mononuclear cells （MNCs） were observed 1 to 3 d post-injury. The AChE-positive cells were mainly fibroblastic cells （FBCs） 5 to 14 d post-injury. The ratio of the AChE-positive cells increased initially 6 h post-injury, and reached the peak at 1 d post-injury. Double immunofluorescent staining showed that the majority of AChE-positive MNCs and FBCs expressed macrophage marker and myofibroblast marker, respectively. Western blotting results showed that the relative expression level of AChE in the incised group was higher than that in the control group averagely, reached the peak at 1 d post-injury, then reached a second peak at 7 d post-injury. Conclusion The expression of AChE is found in PMNs, macrophages and myofibroblast during skin wound healing, which indicates it might be involved in the adjustment of inflammatory response and fibrotic repair after injury. Moreover, combined use of various methods for the detection of the expression of AChE would provide reference for skin wound age estimation.
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Animals , Mice , Acetylcholinesterase/metabolism , Mice, Inbred C57BL , Skin/pathology , Time Factors , Wound Healing/physiologyABSTRACT
Objective To investigate the expression of cannabinoid type 2 receptor （CB2R） at different time points after brain contusion and its relationship with wound age of mice. Methods A mouse brain contusion model was established with PCI3000 Precision Cortical Impactor. Expression changes of CB2R around the injured area were detected with immunohistochemical staining, immunofluorescent staining and Western blotting at different time points. Results Immunohistochemical staining results showed that only a few cells in the cerebral cortex of the sham operated group had CB2R positive expression. The ratio of CB2R positive cells gradually increased after injury and reached the peak twice at 12 h and 7 d post-injury, followed by a decrease to the normal level 28 d post-injury. The results of Western blotting were consistent with the immunohistochemical staining results. Immunofluorescent staining demonstrated that the changes of the ratio of CB2R positive cells in neurons, CB2R positive cells in monocytes and CB2R positive cells in astrocytes to the total cell number showed a single peak pattern, which peaked at 12 h, 1 d and 7 d post-injury, respectively. Conclusion The expression of CB2R after brain contusion in neurons, monocytes and astrocytes in mice suggests that it is likely to be involved in the regulation of the biological functions of those cells. The changes in CB2R are time-dependent, which suggests its potential applicability as a biological indicator for wound age estimation of brain contusion in forensic practice.
Subject(s)
Animals , Mice , Blotting, Western , Brain Contusion/metabolism , Brain Injuries , Forensic Pathology , Muscle, Skeletal/pathology , Receptor, Cannabinoid, CB2/metabolism , Receptors, Cannabinoid , Time Factors , Wound Healing/physiologyABSTRACT
RESUMEN Las enfermedades autoinmunes son un grupo de patologías crónicas en las que factores genéticos, ambientales y hormonales contribuyen a su aparición. Además de tener un amplio espectro clínico, la interpretación de los diversos autoanticuerpos y técnicas utilizadas en el laboratorio también son un reto clínico. Dada la complejidad de estas enfermedades, es muy importante apoyarse en las pruebas de laboratorio para establecer un correcto diagnóstico, seguimiento y, en algunos casos inclusive, establecer pronósticos o predicción de la posible aparición de autoinmunidad. Con todo esto se pretende mejorar la calidad de vida de los pacientes disminuyendo la gran morbimortalidad de este grupo de enfermedades, especialmente al diagnosticarlas en etapas tempranas. La mayoría de las enfermedades reumatológicas se caracterizan por la alta producción de autoanticuerpos y reactantes de fase aguda, los cuales están implicados en su fisiopatología produciendo daño directo a nivel sistémico. Entre estas, el lupus eritematoso sistémico, la artritis reumatoide y el síndrome de Sjögren son las más reconocidas. Portales motivos, el objetivo de este trabajo es hacer una revisión que permita guiar tanto a médicos como a personal de laboratorio en la interpretación de los diferentes autoanticuerpos en enfermedades autoinmunes.
ABSTRACT Autoimmune diseases are a group of chronic diseases in which genetic, environmental, and hormonal factors contribute to their appearance. In addition to having a broad clinical spectrum, the interpretation of the various autoantibodies and techniques used in the laboratory is also a clinical challenge. Given the complexity of these diseases, it is very important to rely on the results of laboratory tests to establish a correct diagnosis and follow-up and, in some cases even to establish a prognosis or prediction of autoimmunity. Taking all this into account, it is intended to improve the quality of life of patients by decreasing the increased morbidity and mortality in this group of diseases, especially by early diagnosis. Most rheumatological diseases are characterised by the high production of auto-antibodies and acute phase reactants, which are involved in their pathophysiology, leading to systemic involvement. Among these, the most recognised are, systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. For these reasons, the objective of this project is to present a review that will help both physicians and laboratory personnel in the interpretation of the different autoantibodies in autoimmune diseases.
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Autoantibodies , Arthritis, Rheumatoid , Quality of Life , Autoimmune Diseases , DiagnosisABSTRACT
Objective To study the expression of S100A8 and the relationship between S100A8 and Toll-like receptor 4 (TLR4) in focal cerebral ischemia reperfusion (I/R) injury.Methods C3H/HeJ mice with TLR4 gene mutation (n =30) and C3H/HeN with normal TLR4 gene mice (n =30) were divided into 4 groups at random (random number),namely C3H/HeJ model group (n =18),C3H/HeJ control group (n =12),and C3H/HeN model group (n =18).C3H/HeN control group (n =12).Middle cerebral artery was occluded to make I/R model in mice by using thread embolism method.Brain tissues were collected after ischemia for one hour and reperfusion for 12 hours.Stroke outcome was evaluated by determination of infarct volume of brain tissue and assessment of neurological scores.And brain injury after cerebral I/R was observed by optical microscope after TTC and HE staining.The immunofluorescence technique and RT-PCR were used to determine the protein level and expression of S100A8 mRNA in damaged brain tissues.Results Compared with C3H/HeN model mice,TLR4-deficient model mice (C3H/ He J) had lower infarct volumes and better outcomes of neurological function after resuscitation for 12 hours.Compared with control groups,the expression of S100A8 mRNA and level of S100A8 protein increased greatly in damaged brain tissues of model mice after I/R injury.In addition,model mice with lacked TLR4 (C3H/HeJ) had lower expression of I/R-induced S100A8 mRNA than C3H/HeN mice in model group,indicating that the close relationship between the levels of S100A8 and TLR4.Conclusions S100A8 interaction with TLR4 might be involved in brain damage and in inflammation triggered by I/R injury.
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Objective To compare the efficiency of explants adherent after enzymatic pre-digestion and traditional typeⅡ?collagenase digestion methods for culturing human nucleus pulposus cells from degenerated intervertebral discs.Methods Human nucleus pulposus tissues collected from patients'degenerated intervertebral discs were divided into Groups A and B.In Group A,the nucleus pulposus tissues were cultured using explants adherent method directly after 0.025 ％ typeⅡ?collagenase digestion for 30 minutes.In Group B,the tissues were firstly digested with 0.025 ％ typeⅡ?collagenase for five hours,and then underwent filtration,centrifugation and inoculation successively Success rate and primary cell fusion time of the two culture methods were compared.Cell morphology and expressions of sex determining region Y-box 9(Sox-9),collagenⅡ?and aggrecan were observed by HE staining,toluidine blue staining and immunofluorescence cell staining.Results There was no significant difference in the successful culture rate between the two groups(P＞0.05).However,the average fusion time of primary passage in Group A was significantly shorter than that in Group B(P＜0.01).In Group A,the nucleus pulposus cells could be stained as azure by toluidine blue and immunofluorescence cell staining showed positive expressions of Sox-9,collagenⅡ?and aggrecan.Conclusions Compared with traditional typeⅡ?collagenase digestion,explants adherent method after typeⅡ?collagenase pre-digestion for the culture of human nucleus pulposus cells from degenerative intervertebral discs has a high success rate and obtains a large number of cells in a short time.Meanwhile,the cells have strong expressions of Sox-9,collagenⅡ?and aggrecan.
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Background Bevacizumab has been widely used in the treatment of new blood vessel disease in ophthalmology.The investigation of the pharmacokinetics and safety after intracameral injection of bevacizumab can offer the basis for the management of iris neovascularization and neovascular glaucoma.Objective The present study was to observe the distribution of bevacizumab(avastin)in eye tissue and toxic effects following the injection of anterior chamber.Methods Twenty-four New Zealand albino rabbits were divided into two groups randomly.0.05 ml (1.25mg)of Bevacizumab was intracamerally injected into the left eyes in the experimental group,and a balanced salt solution of 0.05 ml was injected in the same way into the left eyes of the control group.The anterior segment of eyes and ocular fundus were examined by slit-lamp microscope and direct ophthalmoscope after injection.Intraocular pressure was measured and corneal endothelial microscopy was performed before and after the injections.Five rabbits of the two groups were sacrificed on the first day,the fourth day,the seventh day,the fourteenth day,and the thirtieth day after injection,and the eyeballs were enucleated for histopathological examination.The ultrastructure of eye tissue was observed under the transmission electron microscope on the fourth day and the thirtieth day,and then immunofluorescence staining were performed to assess the distribution of bevacizumab in the eye tissues.This experiment complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission(Version 1988).Results No abnormality in the cornea,lens,vitreous and retina was observed after the injection of bevacizumab under the slit lamp microscope and direct ophthalmoscope.No significant differences were found in intraocular pressure and corneal endothelial cell density in the bevacizumab group compared with the control group before injection and 2 hours,1 day,7 days,14 days,30 days after injection(P =0.760,P =0.956).No histopathological and ultrastructural changes of the cornea,lens,chamber angle,iris,ciliary body and retina were seen after the injection in the experimental group and control group under the light microscope and transmission electron microscope.Bevacizumab was distributed in the anterior chamber angle,iris,ciliary body,choroid and retina in injected eyes and fellow eyes after intracameral injection with red fluorescence and presented the dynamic changes with the lapse of time.The immunofluorescence response of eye tissue to bevacizumab was weaker in the fellow eyes compared with injected eyes.Bevacizumab was mainly distributed in the vessel wall and lumen.Conclusions Bevacizumab can quickly distribute in the vascular tissue of the anterior chamber angle,iris,ciliary body,choroid and retina in injected eyes after intracameral injection without obvious toxic effects to eye tissue.Bevacizumab administered intracamerally may be a new strategy or a joint strategy for iris neovascularisation.
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OBJECTIVE To compare the effectiveness of two laboratory detections methods of Epstein-Barr virns (EBV). METHODS Immuno-fluorescence technique was applied to detect the serum EBV-specific antibody and EBV-DNA was identified by fluorescence quantitative polymerase chain reaction (FQ-PCR) in 124 children infected with EBV. RESULTS The positive rates of VCA-IgM,VCA-IgG,EA-IgG,EBNA-1-IgG and EB-DNA in various samples of 124 cases were 0,89.5%,23.4%,49.2% and 30.3%,respectively,with 21 cases (16.9%) showing simultaneously positive in EB-DNA and EBNA-1-IgG. CONCLUSIONS The two laboratory detections of EBV share their advantages and disadvantages. FQ-PCR is a method for time-saving,accurate and sensitive detection of EBV-DNA,showing clinical significance in the diagnosis of EBV-associated diseases. It's worthy of clinic application.
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Widespread brain-derived neurotrophic factor (BDNF) mRNA expression has been detected in the region of catecholamine groups of the rat lower brainstem, while few BDNF-immunoreactive cells were found in this area. In the present study, a double-color immunofluorescence (IF) technique for BDNF and tyrosine hydroxylase (TH) after colchicine treatment was employed to evaluate the possible presence of BDNF immunoreactivity in the catecholamin-ergic cells of rat lower brainstem. Additionally, a double-color IF technique for BNDF and TH and in situ hybridiza-tion for BDNF mRNA were performed to see effects of hemorrhage on the expression of BDNF and its mRNA. We detected many new BDNF-immunoreactive cells in the A1, A2, A4, A6-A10 and C1-C3 cell groups and in the other lower brainstem nuclei where, without colchicine treatment, BDNF mRNA was expressed, but not BDNF immunoreactivity. In addition, the catecholaminergic neurons were found to express BDNF immunoreactivity with the co-existence being greatest, in percentage terms, in medullary catecholaminergic cell groups. Hypotensive hemorrhage, which activates medullary catecholaminergic neurons, induced the expression of BDNF immunoreactivity in catecholaminergic neurons (A1/C1 and C2) and increased the number of BDNF mRNA-containing neurons in the area. These results demonstrate that BDNF is regulated by activity in medullary catecholaminergic cell groups involved in central cardiovascular regulation.
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Animals , Rats , Brain Stem , Brain-Derived Neurotrophic Factor , Colchicine , Fluorescent Antibody Technique , Hemorrhage , In Situ Hybridization , Neurons , RNA, Messenger , Tyrosine 3-MonooxygenaseABSTRACT
There is a prevailing belief that the tissue culture isolation procedures have their greatest superiority over other procedures in identifying chlamydial infection of the male urethra but the usefulness is limited because the procedures are very complicated and time consuming. Here we presented an experimental results of direct staining of cell scraping with yluorescent antibody and serodiagnosis with indirect microimmunofluorescence technique in male urethra. In terms of sensitivity, specificity and simplicity, the above methods have their merit and demerit in clinical application. 1. The diagnostic sensitivity of direct staining of cell scraping with fluorescent antibody was 64.4%. 2. The diagnostic sensitivity of serodiagnosis with indirect microimmunofluorescent technique was 65.3%.
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Humans , Male , Chlamydia trachomatis , Chlamydia , Diagnosis , Fluorescent Antibody Technique , Sensitivity and Specificity , Serologic Tests , UrethraABSTRACT
The specific IgM (SIgM) antibodies in serum samples from 32 patients with epidemic hemorrhagic fever (EHF) were sequentially determined by IgM antibody capture ELISA (MacELISA) and indirect immunofluorescence technique (IFA) sitnultaneously. Of the samples detected by the two methods, 99.46% had the same positive or negative results. The response curves of SIgM titres separately determined by MacELISA or IFA were parellel, whereas, the SIgM titres detected by MacELISA were comparatively higher than those detected by IFA. During the course of the disease, no significant defference was found between the SIgM titres of defferent illness types at the same illness day.
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Objective To explore the effect of folic acid on neural stem cells(NSCs) proliferation from fetal rats in vitro.Method NSCs were isolated and cultured by microdissection,mechanical blowing and serum-free suspension culture,and identified by immunofluorescent staining using antibody against nestin.BrdU(5’bromo-2’deoxyuridine) was used to mark dividing neural stem cells.Cultured NSCs were divided into four groups:control group,low,high dose group(liquid media with added 4,40 mg/L folic acid),and deficiency group(liquid media with added 0.4 mg/L methotrexate,MTX).Monotetrazolium(MTT) and double-label immunofluorescence technique detected NSCs proliferation under the condition of folic acid.Results In the serum-free suspension medium,neurospheres that consisted of a great number of nestin-positive cells could be obtained.The proliferative ability of NSCs were observed by BrdU labeling methods.MTT assay and double-label immunofluorescence for nestin+BrdU showed that the growth tendency was increased with folate concentration in the medium.Compared with control group,NSCs growth rate of folate group was significantly increased in vitro.Conclusion The culture of NSCs isolated from fetal rats possesses the abilities of proliferation and self-regeneration.Folic acid may stimulate proliferation of NSCs efficiently.