[Role of IL-17A and neutrophilic granulocytes in the pathogenesis of psoriasis]. / Rol' IL-17A i neitrofil'nykh granulotsitov v patogeneze psoriaza.

OBJECTIVE: To study the expression of IL-17A in the inflammatory infiltrating cells in the plaques as one of the key points in the pathological process in moderate to severe psoriasis. MATERIAL AND METHODS: The material obtained from 50 patients with moderate and severe psoriasis was examined by an indirect immunofluorescence assay to determine the composition of cell infiltrate and the type of IL-17A-producing cells in the foci of lesion. The markers of lymphocytes (CD3), dendritic cells (CD11c), neutrophilic granulocytes (Mpo), and mast cells (Trp) were used. The proliferative activity of basal keratinocytes (Ki-67), the expression of IL-17A, and the co-expression of IL-17A, Mpo, and Trp were also studied. RESULTS: A positive correlation was established between the count of neutrophilic granulocytes in the infiltrate, the expression of IL-17A, and the number of neutrophils expressing IL-17A with the duration of the disease and the severity of the patient's condition. CONCLUSION: Neutrophilic granulocytes and their expression of IL-17A play one of the key roles in the pathogenesis of psoriasis.

Rapid and sensitive double-label based immunochromatographic assay for zearalenone detection in cereals.

A double-label immunochromatographic based assay (DL-ICA) was developed to monitor zearalenone (ZEN) levels in cereals, based on Eu3+ nanoparticles (EuNP). The DL-ICA exhibited excellent sensitivity, reliability and selectivity in real samples. It showed low limits of detection (0.21-0.25 µg/kg) and broad analytical ranges (up to 120 µg/kg). The total analytical time, including sample preparation and DL-ICA execution, was reduced by 15 min compared with HPLC. The recovery rates ranged from 95.0-118.4%, with relative standard deviations (RSD) <11.6%. Inter- and intra-day validations were assessed, recovery rates of 89.3-106.9% and RSD of 2.3-9.7% were obtained, suggesting considerable stability and reliability for the assay. An excellent correlation was observed between DL-ICA and a reference HPLC method (R2 = 0.9899). Compared to current immunoassays, the current DL-ICA is inexpensive, highly sensitive, and rapid. Therefore, DL-ICA constitutes a novel tool for monitoring mycotoxins in food and feed to ensure safety.

Coxiella burnetii in Infertile Dairy Cattle With Chronic Endometritis.

Coxiella burnetii is an obligate intracellular pathogen and the cause of Q fever in many animal species and humans. Several studies have reported the association between C. burnetii and abortion, premature delivery, stillbirth, and weak offspring. However, no solid evidence indicates that C. burnetii causes endometritis, subfertility, and retained fetal membranes. For this study, histopathological and PCR evaluation were performed on 40 uterine biopsies from dairy cattle with poor fertility. Uterine swabs were concurrently tested with microbiology assays. The endometrial biopsies of 30 cows did not have any significant lesions, and no pathogens were identified by aerobic bacterial culture and PCR. Ten cows were PCR-positive for C. burnetii and negative for other pathogens by aerobic bacterial culture and PCR. These 10 cases revealed a mild to severe chronic endometritis admixed with perivascular and periglandular fibrosis. Immunohistochemical evaluation of C. burnetii PCR-positive biopsies identified, for the first time, the presence of intralesional and intracytoplasmic C. burnetii in macrophages in the endometrium of cattle.

A double-label time-resolved fluorescent strip for rapidly quantitative detection of carbofuran residues in agro-products.

A rapid and quantitative time-resolved fluorescent immunochromatographic assay (TRFICA) for detecting carbofuran residues in agro-products was reported in this paper. This assay was developed based on double-label immunoprobes, one of which was a carbofuran-specific antibody coupled with europium microbeads for the test (T) line signal while the other was mouse IgG coupled with europium microbeads for the control (C) line signal. Quantitative relationships between carbofuran concentrations and T/C ratios were established to determine the analyte concentration. To increase assay accuracy, four standard curves were established for the agro-products (green bean, cabbage, apple, and pear). The limits of detection (LODs) ranged from 0.04 to 0.76mgL-1. The spiked recoveries of carbofuran in the agro-products were in the range of 81-103%, which was in good agreement with a standard HPLC method. Therefore, we provided a new and reliable method for determination of N-methylcarbamate pesticide carbofuran residues in agro-products including vegetables and fruits.

Immunofluorescence and Immunohistochemical Detection of Keratins.

Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.

Anatomical evidence of pruriceptive trigeminothalamic and trigeminoparabrachial projection neurons in mice.

Itch is relayed to higher centers by projection neurons in the spinal and medullary dorsal horn. We employed a double-label method to map the ascending projections of pruriceptive and nociceptive trigeminal and spinal neurons. The retrograde tracer fluorogold (FG) was stereotaxically injected into the right thalamus or lateral parabrachial area (LPb) in mice. Seven days later, mice received intradermal (id) microinjection of histamine, chloroquine, capsaicin, or vehicle into the left cheek. Histamine, chloroquine, and capsaicin intradermally elicited similar distributions of Fos-positive neurons in the medial aspect of the superficial medullary and spinal dorsal horn from the trigeminal subnucleus caudalis to C2. Among neurons retrogradely labeled from the thalamus, 43%, 8%, and 22% were Fos-positive following id histamine, chloroquine, or capsaicin. Among the Fos-positive neurons following pruritic or capsaicin stimuli, ∼1-2% were retrogradely labeled with FG. Trigeminoparabrachial projection neurons exhibited a higher incidence of double labeling in the superficial dorsal horn. Among the neurons retrogradely labeled from LPb, 36%, 29%, and 33% were Fos positive following id injection of histamine, chloroquine, and capsaicin, respectively. Among Fos-positive neurons elicited by id histamine, chloroquine, and capsaicin, respectively, 3.7%, 4.3%, and 4.1% were retrogradely labeled from LPb. The present results indicate that, overall, relatively small subpopulations of pruriceptive and/or nociceptive neurons innervating the cheek project to thalamus or LPb. These results imply that the vast majority of pruritogen- and algogen-responsive spinal neurons are likely to function as interneurons relaying information to projection neurons and/or participating in segmental nocifensive circuits.

CD3 and CD20 Coexpression in a Case of Canine Cutaneous Epitheliotropic T-Cell Lymphoma (Mycosis Fungoides).

A 14-year-old female spayed Dachshund was presented with generalized scaling, erythema, pruritus, poor quality of hair coat, and progressive weight loss. Cutaneous epitheliotropic T-cell lymphoma (CETCL) was suspected. Skin biopsies were suggestive of CETCL. However, immunohistochemistry revealed the presence of numerous CD20+ and CD3+ cells. Clonality assay demonstrated a clonal T-cell receptor gamma rearrangement and a polyclonal IgH gene rearrangement. Double-label immunofluorescence confirmed coexpression of CD3 and CD20 by neoplastic cells. By double immunohistochemistry, neoplastic cells were CD3+ and PAX5-. The results are compatible with a CD3+, CD20+ CETCL. Coexpression of CD20 and CD3 has been recognized in peripheral T-cell lymphomas. Although documented in human CETCL, it has not been reported in canine CETCL. The pathogenetic basis of CD20 expression in mycosis fungoides is explored.

To Explore the Double-labeling Method of Monitoring the GHRP Regulatory Function on [Ca~(2+)]i and NO on Real Time in Cardiomyocytes Under LSCM / 医学研究杂志

Objective To explore the double-labeling method of monitoring the GHRP regulatory function on [Ca2+]i and NO in cardiomyocytes of rats on real time under LSCM.Methods The reformed constant-flow Langendorff system and enzyme-dissociated was used to isolate cardiomyocytes.[Ca2+]i and NO in the cardiomyocytes of SD rats were double-labeled by their molecular probe Rhod-2/AM and DAF-FM/DA,respectively to monitor the regulatory function of GHRP on [Ca2+]i and NO on real time by LSCM.Results Ca2+ signal showed a red fluorescence and NO showed a green fluorescence while the overlapping of the two signals showed a yellow-green fluorescence by this system,and the similar effect presents in both double-labeled state and the single labeled one:GHRP induced a transient[Ca2+]i increase then followed by a plateau phase while there was not significant change in NO signal system after GHRP stimulation under the LSCM in the cardiomyocytes of rats.Conclusions After having established the double-labeling method we monitored the GHRP regulatory function on [Ca2+]i and NO on real time in cardiomyocytes of rats under LSCM causing the [Ca2+]i biphasic increase while no significant change in NO signal system.

Experimental study of the changes of gap junction in unstable bladder / 中华泌尿外科杂志

Objective To study the quantitative changes of gap junction (GJ) in the detrusors of unstable bladder(USB) in rats,and to deduce the functional changes of GJ,which mediates intercellular communication,so as to demonstrate one of the mechanisms of USB. Methods Thirteen Wistar rats, which were identified to have USB by manometry of filling bladder after establishment of bladder outlet obstruction (BOO) model, served as study,ie,USB group.Another 10 healthy female Wistar rats served as control group.The content and distribution of connexin (Cx) 43 of the detrusors, which were taken from these rats,were quantitatively analyzed by Western blot and laser confocal microscopy with a double-label immunohistochemical technique. Results Cx43 protein was expressed in both control and USB group, the relative molecular weight was 43000.The mean gray levels of the detrusor protein bands in USB group and control group were 31.066 and 11.701,respectively;the difference of the values between the 2 groups was significant (P

EFFECTS OF FOLATE ON PROLIFERATION OF NEURAL STEM CELLS FROM FETAL RATS IN VITRO / 营养学报

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.