Three-color Staining Research Articles

Pharmacodynamic study: Astragaloside IV/chitosan/polylactic acid composite electrospinning scaffold for wound healing in diabetic rats

The wound dressing plays an important role in wound repair as a medical textile that temporarily replaces the skin. However, ordinary wound dressings can only cover wounds and supply a protecting function for wounds, cannot meet some special requirements of chronic wounds, especially diabetic wounds. To overcome these drawbacks, in this study, a novel AS/CS/PLA nanofiber functional dressing was developed to promote diabetic wound healing by electrostatic spinning technology. The pharmacodynamics of AS/CS/PLA nanofiber functional dressings were evaluated. In in vitro pharmacodynamic experiment, the effect of the dressing on cell proliferation was detected by MTT assay. Cell adhesion to the dressing was observed by scanning electron microscopy (SEM), and the cell migration ability promoted by the dressing was evaluated by an in vitro cell scratch test. In in vivo pharmacodynamic experiment, we established a skin wound model in diabetic rats. On the 7th and 15th days, we collected tissue samples from both the surrounding area and the wound area. The expressions of CD31 and TGF-β in wound tissue were detected by H&E and Masson three-color staining, respectively. The results showed that the AS/CS/PLA nanofiber functional dressing could promote cell proliferation, adhesion, and migration. It also increased collagen deposition and granulation tissue formation at wound. Moreover, it promoted the expression of CD31 and TGF-β factors at early stage, thereby accelerating wound healing. Our results indicated that the AS/CS/PLA nanofiber functional dressing have a potential as a treatment material to accelerate diabetic wound healing.

Unraveling T cell mitochondrial dynamics using imaging flow cytometry

Abstract Mitochondria are highly mobile, dynamic subcellular organelles that mediate energy generation in mammalian cells. In response to changes in energy and stress, mitochondria can either join or divide, a process known as fusion or fission, respectively. Mitochondrial morphology plays a critical role in the metabolic state of T cells and can affect T cell function in health and disease. Despite increased interest in mitochondria dynamics, a robust and efficient method to simultaneously image mitochondria morphology, analyze T cell phenotypes and sort cells has not been feasible. Here, we used the state-of-the-art BD CellView™ Image Technology on the BD FACSDiscover™ S8 Cell Sorter to detect changes in mitochondria morphology with high resolution and profile T cell phenotypes associated with specific mitochondrial shapes. With the imaging capabilities of the BD FACSDiscover™ S8 System, we visualized live mitochondria and nuclei using three-color staining in a high-throughput manner. We differentiated effector T cells in vitro and imaged increased fission-like mitochondria in these cells. With the cell sorting feature of the BD FACSDiscover™ S8 System, we sorted effector T cells based on their cell surface phenotype and distinct mitochondrial characteristics and evaluated their mitochondrial gene signature. Thus, we provide an advanced and comprehensive workflow to unravel mitochondrial dynamics and isolate cells with a particular phenotype for downstream in-depth characterization enabled by the BD FACSDiscover™ S8 System. As many cellular processes are linked to mitochondrial dynamics, this technique provides a powerful tool to understand how this critical organelle is regulated in a variety of human diseases.

Abstract 4707: LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment

Abstract Multiplexing in immunofluorescence imaging is important for the spatial profiling of cells and molecules in tumor tissue samples. Cyclic immunofluorescence (IF) methods using oxidants (e.g. hydrogen peroxide) and enzymes (e.g. DNase) localize a great number of cellular makers and proteins in a tissue section while repeating a process of IF staining, imaging, and fluorescence deactivation. However, the repeated use of chemicals and enzymes might cause artifacts in tissue and cell morphologies. Furthermore, these methods are restricted to thin tissue sections (~5 μm thick) which are inappropriate to provide comprehensive structural information on tissue samples. Although reconstruction of two-dimensional (2D) images from serial tissue sections can provide a certain volumetric tissue image, it takes a huge amount of time and effort. Here we introduce a three-dimensional (3D) multiplex IF imaging method using LED photobleaching. We built high-power LED illuminators with 100W warm (emission wavelength: 480-700 nm), green (430-520 nm), and red (600- 680 nm) LED chips, which can efficiently bleach a broad or selected wavelength of fluorescence signals in tissue samples. We integrated this LED photobleaching with the Transparent Tissue Tomography (T3) protocol and created a 3D cyclic IF method involving tissue macrosectioning (400 μm), three-color IF staining, D-fructose-based tissue clearing, 3D confocal fluorescence microscopy, LED photobleaching, tissue washing, and three-color IF staining for other biomarkers, and repeating the process. By applying this method to mouse mammary tumor tissues, we could perform 8-plex fluorescence microscopy for visualizing cell nuclei (DAPI), vascular (CD31, SMA) and structural (ER-TR7) cells, immune cells (CD3, CD8, CD45), and cancer cells (CK8) in the tumor macrosections in 3D at tissue and cellular resolution. To validate the method as an evaluation tool for immunotherapy, we treated the mouse mammary tumor with a STING agonist (DMAXX) intratumorally and collected the tumor tissue 1 day after the treatment, and processed it for the 3D cyclic IF protocol. The quantitative multiplex image data showed immune-driven-cancer eradication and high tumor infiltration of a large number of CD3+CD8+CD45+ cytotoxic T cells. We also examined that Red and Green LED illumination can selectively bleach fluorophores in tissues, which would be useful for patterning fluorescence in tissue as well as studying fluorescent drug-cell interaction in a tissue. In summary, this chemical and enzyme-free 3D cyclic IF imaging method will be a powerful tissue assay tool to provide comprehensive spatial information of tissue (tumor) samples including cell types, cellular and molecular location, and their 3D organization in a tissue sample. Citation Format: Jingtian Zheng, Evan Phillips, Yi-Chien Wu, Steve Seung-Young Lee, Vytautas Bindokas. LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4707.

Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

The main advantage of digital PCR (dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve. Crystal droplet dPCR has a three-color staining detection function, which enables multiplex PCR reaction. In this study, this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 ​× ​MON89788 with stacked traits. Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708 ​× ​MON89788 soybean. Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.

COMPARATIVE ANALYSIS OF MULTIPARAMETRIC ULTRASOUND TECHNIQUES IN THE DIFFERENTIAL DIAGNOSIS OF THYROID CANCER

The aim of the study is to determine the value of multiparametric ultrasound examination in the differential diagnosis of thyroid cancer. The results of a multiparametric ultrasound examination performed in 219 patients with thyroid cancer at the stage of preoperative assessment are analyzed. There were 123 patients with papillary thyroid cancer, 96 patients with follicular thyroid cancer. All patients underwent multiparametric ultrasound examination in grey-scale mode, color Doppler mapping mode and elastography mode: compression elastography and shear wave elastography. In papillary thyroid cancer, two–color staining was found in 13.4% of cases, three–color staining – in 19.3%, four-color staining – in 28.7%, five-color staining – in 26.4%, six–color staining – in 12.2%. The pattern was dominated by "cold" tones. The velocity of the shear wave was 4.61±1.33 m/s (95% CI), Young's modulus was 63.3±14.7 kPa (95% CI), the elasticity index (strain ratio) was 4.89±1.65 (95% CI) c.u. The color scheme of follicular thyroid cancer was characterized by patchiness, the presence of both rigid and elastic areas. In 2 cases (14.3%) there was two-color staining, in 4 (28.6%) – three-color staining, in 8 (57.1%) – four-color and five-color staining. The velocity of the shear wave was 4,53±1,25 m/s (95% CI), Young's modulus was 62,1±12,1 kPa (95% CI), the elasticity index (strain ratio) was 3,76±0,76 (95% CI) c.u. Thus, the study showed that the complex use of multiparametric ultrasound examination techniques (B-mode, color Doppler mapping, compression elastography, shear wave elastography) is highly informative and should be used in the differential diagnosis of thyroid cancer in the preoperative assessment. Thus, the performed examination showed that the complex application of multiparametric ultrasound techniques (V, mode, CDM, CEG, ESP) is highly informative and should be used in the differential diagnosis of thyroid cancer in the preoperative period.

ТРЁХЦВЕТНый FISH-МЕТОД: ТРАНСЛОКАЦИИ В КУЛЬТУРАХ ЛИМФОЦИТОВ ПЕРИФЕРИЧЕСКОЙ КРОВИ ДО И ПОСЛЕ ЛОКАЛЬНОГО Гамма-ОБЛУЧЕНИЯ ПО ПОВОДУ РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ

Purpose: Study of chromosomal aberration yields with a three-color FISH method in the peripheral blood lymphocyte cultures of patients with breast cancer before and after local irradiation. Material and methods: Venous blood was obtained from 24 patients aged from 33 to 79 years which suffering from cancer of the left or right breast after the implementation of a sectoral or radical resection of an affected organ with subsequent local adjuvant distant radiation therapy (DRT) to the scar area. Also it could be preceded by adjuvant polychimotherapy (PCT). A linear accelerator of the Trilogy system based on the Clinac iX platform was used for local irradiation. SOD was installed at 50 Gy per 25 fractions (daily 2 Gy). venous blood sampling produced to PCT and/or DRT and after 1 and/or 3 months after irradiation. A set of DNA probes to 1, 4 and 12 pairs of chromosomes was used for three-color FISH staining. Results: Levels of translocations, dicentrics and acentrics (per 100 cells) in peripheral blood lymphocyte cultures of patients up to DRT did not differ significantly with and without PCT and were amounted to average 0.63 ± 0.10; 0.10 ± 0.04 and 0.17 ± 0.06, respectively. The frequency of these chromosome rearrangements significantly increased after radiation therapy by an average of up to 4.62 ± 0.46; 1.48 ± 0.19 and 0.95 ± 0.14. The differences between 1 and 3 months of taking material after irradiation were absent. One of the patients, unlike the rest, was cytogenetically examined 11 years after a similar local irradiation, and the frequencies of translocations and dicentrics in her lymphocyte culture were 4.56 and 0.19 (per 100 cells), respectively, while they were varied from 1.63 to 10.00 for translocations and from 0.27 (0 in one patient) to 4.06 for dicentrics for other patients. Conclusion: When using a three-color FISH method there was a significant increase in the frequencies of translocations, dicentrics and acentrics in peripheral blood lymphocyte cultures of patients after DRT of breast cancer. At the same time there were more translocations than dicentrics and, apparently, they remain at the initial level for a long time. The frequency of translocations per 1 cell with translocations in the peripheral blood lymphocyte cultures in most cases exceeds 1 after local DRT of the breast cancer. The correlation between average physical doses to the whole body and cytogenetical estimates in individual patients was statistically insignificant.

Influence of hepatotropic and metabiotic correction on the spectrum of free fatty acids in experimental toxic liver damage

Background. Today most of the scientists are studying the processes of hepatocyte damage under the influence of free fatty acids (FFA) in vitro conditions. Therefore, in vivo studies of the spectrum of FFA in liver pathologies of different genesis, including toxic ones, are of considerable interest. Materials and methods. Toxic liver damage was simulated by subcutaneous injection of CCl4 solution in olive oil into rats. The following groups were formed: control (healthy; n=15); I— CCl4-induced liver damage without drug correction (n=6); III— CCl4-induced liver damage + metadoxin (Liveria IC; n=8); III— CCl4-induced liver damage + metabiotic (Hilac forte) (n=7). For morphological assessment of steatosis and fibrosis we used three-color qualitative staining of liver samples by Mallory-Slinchenko. Quantitative content of monounsaturated fatty acids (MUFA) in liver homogenate was determined by gas chromatography. Results. Evaluation of hepatobioptates in group I rats revealed tissue disorganization with macrovesicular steatosis in the cytoplasm of hepatocytes, the formation of interparticle multiple fibrous septa and inflammatory cell infiltration. The use of metadoxin (group II) and metabiotic (group III) improved the morphological picture of the liver, which was damaged by CCl4. Total MUFA content increased significantly in 118 (p<0.001), 34 (p<0.001) and 99 times (p<0.001), respectively, for groups I–III animals relative to control, but in group II— tended to decrease in 3.5 (p=0.430) and 2.9 times (p=0.064), compared to groups I and III, respectively. Conclusions. It was found that correction with methadoxin and metabioticreduced the manifestations of protein-fatty dystrophy in hepatocytes. In all animal research groups, the content of PUFA increased mainly due to significant concentrations of cis-10-pentadecenoic, cis-10-heptadecenoic, trans- and cis-9-octadecenoic and cis-11-eicosenoic acids. MUFA content decreased more under the influence of methadoxin than metabiotic.

APLICACIÓN DE COLORACIONES ESPECIALES EN EL DIAGNÓSTICO HISTOPATOLÓGICO: TINCIÓN TRICRÓMICA DE MASSON

Masson's trichrome (MT) is a three-color staining protocol used in histology. MT allows to show and quantify changes such as tissue repair (healing) and collagen deposition. Also, it can be used to quantify blood vessels, in epithelial dysplasia and squamous cell carcinoma. The objective of this work is to describe the MT staining technique and to exemplify some applications of this technique in routine veterinary histopathological diagnosis. Archived histologic sections were selected from the records of the histopathology laboratory. Tissues were selected in base on theirs structures and lesions that could be evaluated with MT: a rabbit lung with a chronic suppurative bronchopneumonia; a bovine liver with lesions of Echium plantagineum poisoning; and a bovine eyelid with a squamous cell carcinoma. The TM was able to show fibroplasia in the pulmonary interstitium and confirm the presence of a chronic respiratory process, and clearly revealed an abundant fibrovascular tumor stroma, with profuse connective tissue and neovascularization between the tumor cells in deep dermis. In the liver, extensive and marked fibroplasia was confirmed. MT represents a complementary coloration to routine hematoxylin and eosin stain (H&E) and provides accurate information from several pathological processes, mainly those related to fibrovascular proliferation and scarring.

COLORIMETRY OF HISTOLOGICAL MATERIAL

The relevance of the research lies in the use of conventional software products distributed under the GNU license to assess staining of histological material based on the analysis of photomicrographs. The purpose of the study is to demonstrate the opportunities of conventional software distributed under the GNU license, designed for color analysis of photos in histology to identify differences in tinctorial properties of tissues. The authors evaluated the opportunities of the Color Analysis program launched in the Android Nox Player 6.6.1.0 emulator (GNU license) under Windows 10 for color analysis of photomicrographs of various tissues histological sections. Photomicrographs of paraffin sections of 5 microns thick were obtained using a micro-microscope Mikmed-5 using a Levenhuk C800 NG 8M video eyepiece, USB 2.0. A white light lamp Feron G4 4000 K was used as an illuminator. In color analysis of photomicrographs stained with hematoxylin and eosin, or hematoxylin and Congo red, or by Unna, or by the designer three-color amyloid staining in the Color Analysis program, color separation patterns were obtained that reflect the relative area (%) of the field of view staining with shades characteristic for this method of staining. The authors formulate the standard conditions for the preparation of histological specimen and conditions for photographing micro-preparations to obtain comparable results in an experiment with the control-experience design. It is concluded that the program color analysis provides a significant amount of additional information about the histological material, which can be formalized and studied as statistical values.

Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.

The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

Mixed bacterial communities are commonly encountered in microbial infections of humans. Knowledge on the composition of species and viability of each species in these communities allows for a detailed description of the complexity of interspecies dynamics and contributes to the assessment of the severity of infections. Several assays exist for quantification of specific species in mixed communities, including analysis of quantitative terminal restriction fragment length polymorphisms. While this method allows for species-specific cell enumeration, it cannot provide viability data. In this study, flow cytometry was applied to assess the viability of Staphylococcus aureus and Burkholderia cepacia in mixed culture by membrane integrity analysis using SYBR® Green I and propidium iodide staining. Both bacteria are relevant to pulmonary infections of cystic fibrosis patients. Fluorescence staining was optimized separately for each species in pure culture due to differences between species in cell wall structure and metabolic capabilities. To determine viability of species in mixed culture, a protocol was established as a compromise between optimum conditions determined before for pure cultures. This protocol allowed the detection of viable and dead cells of both species, exhibiting an intact and a permeabilized membrane, respectively. To discriminate between S. aureus and B. cepacia, the protocol was combined with Gram-specific fluorescent staining using wheat germ agglutinin. The established three-color staining method was successfully tested for viability determination of S. aureus and B. cepacia in mixed culture cultivations. In addition, growth of both species was monitored by quantitative terminal restriction fragment length polymorphisms. The obtained data revealed alterations in viability during cultivations for different growth phases and suggest interspecies effects in mixed culture. Overall, this method allows for rapid simultaneous Gram-differentiation and viability assessment of bacterial mixed cultures and is therefore suitable for the analysis of dynamics of mixed communities of medical, environmental, and biotechnological relevance.

Reliability of Basophil Activation Test Using CD203c Expression in Diagnosis of Pollen Allergy

CD203c is a basophil surface marker and its expression is rapidly up-regulated after cross-linking of high-affinity immunoglobulin E (IgE) receptor (FcepsilonR1) by an allergen. CD203c basophil activation tests have been studied for the in vitro diagnosis of several allergic conditions. However, there is limited data about its diagnostic usefulness. The optimum allergen concentrations for stimulation and allergen specific cutoff values remain unknown for a number of allergens. This study was designed to investigate the efficacy of basophil activation test via CD203c in the diagnosis of pollen allergy. The CD203c basophil activation was determined in 31 allergic rhinitis patients with pollen allergy and 9 healthy nonatopic controls during the off-season. CD203c expression was evaluated using three-color staining protocol by flow cytometry. After an in vitro stimulation with grass pollen extract, the CD203c assay clearly discriminated pollen-allergic patients from controls (p < 0.001). A dose-dependent increase in the percentages of CD203c-activated basophils was shown in rhinitis patients with pollen allergy (p < 0.001). The sensitivity and specificity was 100% and optimal cutoff values were 14.05 and 10.05% with 45.1 and 4.5 μg/mL Phl p 5 stimulation, respectively. Although the specificity was also 100%, the sensitivity was 93 and 87% and the cutoff values were 5.40 and 5.35% with 4.5 × 10(-4) and 4.5 × 10(-5) micrograms/mL Phl p 5 stimulation, respectively. The CD203c basophil activation test seems to be a reliable tool in the diagnosis of grass pollen allergy. It could be used when conventional diagnostic tests fail or can not be performed.

The influence of puerarin injection combined with irbesartan on atrial fibrillation and r induced by ventricular fibrous ventricular rapid pacing heart failure in dogs

Objective To observe the combined effects of puerarin injection and irbesartan on congestive heartfailure(CHF) in dogs with ventricula fibrous ventricular rapid pacing heart failure.Methods 21 healthy mongrel dogs were randomly divided into normal control group,CI-IF model group and the TMP treated group.CHF was established by right ventricular pacing model of canine.Butst was used to evoke atrial fibrillation (atrial fibrill;tripn,LVEF).Echocardiography was used to detect left ventricular ejection fraction in dogs (Left ventricular ejection fraction,LVEF).Mallory's three-color staining was used to detect the degree of atrial fiber.The level of plasma angiotensin 1,aldosterone concentration,m-type forelimb of the original N-terminal peptide (amino-terllllrlal1 ～ eptldcoftype Ⅲ procollagen,P Ⅲ NP),laminin (1 alTlmln,IN),and hyaluronic acid (hyaltlronlcacid,HA)was detected with radioimmunoassay.Results The LVEF of CHF model group decreased significantly compared with the control group(P ＜0.01).AF incidence ,the incidence of persistent AF and AF duration were significantly increased when compared with the control group(P ＜ 0.01).Left atrial fibrosis were also significantly increased in CHF model group than normal control group (P ＜ 0.01).AF duration was positively correlated with the degree of left atrial fiber (r = 0.84,P = 0.018).plasma angiotensin Ⅱ ,aldosterone,and serurn P Ⅲ NP,HA levels were significantly higher than normal control group(P ＜0.05 or P ＜0.01).There was no statistical difference in LN.Plasma angiotensin Ⅱ and aldosterone levels showed a close positive correlation(r =0.759 ,P =0.182).There is significant improvement in LVEF in treatment group when compared with model group(P ＜ 0.05).The incidence of persistent AF was significantly lower in treated group when compared with CHF model(P ＜ 0.05).The left atrial fibrosis was significantly reduced in treated group when compared with CHF(P ＜ 0.01).Conclusion Puerarin injection combined with irbesartan can reduce II-IF degree of atrial fiber,which may be one of the mechanisms of the reduction of the incidence and duration of CHF and AF. Key words: Puerarin injection; Irbesartan; Joint; Heart failure; Congestive; Atrial fibrillation

Three-color intranuclear staining for measuring mitosis and apoptosis in cells transfected with a GFP-tagged histone

Green fluorescent protein (GFP) has become a powerful tool for monitoring the expression of transfected genes by flow cytometry including GFP-tagged histones for tracking chromatin and elucidating histone function. We describe here a method for simultaneous detection of three nucleus-localized signals: a GFP-tagged histone, DNA content and detection of phosphorylated histone H3, which labels mitotic cells. We also demonstrate another application of this method for simultaneous detection of a GFP-tagged histone, DNA content, and cleaved caspase-3.

Expression of BT-related protein-1 on B lymphocytes in peripheral blood from patients with systemic lupus erythematosus and its relationship with disease activity.

Objeetive To explore the differentiation of B lymphocytes and expression of B7-related protein-1 (B7RP-1)on B lymphocytes in patients with systemic lupus erythematosus(SLE).Methods Three-color immunofluorescent staining and flow cytometric assay were used to analyze the frequency of three types of B lymphocytes,I.e.,plasma cells,memory B lyphocytes and naive B lymphocytes,as well as the expression of B7RP-1 on these cells in peripheral blood from 23 patients with SLE and 16 normal human controls.Clinical data of these patients with SLE were collected.and SLE disease activi index(SLEDAI)was also evaluated.The relationship was assessed between the expression of B7RP-1 and SLEDAI.Results The frequency of plasma cells was highest in patients with active SLE.followed by patients with inactive SLE and normal human controls(P＜0.01).A significant decrease was observed in the frequency of memory B lymphocytes in patients with active SLE compared with normal controls (P＜0.01),but no significant difference was found between patients with inactive SLE and those with active SLE(P＞0.05).Regarding the frequency of naive B lymphocytes,there was no significant difierence among the three groups.Increased frequency of plasma cells was also noted in patients with lupus nephritis(LN)compared with those without LN [(6.15±3.12)%vs(3.31±1.41)%,P＜0.05 ],but no significant difierence was found with regard to the frequency of memory or naive B lymphocytes between these two groups.The expression rate Of B7RP-1 was significantly lower on total lymphocytes from patients with SLE than from normal human controls (46.51%vs 63.75%,P＜0.05),which was the case with B7RP-1 on plasma cells,memory B lyphocytes and naive B lymphocytes (all P＜0.01),whereas no significant difierence was found between patients with inactive SLE and active SLE or between patients with and without LN.In addition.no correlation was found between the expression of B7RP-1 and SLEDAI(r=0.035,P＞0.05).Conclusions In peripheral blood of patients with SLE,the frequency of plasma cells is increased,while the expression of B7RP-1 on lymphocytes is decreased,which may be relevant to the pathogenesis of SLE. Key words: Lupus erythematosus,systemic; Antigens,CD27; B-lymphocytes; B7-related protein-1

Three-color intranuclear staining for measuring mitosis and apoptosis in cells transfected with a GFP-tagged histone

Green fluorescent protein (GFP) has become a powerful tool for monitoring the expression of transfected genes by flow cytometry including GFP-tagged histones for tracking chromatin and elucidating histone function. We describe here a method for simultaneous detection of three nucleus-localized signals: a GFP-tagged histone, DNA content and detection of phosphorylated histone H3, which labels mitotic cells. We also demonstrate another application of this method for simultaneous detection of a GFP-tagged histone, DNA content, and cleaved caspase-3.

Circulating Leukemic Myeloid Dendritic Cells from Patient with Leukemia Elicit CDK2-Specific CTLs from Allogeneic HLA-A24+ Naive CD8+ T Cells.

Aberrantly expressed self-antigens in leukemic cells are a kind of leukemia-associated-antigens (LAAs). Although such self-antigen-derived LAAs potentially induce high avidity CTLs in patients with leukemia, these CTLs usually do not persist in the patients because they undergo apoptosis upon encountering with leukemic cells. In allogeneic stem cell transplant (allo-SCT) recipients with leukemia, LAAs-specific high avidity CTL may be elicited from donor-derived naive T cells that are sensitized by residual leukemic cells. Cyclin-dependent kinase 2 (CDK2) is a cell cycle regulator protein that is aberrantly expressed by leukemia cells. We previously reported that two CDK2-derived peptides (CDK2 158-166, CDK2 178-186) avidly bound to HLA-A24 molecule to elicit each peptide-specific CTLs from HLA identical donor of a AML patient, and CDK2-specific CD8 + T cells preferentially killed the recipient AML cells which aberrantly expressed CDK2 protein (Blood. 108 (11): a3173. 2006). When we assessed cryopreserved PBMCs obtained before and after allo-SCT from 16 HLA-A24+ patients (6 AML, 1 MDS, 1 CML, 2 ALL, 4 NHL and 2 RCC) using CDK2 158/A24 and CDK2 178/A24 pentamers, small populations (0.1–1.0%) of CDK2 158 and CDK2 178-specific CTL were detectable in 6 patients after SCT but not before SCT. All of the 6 patients had MRD at the time of SCT and achieved molecular CR after SCT. None of the 3 patients relapsed after SCT did not show CDK2 immunity. There is no relationship between the appearance of CDK2-specific CTLs and the development of GvHD (Figure). Leukemic myeloid dendritic cells (mDC) are known to present in vivo in patients with leukemia, and be able to trigger a protective antileukemic immune response by allogeneic T-cells. We hypothesized that residual circulating leukemic mDCs may sensitize donor-derived T cells by cross-presenting CDK2-peptides early after allo-SCT and elicit high avidity CTLs specific to leukemic cells. mDCs subset was identified with by three-color staining using mAbs against CD85k, CD33, CD14 and CD16. Circulating mDCs represented 5.4% of PBMCs in a patient with CML-CP at diagnosis. Leukemic mDCs from the patient were purified with CD1c mAb-conjugated maginetic beads and were assessed for their ability to stimulate allogeneic T cells to acquire specific cytotoxicity against CDK2-peptides. The purity of the mDCs as determined by flow cytometry was 92% of living isolated cells. Naive CD8 + T cells isolated from healthy individual were cultured with the leukemic mDCs for 12 days and subjected to pentamers staining. After the coculturing, the proportion of CDK2 158/A24 and CDK2 178/A24 pentamers + CD8 T cells increased 0.6% to 2.2% and 0.6% to 2.6%, respectively. These data suggest that CDK2-specific CTLs can be induced from donor-derived T cells due to in vivo sensitization of donor T cells by residual leukemic mDCs and this may be a mechanism responsible for the generation of CDK2-specific CTLs in allo-SCT recipients with MRD without vaccination of CDK2-peptides. Vaccination with CDK2-peptides after allo-SCT may be useful in both enhancing CDK2-specific immunity in patients with MRD and in generating CDK2 immunity in those without MRD. [Display omitted]

CD40 ligand expression on stimulated T-helper lymphocytes in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is the most common symptomatic primary antibody deficiency, characterized by reduced serum immunoglobulins levels and increased susceptibility to recurrent pyogenic infections. In this study, we evaluated CD40 ligand expression on stimulated versus unstimulated T-helper lymphocytes of nine Common variable immunodeficient patients in comparison with fifteen normal controls. Phorbol myristate acetate (PMA) and Ionomycin were used to stimulate cells in vitro. After six hours stimulation, the cells were subjected to surface staining with three-color staining procedure. Events were analyzed by flow cytometer, using FloMax software. Results were reported as the percentage of lymphocytes expressing CD markers. We did not find any significant statistical difference in CD40 ligand expression between patients and controls (p > 0.05), despite having stimulation documented by CD69 expression as activation marker in each run. The results of this study are in agreement with some other studies, indicating that CD40 ligand expression on stimulated T-helper lymphocytes of Common variable immunodeficiency patients is similar to normal controls.

Resident dendritic cells are the predominant TNF-secreting cell in early renal ischemia–reperfusion injury

Renal ischemia-reperfusion injury (IRI) rapidly induces production of inflammatory mediators including, and in particular, tumor necrosis factor (TNF). Possible sources include resident parenchymal and bone marrow-derived cells as well as recruited leukocytes. Cell suspensions from kidneys subjected to IRI were examined by cell separation followed by in vitro culture and enzyme-linked immunosorbent assay (ELISA), immunoperoxidase and immunofluorescence microscopy, and multicolor flow cytometry to determine the contribution of dendritic cells (DCs) to early production of TNF and other inflammatory mediators. Secretion of TNF, interleukin (IL-6), monocyte chemoattractant protein-1 (MCP-1), and regulated on activation normal T cell expressed and secreted (RANTES) was increased in cell suspensions from IRI compared with control kidneys and was higher in DC-enriched preparations. Immunostaining identified TNF(+ve) cells that coexpressed the DC marker CD11c. Flow cytometry of bone marrow-derived (CD45(+ve)) cell populations at 24 h post-IRI demonstrated that F4/80(+ve)/CD11c(+ve) DCs remained proportionately stable and exhibit higher levels of DC maturation markers, whereas the proportion of F4/80(-ve) DCs, monocytes, neutrophils, and T cells increased. Intracellular staining for TNF confirmed that F4/80(+ve) DCs were the predominant TNF(+ve) cell and expressed higher levels than other TNF(+ve) cells. In vivo depletion of DCs from the kidney substantially attenuated TNF secretion by total and CD45(+ve) cells following IRI. The results uncover a role for resident F4/80(+ve) DCs as the predominant secretors of TNF within 24 h of IRI.

Activation of transforming growth factor-beta1/Smads signal pathway in diabetic cardiomyopathy and effects of valsartan thereon: experiment with rats

To study the activation of transforming growth factor (TGF)-beta(1)/Smads signal pathway in diabetic cardiomyopathy (DCM) and effects of valsartan thereon. 40 male Wistar rats were randomly divided into 3 groups: DCM group (n = 16, fed with high-calorie fat diet for 4 weeks, injected intraperitoneally with streptozocin so as to establish DCM model, and then perfused into the stomach with normal saline once daily since the injection of STZ for 16 weeks), valsartan group (n = 16, perfused into the stomach with valsartan at the dose of 30 mg/kg once a day for 16 weeks after the establishment of DCM model), and control group (n = 8, fed with normal diet and perfused into the stomach with normal saline once daily for 16 weeks). At the end of the experiment, the contents of fast blood-glucose (FBG), fast insulin (FIN), serum cholesterol, and triglyceride were detected, and insulin sensitivity index (ISI) was calculated. Cardiac catheterization was performed to measure the hemodynamics indexes: left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and maximal rise/fall velocity of ventricular pressure (+/- dp/dt(max)), and the left ventricular diastolic function (T) was calculated. Pieces of myocardium tissue were taken out to undergo ultrastructural histopathological examination by transmission electron microscopy. The content of collagen was quantified by Masson three-color staining. Real-time RT-PCR and Western-blotting were used to detect the mRNA expression and protein expression of TGFbeta(1), TGFbetaRII, Smad2, Smad3, and Smad7. By the end of experiment the levels of FBG, triglyceride, and cholesterol increased and the ISI decreased significantly in the DCM and valsartan groups (all P < 0.01). Compared with the valsartan and control groups the levels of LVEDP and T significantly increased, and the levels of LVSP and +/- dp/dt(max), significantly decreased (all P < 0.01); and the LVEDP of the valsartan group was significantly higher than that of the control group and the +/- dp/dt(max) of the valsartan group was significantly lower than that of the control group (P < 0.01). The volume of collagen in the myocardial tissue of the DCM group was 17% +/- 3%, significantly higher than that of the control group (11% +/- 3%, P < 0.01). The content of collagen in the myocardial tissue of the valsartan group was lower significantly than that of the DCM group. The levels of mRNA expression of TGFbeta(1), TGFbetaRII, Smad2, and Smad3 were 0.0126 +/- 0.0057, 0.0877 +/- 0.0272, 0.0884 +/- 0.0146, and 0.012 +/- 0.0048 respectively, all significantly higher than those of the control group (0.0054 +/- 0.0009, 0.0523 +/- 0.0218, 0.0413 +/- 0.0186, and 0.0064 +/- 0.0021 respectively, all P < 0.05 - 0.01). The ratios Smad2/Smad7 and Smad3/Smad7 of the DCM group were significantly higher than those of the control group (both P < 0.05). The protein levels of TGFbeta(1), P-Smad2, and P-Smad3 were 143 +/- 17, 212 +/- 43, and 151 +/- 32 respectively, all significantly higher than those of the control group (103 +/- 18, 107 +/- 21, and 89 +/- 17 respectively, P < 0.01). The P-Smad2/Smad7 and P-Smad3/Smad7 of the DCM group were significantly higher than those of the control group (both P < 0.05), The Smad2/Smad7 (or P-Smad2/Smads7) and Smad3/Smad7 (or P-Smad3/Smads7) of the valsartan group was significantly lower than those of the DCM group (both P < 0.05). Activation of TGFbeta(1)/Smads signal pathway and imbalance between Smad2, 3 and Sma7 may be one of the mechanisms of myocardial interstitial fibrosis in DCM. Valsartan can prevent myocardium from damage by blocking the signal pathway.