Mean Fluorescence Intensity Research Articles

Reduced expressions of apoptosis-related proteins TRAIL, Bcl-2, and TNFR1 in NK cells of juvenile-onset systemic lupus erythematosus patients: relations with disease activity, nephritis, and neuropsychiatric involvement.

Lupus pathogenesis is mainly ascribed to increased production and/or impaired clearance of dead cell debris. Although self-reactive T and B lymphocytes are critically linked to lupus development, neutrophils, monocytes, and natural killer (NK) cells have also been implicated. This study assessed apoptosis-related protein expressions in NK cells of patients with juvenile-onset systemic lupus erythematosus (jSLE) and relations to disease activity parameters, nephritis, and neuropsychiatric involvement. Thirty-six patients with jSLE, 13 juvenile dermatomyositis (JDM) inflammatory controls, and nine healthy controls had Fas, FasL, TRAIL, TNFR1, Bcl-2, Bax, Bim, and caspase-3 expressions in NK cells (CD3-CD16+CD56+) simultaneously determined by flow cytometry. Disease activity parameters included Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score, erythrocyte sedimentation rate, C-reactive protein level, anti-double strain DNA antibody level, complement fractions C3 and C4 levels. Patients with jSLE had a profile of significantly reduced expression of TRAIL, Bcl-2, and TNFR1 proteins in NK cells when compared to healthy controls. Similar profile was observed in patients with jSLE with active disease, positive anti-dsDNA, nephritis, and without neuropsychiatric involvement. Patients with jSLE with positive anti-dsDNA also had reduced expression of Bax in NK cells when compared healthy controls and to those with negative anti-dsDNA. Yet, patients with jSLE with negative anti-dsDNA had reduced mean fluorescence intensity (MFI) of Bim in NK cells compared to healthy controls. Patients with jSLE with nephritis also had reduced MFI of Fas in NK cells when compared to those without nephritis. In addition, in patients with jSLE, the proportion of FasL-expressing NK cells directly correlated with the SLEDAI-2K score (rs = 0.6, p = 0.002) and inversely correlated with the C3 levels (rs = -0.5, p = 0.007). Moreover, patients with jSLE had increased NK cell percentage and caspase-3 protein expression in NK cells when compared to JDM controls. This study extends to NK cells an altered profile of TRAIL, Bcl-2, TNFR1, Fas, FasL, Bax, Bim, and caspase-3 proteins in patients with jSLE, particularly in those with active disease, positive anti-dsDNA, nephritis, and without neuropsychiatric involvement. This change in apoptosis-related protein expressions may contribute to the defective functions of NK cells and, consequently, to lupus development. The full clarification of the role of NK cells in jSLE pathogenesis may pave the way for new therapies like those of NK cell-based.

Comparing fluorescent contrast agents for fluorescence guided surgery using 3-D cryo-imaging.

Fluorescence cryo-imaging is a high-resolution optical imaging technique that produces 3-D whole-body biodistributions of fluorescent molecules within an animal specimen. To accomplish this, animal specimens are administered a fluorescent molecule or reporter and are frozen to be autonomously sectioned and imaged at a temperature of -20°C or below. Thus, to apply this technique effectively, administered fluorescent molecules should be relatively invariant to low temperature conditions for cryo-imaging and ideally the fluorescence intensity should be stable and consistent in both physiological and cryo-imaging conditions. Herein, we assessed the mean fluorescence intensity of 11 fluorescent contrast agents as they are frozen in a tissue-simulating phantom experiment and show an example of a tested fluorescent contrast agent in a cryo-imaged whole pig brain. Most fluorescent contrast agents were stable within ~25% except for FITC and PEGylated FITC derivatives, which showed a dramatic decrease in fluorescence intensity when frozen.

Histology and Lung Nodule Fluorescence in Intraoperative Molecular Imaging With Pafolacianine

BackgroundIntraoperative molecular imaging (IMI) uses a cancer-targeted fluorescent agent injected into patients to localize tumor nodules. Pafolacianine is a folate receptor (FR)–targeted near-infrared fluorescent probe. Almost 10% of patients have false negative fluorescence findings intraoperatively. We hypothesized that tumor histology explains why lung cancer may not fluoresce. MethodsAdenocarcinoma (AC) (A549, LKR) and squamous cell carcinoma (SCC) (H127, H1264) cell lines were stained with pafolacianine. Near-infrared fluorescent microscopy was used to quantify mean fluorescence intensity. Tissue microarray slides of patients with AC and SCC were evaluated by immunohistochemistry for FR alpha (FRα) and beta (FRβ) expression. Finally, we retrospectively analyzed IMI data from clinical trials of patients with AC and SCC receiving pafolacianine. ResultsAC (intensity 30.31) cell lines have a higher fluorescence intensity than SCC cell lines (intensity 5.4) (P < .001). On slide analysis, 93.8% of ACs expressed FRα compared with 44.4% of SCCs (P = .002). Finally, there were 326 patients enrolled in clinical trials: 211 had lesions localized in vivo, and 134 of these patients had pure AC or SCC. All 9 patients with SCC have a positive smoking history and a mean pack-year of 60.2 (SD 3,6), whereas 76% of patients with AC have a history of smoking and a mean pack-year of 29.3 (P = .02). The odds ratio for fluorescence of (AC/SCC) was 2.05 (P = .004) and 2.01 (P = .02) on univariate and multivariate logistic regression, respectively. ConclusionsDuring IMI with pafolacianine, a nonfluorescent nodule is more likely to be SCC than AC. AC has a high probability of fluorescing because of higher expression of FRα or FRβ, or both.

Lower NKG2D expression in hepatic natural killer cells predicts poorer prognosis for chronic hepatitis B patients with cirrhosis

ObjectiveNatural killer cells (NK) acts a central player of the immune system in liver cirrhosis. The aim of this study was to examine the expression of activating intra-hepatic NK cell group 2D (NKG2D) in patients with chronic hepatitis B (CHB) and analyzed the correlation between NKG2D expression and prognosis of liver cirrhosis in these patients. MethodsThis was a cross-section study. Subjects with liver biopsy or sponge hemangioma surgery were included. The primary outcome was the NKG2D expression on intra-hepatic NK cells and their subtype cells in patients with CHB-related liver cirrhosis. Subsequently, the correlation of expression of NKG2D and clinical characteristic indicators were assayed ResultsAmong 38 subjects, 11 (28.95%) normal liver sections adjacent the sponge hemangioma (healthy group) were collected during surgery, and 27 (71.05%) CHB-cirrhosis tissues (Cirrhosis group) were preserved after liver biopsy. Compared with healthy group, sections from cirrhosis group revealed more severe inflammation and collagen deposition and lower NKG2D expression in hepatic NK cells. The proportion of hepatic NK cells and the mean fluorescence intensity (MFI) of NKG2D on hepatic NK cells showed a positive correlation with serum albumin (Alb) level, platelet (Plt) count. Moreover, they had a significantly negative correlation with patient prothrombin time (PT), international standardized ratio (INR), the sirius red positive stained area and fibrosis stages. ConclusionsLower NKG2D expression in intra-hepatic NK cells may be predictive of poorer prognosis of CHB patients with cirrhosis.

Donor specific anti-HLA antibodies in hematopoietic stem cell transplantation. Single Center prospective evaluation and desensitization strategies employed.

In the setting of mismatched-hematopoietic stem cells transplantation, the detection of antibodies directed against donor-specific HLA allele(s) or antigen(s) (DSA) represents a barrier for engraftment. It is thus necessary to plan an immunosuppressive strategy, or to select an alternative donor. This prospective study aimed at evaluating the efficacy of our strategy for testing DSAs and the efficacy of the desensitization strategy (DS) employed between November 2017 and November 2020. The anti-HLA antibody search was performed using the Luminex bead assays (Lifecode ID and LSA I/II-Immucor) and expressed as mean fluorescence intensity (MFI >1,000 positive). If the patient had DSAs and no alternative donors, a DS was employed with rituximab (day -15), 2 single volume plasmaphereses (PP; days -9 and -8), intravenous immunoglobulins (day -7) and infusion of HLA selected platelets, if persistent DSAs were directed against class I HLA. DS was scheduled with or without PP, according to the DSA MFI (>1,000 or <5,000) and FCXM (flow cytometry crossmatch). Twenty-two out of 126 patients (17.46%) showed anti-HLA antibodies, 5 of them DSAs (3.97% of total); 3 patients underwent DS obtaining engraftment. Female gender (p=0.033) and a history of previous pregnancies or miscarriages (p=0.009) showed a statistically significant impact on alloimmunization. Factors associated with a delayed neutrophil engraftment were patient's female gender (p=0.039), stem cell source (p=0.025), and a high HSCT-specific comorbidity index (p=0.028). None of the analyzed variables, including the DSA detection, influenced engraftment. Our study confirms the importance to test DSAs in mismatched-hematopoietic stem cells transplantation The DS used proved successful in removing DSAs. Prospective multicenter studies are needed to better define and validate consensus strategies on DSA management in HSCT.

Decreased human leukocyte antigen-D–related expression on CD14+ monocytes in patients with out-of-hospital cardiac arrest provided target temperature management therapy: a prospective observational study

Abstract Background Post–cardiac arrest syndrome involves systemic inflammation, which causes subsequent neurological impairments. We investigated the influence of targeted temperature management (TTM) therapy in patients with out-of-hospital cardiac arrest (OHCA) after return of spontaneous circulation (ROSC) by observing the changes in circulating CD14+ monocytes and the expression of human leukocyte antigen D–related (HLA-DR) and programmed cell death ligand 1 (PD-L1) in CD14+ monocytes. Methods Adult patients admitted to the emergency department of Beijing Chao-Yang Hospital after OHCA between January 2017 and March 2018 were included in this study. Thirty control subjects, 10 patients with OHCA, and 37 patients with OHCA who received 72 hours of TTM therapy were enrolled. Peripheral blood samples of patients in the OHCA and TTM groups were collected on Days 1 and 3 (D1 and D3) after ROSC and evaluated for HLA-DR and PD-L1 expression on CD14+ monocytes using flow cytometry. Results Compared with control subjects, the percentage of circulating CD14+ monocytes, HLA-DR+/CD14+ monocyte ratios, and mean fluorescence intensity were significantly decreased in patients with OHCA. After ROSC, HLA-DR expression in CD14 + monocytes in the TTM group was lower than that in patients with OHCA. However, there were no significant differences in the percentage of PD-L1+/CD14+ monocytes or the mean fluorescence intensity between patients with OHCA and healthy volunteers. Conclusion After ROSC, circulating CD14+ monocytes and HLA-DR+/CD14+ monocyte ratios decreased significantly in patients with OHCA. Human leukocyte antigen D–related expression in CD14+ monocytes was lower in patients treated with TTM.

NG2 Molecule Expression in Acute Lymphoblastic Leukemia B Cells: A Flow-Cytometric Marker for the Rapid Identification of KMT2A Gene Rearrangements.

B-lineage acute lymphoblastic leukemias (B-ALL) harboring rearrangements of the histone lysine [K]-Methyltransferase 2A (KMT2A) gene on chromosome 11q23 (KMT2A-r) represent a category with dismal prognosis. The prompt identification of these cases represents an urgent clinical need. Considering the correlation between rat neuron glial-antigen 2 (NG2) chondroitin-sulfate-proteoglycan molecule expression and KMT2A-r, we aimed to identify an optimized cytofluorimetric diagnostic panel to predict the presence of KMT2A-r. We evaluated 88 NG2+ B-ALL cases identified with an NG2 positivity threshold >10% from a cohort of 1382 newly diagnosed B-ALLs referred to the Division of Hematology of 'Sapienza' University of Rome. Eighty-five of 88 (96.6%) NG2+ B-ALLs harbored KMT2A-r and were mainly pro-B ALL (77/85; 91%). Only 2 B-ALLs with KMT2A-r showed NG2 expression below 10%, probably due to the steroid therapy administered prior to cytofluorimetric analysis.Compared to KMT2A-r-cases, KMT2A r+ B-ALLs showed a higher blast percentage, significantly higher mean fluorescence intensity (MFI) of CD45, CD38, and CD58, and significantly lower MFI of CD34, CD22, TdT, and CD123.The study confirmed differences in CD45, CD34, CD22, and TdT MFI within the same immunologic EGIL group (European Group for the immunological classification of leukemias), indicating no influence of the B-ALLs EGIL subtype on the KMT2A-r+ B-ALLs immunophenotype. Our data demonstrate the association between NG2 and KMT2A-r in B-ALLs identify a distinctive immunophenotypic pattern, useful for rapid identification in diagnostic routines of these subtypes of B-ALLs with a poor prognosis that benefits from a specific therapeutic approach.

Quantitative pharmacokinetic and biodistribution studies for fluorescent imaging agents.

Pharmacokinetics and biodistribution studies are essential for characterizing fluorescent agents in vivo. However, few simple methods based on fluorescence imaging are available that account for tissue optical properties and sample volume differences. We describe a method for simultaneously quantifying mean fluorescence intensity of whole blood and homogenized tissues in glass capillary tubes for two fluorescent agents, ABY-029 and IRDye 680LT, using wide-field imaging and tissue-specific calibration curves. All calibration curves demonstrated a high degree of linearity with mean R2 = 0.99 ± 0.01 and RMSE = 0.12 ± 0.04. However, differences between linear regressions indicate that tissue-specific calibration curves are required for accurate concentration recovery. The lower limit of quantification (LLOQ) for all samples tested was determined to be < 0.3 nM for ABY-029 and < 0.4 nM for IRDye 680LT.

Effect of electroacupuncture of Governor Vessel on mitochondrial fusion and proliferation and differentiation of neural stem cells in spinal cord injury rats.

To observe the effect of electroacupuncture (EA) at "Dazhui" (GV14) and "Jizhong"(GV6) of the Governor Vessel (GV) on mitochondrial fusion and neural stem cell (NSC) proliferation and differentiation in the spinal cord of rats with spinal cord injury (SCI), so as to investigate its mechanisms underlying improvement of SCI. SD rats were randomly divided into sham operation, model and EA groups, with 15 rats in each group. The SCI model was established by using a precision impactor. EA (20 Hz/100 Hz, 1-2 mA) was applied to GV14 and GV6 for 30 min, once daily for 14 days. The rats' hindlimb locomotor function in each group was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor scale. Histopathological changes of the injured spinal cord tissue and the number of neurons were evaluated after H.E. staining and Nissl staining. The expressions of Nestin, mitochondrial fusion-related protein optic atrophy-1 (OPA1) and NSC markers sex-determining region Y-box 2 (SOX2) in the injured spinal cord tissue were detected by immunofluorescence staining. The protein and mRNA expression levels of Nestin in the spinal cord tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the sham operation group, the BBB scores after modeling, and the number of neurons were significantly decreased (P<0.001), while the mean fluorescence intensity values of Nestin, SOX2 and OPA1, and the expressions of Nestin mRNA and protein considerably increased (P<0.001, P<0.01, P<0.05) in the model group. After EA intervention and in comparison with the model group, the BBB scores at the 7th and 14th day, the number of neurons, the mean fluorescence intensity values of Nestin, SOX2 and OPA1, and the expressions of Nestin mRNA and protein were strikingly increased (P<0.05, P<0.01, P<0.001) in the EA group. H.E. staining showed swollen, ruptured and necrotic neurons of the spinal cord, with a large number of vacuoles and severe inflammatory cell infiltration after modeling, which was relatively milder in the EA group. EA stimulation of GV14 and GV6 can promote the recovery of motor function in rats with SCI, which may be related to its effects in promoting mitochondrial fusion and enhancing the proliferation and differentiation of NSCs.

Role of HLA matching and donor specific antibody development in long-term survival, acute rejection and cardiac allograft vasculopathy

Although there are different data supporting benefits of HLA matching in kidney transplantation, its role in heart transplantation is still unclear. HLA mismatch (MM) between donor and recipient can lead to the development of donor-specific antibodies (DSA) which produces negative events on the outcome of heart transplantation. Moreover, DSAs are involved in the development of antibody-mediated rejection (AMR) and are associated with an increase in cardiac allograft vasculopathy (CAV). In this study it is analyzed retrospectively the influence of HLA matching and anti-HLA antibodies on overall survival, AMR and CAV in heart transplantation. For this retrospective study are recruited heart transplanted patients at the Cardiac Transplantation Centre of Naples between 2000 and 2019. Among the 155 heart transplant patients, the mean number of HLA-A, B, -DR MM (0 to 6) between donor and recipient was 4.5 ± 1.1. The results show a negative association between MM HLA-DR and survival (p = 0.01). Comparison of patients with 0–1 MM at each locus to all others with 2 MM, for both HLA class I and class II, has not showed significant differences in the development of CAV. Our analysis detected DSA in 38.1% of patients. The production of de novo DSA reveals that there is not an influence on survival (p = 0.72) and/or AMR (p = 0.39). Instead, there is an association between the production of DSA class II and the probability of CAV development (p = 0.03). Mean fluorescence intensity (MFI) values were significantly higher in CAV-positive patients that CAV-negative patients (p = 0.02). Prospective studies are needed to evaluate HLA class II matching as an additional parameter for heart allocation, especially considering the increment of waiting list time.

Radiation exposure lymphocyte damage assessed by γ-H2AX level using flow cytometry

DNA double-strand breaks (DSBs) are considered the most relevant lesions to the DNA damage of ionizing radiation (IR), and γ-H2AX foci in peripheral blood lymphocytes are regarded as an adequate marker for DSB quantitative studies. This study aimed to investigate IR-induced DNA damage in mice through γ-H2AX fluorescence analyses by flow cytometry (FCM). The levels of γ-H2AX in CD4/CD8/B220-positive lymphocytes were quantified by FCM through mean fluorescence intensity (MFI) values. Peripheral venous blood samples were collected for evaluation, and all the control groups were restrained from irradiation. For external irradiation experiments, the dose-dependency of MFI values and temporal alternations were assessed both in vitro and in vivo. External radiation exposure damage was positively correlated with the absorbed radiation dose, and the lymphocyte recovered from damage within 3 days. I-131 sodium iodide solution (74 MBq) was injected into the mice intraperitoneally for internal irradiation experiments. Gamma counting and γH2AX foci analyses were performed at 1 h and 24 h by the group. The blood-to-blood S values (Sblood←blood) were applied for the blood-absorbed dose estimation. Internal low-dose-irradiation-induced damage was proved to recover within 24 h. The FCM method was found to be an effective way of quantitatively assessing IR-induced DNA damage.

Quantitative assessment of glomerular basement membrane collagen IV α chains in paraffin sections from patients with focal segmental glomerulosclerosis and Alport gene variants

Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in COL4A3/A4/A5 genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α 3 α 4 α5(IV) and α 1 α 1 α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV): α1/2(IV) mean fluorescence intensities ( α5: α1/2 intensity ratios) and thicknesses ( α5: α1/2 thickness ratios) were calculated to represent the levels of collagen α 3 α 4 α5(IV) relative to α 1 α 1 α2(IV). The α5: α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport COL4A variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic COL4A variants who could benefit from genetic testing.

The impact of ageing on the distribution of preformed anti-HLA and anti-MICA antibody specificities in recipients from eastern China prior to initial HSCT

BackgroundWith the development of Hematopoietic Stem Cell Transplantation (HSCT) technology, increasing numbers of elderly patients were undergoing allogeneic HSCT and elderly patients with hematologic malignancies could benefit most from it. Preformed donor-specific human leukocyte antigen (HLA) antibodies (DSA) were associated with graft failure in HLA-mismatched allogeneic HSCT and the absence of DSA was the main criterion of selecting the donor. Except for sensitization events such as transfusion, pregnancy or previous transplantation, ageing affects the humoral immune response both quantitatively and qualitatively. To evaluate the prevalence and distribution of anti-HLA and antibodies of MHC class I chain related antigens A (MICA) specificities in different age groups before initial HSCT would provide HLA and MICA specific antibody profiles under the impact of ageing, which could provide meaningful information in the process of selecting suitable HLA-mismatched donors by avoiding preformed DSA.ResultsThere were no significant differences in the distribution of anti-HLA class I, class II and anti-MICA antibodies among the three age groups in this study except that a significant lower negative ratio of anti-HLA class I, class II antibodies and higher positive rate of MICA antibodies with maximum mean fluorescent intensity (MFI) > 5000 in the elderly than in young age group. The distribution of antibody specificities against HLA -A, -B, -C, -DR, -DQ, -DP and MICA antigens in the three age groups were generally consistent. The anti-HLA class I antibody specificities with higher frequencies were A80,A68;B76,B45;Cw17, which were unlikely to become DSA in Chinese. Anti-HLA class II antibody specificities were more likely to become potential DSA than class I.DR7, DR9, DQ7, DQ8 and DQ9 were most likely to become potential DSA.ConclusionsThe prevalence of anti-HLA and anti-MICA antibodies increased slightly as age increased. While ageing had a small impact on the distribution of antibody specificity frequencies against HLA-A, -B, -C, -DR,-DQ, -DP and MICA antigens in recipients awaiting initial HSCT from East China. The risk of developing preformed DSA was basically consistent in the three age groups and the elderly group might be more favorable in HLA-mismatched HSCT due to higher positive rate of anti-MICA antibody.

Predicting flow cytometry crossmatch results from single-antigen bead testing.

The aim of this study was to devise an algorithm that would predict flow cytometry crossmatch (FCXM) results using single-antigen bead (SAB) mean fluorescent intensity (MFI) levels using samples received through the National External Quality Assurance Scheme (NEQAS) 2B external proficiency testing scheme between 2019 and 2023. A total of 159 serum samples were retrospectively screened using LABScreen Single Antigen Class I and II (SAB), and 40 peripheral blood samples were human leucocyte antigen (HLA) typed with LABType SSO. Donor-specific antibodies were identified for each cell-serum combination tested, and cumulative MFI values were calculated for each test before correlating the screening result with the consensus crossmatch results for this scheme. HLA Class I MFIs were combined to predict the T cell crossmatch. For the B cell crossmatch prediction, two options were considered: (i) HLA Class II MFI values alone and (ii) HLA Class I+Class II MFIs. Receiver operating characteristic analysis was carried out to identify the combined MFI threshold that predicted NEQAS consensus results with the greatest sensitivity and specificity. HLA Class I combined MFI >5000 predicted T cell crossmatch results with 96% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 92% negative predictive value (NPV). For B cell results, HLA Class I+Class II combined MFIs >11,000 gave the best model, showing 97% sensitivity, 82% specificity, 96% PPV and 85% NPV. However, for samples with only HLA Class II sensitization, combined MFIs >13,000 improved the B cell crossmatch predictions: 92% sensitivity, 95% specificity, 96% PPV and 91% NPV. Using this model, combined MFI can be used to predict the immunological risk posed by donor-specific antibodies when it is not possible to carry out an FCXM.

Detection of effusion tumor cells under different storage and processing conditions.

Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Intrahepatic IgA complex induces polarization of cancer-associated fibroblasts to matrix phenotypes in the tumor microenvironment of HCC.

Cancer-associated fibroblasts (CAFs) play key roles in the tumor microenvironment. IgA contributes to inflammation and dismantling antitumor immunity in the human liver. In this study, we aimed to elucidate the effects of the IgA complex on CAFs in Pil Soo Sung the tumor microenvironment of HCC. CAF dynamics in HCC tumor microenvironment were analyzed through single-cell RNA sequencing of HCC samples. CAFs isolated from 50 HCC samples were treated with mock or serum-derived IgA dimers in vitro. Progression-free survival of patients with advanced HCC treated with atezolizumab and bevacizumab was significantly longer in those with low serum IgA levels ( p <0.05). Single-cell analysis showed that subcluster proportions in the CAF-fibroblast activation protein-α matrix were significantly increased in patients with high serum IgA levels. Flow cytometry revealed a significant increase in the mean fluorescence intensity of fibroblast activation protein in the CD68 + cells from patients with high serum IgA levels ( p <0.001). We confirmed CD71 (IgA receptor) expression in CAFs, and IgA-treated CAFs exhibited higher programmed death-ligand 1 expression levels than those in mock-treated CAFs ( p <0.05). Coculture with CAFs attenuated the cytotoxic function of activated CD8 + T cells. Interestingly, activated CD8 + T cells cocultured with IgA-treated CAFs exhibited increased programmed death-1 expression levels than those cocultured with mock-treated CAFs ( p <0.05). Intrahepatic IgA induced polarization of HCC-CAFs into more malignant matrix phenotypes and attenuates cytotoxic T-cell function. Our study highlighted their potential roles in tumor progression and immune suppression.

Calycosin enhances Treg differentiation for alleviating skin inflammation in atopic dermatitis

Ethnopharmacological relevanceAtopic dermatitis (AD) is a prevalent chronic inflammatory skin disorder that poses a significant global health challenge. There is a lack of safe and effective medications to treat AD. Astragalus membranaceous is a traditional Chinese medicine widely used in clinical treatment of skin diseases. Calycosin (CA), derived from the root of Astragalus membranaceous, exhibits dual attributes of anti-inflammatory and antioxidant properties, suggesting its promise for addressing cutaneous inflammation. Nonetheless, the precise mechanisms underlying CA's therapeutic actions in AD remain elusive. Aim of the studyThis study aimed to evaluate the efficacy and safety of CA in treating AD while also delving into the mechanistic underpinnings of CA's action in AD. Materials and methodsThe cell viability and anti-inflammatory impacts of CA in vitro were first gauged using CCK-8 and RT-qPCR. The potential mechanisms of CA were then probed using modular pharmacology. Flow cytometry was employed to ascertain the differentiation of Treg and Th17 cells derived from naïve T cells, as well as the proportions and mean fluorescence intensity (MFI) of human iTreg cells. The expressions of IL-10 and TGF-β1 were measured and Treg suppression assay was performed. The in vivo therapeutic efficacy of topical CA application was assessed using a calcipotriol (MC903)-induced AD mouse model. The expression metrics of inflammatory cytokines, IL-17A, FOXP3, and RORγt were authenticated via immunohistochemistry, RT-qPCR, Western blot, and ELISA. ResultsCA exhibited a favorable safety profile and reduced the mRNA expressions of Th2 inflammatory cytokines in HaCaT cells. Modular pharmacology analysis pinpointed Th17 differentiation as the pivotal mechanism behind CA's therapeutic effect on AD. In vitro, CA fostered the differentiation of naïve T cells into Tregs while inhibiting their differentiation into Th17 cells. Furthermore, CA augmented the proliferation of human iTregs. In vivo, CA alleviated skin manifestations and decreased the levels of inflammatory mediators (IL-4, IL-5, IL-13, TSLP, and NF-κB related cytokines) in AD-like mouse models. Simultaneously, it regulated Treg/Th17 balance through suppressing IL-17A and RORγt expressions and bolstering FOXP3 expression. ConclusionsThe study provides insights into the mechanistic pathways through which CA exerts its anti-inflammatory effects, particularly through promoting Treg cell differentiation and inhibiting Th17 cell differentiation. Furthermore, CA emerges as an alternative or adjunctive treatment strategy for managing AD.

Establish a Graded Method to Avoid HLA Class I Antibodies Corresponding Antigen and Combining HLAMatchmaker Application in Improving the CCI Value after Platelet Transfusion for Patients with IPTR

To establish a graded method to avoid mean fluorescence intensity (MFI) threshold of HLA Class I antibodies corresponding antigen, and the HLAMatchmaker program has been used to select the minimum mismatch value of donor-patient epitopes. Evaluate the application value of combining both methods in selecting HLA compatible platelets (PTL) for patients with immune platelet transfusion failure (IPTR) in improving platelet the corrected count increment (CCI). A total 7 807 PLT cross-matching compatible were performed by the solid-phase red cell adherence (SPRCA) method for 51 IPTR patients. The Luminex single antigen flow cytometry was used to detect HLA Class I antibodies in patients, and detected the MFI value for different specificity antigens of HLA Class I antibodies, was graded into strong positive group (MFI>4 000, level 1), medium positive group (1 000< MFI≤4 000, 2), weak positive group (500< MFI≤1 000, 3), and one negative control group (MFI≤500). The results of 7 807 SPRCA their negative/positive reaction wells were enrolled and statistically analyzed in different grades and the four groups, the statistical differences between the four groups were compared. Multiple applications for the select HLA Class I compatible donor events were made for patients in two cases, and HLAMatchmaker program was used to calculate the number of HLA Class I epitopes mismatches between the donors and patients. The donor with the minimum number of epitopes mismatches was selected, while avoiding the corresponding antigens of HLA Class I antibodies in levels 1 and 2, the provision of HLA compatible platelets for IPTR. After the transfusions, the CCI value of the platelet transfusion efficacy evaluation index was calculated, and the clinical evaluation of the transfusion effect was obtained through statistical analysis. There were statistically significant differences in the positive results of SPRCA immunoassay among the strong positive group, medium positive group, and weak positive group of 51 IPTR patients with different specific of HLA -I class antibodies and corresponding antigens(all P <0.001). The positive results showed a range from high to low, with strong positive group>medium positive group>weak positive group. There were a statistical difference among between the strongly positive or moderately positive groups and the negative control group(P <0.001). There was no statistical difference between the weakly positive group and the negative control group(P >0.05). The strong positive group was set as the corresponding specific HLA Class I site corresponding antigen grade 1 avoidance threshold, the medium positive group as the grade 2 avoidance thresholds, and the weak positive group as the grade 3 avoidance threshold. In the case of donor platelet shortage, it is not necessary to avoid the weak positive group. Avoiding the strategy of donor antigens and HLAMatchmaker program scores ≤7 corresponding to HLA Class I antibodies of levels 1 and 2, with CCI values>4.5×109/L within 24 hours, can obtain effective clinical platelet transfusion conclusions. When selecting HLA Class I compatible donors for IPTR patients, the grading avoids HLA Class I antibodies corresponding to donor antigens, and the donor selection strategy with the minimum scores of HLAMatchmaker program is comprehensively selected. The negative result confirmed by platelet cross-matching experiments has certain practical application value for improving platelet count in IPTR patients.

Determination of lead(II) in food samples using a functionalized paper-based fluorescent sensor modified by carbon dots.

This work discusses surface modification of cellulose paper specimens for compatibility with nitrogen and sulfur co-doped carbon dots (NSCDs) for lead ion sensing. The interaction of carbon dots (CDs) and cellulose fibers was investigated using silane or chitosan-modified cellulose papers. It was found that modified papers could reduce undesirable redistribution of CDs, during paper drying. Also, only chitosan-modified filter paper was suitable for the successful immobilization of NSCDs. The effect of paper type, chitosan amount, pH, and NSCDs concentration was also studied, and a Whatman No. 42 filter paper modified with chitosan (1% w/v), pH8.0, and an NSCD concentration of 2.5g L-1 being selected for further studies. The sensor exhibited high selectivity for lead(II) compared with other metal ions because lead(II) resulted in the most significant changes in the emitted light intensity. Variations in NSCDs fluorescence were measured using a fluorescence imaging system. The NSCDs-paper sensor showed a linear relationship between mean fluorescence intensity and lead(II) in the concentration range of 5.00-1.25 × 102 μmol L-1 with a correlation coefficient (R2 ) of 0.9988 and a detection limit of 4.50 μmol L-1 . The suggested method showed satisfying results for lead(II) determination in different samples as a fast and low-cost approach with on-site application.

Identification of Optimal Tissue-Marking Dye Color for Pathological Evaluation in Fluorescence Imaging Using IRDye800CW.

Fluorescence-guided surgery using a tumor-specific antibody-dye conjugate is useful in various cancer types. Fluorescence imaging is a valuable tool both intraoperatively and postoperatively for ex vivo imaging. The color of inks used for tumor specimens during ex vivo specimen processing in pathology is an important consideration for fluorescence imaging since the absorption/emission of the dyes may interfere with the fluorescent dye. This study assesses suitable ink colors for use specifically with IRDye800CW fluorescence imaging. Eight tissue-marking inks or dyes (TMDs) commonly used for pathological evaluation were assessed. Agarose tissue-mimicking phantoms containing Panitumumab-IRDye800CW were used as an initial model. Mean fluorescence intensity was measured at 800nm using both Pearl Trilogy as a closed-field fluorescence imaging system and pde-neo II as an open-field fluorescence imaging system before and after TMD application. An in vivo mouse xenograft model using the human head and neck squamous cell carcinoma FaDu cell line was then used in conjunction with TMDs. The retained IRDye800CW fluorescence on Pearl Trilogy was as follows: yellow at 91.0 ± 4.5%, red at 90.6 ± 2.7%, orange at 88.2 ± 2.2%, violet at 56.6 ± 1.1%, lime at 40.9 ± 1.8%, green at 19.3 ± 2.8%, black at 13.3 ± 0.6%, and blue at 8.1 ± 0.2%. The retained IRDye800CW fluorescence on pde-neo II was as follows: yellow at 86.5 ± 6.4%, red at 77.0 ± 6.2%, orange at 76.9 ± 2.8%, lime at 72.5 ± 9.5%, violet at 59.7 ± 0.4%, green at 30.1 ± 6.9%, black at 17.0 ± 2.7%, and blue at 6.7 ± 1.7%. The retained IRDye800CW fluorescence in yellow and blue TMDs was 42.1 ± 14.9% and 0.2 ± 0.2%, respectively in the mouse experiment (p = 0.039). Yellow, red, and orange TMDs should be used, and blue and black TMDs should be avoided for evaluating tumor specimens through fluorescence imaging using IRDye800CW.