Assessment of Adenosine Triphosphatase Phospholipid Transporting 8B1 (ATP8B1) Function in Patients With Cholestasis With ATP8B1 Deficiency by Using Peripheral Blood Monocyte-Derived Macrophages.

Adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) deficiency, an ultrarare autosomal recessive liver disease, includes severe and mild clinical forms, referred to as progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1), respectively. There is currently no practical method for determining PFIC1 or BRIC1 at an early disease course phase. Herein, we assessed the feasibility of developing a diagnostic method for PFIC1 and BRIC1. A nationwide Japanese survey conducted since 2015 identified 25 patients with cholestasis with ATP8B1 mutations, 15 of whom agreed to participate in the study. Patients were divided for analysis into PFIC1 (n = 10) or BRIC1 (n = 5) based on their disease course. An in vitro mutagenesis assay to evaluate pathogenicity of ATP8B1 mutations suggested that residual ATP8B1 function in the patients could be used to identify clinical course. To assess their ATP8B1 function more simply, human peripheral blood monocyte-derived macrophages (HMDMs) were prepared from each patient and elicited into a subset of alternatively activated macrophages (M2c) by interleukin-10 (IL-10). This was based on our previous finding that ATP8B1 contributes to polarization of HMDMs into M2c. Flow cytometric analysis showed that expression of M2c-related surface markers cluster of differentiation (CD)14 and CD163 were 2.3-fold and 2.1-fold lower (95% confidence interval, 2.0-2.5 for CD14 and 1.7-2.4 for CD163), respectively, in patients with IL-10-treated HMDMs from PFIC1 compared with BRIC1. Conclusion: CD14 and CD163 expression levels in IL-10-treated HMDMs may facilitate diagnosis of PFIC1 or BRIC1 in patients with ATP8B1 deficiency.

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor α) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Efficiency and Impact of Positive and Negative Magnetic Separation on Monocyte Derived Dendritic Cell Generation.

BACKGROUND: The immunomagnetic separation technique is the basis of monocyte isolation and further generation of monocyte-derived dendritic cells. OBJECTIVE: To compare the efficiency of monocyte positive and negative separation, concentration of beads, and their impact on generated dendritic cells. METHODS: Monocytes were obtained using monoclonal antibody-coated magnetic beads followed the Ficoll-Paque gradient separation of mononuclear cell fraction from the peripheral blood of 6 healthy volunteers. CD14 expression was analyzed by flow cytometry. CONCLUSIONS: Both types of magnetic separation including recommended and reduced concentrations of beads did not affect the yield and the purity of monocytes and their surface CD14 expression. However, DCs originated from the "positively" separated monocytes had noticeable higher expression of CD80.

Immunohistochemical detection of CD14 and combined assessment with CD32B and CD68 for wound age estimation.

Estimation of wound age is a major topic of study for forensic pathologists, but few markers exist that can indicate a specific period 1-5 days postinfliction, and a method to estimate wound age with high accuracy has not yet been established. This study examined CD14 as such a marker in mouse skin wounds of different ages (0min and 1, 2, 3, 5, 7, and 9 days) and in human subjects (group 1, 0-1 day; group 2, 1-5 days; group 3, >7 days) using Western blot analysis and/or immunohistochemical staining. In addition, we evaluated a combination of immunohistochemical markers in human skin wounds using transmembrane proteins, CD14, CD32B, and CD68, expressed on inflammatory cells. The expression of CD14 was detected only during 1-5 days postinfliction and, thus, the evaluation of CD14-expressing cells could specify wound age during 1-5 days postinfliction in mouse skin wounds. The ratio of samples assessed to be CD14(+) was significantly high in human skin wounds in group 2. Combined assessment using the three markers increased the specificity of diagnosis and shortened the range of wound age, compared with the assessment using a single marker. Our results indicate that CD14 may be a useful marker of wound age, 1-5 days postinfliction, and that combined assessment with CD14, CD32B, and CD68 may be a good method for the accurate estimation of wound age.

IL22 regulates human urothelial cell sensory and innate functions through modulation of the acetylcholine response, immunoregulatory cytokines and antimicrobial peptides: assessment of an in vitro model.

Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.

Assessment of the number and function of macrophages in the placenta of gestational diabetes mellitus patients.

In order to assess the number and function of macrophages in the placenta of pregnancy complicated with gestational diabetes mellitus (GDM) as well as those of normal pregnancies, placenta samples were collected from 15 GDM patients (GDM group) and 10 normal pregnant women (control group). The expression levels of macrophage markers (CD68/CD14) and inflammatory cytokines (IL-6/TNF-α) in placenta were detected using immunohistochemistry and PCR. The results showed that the number of CD68+ or CD14+ cells in the GMD group was remarkably higher than that in the control group (P<0.05), indicating that the number of macrophages in the GDM group was significantly greater than that in the control group. The mRNA expression levels of CD68+, IL-6 and TNF-α were higher in the GMD group than in the control group. In conclusion, more macrophages accumulate in placenta of pregnancy complicated with GDM, and the expression levels of pro-inflammation factors are also increased in GDM pregnancies, suggesting that macrophages and inflammatory mediators (IL-6 and TNF-α) may play an important role in GDM.

A quantitative comparison of single-dye tracking analysis tools using Monte Carlo simulations.

Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-ß-cyclodextrin and latrunculin B.

Quantitative assessment of cell viability based on flow cytometry and microscopy.

We compare flow cytometric and microscopic determination of cell viability by fluorescence labeling using calcein acetoxy-methyl-ester and ethidium homodimer-1 as live and dead stain, respectively. Peripheral blood monocytes served as model system and were accumulated applying density gradients. Subsequently, monocytes were further enriched by magnetic-activated or fluorescence-activated cell sorting (MACS, FACS) targeting the antigen CD14. Identical samples were used for flow cytometric and microscopic analysis to allow direct comparison of both analysis methods. More than 1,000 cells were measured for each sample to minimize the measurement uncertainty caused by counting statistics. We observed good agreement of flow cytometric and microscopic viability measurements. On average, the difference in viability measured by flow cytometry and microscopy amounted to (2.7 ± 1.4)% for live staining and (1.7 ± 1.2)% for dead staining. These deviations were similar to the uncertainty of measurement for cell viability, thus demonstrating that both methods delivered equal results. Besides monocytes, comparison of flow cytometric and microscopy viability for MACS enriched CD34-positive cells also showed consistent results.

Diagnosing symptomatic HIV-associated neurocognitive disorders: self-report versus performance-based assessment of everyday functioning.

Three types of HIV-associated neurocognitive disorders (HAND) exist that are distinguished by presence and severity of impairment in cognitive and everyday functioning. Although well-validated neurocognitive measures exist, determining impairment in everyday functioning remains a challenge. We aim to determine whether Self-Report measures of everyday functioning are as effective in characterizing HAND as Performance-Based measures. We assessed 674 HIV-infected participants with a comprehensive neurocognitive battery; 233 met criteria for a HAND diagnosis by having at least mild neurocognitive impairment. Functional decline was measured via Self-Report and Performance-Based measures. HAND diagnoses were determined according to published criteria using three approaches to assess functional decline: (1) Self-Report measures only, (2) Performance-Based measures only, and (3) Dual-method combining Self-Report and Performance-Based measures. The Dual-method classified the most symptomatic HAND, compared to either singular method. Singular method classifications were 76% concordant with each other. Participants classified as Performance-Based functionally impaired were more likely to be unemployed and more immunosuppressed, whereas those classified as Self-Report functionally impaired had more depressive symptoms. Multimodal methods of assessing everyday functioning facilitate detection of symptomatic HAND. Singular Performance-Based classifications were associated with objective functional and disease-related factors; reliance on Self-Report classifications may be biased by depressive symptoms.

Oxidative burst assessment and neutrophil-platelet complexes in unlysed whole blood.

BACKGROUND: Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation. METHODS: To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose-response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated. RESULTS: With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not. CONCLUSIONS: This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation.

Mite allergen induces nitric oxide production in alveolar macrophage cell lines via CD14/toll-like receptor 4, and is inhibited by surfactant protein D.

BACKGROUND: Previously, we have found that dust mite allergens can directly activate alveolar macrophages (AMs), induce inflammatory cytokines, and enhance T-helper type 2 cytokine production. A molecule of innate immunity in the lung, surfactant protein D (SP-D), is able to bind mite allergens and alleviates allergen-induced airway inflammation. OBJECTIVES: This study was aimed at investigating the activation pathway of mite allergen (Dermatophagoides pteronyassinus, Der p)-induced nitric oxide (NO) production by AMs, and the role of SP-D in the modulation of activated AMs by mite allergens. METHODS: Porcine SP-D was purified from bronchoalveolar lavage fluids of Lan-Yu mini-pigs, by affinity chromatography on maltose-sepharose. NO production, inducible expression of lipopolysaccharides (LPS)-related binding and responding surface receptors complex, CD14 and toll-like receptor 4 (TLR4), as well as inducible NO synthase (iNOs) and nuclear factor-kappaB activation were studied in two AMs cell lines, MH-S (BALB/c strain),and AMJ2-C11 (C57BL/6 strain), and one peritoneal macrophage cell line (RAW264.7), after stimulation with LPS, or Der p. RESULTS: LPS and Der p elicited different responses of NO production in the different cell lines, and the response might depend upon the expression of the cell surface CD14/TLR4 complex in different genetic backgrounds of macrophage cell lines. Pretreatment of macrophages with SP-D could inhibit NO production from Der p or LPS-stimulated alveolar macrophages. CONCLUSION: Mite allergen-induced alveolar macrophage activation is mediated by CD14/TLR4 receptors and can be inhibited by SP-D; it further supports the concept that SP-D may be an important modulator of allergen-induced pulmonary inflammation.