A Method for Identification and Analysis of Non-Overlapping Myeloid Immunophenotypes in Humans

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN^-CD33⁺HLA-DR^- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies.     

An Age-Wise Comparison of Human Airway Smooth Muscle Proliferative Capacity

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca²⁺]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.     

Data-Driven Method to Estimate Nonlinear Chemical Equivalence

There is great need to express the impacts of chemicals found in the environment in terms of effects from alternative chemicals of interest. Methods currently employed in fields such as life-cycle assessment, risk assessment, mixtures toxicology, and pharmacology rely mostly on heuristic arguments to justify the use of linear relationships in the construction of “equivalency factors,” which aim to model these concentration-concentration correlations. However, the use of linear models, even at low concentrations, oversimplifies the nonlinear nature of the concentration-response curve, therefore introducing error into calculations involving these factors. We address this problem by reporting a method to determine a concentration-concentration relationship between two chemicals based on the full extent of experimentally derived concentration-response curves. Although this method can be easily generalized, we develop and illustrate it from the perspective of toxicology, in which we provide equations relating the sigmoid and non-monotone, or “biphasic,” responses typical of the field. The resulting concentration-concentration relationships are manifestly nonlinear for nearly any chemical level, even at the very low concentrations common to environmental measurements. We demonstrate the method using real-world examples of toxicological data which may exhibit sigmoid and biphasic mortality curves. Finally, we use our models to calculate equivalency factors, and show that traditional results are recovered only when the concentration-response curves are “parallel,” which has been noted before, but we make formal here by providing mathematical conditions on the validity of this approach.     

An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Fungal Bioweathering of Mimetite and a General Geomycological Model for Lead Apatite Mineral Biotransformations

Fungi play important roles in biogeochemical processes such as organic matter decomposition, bioweathering of minerals and rocks, and metal transformations and therefore influence elemental cycles for essential and potentially toxic elements, e.g., P, S, Pb, and As. Arsenic is a potentially toxic metalloid for most organisms and naturally occurs in trace quantities in soil, rocks, water, air, and living organisms. Among more than 300 arsenic minerals occurring in nature, mimetite [Pb₅(AsO₄)₃Cl] is the most stable lead arsenate and holds considerable promise in metal stabilization for in situ and ex situ sequestration and remediation through precipitation, as do other insoluble lead apatites, such as pyromorphite [Pb₅(PO₄)₃Cl] and vanadinite [Pb₅(VO₄)₃Cl]. Despite the insolubility of mimetite, the organic acid-producing soil fungus Aspergillus niger was able to solubilize mimetite with simultaneous precipitation of lead oxalate as a new mycogenic biomineral. Since fungal biotransformation of both pyromorphite and vanadinite has been previously documented, a new biogeochemical model for the biogenic transformation of lead apatites (mimetite, pyromorphite, and vanadinite) by fungi is hypothesized in this study by application of geochemical modeling together with experimental data. The models closely agreed with experimental data and provided accurate simulation of As and Pb complexation and biomineral formation dependent on, e.g., pH, cation-anion composition, and concentration. A general pattern for fungal biotransformation of lead apatite minerals is proposed, proving new understanding of ecological implications of the biogeochemical cycling of component elements as well as industrial applications in metal stabilization, bioremediation, and biorecovery.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.