Cells In Solid Tissues Research Articles

Identification of neutrophils and eosinophils in upper airway mucosa with immunofluorescence multiplex image cytometry.

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4',6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2mm2 and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry.

Retrieval of vector integration sites from cell-free DNA.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines.

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs.

(Invited) Multifunctional Surface-Enhanced Raman Spectroscopy (SERS) Probes for Targeted Detection of Cancer Cells

Cancer continues to raise global concerns with a significant mortality rate and an equally substantial socio-economic burden. As is well-known, cancer begins with out-of-control cell growth. To effectively screen and treat cancer, it is essential to identify and distinguish various subtypes of cancer cells as well as normal cells in solid tissues or body fluids, which will significantly improve personalized individual medical treatment. This talk will present our recent studies for identification and discrimination of subtypes of cancerous cells using surface-enhanced Raman spectroscopy (SERS). The philosophy in our work is to actively target the overexpressed biomarkers on the surface of cancer cells with our functionalized SERS probes. The urea-based small-molecule inhibitor is grafted onto the SERS probe to endow with specificity for prostate-specific membrane antigen (PSMA) overexpressed on the surface of bone metastasis-derived, androgen independent isogenic PC3-PIP cells. Our finding shows high-resolution targeted SERS imaging of PC3-PIP cells in contrast to its phenotypically identical PC3-Flu cells with negatively-expressed PSMA. The significantly amplified SERS signal in the SERS probe endows the extraordinary sensitivity. The developed SERS routine is able to identify PC3-PIP cells in body fluids and solid tissues, providing an effective avenue for assessment of prostate cancer burden and image-guided surgery/treatment. Following this, a ratiometric SERS strategy is developed to discriminate breast cancer cells—MDA-MB-231 and MCF-7 cells. Two different SERS probes with distinct SERS signals are functionalized with two peptides that are able to specifically target the urokinase plasminogen activation receptor (uPAR) and epidermal growth factor receptor (EGFR), respectively. Our results show that we can distinguish MDA-MB-231 cells from MCF-7 cells by the SERS intensity ratio. The present finding offers a compelling approach to discriminate subtypes of cancerous cells to assess the progression and stage of cancer. More importantly, the present SERS technique has potential for the rapid identification and discrimination of subtypes of multiple cancerous cells owing to the powerful multiplexing capability in SERS.

Defining cell-matrix combination products in the era of pluripotency

Human pluripotent stem (hPS) cells provide an attractive opportunity for the manufacture of a wide array of therapeutic cell types. The challenges to commercialization include the thousand-fold diversity of cell types emerging from hPS cells and the associated difficulties in validating processes to reliably generate cells with precise identity and purity. Improved methods of controlling the dosage and migration of hPS-derived cells in solid tissues are also needed. To directly address these issues, we clonally expanded proliferating lineages of cells that were intermediate in regard to their state of differentiation between hPS and terminally differentiated cells. These cells called monoclonal embryonic progenitors (hEP), are expandable mortal lineages with diverse site-specific homeobox gene expression and multipotentiality. In this review, we discuss methods of generating combination products wherein the fate space of precisely identified monoclonal hEP cells is mapped by differentiating the cells in vitro in HyStem-3D bead arrays in the presence of diverse growth factors. This combination of discovery processes has the potential to translate directly into cell-matrix formulations that can be used to generate pre-clinical data leading to human clinical trials and potentially new medical therapies.

B-cell subpopulations in humans and their differential susceptibility to depletion with anti-CD20 monoclonal antibodies

In humans, different B-cell subpopulations can be distinguished in peripheral blood and other tissues on the basis of differential expression of various surface markers. These different subsets correspond to different stages of maturation, activation and differentiation. B-cell depletion therapy based on rituximab, an anti-CD20 mAb, is widely used in the treatment of various malignant and autoimmune diseases. Rituximab induces a very significant depletion of B-cell subpopulations in the peripheral blood usually for a period of 6 to 9 months after one cycle of therapy. Cells detected circulating during depletion are mainly CD20 negative plasmablasts. Data on depletion of CD20-expressing B cells in solid tissues are limited but show that depletion is significant but not complete, with bone marrow and spleen being more easily depleted than lymph nodes. Factors influencing depletion are thought to include not only the total drug dose administered and distribution into various tissues, but also B-cell intrinsic and microenvironment factors influencing recruitment of effector mechanisms and antigen and effector modulation. Available studies show that the degree of depletion varies between individuals, even if treated with the same dose, but that it tends to be consistent in the same individual. This suggests that individual factors are important in determining the final extent of depletion.

Different Ca2+-sensitivities between the EF-hands of T- and L-plastins

Plastins are Ca(2+)-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells. Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca(2+), but the sensitivity to Ca(2+) is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons.

MMP-2 genetic variant and plaque features of instability

More than 80 different proteins, glycoproteins, collagens, proteoglycans and glycosaminoglycans, constitute the extra cellular matrix (ECM), the complex structure surrounding cells in solid tissues and organs. Tissue remodeling, i.e. the changes in amount, type and architecture of these proteins is critical in important physiological events like growth, development and reproduction as in disease. ECM protein degradation is largely (but not exclusively) dependent on a class of neutral endoproteases called MMPs.

Gap Junctions

Gap junctions are aggregates of intercellular channels that permit direct cell-cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology.

Extracellular Space Volume Measured by Two-Color Pulsed Dye Infusion with Microfiberoptic Fluorescence Photodetection

The extracellular space (ECS) is the aqueous matrix surrounding cells in solid tissues. The only method to measure ECS volume fraction ( α) in vivo has been tetramethylammonium iontophoresis, a technically challenging method developed more than 25 years ago. We report a simple, quantitative method to measure α by microfiberoptic fluorescence detection of a self-quenched green dye, calcein, and a reference red dye, sulforhodamine 101, after pulsed iontophoretic infusion. The idea is that the maximum increase in calcein fluorescence after iontophoresis is proportional to the aqueous volume into which the dye is deposited. We validated the method theoretically, and experimentally, using cell-embedded gels with specified α and ECS viscosity. Measurements in living mice gave α of 0.20 ± 0.01 in brain, 0.13 ± 0.02 in kidney and 0.074 ± 0.01 in skeletal muscle. The technical simplicity of the ”pulsed-infusion microfiberoptic photodetection” method developed here should allow elucidation of the relatively understudied biological roles of the ECS.

How deep cells feel: Mean-field Computations and Experiments

Most cells in solid tissues exert contractile forces that mechanically couple them to elastic surroundings and that significantly influence cell adhesion, cytoskeletal organization, and even cell differentiation. However, strains within the depths of matrices are often unclear and are likely relevant not only to the fact that some matrices such as so-called basement membranes are thin relative to cell dimensions but also to defining how far cells can ‘feel’. Here we present experimental results for cell spreading on thin, ligand-coated gels and for prestress in stem cells in relation to gel stiffness. Spread area on thin and soft gels was found to resemble cells on thick and stiff gels. Matrix thickness also affects focal adhesions and cytoskeleton organization in stem cells, which we will compare to differentiated cells. We introduce a finite element computation in which a cell is placed on an elastic matrix, while matrix elasticity and thickness are varied in order to compute and compare elastostatic deformations within the matrix. Average interfacial strains between cell and matrix show large deviations only when soft matrices are a fraction of the height and width of a cell, proving consistent with experiments. Three-dimensional (3D) cell morphologies that model stem cell-derived neurons, myoblasts, and osteoblasts show that a cylinder-shaped myoblast induces the highest strains, consistent with the prominent contractility of muscle. Groups of such cells show a weak crosstalk in matrix strains, but the cells must be much closer than a cell-width - experimental tests of this are emerging. Cells thus feel on length scales closer to that of adhesions than on cellular scales or larger.

Utility of CD26 in flow cytometric immunophenotyping of T‐cell lymphomas in tissue and body fluid specimens

CD26 is expressed by most CD4+ T cells in normal peripheral blood specimens. Neoplastic T cells are frequently CD26- in mycosis fungoides/Sezary syndrome involving the peripheral blood. However, CD26 expression by reactive and neoplastic T cells in solid tissues and body fluids has not been fully characterized by flow cytometry (FC). Solid tissue and body fluid specimens were assayed for CD26 expression using four-color FC immunophenotyping, by qualitative assessment of population clusters, and by quantitation with comparison with isotype controls. Benign T cells were studied in reactive tissues and in the background of other malignancies. Many T-cell lymphomas were dim or negative for CD26, whereas a few were brightly positive. In the majority of T-cell lymphomas, CD26 expression could potentially help identify aberrant population clusters. T cells in reactive tissue specimens and tumor-infiltrating T cells were commonly dim to negative for CD26. Both T-cell lymphomas and reactive T cells in tissue and body fluid specimens often show low levels of CD26 expression. Therefore, quantitative methods may not reliably distinguish benign from neoplastic T cells in these specimens. However, CD26, in combination with other T-cell markers, can be helpful for identifying aberrant population clusters in T-cell lymphomas.

Age‐related accumulation of autosomal mutations in solid tissues of the mouse is gender and cell type specific

Most cancers in solid tissues increase with age and invariably contain causal mutations eliminating expression of one or more autosomal tumor suppressor genes. However, very little is known about the effect of age on autosomal mutations, often large in size, in cells of solid tissues. In this study, the frequency and spectrum of autosomal mutations were examined as a function of age for kidney epithelial cells and ear mesenchymal cells in B6D2F1 mice heterozygous for the selectable Aprt locus. Aprt mutant frequencies were found to increase with age in the kidneys of both male and female mice, but at all ages the mutant frequencies were approximately twice as high in the females, which in this strain have shorter lifespans than the males. An age-related increase in Aprt mutant frequencies was also observed for ear cells from female mice, but no significant increases in mutant frequencies were observed for the ear cells of male mice. A molecular analysis showed that the kidney and ear mutational spectra were distinct and that the age-related increases in mutant frequencies did not involve significant shifts in the mutational spectra. In total, the results demonstrate both gender and cell-type-specific patterns of autosomal mutational accumulation as a function of age in two solid tissues of the mouse.

AtmHaploinsufficiency does not Affect Ionizing Radiation Mutagenesis in Solid Mouse Tissues

Ataxia telangiectasia (AT) is a hereditary disease with autosomal recessive inheritance of ATM (ataxia telangiectasia mutation) alleles. AT is associated with severe sensitivity to ionizing radiation and a strong predisposition to develop cancer. A modest increase in cancer, particularly for the breast, has been shown for ATM carriers (i.e. heterozygotes), and a modest increase in radiation sensitivity has also been shown for those patients and their cells. However, the extent of these effects is unclear. Based on the well-established relationship between cancer and mutation, we used a mouse model for Atm haploinsufficiency to ask whether partial loss of Atm function could lead to an increased mutagenic response for solid tissues of mice exposed to radiation. The autosomal mouse Aprt gene was used as the mutational target and kidney and ear as the target tissues in B6D2F1 hybrids. Although induction of autosomal mutations was readily demonstrated in both tissues, a comparison of these data with those from an identical study performed with B6D2F1 mice that were wild-type for Atm (Cancer Res. 62, 1518-1523, 2002) revealed that Atm haploinsufficiency did not alter the radiation mutagenic response for the cells of either tissue. Moreover, no effect of Atm haploinsufficiency on reduced cellular viability due to radiation exposure was observed. The results demonstrate that Atm haploinsufficiency does not alter the radiation mutagenic response or decrease viability for normally quiescent cells in solid tissues of the mouse.

Generation and Characterization of Monoclonal Antibodies That Specifically Recognize p65/L-Plastin Isoform but Not T-Plastin Isoform

The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca(2+)-, calmodulin-, and beta-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.

A simple method for unbiased quantitation of adoptively transferred cells in solid tissues

In a mouse model, we demonstrate how to obtain a direct, unbiased estimate of the total number of adoptively transferred cells in a variety of organs at different time points. The estimate is obtained by a straightforward method based on the optical fractionator principle. Specifically, non-stimulated C57BL/6J mouse splenocytes were labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and adoptively transferred to normal C57BL/6J mice by intravenous injection. The total number of CFSE-positive cells was subsequently determined in lung, spleen, liver, kidney, and inguinal lymph node at six different time points following adoptive transfer (from 60 s to 1 week), providing a quantitative estimate of the organ distribution of the transferred cells over time. These estimates were obtained by microscopy of uniform samples of thick sections from the respective organs. Importantly, the samples were chosen and prepared in accordance with the optical fractionator principle. We demonstrate that the method is simple, precise, and well suited for quantitative immunological studies.

Production of Monoclonal Antibodies Against ADAMTS13 Determining Its Expression in Different Human Tissues.

von Willebrand factor-cleaving protease/ADAMTS13 is a plasma metalloproteinase that degrades unusually large von Willebrand factor multimers (UL-vWF) derived from endothelial cells (ECs) and megakaryocytes (MCs) into small peptides circulating in blood. It is documented that transcript mRNAs of the protease are present in many human tissues; however, the protein expression of ADAMTS13 remains to be elucidated. In the present work, the gene of metalloproteinase domain of human ADAMTS13 was cloned into the multiclone site of pET28a(+). After induced by IPTG, the recombinant protein was purified using a Ni-NTA column and the Bal b/c mice were immunized with the protein. Screened with ELISA, three monoclonal antibodies against the metalloproteinase domain of ADAMTS13 were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belonged to IgG1 subclass. The quantity of them in ascites were 4 mg/ml, and their titers were as high as 1×10−5. The data of competitive ELISA showed that SZ-112 and SZ-113 recognized different epitopes of the recombinant protein. Western blot results demonstrated that SZ-112 not only reacted against the recombinant protein, but also recognized the full-length recombinant ADAMTS13 protein that expressed in CHO cell line (the vectors containing the ADAMTS13 cDNA sequences were provided by Prof. Sadler JE). The immunoprecipitation results showed that the two antibodies could react to an approximately 200 KDa protein in platelet lysate. Then, the expression panels of ADAMTS13 in human normal tissues were investigated using immunohistochemistry with the monoclonal antibodies. And the protease was found to be present in many kinds of tissues such as liver, spleen, ovary, prostate, bladder, small intestine, thyroid and thymus with significantly positive staining. The protease was also present in lung, uterus, large intestine and heart but stained weakly. We did not found the protease in brain. In most of these organs, the protease was expressed in epithelium of the tissues. While in liver, spleen and thymus, it was mainly presented in a subgroup of the solid tissue cells. Moreover, the preliminary results showed that the expression of ADAMTS13 slightly decreased in liver tissues of patients suffering form hepatitis type B and cirrhosis. In conclusion, our data indicated that two novel monoclonal antibodies against the metalloproteinase domain of human ADAMTS13 were successfully prepared, and the expression of ADAMTS13 in different tissue and specific locality might be associated with the regulation and function of the protease, which would contribute to the further research of the deficiency mechanism of the proteases in some disorders.

Persistence of chromatid aberrations in the cells of solid mouse tissues exposed to 137Cs gamma radiation.

Primary mouse ear and kidney cultures were established for determination of cytogenetic aberrations at short (3 days to 1 month) and long (12-23 months) times after exposure of their right sides to 7.5 Gy of (137)Cs gamma radiation. In every case, higher levels of aberrations were observed in primary cultures established from the irradiated tissues than in those established from the contralateral tissues. The most common aberrations in the contralateral tissues and those from nonirradiated mice were chromatid and isochromatid breaks and small chromatid fragments. Primary cells from irradiated tissues removed from animals within a month of exposure displayed a variety of unstable chromosome-type aberrations characteristic of recent exposure to ionizing radiation including rings, dicentrics, double minutes, and large acentric fragments. The percentages of cells exhibiting chromatid breaks and small chromatid fragments were also markedly elevated. Although the levels of chromosome-type aberrations found in primary cells from irradiated tissues dropped to near background levels a year or more after exposure, chromatid-type aberrations remained elevated. These results are consistent with long-term persistence of damage in the genomes of ionizing radiation-exposed cells in solid tissues and the induction of genomic instability in vivo.

Identifying and tracking neural stem cells

Hematopoietic stem cells, unlike neural stem cells, can be readily identified and isolated from developing and adult cell populations using positive and negative selection criteria. Isolating stem cells and progenitor cells from neural tissue has been more difficult because of difficulties in separating cells in solid tissue, the limited numbers of stem cells that persist in the adult, and the paucity of rigorously characterized markers. Nevertheless, strategies that have worked successfully in hematopoietic stem cell isolation can be adapted to isolate multiple classes of stem and progenitor cells from neural tissue. Neural stem cells also share cellular and molecular properties with other stem cell populations that may serve as surrogate identifiers of multipotentiality. Such potential markers are described. Unlike hematopoietic stem cells, tracking neural cells after transplantation is both necessary and more difficult. It will therefore be necessary to develop invasive and non-invasive strategies to follow transplanted cells and develop useful quantifiable readouts. Some potential strategies are described and current results are discussed.

Cannabinoids inhibit gap junctional intercellular communication and activate ERK in a rat liver epithelial cell line.

Many tumor promoters suppress the immune system; however, the direct effect of immunosuppressants on the tumorigenic pathways of nonimmune cells in solid tissue has not been well documented. Cannabinoids were chosen to explore this question further. Cannabinoids are immune modulators that affect specific intracellular signaling pathways in leukocytes. Since these compounds are nongenotoxic, any tumorigenic effect that might be associated with these compounds would need to occur through an epigenetic mechanism. Therefore, we determined the effect of Delta(9)-THC and CBN, 2 plant-derived cannabinoids, on 2 key epigenetic markers of tumor promotion: inhibition of GJIC, which is essential in removing a cell from growth suppression, and activation of the ERK-MAPK pathway, which is crucial in activating the appropriate genes for mitogenesis. Both Delta(9)-THC and CBN reversibly inhibited GJIC at noncytotoxic doses (15 microM) in a normal diploid WB rat liver epithelial oval cell line within 20 min and activated ERK1 and ERK2 within 5 min. Inhibition of MEK with PD98059 prevented the inhibition of GJIC by either cannabinoid, suggesting that inhibition of GJIC was MEK-dependent. Based on RT-PCR analysis and employment of an antagonist of CB1 and CB2, the effects on GJIC and MAPK were independent of both cannabinoid receptors. Cannabinoids affected crucial epigenetic pathways associated with cell proliferation in a rodent liver epithelial cell model system.