Relationships among cell morphology,intrinsic cell stiffness and cell–substrate interactions

Cell modulus (stiffness) is a critical cell property that is important in normal cell functions and increasingly associated with disease states, yet most methods to characterize modulus may skew results. Here we show strong evidence indicating that the fundamental nature of free energies associated with cell/substrate interactions regulates adherent cell morphology and can be used to deduce cell modulus. These results are based on a mathematical model of biophysics and confirmed by the measured morphology of normal and cancerous liver cells adhered on a substrate. Cells select their final morphology by minimizing the total free energy in the cell/substrate system. The key mechanism by which substrate stiffness influences cell morphology is the energy tradeoff between the stabilizing influence of the cell-substrate interfacial adhesive energy and the destabilizing influence of the total elastic energies in the system. Using these findings, we establish a noninvasive methodology to determine the intrinsic modulus of cells by observing global changes in cell morphology in response to substrate stiffness. We also highlight the importance of selecting a relevant morphological index, cell roundness, that reflects the interchange between forms of energy governing cell morphology. Thus, cell-substrate interactions can be rationalized by the underlying biophysics, and cell modulus is easily measured.     

Impaired T cell receptor signaling and development of T cell–mediated autoimmune arthritis

Mutations of the genes encoding T-cell receptor (TCR)-proximal signaling molecules, such as ZAP-70, can be causative of immunological diseases ranging from T-cell immunodeficiency to T-cell–mediated autoimmune disease. For example, SKG mice, which carry a hypomorphic point mutation of the Zap-70 gene, spontaneously develop T-cell–mediated autoimmune arthritis immunopathologically similar to human rheumatoid arthritis (RA). The Zap-70 mutation alters the sensitivity of developing T cells to thymic positive/negative selection by self-peptides/MHC complexes, shifting self-reactive TCR repertoire to include a dominant arthritogenic specificity and also affecting thymic development and function of autoimmune suppressive regulatory T (Treg) cells. Polyclonal self-reactive T cells, including potentially arthritogenic T cells, thus produced by the thymus recognize self-peptide/MHC complexes on antigen-presenting cells (APCs) in the periphery and stimulate them to produce cytokines including IL-6 to drive the arthritogenic T cells to differentiate into arthritogenic T-helper 17 (Th17) cells. Insufficient Treg suppression or activation of APCs via microbial and other environmental stimuli evokes arthritis by activating granulocyte-macrophage colony-stimulating factor-secreting effector Th17 cells, mediating chronic bone-destructive joint inflammation by activating myeloid cells, innate lymphoid cells, and synoviocytes in the joint. These findings obtained from the study of SKG mouse arthritis are instrumental in understanding how arthritogenic T cells are produced, become activated, and differentiate into effector T cells mediating arthritis, and may help devising therapeutic measures targeting autoimmune pathogenic Th17 cells or autoimmune-suppressing Treg cells to treat and prevent RA.     

Dendritic cell–epithelial cell crosstalk in the gut

Intestinal epithelial cells are fundamental to maintain barrier integrity and to participate in food degradation and absorption, but they can also decipher signals coming from the outside world and ‘educate’ the immune system accordingly. In particular, they interact with dendritic cells (DCs) and other intraepithelial immune cells to drive tolerogenic responses under steady state, but they can also release immune mediators to recruit inflammatory cells and to elicit immunity to infectious agents. When these interactions are deregulated, immune disorders can develop. In this review, we discuss some important features of epithelial cells and DCs and their fruitful interactions.     

The enhancement of dermal papilla cell aggregation by extracellular matrix proteins through effects on cell–substratum adhesivity and cell motility

Generally, cells tend to aggregate on a substratum with lower cell adhesivity. However, it also leads to compromised cell growth and higher cell loss after seeding. This study is aimed at tackling this dilemma by extracellular matrix (ECM) protein coating of a lower adhesive substratum poly(ethylene-co-vinyl alcohol) (EVAL) that has been shown to facilitate hair follicle dermal papilla (DP) spheroid formation. We found that coating with either fibronectin (Fn), collagen I, or collagen IV yields higher adhesivity and cell growth than that with laminin. However, cells can only aggregate on uncoated or Fn-coated EVAL. Quantitatively, Fn coating increases the number of spheroids by 67%. Analysis of cell migration reveals that collagen I, collagen IV and laminin coatings reduce cell motility, while Fn coating keeps cells highly motile. Inhibition of cell migration hinders spheroid formation. In addition, disruption of Fn function does not significantly compromise intercellular adhesion. Hence, Fn enhances cell aggregation by enhancing cell attachment, cell growth and cell motility. Our study demonstrates that intercellular organization as spheroids or flat monolayers is switchable by specific ECM protein coating and preserving cell motility is vital to cell aggregation. In addition to generation of spheroidal DP microtissues for hair follicle regeneration and large-scale production of aggregates of other cells, this strategy can help to regulate the tissue–substrate adhesivity and tissue spreadibility on the surface of implantable materials.     

Electrospun collagen–chitosan nanofiber: A biomimetic extracellular matrix for endothelial cell and smooth muscle cell

Electrospinning of collagen and chitosan blend solutions in a 1,1,1,3,3,3-hexafluoroisopropanol/trifluoroacetic acid (v/v, 90/10) mixture was investigated for the fabrication of a biocompatible and biomimetic nanostructure scaffold in tissue engineering. The morphology of the electrospun collagen–chitosan nanofibers was observed by scanning electron microscopy (SEM) and stabilized by glutaraldehyde (GTA) vapor via crosslinking. Fourier transform infrared spectra analysis showed that the collagen–chitosan nanofibers do not change significantly, except for enhanced stability after crosslinking by GTA vapor. X-ray diffraction analysis implied that both collagen and chitosan molecular chains could not be crystallized in the course of electrospinning and crosslinking, and gave an amorphous structure in the nanofibers. The thermal behavior and mechanical properties of electrospun collagen–chitosan fibers were also studied by differential scanning calorimetry and tensile testing, respectively. To assay the biocompatibility of electrospun fibers, cellular behavior on the nanofibrous scaffolds was also investigated by SEM and methylthiazol tetrazolium testing. The results show that both endothelial cells and smooth muscle cells proliferate well on or within the nanofiber. The results indicate that a collagen–chitosan nanofiber matrix may be a better candidate for tissue engineering in biomedical applications such as scaffolds.     

Micropatterning–retinoic acid co-control of neuronal cell morphology and neurite outgrowth

Creating physical–biochemical superposed microenvironments optimal for stimulating neurite outgrowth would be beneficial for neuronal regenerative medicine. We investigated potential co-regulatory effects of cell micropatterning and retinoic acid (RA) soluble factor on neuronal cell morphology and neurite outgrowth. Human neuroblastoma (SH-SY5Y) cell patterning sensitivity could be enhanced by poly-l-lysine-g-polyethylene glycol cell-repellent back-filling, enabling cell confinement in lanes as narrow as 5 μm. Cells patterned on narrow (5 and 10 μm) lanes showed preferred nucleus orientation following the patterning direction. These cells also showed high nucleus aspect ratio but constrained nucleus spreading. On the other hand, cells on wide (20 μm and above) lanes showed random nucleus orientation and cell and nucleus sizes similar to those on unpatterned controls. All these changes were generally maintained with or without RA. Confining cells on narrow (5 and 10 μm) lanes, even without RA, significantly enhanced neurite extension relative to unpatterned control, which was further stimulated by RA. Interestingly, cell patterning on 5 and 10 μm lanes without RA produced longer neurites relative to the RA treatment alone case. Our data on the potential interplay between microscale physical cell confinement and RA-soluble stimulation may provide a new, integrative insight on how to trigger neurite/axon formation for neuronal regenerative medicine.     

Ticagrelor inhibits platelet–tumor cell interactions and metastasis in human and murine breast cancer

Activated platelets promote the proliferation and metastatic potential of cancer cells. Platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets. We examined the potential of the reversible P2Y12 inhibitor ticagrelor, an agent used clinically to prevent cardiovascular and cerebrovascular events, to reduce tumor growth and metastasis. In vitro, MCF-7, MDA-MB-468, and MDA-MB-231 human mammary carcinoma cells exhibited decreased interaction with platelets treated with ticagrelor compared to untreated platelets. Prevention of tumor cell–platelet interactions through pretreatment of platelets with ticagrelor did not improve natural killer cell-mediated tumor cell killing of K562 myelogenous leukemia target cells. Additionally, ticagrelor had no effect on proliferation of 4T1 mouse mammary carcinoma cells co-cultured with platelets, or on primary 4T1 tumor growth. In an orthotopic 4T1 breast cancer model, ticagrelor (10 mg/kg), but not clopidogrel (10 mg/kg) or saline, resulted in reduced metastasis and improved survival. Ticagrelor treatment was associated with a marked reduction in tumor cell–platelet aggregates in the lungs at 10, 30 and 60 min post-intravenous inoculation. These findings suggest a role for P2Y12-mediated platelet activation in promoting metastasis, and provide support for the use of ticagrelor in the prevention of breast cancer spread.     

Tumor cell surface β1–6 branched oligosaccharides and lung metastasis

NIH3T3 cells transfected with an activated Ha-ras oncogene were treated with L-PHA, the leukoagglutinin from red kidney beans. Cell lines resistant to L-PHA-mediated cytotoxicity were isolated and found to contain reduced levels of L-PHA-binding oligosaccharides. The levels of N-acetylglucosaminyltransferase V, the enzyme responsible for the initiation of the1–6 branch, were reduced in L-PHA-resistant cells. Tumorigenicity in nude mice was unchanged by the change in oligosaccharide expression, but the ability to form lung tumors after intravenous injection was significantly reduced. These results demonstrate that the ability of NIH3T3 cells transfected with an activated Ha-ras oncogene to form lung tumors after intravenous injection into nude mice is reduced in all six L-PHA selected cell lines containing a reduction in1–6 branched Asn-linked oligosaccharides.     

Effects of xylazine–ketamine–diazepam anesthesia on blood cell counts and plasma biochemical values in sheep and goats

To examine the effects of xylazine, ketamine, diazepam anesthesia on clinical pathology parameters in sheep and goats, seven adult, healthy, non-pregnant Awassi sheep weighing 40–60 kg and seven adult, healthy non-pregnant Damascus breed goats weighing 35 to 55 kg were used. Anesthesia was induced using 0.1 mg/kg xylazine, 5 mg/kg ketamine, and 0.25 mg/kg diazepam as a single intravenous injection. Blood cell counts and plasma biochemical analysis were performed before anesthesia (T0), 2 h after recovery, 24 h after recovery, and 5 days later. Hematological analysis indicated that there was a significant increase in neutrophil percentages (P ≤ 0.05) in sheep and goats at 2 h and 24 h after recovery. There was a mild lymphopenia and a significant leukocytosis at 24 h after recovery in both species. Packed cell volume was significantly increased at 24 h and at 2 h and 24 h after recovery in sheep and goats, respectively, while total red blood cell count was significantly decreased at 24 h after recovery in sheep and goats. There were no significant changes in hemoglobin, blood urea nitrogen, creatinine, total protein, and albumin concentrations. Glucose was significantly elevated at 2 h after recovery in goats only. Aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transpeptidase were not significantly changed at any sampling point. It is therefore concluded that a combination of xylazine, ketamine, and diazepam can be used to induce short term anesthesia in sheep and goats with minimum effects on clinical laboratory parameters.     

Aberrant acinar cell CA 19–9 expression and peri-insular acinar cell alterations in an adult human pancreas

We herein report on a 23-year-old female patient who suffered from Crohns disease and anorexia nervosa and died after long-term malnutrition and a perforated colitis. At autopsy, her pancreas displayed two peculiar findings. First, there was a constant and aberrant expression of CA 19–9 in the acinar cells. The expression of the carbohydrate antigen CA 19–9 is normally confined to duct-like epithelia, including centroacinar cells, while islet and acinar cells have repeatedly been reported to be immunohistochemically negative. Thus, to the best of our knowledge, our case is the first to show aberrant acinar CA 19–9 expression, and its potential meaning is discussed. Second, the pancreas showed a heterogeneity in acinar morphology, with peri-insular acini being considerably larger than tele-insular acini. The existence of enlarged peri-insular acini, mainly in animals, has occasionally been reported. Its origin, however, is still unclear. Some authors have proposed an influence of high insulin concentrations, exerted via the insulo-acinar capillary axis. We agree with the concept of an islet-derived mechanism. However, as we have observed a similar heterogeneity in streptozotocin- and autoimmune-diabetic rats, we presume that other islet hormones, in particular glucagon, might be more important for this phenomenon in the animals, as well as in the present case.     

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell–cell adhesion of colorectal cancer cells

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-α) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-α and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.     

Sertoli–Leydig cell tumours of the ovary and testis: a CGH and FISH study

We present two malignant cases of Sertoli–Leydig cell tumours (SLCT) of the testis and one ovarian SLCT with benign behaviour. The DNA copy number changes affected chromosome 1, 8, 9p, 10, 11, 12, 16, 19, 22 and X. The present study is the first molecular–cytogenetic analysis of Sertoli–Leydig cell tumours of the testis.     

A tunable silk–alginate hydrogel scaffold for stem cell culture and transplantation

One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk–alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk–alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk–alginate ratio and the concentration of crosslinker – a characteristic that is very hard to accomplish in current hydrogels.     

The concentration of intracellular nickel in Haemophilus influenzae is linked to its surface properties and cell–cell aggregation and biofilm formation

Of the known proteins which use nickel as a co-factor, Haemophilus influenzae contains only urease and glyoxalase I (gloA). We have recently reported that this pathogen harbours a unique nickel uptake system (nikKLMQO–nimR). Unusually, the disruption of the nickel uptake system (nikQ or nimR mutants) resulted in cells that aggregated and formed an increased biofilm compared to the wild type cells. Using a gloA mutant strain and urease-specific inhibitor we showed that this phenotype is not due to the loss-of-function of these enzymes. By generating H. influenzae “resting cells” which are enzymatically inactive but maintain their structural integrity we have shown that the cell aggregation in the nikQ/nimR mutants is not due to the loss of enzymatic function. The nikQ mutant was unable to accumulate nickel but the addition of excess nickel did restore intracellular nickel levels and this resulted in the nikQ mutant returning to the wild type “free-living” phenotype; cells with no aggregation and no biofilm formation. We used a range of techniques which showed that the nikQ mutant possesses changes to its cell surface properties. The mutant was more negatively charged than wild type cells as well as being more hydrophobic. Analysis of the outer membrane constituents showed that there were molecular differences. Although the nikQ mutant appears to grow the same as its wild type cell we have shown that there is a change in the “lifestyle” of these nickel limited cells and this induces changes to the surface of the cell to promote cell–cell aggregation and biofilm formation.     

β1–4-galactosyltransferase gene expression is regulated during entry into the cell cycle and during the cell cycle

Mammalian glycosyltransferases have been implicated in a wide variety of functions besidesN-linked glycosylation, including developmental processes. For this reason, we studied the effects of cell cycle and entry into the cell cycle on 1–4-galactosyltransferase gene expression. In this study we report that 1–4-galactosyltransferase (GalTase) gene expression is, indeed, regulated during the normal cell cycle, peaking during late G₁-, S, and early G₂ phase of the cell cycle. In addition, GalTase gene expression is regulated in a manner that resembles other early response genes such asjun andfos upon reentry into the cell cycle from quiescence. Finally, we show that the GalTase gene is differentially expressed during murine embryogenesis and in terminally differentiated adult tissues. It is most abundant in testis, followed by skeletal muscle and spleen. The reasons for this pattern of differential expression in adult tissues are unknown. These studies should provide important new information regarding GalTase gene expression, its regulation, and its potential link to other developmental functions.