Phase Contrast Light Microscopy Research Articles

Single‐step mineralization of woodpile chitosan scaffolds with improved cell compatibility

A facile and efficient single-step mineralization approach was exploited for achieving nanoscopic hydroxyapatite (HAP) crystal layer in chitosan porous matrix, wherein a mixed water-ethanol solvent was used to control the growth of minerals. The crystallographic structure, morphology, and mechanical properties of the scaffold were analyzed with XRD, FTIR, environmental scanning electric microscopy (ESEM), TEM, and compression tests. The behaviors and responses of MC3T3-E1 pre-osteoblast cells on the scaffolds were studied as well. The results showed that the scaffolds kept woodpile structure with predefined and controlled hierarchical structure after mineralization. The inorganic phase in the mineralized chitosan scaffolds was determined as pure rod-like HAP, which settled densely on the matrix. The compression strength and compressive modulus of the scaffolds increased dramatically to 0.54 ± 0.005 MPa and 5.47 ± 0.65 MPa, respectively. During a culture period of 2 and 3 weeks, cell proliferation and in-growth were observed by phase contrast light microscopy and SEM. The alkaline phosphatase (ALP) activity increased after 1 week. Cell viability and cell proliferation index (PI) obtained higher values than that of the chitosan scaffolds. The novel single-step mineralization approach and the porous hybrid scaffolds would be a promising method for designing hybrid bone graft.

X-Ray Phase Contrast Imaging of Freestanding Lipid Model Membranes

Membranes are considered as the most important interfaces in biology, and can be visualized under physiological conditions by optical techniques such as phase contrast and fluorescence light microscopy. While the contour lines and large lateral domains of biological membranes can be imaged, the density profile of the membrane and associated changes cannot be resolved by visible light. We report on hard x-ray phase contrast imaging of black lipid membranes (BLMs), which are freely suspended over a micro machined aperture in an aqueous solution. This new way of membrane structure analysis allows investigating bio molecular and organic sub-stances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-keV x-ray radiation. The width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the Fresnel diffraction regime down to its native thickness of about 5nm. We have put forward a simplified but extendable model, which enables the theoretical description of image formation and charac-terization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. The structural changes can be obtained from both the loss of contrast and the asymmetry of the detected Fresnel fringes. On the basis of the recent experiments, future investigations will be performed to study the inte-ractions of membranes, as they are for example known from synaptic fusion, with high spatial resolution.

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Sapstain of Japanese Black Pine Caused by Ophiostoma piceae in Korea

Sapstain fungi that cause blue or black discolorations of sapwood indiverse trees are one of the important fungal groups in forest healthas tree pathogens, and in forest products industry as wood-stainers(Seifert, 1993). Sapstain, caused by fungi, is the cosmetic damage ofwood that reduces wood quality and the values of commercial woodproducts. In July 2006, bluish or brown colored signs of sapstainwere found on cut sapwood of Japanese black pine (Pinusthunbergii) in Taean, Korea (Fig. 1A). The signs of sapstain werealso found on cut sapwood of Japanese black pine infected withpinewood nematode in Jinju in June 2007. Voucher specimens weredeposited at Dankook University Culture Collection, Cheonan,Korea (DKM 0510, 0410, 0412, 0413, 0601).The fungal colony was brown and dark on MEA, light brown onPDA and black on oat meal agar (Fig. 1B). Optimal growthtemperature and pH for the fungus was 22 and pH 6. The fungusshowed tolerance to cycloheximide (250 μg/ml). The optimal pHfor growth was on PDA and MEA. Observation of morphologicalfeatures on a phase-contrast light microscope (Karl Zeiss Axioskop40) and a scanning electron microscope (Hitachi S-4200) revealedthat the fungus has Sporothrix and Pesotum anamorph of O. piceae-complex. Conidial mass on apex of synnema was milky color.Synnema were dark brown and 500-1300 ( −1600)μm. Synemmat-ousconidia were hyaline and 5-15×1-3.5 μ m (Fig. 1C-E). Peritheciaand ascospores were not found in our cultures. To confirm the identity of this fungus, partial β-tubulin gene wasamplified with primers T10 and BT12 (Kim et al., 2003) and directlysequenced. The determined nucleotide sequence was deposited inGenBank (EU012505). The determined sequence shared 100%sequence identity with those of O. piceae. Based on β-tubulin genesequence, a cladogram was constructed using PAUP by the neigh-bor-joining method with the heuristic search option (Swofford,2002). The fungus grouped with O. piceae (Fig. 2). This moleculardata confirmed the fungus is O. piceae. An inoculation test on woodchips of Japanese pine displayed that the fungus colonized anddeveloped dark and/or brown stain in the chips (Fig. 3B). On theinoculated chip, the fungus formed its typical brown pigmentedsynnema having a droplet of conidial mass (Fig. 3C). So far m, ore than 150 species of Ophiostoma have been descrbed,ibut their occurrence has rarely been studied in Korea. This is the firstreport of O. piceae in Korea. Since we isolated O. piceae from a pinediseased by pinewood nematode that recently has devastated pinetrees in Korea, further study on Ophiostoma fungi should be done toexplore their impact on forest health and wood discoloring damage.

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1–2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.

Redescription of the immatures stages of Forcipomyia (Phytohelea) bromelicola (Lutz, 1914) (Diptera: Ceratopogonidae)

The fourth instar larva and pupa of Forcipomyia (Phytohelea) bromelicola (Lutz) are redescribed and illustrated, and compared with immatures of F. (P.) musae Clastrier and Delécolle and F. (P.) dominicana de Meillon and Wirth. Both life stages were photographed using scanning electron microscopy and phase-contrast light microscopy. The immatures were collected from bromeliads in Florida, USA.

Proliferation of the hyperthermophilic archaeon Pyrobaculum islandicum by cell fission

Pyrobaculum islandicum is a hyperthermophilic archaeon. P. islandicum cells have been suggested to multiply by constriction, budding and branching, as no septa were observed in cells by phase-contrast light microscopy. In this study, we observed the cells using transmission electron microscopy, scanning electron microscopy, and light microscopy with dark-field image analyses, and we report binary fission via septum formation to be the main mode of P. islandicum's proliferation. "Long cells" reported previously were found to comprise several cylindrical cells that align in tandem.

Spiroplasma eriocheiris sp. nov., associated with mortality in the Chinese mitten crab, Eriocheir sinensis

A motile bacterium, designated strain TDA-040725-5(T), was isolated from the haemolymph of a Chinese mitten crab, Eriocheir sinensis, with tremor disease. Based on 16S rRNA gene sequence analysis, the strain was phylogenetically distinct from other spiroplasmas but was closely related to Spiroplasma mirum ATCC 29335(T). Cells of strain TDA-040725-5(T) were variable in length and shape, helical and motile, as determined by phase-contrast light microscopy. Examination by electron microscopy revealed wall-less cells delimited by a single membrane. The strain grew in M1D or R-2 liquid media at 20-40 °C, with optimum growth at 30 °C. Doubling time at the optimal temperature was 24 h. The strain catabolized glucose and hydrolysed arginine but did not hydrolyse urea. The DNA G+C content was 29.7±1 mol%. The genome size was ~1.4-1.6 Mbp. Serological analysis, performed using the deformation test, did not reveal any reciprocal titres ≥320, indicating that strain TDA-040725-5(T) had minimal cross-reactivity to strains of recognized species of the genus Spiroplasma. Based on this evidence, strain TDA-040725-5(T) ( = CCTCC M 207170(T) = DSM 21848(T)) represents a novel species of the genus Spiroplasma, for which the name Spiroplasma eriocheiris sp. nov. is proposed, belonging to the novel Spiroplasma serological group XLIII.

Effect of tension on collagen arrangement

Objective To study the effect of mechanical tension on collagen arrangement and illustrate the relationship between tissue architecture and tension properties. Methods Cell morphologies, orientation and collagen arrangement of fibroblasts cultured in three different types of collagen gels with variation of mechanical tension were observed by phase contrast photomicrographs, light microscopy and transmission electron microscopy. Expression and distribution of a-smooth muscle actin (α-SMA) were investigated by immunohistochemistry. Results Phase contrast photomicrographs, light microscopy and transmission electron microscopy showed high level of tension distributed anisotropically in the monolayer gels and the anchored collagen gels, with bipolar shape of the fibroblasts, obvious polarity, arrangement of exogenous collagen fibres parallel to the long axis of the fibroblasts, especially prominent in monolayer gels. Low level of tension distributed isotropically was observed in floating collagen gels, with stellate morphology and arrangement of exogenous collagen fibres in a reticular array. Immunofluorescence showed that fibroblasts expressed high level of α-SMA protein distributed along the long axis of fibroblasts in the monolayer gels and the anchored collagen gels, especially in former ones. In contrast, few expression of α-SMA protein was found in floating collagen gels. Cell morphologies and orientation, expression and distribution of α-SMA as well as collagen arrangement of fibroblasts in the monolayer gels and the anchored collagen gels were similar to those in granulation tissue, whereas floating collagen gels resembled normal dermis or remodelled tissues. Conclusions Tissue architecture or morphology of the dermis are corresponding to tension proporties. Different tissue architectures are closely correlated with particular tension proporties. Key words: Tensile strength; Fibroblasts; Collagen; Cytoskeleton

Structure and dynamics of an Arp2/3 complex‐independent component of the lamellipodial actin network

Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow transmission electron microscopy methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of elongate actin filaments organized into concave arcs that displayed retrograde flow at approximately one quarter the normal rate. Actin polymerization inhibitor experiments indicated that these arcs were generated by polymerization at the cell edge, while active myosin-based contraction in BDM treated cells was demonstrated by localization with antiphospho-myosin regulatory light chain (MRLC) antibody, the retraction of the cytoskeleton in the presence of BDM, and the response of the BDM arcs to laser-based severing. The results suggest that BDM treatment reveals an Arp2/3 complex-independent actin structure in coelomocytes consisting of elongate filaments integrated into the LP network and that these filaments represent a potential connection between the LP network and the central cytoskeleton.

Asbestos bodies in lung tissue are associated with pathologic features and patient outcome for epithelial but not non‐epithelial mesothelioma treated by extrapleural pneumonectomy

We examined patient survival and pathologic tumor features in relation to asbestos body (AB) counts in lung tissue from patients with malignant pleural mesothelioma (MPM).Lung tissue was digested with Clorox® by standard methods. AB counts were made using phase contrast light microscopy. Kaplan‐Meier estimates of overall survival were compared using logrank tests for dichotomized AB count in WHO epithelial (E) and non‐epithelial (NE) MPM subgroups. Association of AB count with reported pathological features was assessed using Fisher's Exact test.From 1988 to 2005, 365 E (72% male; median age 57) and 194 NE patients (90% male; median age 60) were treated. Of these, 234 (64%) E and 142 (73%) NE had AB evaluation. Median AB count for E was 265/g (wet weight) vs. 1084/g for NE and 20/g control AB count. Among E patients, 105 with AB≤200/g had 27‐mo. median survial vs 17.1 mo. for 129 with >200 AB/g (p=0.0003). By contrast, survival for 32 NE with AB≤200/g (9.5 mo.) did not differ from 109 NE with >200 AB/g (12.7 mo.;p=0.58). AB counts above median were significantly associated with involvement of lung parenchema (p=0.0055) and intralobar fissure (p=0.0176) for E but not NE. No relation of AB to diaphragm, pericardium or chest wall involvement was evident.We conclude that AB has prognostic significance in MPM and that E and NE represent distinct disease entities.Supported by the International Mesothelioma Program

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization.

BackgroundGemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy.ResultsUsing phase contrast and fluorescence light microscopy, and transmission electron microscopy, the cell division cycle of G. obscuriglobus was determined. During the budding process, the bud was formed and developed in size from one point of the mother cell perimeter until separation. The matured daughter cell acted as a new mother cell and started its own budding cycle while the mother cell can itself initiate budding repeatedly. Fluorescence microscopy of DAPI-stained cells of G. obscuriglobus suggested that translocation of the nucleoid and formation of the bud did not occur at the same time. Confocal laser scanning light microscopy applied to cells stained for membranes as well as DNA confirmed the behaviour of the nucleoid and nucleoid envelope during cell division. Electron microscopy of cryosubstituted cells confirmed deductions from light microscopy concerning nucleoid presence in relation to the stage of budding, and showed that the nucleoid was observed to occur in both mother and bud cells only at later budding stages. It further suggested that nucleoid envelope formed only after the nucleoid was translocated into the bud, since envelopes only appeared in more mature buds, while naked nucleoids occurred in smaller buds. Nucleoid envelope appeared to originate from the intracytoplasmic membranes (ICM) of both mother cell and bud. There was always a connecting passage between mother cell and bud during the budding process until separation of the two cells. The division cycle of the nucleated planctomycete G. obscuriglobus appears to be a complex process in which chromosomal DNA is transported to the daughter cell bud after initial formation of the bud, and this can be performed repeatedly by a single mother cell.ConclusionThe division cycle of the nucleated planctomycete G. obscuriglobus is a complex process in which chromosomal nucleoid DNA is transported to the daughter cell bud after initial formation of a bud without nucleoid. The new bud nucleoid is initially naked and not surrounded by membrane, but eventually acquires a complete nucleoid envelope consisting of two closely apposed membranes as occurs in the mother cell. The membranes of the new nucleoid envelope surrounding the bud nucleoid are derived from intracytoplasmic membranes of both the mother cell and the bud. The cell division of G. obscuriglobus displays some unique features not known in cells of either prokaryotes or eukaryotes.

Mechanical and in vitro evaluations of composite PLDLLA/TCP scaffolds for bone engineering

Bone tissue engineering scaffolds have two challenging functional tasks to play; to be bioactive by encouraging cell proliferation and differentiation, and to provide suitable mechanical stability upon implantation. Composites of biopolymers and bioceramics unite the advantages of both materials, resulting in better processability, enhanced mechanical properties through matrix reinforcement and osteoinductivity. Novel composite blends of poly(L-lactide-co-D,L-lactide)/tricalcium phosphate (PLDLLA/TCP) were fabricated into scaffolds by an extrusion deposition technique customised from standard rapid prototyping technology. PLDLLA/TCP composite material blends of various compositions were prepared and analysed for their mechanical properties. PLDLLA/TCP (10%) was optimised and fabricated into scaffolds. Compressive mechanical properties for the composite scaffolds were measured. In vitro studies were conducted using porcine bone-marrow stromal cells (BMSCs). Cell-scaffold constructs were induced using osteogenic induction factors for up to 8 weeks. Cell proliferation, viability and differentiation capabilities were assayed using phase-contrast light microscopy, scanning electron microscopy, DNA quantification (Pico Green), Alamar Blue metabolic assay; FDA/PI fluorescent assay and western blot analysis for osteopontin. Microscopy observations showed BMSCs possessed high proliferative capabilities and demonstrated bridging across the pores of the scaffolds. FDA/PI staining as well as Alamar Blue assay showed high viability of BMSCs cultured on the composite scaffolds. Cell numbers, based on DNA quantitation, were observed to increase continuously up to the eighth week of study. Western blot analysis showed increased osteopontin synthesis on the scaffolds compared to tissue culture plastic. Based on our results the PLDLLA/TCP scaffolds exhibited good potential and biocompatibility for bone tissue engineering applications.

Differential influences of bucillamine and methotrexate on the generation of fibroblast-like cells from bone marrow CD34+ cells of rheumatoid arthritis patients

We have recently demonstrated that bone marrow CD34+ cells from rheumatoid arthritis (RA) patients displayed abnormal capacities to respond to TNF-α and to differentiate into fibroblast-like cells producing MMP-1 (type B synoviocyte -like cells). The current study examined the effects of representative potent disease-modifying antirheumatic drugs, including bucillamine (BUC) and methotrexate (MTX) on the in vitro generation of fibroblast-like cells from RA bone marrow CD34+ cells. CD34+ cells purified from bone marrow specimens of 8 patients with active RA were cultured in the presence or absence of pharmacologically attainable concentrations of intramolecular disulfide form of bucillamine (BUC-ID, 3 μM), a major metabolite of BUC or MTX (20 nM). After incubation for 28 days, the generation of fibroblast-like cells was assessed under phase-contrast light microscopy and the concentrations of MMP-1 and VEGF in the culture supernatants were measured by ELISA. BUC-ID, but not MTX, significantly suppressed the generation of fibroblast-like cells from RA bone marrow CD34+ cells stimulated with SCF, GM-CSF and TNF-α ( p = 0.024 as determined by Wilcoxon signed rank test). Accordingly, BUC-ID, but not MTX, significantly suppressed the production of MMP-1 ( p = 0.017) and VEGF ( p = 0.017) by RA bone marrow CD34+ cells, without inhibition of β2-microglobulin production. These results demonstrate that BUC-ID, but not MTX, is a potent inhibitor of differentiation of fibroblast-like cells from RA bone marrow CD34+ cells. Since MTX, but not BUC, has been previously shown to influence on type A synoviocytes, the data provide rationale of combination of BUC and MTX in the treatment of RA.

Irradiation Is an Early Determinant of Endothelial Injury During Hematopoietic Stem Cell Transplantation

Objective We investigated the degree and the time course of endothelial injury in mice pretreated with lethal or reduced-intensity irradiation administered before transplantation. Materials and Methods Six- to eight-week-old female mice were randomly allocated into three groups: lethal-intensity irradiation (8.5 Gy, group 1), reduced-intensity irradiation (5.0 Gy, group 2), or nonirradiated controls (group 3). After conditioning, circulating endothelial cells (CD31 +, CD133 −, and CD45 low) and peripheral blood CD4 + or CD8 + T-lymphocyte subpopulations were enumerated using flow cytometry at various times. The morphologic changes in endothelium were examined at phase-contrast light microscopy. Results Circulating endothelial cells showed an earlier and higher peak in the lethal irradiation group compared with the reduced-intensity irradiation group, which exhibited a protean elevation in cell numbers. There were no visible histopathologic changes during the early stage of endothelial damage. Conclusions Lethal and reduced doses of irradiation induced endothelial injury in a dose-dependent manner. Endothelial damage may occur before graft-vs-host disease and its related complications.

Endothelial Injury, an Intriguing Effect of Methotrexate and Cyclophosphamide During Hematopoietic Stem Cell Transplantation in Mice

Objective Elevated circulating endothelial cells (EC) in peripheral blood are an important indicator of endothelial damage and graft-versus-host disease (GVHD). However, the injured endothelial vasculature may in turn promote GVHD. In this study, we investigated whether methotrexate or cyclophosphamide, two conventional chemotherapeutic agents used in hematopoietic stem cell transplantation, caused endothelial injury and what were the functional consequences of this injury on GVHD. Methods Six to 8-week-old female mice were randomly separated into three groups, including methotrexate (15 mg/m 2 on day 1 and 10 mg/m 2 on days 3, 6, and 11), cyclophosphamide (60 mg/kg; days 1, 2), and PBS saline alone (control). Circulating EC (CD31 +CD133 −CD45 low) and the CD4 +/CD8 + T lymphocytes in the peripheral blood were estimated by flow cytometry on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. The morphologic changes of the endothelium were examined by phase contrast light microscopy to determine the integrity of the endothelial vasculature. Results Elevated EC were detected at day 1 or 3 in mice receiving cyclophosphamide or methotrexate, respectively, with a peak increase at day 5 or day 3, respectively. The ratio of CD4 +/CD8 + T lymphocytes showed a delayed increase in the peak to day 11 for both groups. In the cyclophosphamide group, there was significant apomorphosis with necrosis/thrombosis under light microscopy, whereas only apomorphosis was noticed in the methotrexate group. In both groups, EC showed hydropsia and cytomembrane damage. Conclusions Circulating EC increase during the early phases of cyclophosphamide or methotrexate conditioning, suggesting that both chemotherapeutic drugs induce endothelial damage, which occurs a little earlier than suppression of the immune system.

Substance P-Mediated Expression of the Pro-Angiogenic Factor CCN1 Modulates the Course of Colitis

Substance P (SP) regulates important intestinal functions, such as mucosal permeability, motility, chloride secretion, and inflammation via the neurokinin-1 receptor (NK-1R). Previous reports showed that vascularization and expression of angiogenic factors are evident in the colonic mucosa of rats with colitis and patients with inflammatory bowel disease. Here we determined whether SP is associated with angiogenesis. Human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and mice with dextran sodium sulfate-induced colitis were used. We found that expression of the angiogenic factor CCN1 was increased in the colons of patients with Crohn's disease and ulcerative colitis. Mucosal extracts from inflammatory bowel disease patients induced human intestinal microvascular endothelial cell migration that was inhibited by blockade of CCN1 and its receptor integrin alphavbeta3. Both the degree of angiogenesis and CCN1 expression were elevated in the colons of mice with dextran sodium sulfate-induced colitis, which was reduced by treatment with the NK-1R antagonist CJ-12255. SP also increased CCN1 expression in NCM460-NK-1R colonocytes. SP exposure to human intestinal microvascular endothelial cells co-cultured with NCM460-NK-1R cells induced angiogenic activity that was inhibited by CCN1 silencing. In addition, intracolonic overexpression of CCN1 induced angiogenesis in mouse colon. Thus, SP mediates angiogenesis via CCN1 during colitis.

Cytotoxicity of single-walled carbon nanotubes suspended in various surfactants

The cytotoxicity of single-walled carbon nanotubes (SWCNTs) suspended in varioussurfactants was investigated by phase contrast light microscopy characterization incombination with an absorbance spectroscopy cytotoxicity analysis. Our data indicate thatindividual SWCNTs suspended in the surfactants, sodium dodecyl sulfate (SDS) andsodium dodecylbenzene sulfonate (SDBS), were toxic to 1321N1 human astrocytoma cellsdue to the toxicity of SDS and SDBS on the nanotube surfaces. This toxicity was observedwhen cells were exposed to an SDS or SDBS solution having a concentration as low as0.05 mg ml−1 for 30 min. The proliferation and viability of the cells were not affected by SWCNTs aloneor by conjugates of SWCNTs with various concentrations of sodium cholate (SC) orsingle-stranded DNA. The cells proliferated similarly to untreated cells when surrounded bySWCNTs as they grow, which indicated that the nanotubes did not affect cells adversely.The cytotoxicity of the nanotube–surfactant conjugates was controlled in these experimentsby the toxicity of the surfactants. Consequently, when evaluating a surfactant to beused for the dispersion of nanoscale materials in applications such as nanoscaleelectronics or non-viral biomolecular transporters, the cytotoxicity needs to beevaluated. The methodology proposed in this study can be used to investigate thecytotoxicity of other nanoscale materials suspended in a variety of surfactants.

Characterization of a Spontaneous Mouse Retinal Pigment Epithelial Cell Line B6-RPE07

A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel-coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and beta-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.