Trafficking and Association of Plasmodium falciparum MC-2TM with the Maurer's Clefts.

In this study, we investigated stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in P. falciparum (3D7) infected erythrocytes. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence with antibodies reactive with PfMC-2TM. PfMC-2TM is sensitive to BFA treatment and permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associated with the MC and infected erythrocyte membrane and was resistant to extraction with alkaline sodium carbonate, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM is an additional marker of the nascent MCs.

IL-1 induces mitochondrial translocation of IRAK2 to suppress oxidative metabolism in adipocytes.

Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R-MyD88-IRAK2-PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity.

RhopH3, rhoptry gene conserved in the free-living alveolate flagellate Colpodella sp. (Apicomplexa).

In this study, we investigated morphological, immunological and molecular characteristics of Colpodella sp. (American Type Culture Collection 50594) in a diprotist culture containing Bodo caudatus as prey using Plasmodium rhoptry specific antibodies and oligonucleotide primers targeting Plasmodium falciparum rhoptry genes. In culture, Colpodella sp. attached to its prey using the apical end with attachment lasting for approximately 20 min while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella sp. Encystment of Colpodella sp. was observed following feeding. Indirect immunofluorescence assay (IFA) and confocal microscopy using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella sp. Bodo caudatus from diprotist and monoprotist (ATCC 30395) cultures showed weak background reactivity. Giemsa staining permitted differentiation of both protists. Genomic DNA isolated from the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers targeting the P. falciparum rhoptry genes RhopH3, RhopH1/Clag3.2 and RAMA. Primers targeting exon 7 of the P. falciparum RhopH3 gene amplified an approximately 2 kb DNA fragment from the diprotist DNA template. DNA sequence and BLAST search analysis of the amplified product from diprotist DNA identified the RhopH3 gene demonstrating that the RhopH3 gene is conserved in Colpodella sp.

Folic acid conjugated polymeric drug delivery vehicle for targeted cancer detection in hepatocellular carcinoma.

Targeted therapies provide increased efficiency for the detection and treatment of cancer with reduced side effects. Folate receptor (alpha subunit) is overexpressed in multiple tumors including liver cancer. In this study, we evaluated the specificity and toxicity of a folic acid-containing drug delivery vehicle (DDV) in a hepatocellular carcinoma (HCC) model. The DDV was prepared with two units each of folic acid (FA) and fluorescein isothiocyanate (FITC) molecules and conjugated to a central poly (ethylene glycol) (PEG) core via a modified chemo-enzymatic synthetic process. Rat hepatoma (N1S1) and human monocytic (U937) cell lines were used for cell culture-based assays and tested for DDV uptake and toxicity. Folate receptor expressions in liver tissues and cell lines were verified using standard immunohistochemistry techniques. Rat HCC model was used for in vivo assessment. The DDV was injected via intra-arterial or intravenous methods and imaged with IVIS spectrum in vivo imaging system. Strong signals of FITC in the liver tumor region correlated to targeted DDV uptake. The use of PEG enhanced water-solubility and provided flexibility for the interaction of FA ligands with multiple cell surface folate receptors that resulted in increased specific uptake. Our study suggested that PEG incorporation and folate targeting via intra-arterial approach is an efficient strategy for targeted delivery in HCC therapy.

ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation.

Focal adhesions anchor cells to extracellular matrix (ECM) and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC) focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM.

Quantity of Lymph Nodes in the Vascularized Lymph Node Transfer Influences Its Lymphaticovenous Drainage.

BACKGROUND: The purpose of this study was to: (1) evaluate the mechanism of lymph drainage through a vascularized lymph node (VLN) flap, and (2) investigate if the number of VLNs impacts lymph transit time through the flap. METHODS: Twenty-seven axillary VLN flaps were elevated in 14 Sprague-Dawley rats and divided into three groups (n = 9 each) based on the number of lymph nodes present: group 1 (0-VLNs), group 2 (2-VLNs), and group 3 (4-VLNs). Indocyanine green (n = 8/group) and Alexa680-albumin (n = 1/group) were injected into the edge of flaps and the latency period between injection and fluorescence in the axillary vein was recorded. Stereomicroscopic fluorescent lymphography was performed to directly visualize lymphatic transit through VLNs. RESULTS: Fluorescence was detected in the axillary vein after 229s [47-476], 79s [15-289], and 56s [16-110] in group 1, 2, and 3, respectively (p < 0.01). There was a negative correlation between the number of VLNs in the flap and the latency period (r = -0.59; p < 0.05). Median flap weights were comparable in group 1, 2, and 3 (258 mg [196-349], 294 mg [212-407], 315 mg [204-386], respectively; p = 0.54). Stereoscopic lymphography allowed direct visualization of lymphatic fluid transit through VLNs. CONCLUSION: Lymphatic fluid in VLN flaps drains into the venous system mainly by passing through the afferent lymphatics and lymph nodes. A secondary mechanism appears to be the diffusion of fluid into the venous system via intratissue lymphaticovenous connections created during flap elevation. Increasing the number of lymph nodes in the flap is associated with a more rapid transit of fluid.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel roll-up method, we have developed large scale MSC based tissue-engineered cartilage that shows microscale structural organization and enhanced compressive properties compared to current tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineered cartilage constructs made with human mesenchymal stem cells (hMSCs), scaffolds and bioactive factors are a promising solution to treat cartilage defects. A major disadvantage of these constructs is their inferior mechanical properties compared to the native tissue, which is likely due to the lack of structural organization of the extracellular matrix of the engineered constructs. In this study, we developed three-dimensional (3-D) cartilage constructs from rectangular scaffold sheets containing hMSCs in micro-guidance channels and characterized their mechanical properties and metabolic requirements. The work led to a novel roll-up method to embed 2-D microscale structures in 3-D constructs. Further, micro-guidance channels incorporated within the 3-D cartilage constructs led to the production of aligned cell-produced matrix and enhanced mechanical function.

Control of organization and function of muscle and tendon by thrombospondin-4.

Thrombospondins (TSPs) are multifunctional proteins that are deposited in the extracellular matrix where they directly affect the function of vascular and other cell types. TSP-4, one of the 5 TSP family members, is expressed abundantly in tendon and muscle. We have examined the effect of TSP-4 deficiency on tendon collagen and skeletal muscle morphology and function. In Thbs4(-/-) mice, tendon collagen fibrils are significantly larger than in wild-type mice, and there is no compensatory over-expression of TSP-3 and TSP-5, the two TSPs most highly homologous to TSP-4, in the deficient mice. TSP-4 is expressed in skeletal muscle, and higher levels of TSP-4 protein are associated with the microvasculature of red skeletal muscle with high oxidative metabolism. Lack of TSP-4 in medial soleus, red skeletal muscle with predominant oxidative metabolism, is associated with decreased levels of several specific glycosaminoglycan modifications, decreased expression of a TGFß receptor beta-glycan, decreased activity of lipoprotein lipase, which associates with vascular cell surfaces by binding to glycosaminoglycans, and decreased uptake of VLDL. The soleus muscle is smaller and hind- and fore-limb grip strength is reduced in Thbs4(-/-) mice compared to wild-type mice. These observations suggest that TSP-4 regulates the composition of the ECM at major sites of its deposition, tendon and muscle, and the absence of TSP-4 alters the organization, composition and physiological functions of these tissues.

Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin).

BACKGROUND: Macroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines. METHODS: Immunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting. RESULTS: We found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation. CONCLUSIONS: We show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.

Oxidized LDL/CD36 interaction induces loss of cell polarity and inhibits macrophage locomotion.

Cell polarization is essential for migration and the exploratory function of leukocytes. However, the mechanism by which cells maintain polarity or how cells revert to the immobilized state by gaining cellular symmetry is not clear. Previously we showed that interaction between oxidized low-density lipoprotein (oxLDL) and CD36 inhibits macrophage migration; in the current study we tested the hypothesis that oxLDL/CD36-induced inhibition of migration is the result of intracellular signals that regulate cell polarity. Live cell imaging of macrophages showed that oxLDL actuated retraction of macrophage front end lamellipodia and induced loss of cell polarity. Cd36 null and macrophages null for Vav, a guanine nucleotide exchange factor (GEF), did not show this effect. These findings were caused by Rac-mediated inhibition of nonmuscle myosin II, a cell polarity determinant. OxLDL induced dephosphorylation of myosin regulatory light chain (MRLC) by increasing the activity of Rac. Six-thioguanine triphosphate (6-thio-GTP), which inhibits Vav-mediated activation of Rac, abrogated the effect of oxLDL. Activation of the Vav-Rac-myosin II pathway by oxidant stress may induce trapping of macrophages at sites of chronic inflammation such as atherosclerotic plaque.

CXCL12-induced monocyte-endothelial interactions promote lymphocyte transmigration across an in vitro blood-brain barrier.

The accumulation of inflammatory cells in the brain parenchyma is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Chemokines and adhesion molecules orchestrate leukocyte transmigration across the blood-brain barrier (BBB), but the dynamics of chemokine receptor expression during leukocyte transmigration are unclear. We describe an in vitro BBB model system using human brain microvascular endothelial cells that incorporates shear forces mimicking blood flow to elucidate how chemokine receptor expression is modulated during leukocyte transmigration. In the presence of the chemokine CXCL12, we examined modulation of its receptor CXCR4 on human T cells, B cells, and monocytes transmigrating across the BBB under flow conditions. CXCL12 stimulated transmigration of CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes. Transmigration was blocked by CXCR4-neutralizing antibodies. Unexpectedly, CXCL12 selectively down-regulated CXCR4 on transmigrating monocytes, but not T cells. Monocytes underwent preferential CXCL12-mediated adhesion to the BBB in vitro compared with lymphocytes. These findings provide new insights into leukocyte-endothelial interactions at the BBB under conditions mimicking blood flow and suggest that in vitro BBB models may be useful for identifying chemokine receptors that could be modulated therapeutically to reduce neuroinflammation in diseases such as MS.

Autophagy-dependent senescence in response to DNA damage and chronic apoptotic stress.

Autophagy regulates cell survival and cell death upon various cellular stresses, yet the molecular signaling events involved are not well defined. Here, we established the function of a proteolytic Cyclin E fragment (p18-CycE) in DNA damage-induced autophagy, apoptosis, and senescence. p18-CycE was identified in hematopoietic cells undergoing DNA damage-induced apoptosis. In epithelial cells exposed to DNA damage, chronic but not transient expression of p18-CycE leads to higher turnover of LC3 I/II and increased emergence of autophagosomes and autolysosomes. Levels of p18-CycE, which was generated by proteolytic cleavage of endogenous Cyclin E, were greatly increased by chloroquine and correlated with LC 3II conversion. Preventing p18-CycE genesis blocked conversion of LC3 I to LC3 II. Upon DNA damage, cytoplasmic ataxia-telangiectasia-mutated (ATM) was phosphorylated in p18-CycE-expressing cells resulting in sustained activation of the adenosine-mono-phosphate-dependent kinase (AMPK). These lead to sustained activation of mammalian autophagy-initiating kinase ULK1, which was abrogated upon inhibiting ATM and AMPK phosphorylation. Moreover, p18-CycE was degraded via autophagy followed by induction of senescence. Both autophagy and senescence were prevented by inhibiting autophagy, which leads to increased apoptosis in p18-CycE-expressing cells by stabilizing p18-CycE expression. Senescence was further associated with cytoplasmic co-localization and degradation of p18-CycE and Ku70. In brief, chronic p18-CycE expression-induced autophagy leads to clearance of p18-CycE following DNA damage and induction of senescence. Autophagy inhibition stabilized the cytoplasmic p18-CycE-Ku70 complex leading to apoptosis. Thus, our findings define how chronic apoptotic stress and DNA damage initiate autophagy and regulate cell survival through senescence and/or apoptosis.

Actin cytoskeleton remodeling by the alternatively spliced isoform of PDLIM4/RIL protein.

RIL (product of PDLIM4 gene) is an actin-associated protein that has previously been shown to stimulate actin bundling by interacting with actin-cross-linking protein α-actinin-1 and increasing its affinity to filamentous actin. Here, we report that the alternatively spliced isoform of RIL, denoted here as RILaltCterm, functions as a dominant-negative modulator of RIL-mediated actin reorganization. RILaltCterm is regulated at the level of protein stability, and this protein isoform accumulates particularly in response to oxidative stress. We show that the alternative C-terminal segment of RILaltCterm has a disordered structure that directs the protein to rapid degradation in the core 20 S proteasomes. Such degradation is ubiquitin-independent and can be blocked by binding to NAD(P)H quinone oxidoreductase NQO1, a detoxifying enzyme induced by prolonged exposure to oxidative stress. We show that either overexpression of RILaltCterm or its stabilization by stresses counteracts the effects produced by full-length RIL on organization of actin cytoskeleton and cell motility. Taken together, the data suggest a mechanism for fine-tuning actin cytoskeleton rearrangement in response to stresses.

Viewing hyaluronan: imaging contributes to imagining new roles for this amazing matrix polymer.

Hyaluronan (HA) is an ubiquitous extracellular matrix polymer that plays many roles in health and disease. The ability to view the spatial and temporal expression of HA in tissues and on/in cells has provided researchers with insights into the tremendously diverse biological processes in which HA is involved. Biochemical extraction, quantity, and size measurement of HA can tell part of the story, but these techniques are incomplete in placing HA at the scene of a biological event and determining which other molecules are likely to be cooperating. HA, however, is not immunogenic, so preparing antibodies for histochemistry is problematic. Fortunately, a probe for HA was devised based on the HA binding region of aggrecan, and today this probe is commercially available and very useful for histochemistry. This article discusses the conditions and considerations that the authors' lab and others have developed for optimal HA staining in many tissues and cell types.

A simple and efficient method for preparation of isolated ovarian follicles for transmission electron microscopy.

OBJECTIVE: A simple method for preparation of isolated ovarian follicles for transmission electron microscopy (TEM) using transwell inserts is described. MATERIALS AND METHODS: Pre-antral follicles were enzymatically isolated from mouse ovaries and cultured overnight on transwell insert polyester membranes. The following day, isolated ovarian follicles were processed for TEM by moving the transwell insert through successive wells containing the fixation and embedding reagents. After polymerization of the resin, the polyester membrane with the follicles embedded in the resin was disengaged from the transwell unit. The resin was sectioned. Semi-thin sections were stained with toluidine blue while ultra-thin sections were stained by uranyl acetate and examined by light microscopy and TEM, respectively. RESULTS: Isolated ovarian follicles were easily processed in groups for TEM. Follicles were well embedded and there appeared to be no loss of tissue during processing. The ultra-structure of processed isolated ovarian follicles was well preserved with little evidence of processing artifacts. CONCLUSIONS: In situ processing and preparation of isolated ovarian follicles by first allowing attachment on transwell insert membranes was shown to be a simple, rapid and effective method for TEM.

A new bioartificial pancreas utilizing amphiphilic membranes for the immunoisolation of porcine islets: a pilot study in the canine.

We have developed a replaceable bioartificial pancreas to treat diabetes utilizing a unique cocontinous amphiphilic conetwork membrane created for macroencapsulation and immunoisolation of porcine islet cells (PICs). The membrane is assembled from hydrophilic poly(N,N-dimethyl acrylamide) and hydrophobic/oxyphilic polydimethylsiloxane chains cross-linked with hydrophobic/oxyphilic polymethylhydrosiloxane chains. Our hypothesis is that this membrane allows the survival of xenotransplanted PICs in the absence of prevascularization or immunosuppression because of its extraordinarily high-oxygen permeability and small hydrophilic channel dimensions (3-4 nm). The key components are a 5-10 microm thick semipermeable amphiphilic conetwork membrane reinforced with an electrospun nanomat of polydimethylsiloxane-containing polyurethane, and a laser-perforated nitinol scaffold to provide geometric stability. Devices were loaded with PICs and tested for their ability to maintain islet viability without prevascularization, prevent rejection, and reverse hyperglycemia in three pancreatectomized dogs without immunosuppression. Tissue tolerance was good and there was no systemic toxicity. The bioartificial pancreas protected PICs from toxic environments in vitro and in vivo. Islets remained viable for up to 3 weeks without signs of rejection. Neovascularization was observed. Hyperglycemia was not reversed, most likely because of insufficient islet mass. Further studies to determine long-term islet viability and correction of hyperglycemia are warranted.

Collagen remodeling and suburethral inflammation might account for preserved anti-incontinence effects of cut polypropylene sling in rat model.

OBJECTIVES: To determine whether the inflammatory responses or collagen remodeling in suburethral tissue could have contributed to the preserved anti-incontinence effects of a cut polypropylene sling. METHODS: Stress urinary incontinence was created in 60 age-matched female Sprague-Dawley rats that were subsequently randomized into 3 equal-size groups according to surgical procedure: placement of a vaginal suburethral sling, placement of a vaginal suburethral sling in which the suburethral portion of the sling was immediately cut, and sham surgery without placement of the sling. In a previous study, the leak point pressure measurements were obtained on these rats 6 weeks after surgery. The rats were then killed, the urethrovaginal tissue was harvested (cross-section of the entire urethra and anterior vagina) from 30 animals (10 from each experimental group), and the tissue was stained with hematoxylin-eosin and Masson's trichrome for histopathologic studies and picrosirius red for collagen fibers. RESULTS: As previously published, the median leak point pressures were similarly and significantly increased in the rats in the intact and cut sling groups compared with those in the sham surgery group. The inflammatory mediators and interstitial edema were similarly increased in the intact and cut sling specimens compared with the sham surgery specimens. Under polarized light, picrosirius red-stained specimens from the sham surgery animals appeared to be composed of collagen that predominately birefringed red to yellow (typical of type I collagen). The picrosirius red-stained cut and intact sling specimens appeared to contain collagen fibers that predominately birefringed green (typical of type III collagen). CONCLUSIONS: Histologic changes, including inflammation, localized edema, and differential collagen remodeling, might contribute to the preserved anti-incontinence mechanisms of cut or intact polypropylene slings observed clinically.

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria.

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.

Differentiated murine airway epithelial cells synthesize a leukocyte-adhesive hyaluronan matrix in response to endoplasmic reticulum stress.

In this report, we describe a novel method for culturing murine trachea epithelial cells on a native basement membrane at an air-liquid interface to produce a pseudostratified, differentiated airway epithelium composed of ciliated and nonciliated cells. This model was used to examine hyaluronan synthesis by the airway epithelial cells (AECs) in response to poly(I,C) and tunicamycin. The former induces a response similar to viral infection, and the latter is a bacterial toxin known to induce endoplasmic reticulum (ER) stress. We found significant accumulation of hyaluronan on the apical surface of the AECs in response to ER stress, but, unlike previously reported results with smooth muscle cells, no increase in hyaluronan was observed in response to poly(I,C). Monocytic U937 cells adhered at 4 degrees C to the apical surface of the AECs subjected to ER stress by a mechanism almost entirely mediated by hyaluronan. The U937 cells spontaneously released themselves from the abnormal hyaluronan matrix when their metabolism was restored by shifting the temperature from 4 to 37 degrees C in a custom-made flow chamber. Time lapse confocal microscopy permitted live imaging of this interaction between the U937 cells and the hyaluronan matrix and their subsequent response at 37 degrees C. Within 45 min, we observed dynamic protrusions of the U937 cell plasma membrane into nearby hyaluronan matrix, resulting in the degradation of this matrix. Simultaneously, we observed some reorganization of the hyaluronan matrix, from a generalized, apical distribution to localized regions around the AEC tight junctions. We discuss the implications these results might have for the airway epithelium and its relation to airway inflammation and hyperresponsiveness associated with asthma and other airway diseases.

Circulating angiogenic precursors in idiopathic pulmonary arterial hypertension.

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34+ CD133+ bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34+ CD133+ bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-beta or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.