A Mathematical Model for Simultaneous Spatio-Temporal Dynamics of Calcium and Inositol 1,4,5-Trisphosphate in Madin–Darby Canine Kidney Epithelial Cells

The landmark paper by Hirose et al. (Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., Iino, M., Science 284:1527–1530, 1999) presented experimental investigations to show that not only can calcium upregulate IP₃, but that it can also have an inhibitory effect on IP₃. In this paper, we present a preliminary model, which is consistent with these experiments. This model includes positive and negative feedback between calcium and IP₃ and is able to reproduce more precisely the data presented in Hirose et al. (Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., Iino, M., Science 284:1527–1530, 1999). In the second part of the paper, the intracellular and intercellular calcium movement in Madin–Darby canine kidney epithelial cells is investigated. With the aid of the model we are able to identify the aspects of IP₃ and calcium signalling, which should be studied further experimentally before refining the model.     

The Occurrence of Matriptase C-Terminal Fragments on the Apical and Basolateral Sides of Madin–Darby Canine Kidney Epithelial Cells

Matriptase is a type II transmembrane serine protease. In the present study, matriptase C-terminal fragments containing the catalytic serine protease domain were found to occur on the apical and basolateral sides of Madin–Darby canine kidney epithelial cells transfected with a cDNA encoding the protease. This suggests that matriptase interacts with various potential substrates when expressed in simple epithelia.     

Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin–Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La³⁺, a non-selective cation channel (NSCC) blocker, and by K⁺ channel blockers Ba²⁺ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl^? channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTR_inh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V _r) ~?80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba²⁺ or clotrimazole, and absent in elevated [Ca²⁺]_i, but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTR_inh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca²⁺. Ba²⁺ and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current V _r near ?85 mV, and reduced GTTR uptake by ~20 %. La³⁺ alone hyperpolarized the cells by ~?14 mV, reduced the I/V slope with a net current V _r near ?10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La³⁺, bumetanide-caused negligible change in potential or I/V. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca²⁺ and thus facilitating activation of the intermediate conductance Ca²⁺-activated K⁺ channels.     

Retromer Regulates Postendocytic Sorting of β-Secretase in Polarized Madin-Darby Canine Kidney Cells

β-Amyloid (Aβ) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the β-APP cleaving enzyme (BACE or β-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aβ peptides.     

Epithelial–Mesenchymal Transition in Kidney Tubular Epithelial Cells Induced by Globotriaosylsphingosine and Globotriaosylceramide

Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (α-gal A), which results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. Globotriaosylsphingosine (lyso-Gb3), a deacylated Gb3, is also increased in the plasma of patients with Fabry disease. Renal fibrosis is a key feature of advanced Fabry disease patients. Therefore, we evaluated the association of Gb3 and lyso-Gb3 accumulation and the epithelial–mesenchymal transition (EMT) on tubular epithelial cells of the kidney. In HK2 cells, exogenous treatments of Gb3 and lyso-Gb3 increased the expression of TGF-β, EMT markers (N-cadherin and α-SMA), and phosphorylation of PI3K/AKT, and decreased the expression of E-cadherin. Lyso-Gb3, rather than Gb3, strongly induced EMT in HK2 cells. In the mouse renal mesangial cell line, SV40 MES 13 cells, Gb3 strongly induced phenotype changes. The EMT induced by Gb3 was inhibited by enzyme α-gal A treatment, but EMT induced by lyso-Gb3 was not abrogated by enzyme treatment. However, TGF-β receptor inhibitor (TRI, SB525334) inhibited the activation of TGF-β and EMT markers in HK2 cells with Gb3 and lyso-Gb3 treatments. This study suggested that increased plasma lyso-Gb3 has a crucial role in the development of renal fibrosis through the cell-specific induction of the EMT in Fabry disease, and that TRI treatment, alongside enzyme replacement therapy, could be a potential therapeutic option for patients with Fabry disease.     

αB-Crystallin is Associated with Intermediate Filaments in Astrocytoma Cells

B-crystallin, a major protein of the vertebrate lens and a member of the small heat shock protein family, is expressed in non-lenticular tissues, including the central nervous system, where it is found mainly in glia. In Rosenthal fibers (RF), astrocytic inclusions that accumulate in Alexander's Disease, B-crystallin is found with hsp27 and skeins of intermediate filaments (IF) of the GFAP and vimentin types. We have investigated the association between IF and B-crystallin in a human astrocytoma cell line, U-373MG, which expresses B-crystallin. Cytoskeletal preparations contained B-crystallin, and a filamentous pattern in which B-crystallin co-localized with GFAP and vimentin by double label immunofluorescence. Immuno-electronmicroscopy confirmed the localization to IF. GFAP isolated from bovine brain and re-assembled, was associated with B-crystallin. Thus, a proportion of B-crystallin in astroglia is associated with IF, and this association may be critical in the formation of RF.     

Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells

Background

We recently reported that colon tumor cells stimulate macrophages to release IL-1β, which in turn inactivates GSK3β and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.

Principal Findings

Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1β by neutralizing IL-1β antibody, or silencing of IL-1β in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1β was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Δψ) and activation of caspases were prevented by macrophages or by recombinant IL-1β. Pharmacological inhibition of IL-1β release from macrophages by vitamin D₃, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1β failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIκB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1β stabilized Snail in tumor cells in an NF-κB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1β, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.

Significance

We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1β, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D₃ halts this amplifying loop by interfering with the release of IL-1β from macrophages. Accordingly, vitamin D₃ sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent.     

Renoprotective Effect of Human Umbilical Cord–Derived Mesenchymal Stem Cells in Immunodeficient Mice Suffering from Acute Kidney Injury

It is unknown whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) can improve the renal function of patients suffering from acute kidney injury. Moreover, before beginning clinical trials, it is necessary to investigate this renoprotective effect of hUC-MSCs in a xenogeneic model of acute kidney injury. However, no previous studies have examined the application of hUC-MSCs to immunodeficient mice suffering from acute kidney injury. The objectives of this study were to examine whether hUC-MSCs could improve renal function in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice suffering from acute kidney injury, and to investigate the mechanism(s) for hUC-MSCs to improve renal function in this xenogeneic model. Early (3 hr) and late (12 hr) administrations of hUC-MSCs (10⁶ cells) were performed via the external jugular vein into NOD-SCID mice suffering from either folic acid (FA) (250 mg/kg body weight) or vehicle. The results showed that early administration of hUC-MSCs improved the renal function of NOD-SCID mice suffering from FA-induced acute kidney injury, as evidenced by decreased serum urea nitrogen and serum creatinine levels, as well as a reduced tubular injury score. The beneficial effects of hUC-MSCs were through reducing apoptosis and promoting proliferation of renal tubular cells. These benefits were independent of inflammatory cytokine effects and transdifferentiation. Furthermore, this study is the first one to show that the reduced apoptosis of renal tubular cells by hUC-MSCs in this xenogeneic model is mediated through the mitochondrial pathway, and through the increase of Akt phosphorylation.     

Use of Low-Molecular�CWeight Heparin to Decrease Mortality in Mice after Intracardiac Injection of Tumor Cells

Intracardiac injection of human tumor cells into anesthetized nude mice is an established model of bone metastasis. However, intracardiac injection of some human tumor cell lines cause acute neurologic signs and high mortality, making some potentially relevant tumor cell lines unusable for investigation. We showed that intracardiac injection of tumor cells can induce a hypercoagulable state leading to platelet consumption and thromboemboli formation and that pretreatment with intravenous injection of low-molecular–weight heparin (LMWH; enoxaparin) blocks this state. In addition, intravenous injection of enoxaparin before intracardiac injection with 2 different small-cell lung carcinoma lines, H1975 and H2126, dramatically decreased mouse mortality while still generating bone metastases. Therefore, reduction of mortality by pretreatment with LMWH increases the types of cells that can be studied in this metastasis model and decreases the number of animals used.Abbreviations: APTT, activated partial thromboplastin time; BLI, bioluminescent imaging; CBC, complete blood count; DIC, disseminated intravascular coagulation; H1975luc, H1975 cell line tagged with lucerifase–green fluorescent protein; H2126luc, H2126 cell line tagged with lucerifase–green fluorescent protein; LMWH, low-molecular–weight heparin; PT, prothrombin time; UFH, unfractionated heparinResearch using animal models mimicking the metastasis of human tumors to bone is critical for the development of cancer therapeutics. Bone metastases are present in almost all people who die of cancer and are more likely to occur with breast, prostate, lung, kidney, and thyroid cancers.^1,24 In patients with advanced breast and prostate cancers, much of the tumor burden at the time of death will be found in bone.²⁰ The pattern of bone metastases can range from purely destructive (osteolytic) to mostly osteoblastic (bone-forming) lesions. Osteolysis is accompanied by pain, bone fragility, and increased susceptibility to pathologic fracture. In osteolytic metastasis, a 2-way interaction between tumor cells and osteoclasts in the bone microenvironment leading to continued osteolysis and tumor growth is suspected.²⁰ Current therapies for bone metastases, such as bisphosphonates, are directed at inhibiting bone resorption, but other therapies are in development that specifically target tumor cell or osteoclast factors involved in the 2-way cycle between tumor growth and osteolysis.¹⁸Bone metastasis is rare in mouse models of spontaneous mammary and prostate carcinomas, experimentally implanted animal tumor models (such as syngeneic and xenograft tumors), and chemical or transgenic induction of mammary and prostate carcinomas. To increase the frequency of bone metastases, injection techniques using either orthotopic tumor cell injection into mammary glands or prostate or intracardiac injection of human tumor cell lines into the left ventricle of nude mice have been developed.^5,14,25,31 In contrast to the late stage, low incidence of metastasis after orthotopic injection, intracardiac injection of human tumor cell lines results in much higher rates of bone metastasis at an early stage in the disease, with osteolytic metastases to the metaphyses of long bones.^6,23 Development of osteolytic lesions in this model can be monitored by various methods, including radiography and, more recently, in vivo bioluminescent imaging (BLI) using lucerifase-tagged tumor cells. Bioluminescent imaging detects micrometastatic lesions and allows for serial in vivo monitoring of bone metastases.^9-11 After a BLI study, bone metastases can be assessed histologically, with tumor foci typically seen in the femur or tibia.Bone metastasis models using the intracardiac tumor injection technique have been primarily focused on a few breast (for example, MDA-MB-231) and prostate models (for example, PC3), but additional models of other tumors that interact with bone (especially lung carcinomas) need to be developed.^24,30 Intracardiac injection of some nonsmall cell lung carcinoma tumor cell lines have led to stroke-like clinical signs, including head tilt, spinning, and failure to recover from anesthesia after intracardiac injection.¹⁵ We postulated that the stroke-like clinical signs and mortality were due to thromboembolism formation immediately after intracardiac tumor cell injection.Tumor cells have procoagulant activity. Procoagulants, such as tissue factor, may be increased on the surface of or secreted into the blood by cancer cells, leading to changes in the clotting cascade.¹³ Approximately 15% of all cancer patients are affected by thromboembolic disease, including superficial and deep-vein thrombosis, arterial thrombosis and embolism, pulmonary emboli, and thrombosis of venous access devices.^12,13 Anticoagulant treatments used clinically to prevent thrombi and thromboemboli include warfarin, unfractionated heparin (UFH), and low-molecular–weight heparins (LMWH), such as enoxaparin (Lovenox, Sanofi Aventis, Bridgewater, NJ) and dalteparin (Fragmin, Pfizer, New York, NY). LMWHs are prepared through chemical, hydrolytic, or enzymatic degradation of unfractionated heparin.¹³ Both UFH and LMWH exert their anticoagulant effects by binding to antithrombin and causing a confrontational change. This change increases the interaction of antithrombin with thrombin (IIa) and activated factors X (Xa) and IX (IXa), leading to inhibition of clotting.^8,28LMWHs decrease the formation of thromboembolism and subsequent mortality in several murine models of thromboembolism and disseminated intravascular coagulation (DIC). In the murine model of thrombin-induced thromboembolism, massive deposition of intravascular fibrin—mainly within the pulmonary arteries—causes death within 5 minutes after thrombin injection.^16,22 Both UFH and LMWH inhibit thrombin and prevent mortality in this model, but bleeding times and activated partial prothrombin time (APPT) are less prolonged with LMWH.¹⁶ LMWH is also effective in preventing murine DIC in a lipopolysaccharide model, in which mice given 2 injections of lipopolysaccharide develop DIC, multiple organ failure, and die. Mice given LMWH before lipopolysaccharide administration have fewer lung and liver microthrombi and greater survival than do mice not given LMWH.^26,27Here, we evaluated the use of LMWH in mice to prevent morbidity and mortality associated with intracardiac injection of human tumor cell lines. We determined that thromboembolism occurred in intracardiac tumor-challenged mice and that LMWH blocked thromboembolism. We also determined the effect of LMWH on animal survival and subsequent development of bone metastasis in this mouse model.     

1H NMR Based Serum Metabolic Profiles Associated with Pathological Progression of Pancreatic Islet β Cell Tumor in Rip1-Tag2 Mice

Pancreatic islet β cell tumor is the most common islet cell tumor. A well-characterized tumor progression in Rip1-Tag2 mice undergoes five stages, involving normal, hyperplasia, angiogenic islets, tumorigenesis and invasive carcinoma. ¹H NMR based metabonomics was applied to identify potential biomarkers for monitoring pancreatic islet β cell tumor progression in Rip1-Tag2 mice. Multivariate analysis results showed the serum metabonome at hyperplasia stage shared the similar characteristics with the ones at normal stage as a result of slight proliferation of pancreatic islet β cells. At angiogenic islets stage, the up-regulated glycolysis, disturbed choline and phospholipid metabolism composed the metabolic signature. In addition to the changes mentioned above, several metabolites were identified as early biomarkers for tumorigenesis, including increased methionine, citrate and choline, and reduced acetate, taurine and glucose, which suggested the activated energy and amino acid metabolism. All the changes were aggravated at invasive carcinoma stage, coupled with notable changes in alanine, glutamate and glycine. Moreover, the distinct metabolic phenotype was found associated with the implanting of SV40 large T antigen in Rip1-Tag2 mice. The combined metabolic and multivariate statistics approach provides a robust method for screening the biomarkers of disease progression and examining the association between gene and metabolism.     

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Endothelial Dysfunction in Tristetraprolin-deficient Mice Is Not Caused by Enhanced Tumor Necrosis Factor-α Expression

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP^−/− mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP^−/− mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP^−/− mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP^−/− mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP^−/− animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP^−/− animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.     

Increased α-Defensins 1-3 Production by Dendritic Cells in HIV-Infected Individuals Is Associated with Slower Disease Progression

Background

Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.

Methodology/Principal Findings

Monocyte-derived DC (MDDC) were analyzed comparatively in healthy controls (HC) and HIV-infected patients, including untreated “elite” and “viremic” controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of α-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05). Secreted α-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher α-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm³, lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters.

Conclusions/Significance

High production of α-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of α-defensins1-3 to damage the virions prior and/or after their internalization by immature DC, and hence favoring a more efficient viral processing and presentation to HIV-specific CD4+ T cells, without or with a minor rate of transmission of infectious HIV-1 virions.     

Impaired Clearance of Early Apoptotic Cells Mediated by Inhibitory IgG Antibodies in Patients with Primary Sj?gren's Syndrome

Objectives

Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren''s syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis).

Design

ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD.

Results

Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients'' serum-free conditions.

Conclusion

Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes'' dysfunction.     

Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-β Production and Th17 Differentiation

It has recently been reported that the CD40-CD40 ligand (CD40L) interaction is important in Th17 development. In addition, transforming growth factor—beta (TGF-β) promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-β. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-β production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-β production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells.