bluuebunny/crossref_metadata_2025 huggingface dataset:
A Transformed Cell Population Derived from Cultured Mesenchymal Stem Cells has no Functional Effect after Transplantation into the Injured Heart
Authors: Furlani Dario , Li Wenzhong , Pittermann Erik , Klopsch Christian , Wang Liang , Knopp Agnes , Jungebluth Philipp , Thedinga Elke , Havenstein Carolin , Westien Ingeborg , Ugurlucan Murat , Li Ren-Ke , Ma Nan , Steinhoff Gustav
Published: June 2009

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells characterized by their self-renewal and differentiation potential. Accumulating clinical and preclinical evidence indicate MSCs are a promising cell source for regenerative medical therapies. However, undesirable immortalization, spontaneous transformation, and tumorigenic potential from long-term cultured MSCs have been reported in human and mouse. We report rat MSCs isolated from young donors could undergo transformation in early passage culture. We aimed to characterize the transformed population and determine their therapeutic effects after intracardiac transplantation in the infarcted myocardium. MSCs were isolated from bone marrow of Lewis rats according to standard protocols and cultured under standard conditions. Phenotype of growing cells was assessed by flow cytometry. Following acute myocardial infarction in rats, cells were delivered by intracardiac injection. Cardiac functions were assessed by pressure–volume loops. Infarction size and pathologic effects were evaluated after 6 weeks. The abnormal colonies were detected in culture as early at passage 3. They were noted to appear as distinctly different morphology from typical MSCs, which changed from a normal elongated spindle shape to a compact abnormal morphology. They exhibited rapid cell proliferation. Some subclones lost contact inhibition of cell division and formed multilayer aggregates. Chromosomal instability was detected. They were devoid of surface markers CD29, CD44, CD90, and CD117. Furthermore, there was no significant improvement on infarction size and cardiac function 6 weeks after cell transplantation. Our study highlights the need for establishment of biosafety criteria in regulating culture-expanded MSCs to achieve the full clinical therapeutic benefits.
Percutaneous Cell Delivery into the Heart Using Hydrogels Polymerizing in Situ
Authors: Martens Timothy P. , Godier Amandine F. G. , Parks Jonathan J. , Wan Leo Q. , Koeckert Michael S. , Eng George M. , Hudson Barry I. , Sherman Warren , Vunjak-Novakovic Gordana
Published: June 2009

Heart disease is the leading cause of death in the US. Following an acute myocardial infarction, a fibrous, noncontractile scar develops, and results in congestive heart failure in more than 500,000 patients in the US each year. Muscle regeneration and the induction of new vascular growth to treat ischemic disorders of the heart can have significant therapeutic implications. Early studies in patients with chronic ischemic systolic left ventricular dysfunction (SLVD) using skeletal myoblasts or bone marrow-derived cells report improvement in left ventricular ejection function (LVEF) and clinical status, without notable safety issues. Nonetheless, the efficacy of cell transfer for cardiovascular disease is not established, in part due to a lack of control over cell retention, survival, and function following delivery. We studied the use of biocompatible hydrogels polymerizable in situ as a cell delivery vehicle, to improve cell retention, survival, and function following delivery into the ischemic myocardium. The study was conducted using human bone marrow-derived mesenchymal stem cells and fibrin glue, but the methods are applicable to any human stem cells (adult or embryonic) and a wide range of hydrogels. We first evaluated the utility of several commercially available percutaneous catheters for delivery of viscous cell/hydrogel suspensions. Next we characterized the polymerization kinetics of fibrin glue solutions to define the ranges of concentrations compatible with catheter delivery. We then demonstrate the in vivo effectiveness of this preparation and its ability to increase cell retention and survival in a nude rat model of myocardial infarction.
Two-Dimensional Speckle Tracking Strain Analysis for Efficacy Assessment of Myocardial Cell Therapy
Authors: Nasseri Boris A. , Kukucka Marian , Dandel Michael , Knosalla Christoph , Choi Yeong-Hoon , Ebell Wolfram , Hetzer Roland , Stamm Christof
Published: June 2009

The subtle effects of transplanted bone marrow cells (BMC) on regional myocardial behavior in patients with ischemic heart disease are difficult to assess. Novel echocardiographic techniques can quantify regional myocardial deformation (strain) and distinguish between passive and active wall motion. We hypothesized that this technique may help delineate cell therapy-induced changes in regional LV contractility that escape clinical routine studies. Twelve patients with coronary artery disease and impaired LV function (LVEF &<35%) underwent CABG surgery plus intramyocardial injection of autologous bone marrow mononuclear cells. Between two and five predefined segments of ischemic myocardium per patient received BMCs, and untreated ischemic segments served as internal controls. Segmental echocardiographic analysis of peak systolic strain by speckle tracking was performed before and 1 year after surgery and compared with standard wall motion analysis. Two patients died during the follow-up period. In the remaining 10 patients, mean LVEF increased from 24.5 ± 10% to 32.1 ± 11% ( p = 0.02). A moderate improvement of systolic function was noted in ischemic control segments by both wall motion score (WMS) and 2D strain echocardiography (2DSE). In BMC-treated segments, WMS improved slightly, but the data failed to reach statistical significance. As assessed by 2DSE, however, systolic function of BMC-treated segments improved by nearly 100%. 2DSE proved to detect BMC-induced change with 30-fold higher sensitivity than WMS, and the Receiver Operating Characteristic curve (ROC) confirmed the diagnostic precision of 2DSE (area-under-the-ROC = 0.87). We conclude that echocardiographic speckle tracking two-dimensional strain analysis can detect cell therapy-induced changes in regional contractile function that may escape detection by standard wall motion assessment. Thus, 2DSE may be a useful tool for the further development of clinical cardiac cell therapy.
Myoblast Transfer in Ischemic Heart Failure: Effects on Rhythm Stability
Authors: Sherman Warren , He Kun-Lun , Yi Geng-Hua , Wang Jie , Harvey Jack , Lee Myung J. , Haimes Howard , Lee Paul , Miranda Emma , Kanwal Sunil , Burkhoff Daniel
Published: June 2009

Skeletal myoblast (SM) implantation promotes recovery of myocardial function after ischemic injury. Clinical observations suggest an association of SM implantation and ventricular arrhythmias. Support for this link has been sought in animal studies, but none employing models of congestive heart failure. In a canine model of postinfarction congestive heart failure (CHF) we compared the frequency of rhythm disturbances using ambulatory electrocardiography monitoring following skeletal myoblast or saline (SAL) implantation. In 19 mongrel dogs ischemic injury and CHF were induced by intracoronary microsphere infusions. Direct intramyocardial injection of autologous skeletal myoblasts (ASM) (2.7–8.3 × 108 cells) or SAL controls was administered to 11 and 8 dogs, respectively. Serial echocardiography and 24-h ambulatory electrocardiography were recorded at baseline (after CHF induction) and at 4 weeks and at 8–10 weeks after injection. Comparisons between groups of left ventricular ejection fraction (LVEF) and the frequency of ventricular arrhythmias, supraventricular arrhythmias, and measures of heart rate variability (HRV) were made at each of the three time points. LVEF increased from 41 ± 6% to 47 ± 2% ( p < 0.03) in the ASM group, and did not change (42 ± 6% to 40 ± 2%, p = ns) in SAL. After injection, no differences were seen in the number of dogs demonstrating ventricular tachycardia ( n = 3 vs. n = 2, p = ns) or frequent PVCs ( n = 3 vs. n = 3, p = ns) in the ASM versus SAL groups, respectively. Significant changes were observed in a time-domain measure of HRV, standard deviation of normal-to-normal RR interval (in ms: 4 weeks 174 ± 95 vs. 242 ± 19; 8 weeks 174 ± 78 vs. 276 ± 78, ASM vs. SAL), but not in other time domain parameters. In this canine model of ischemic CHF, ASM implantation did not result in a significant increase in ventricular arrhythmias compared to controls animals. The potential for ASM implantation to affect time–domain parameters of HRV merits further study.
Intramyocardial Injection of Autologous Platelet-Rich Plasma Combined with Transmyocardial Revascularization
Authors: Wehberg K. E. , Answini G. , Wood D. , Todd J. , Julian J. , Ogburn N. , Allen K. B.
Published: June 2009

Transmyocardial revascularization (TMR) can improve refractory angina but does not consistently demonstrate an effect on myocardial function. Recent studies suggest a synergistic effect between TMR and exogenously supplied growth factors. We evaluated the clinical role of intramyocardial injection of autologous platelet-rich plasma (PRP) in conjunction with TMR. Twenty-five nonrevascularizable patients with class III/IV angina underwent minimally invasive sole therapy TMR during a 5-year period at a single institution. Group 1 (14 patients) underwent TMR alone while group 2 (11 patients) underwent TMR plus injection of PRP (Magellan plasma separator) between TMR channels. Blinded angina assessment and ejection fraction (EF) were measured preoperatively and at 6 months postoperatively. Baseline EF (57 ± 10% vs. 50 ± 7%), angina class (3.7 ± 0.5 vs. 3.7 ± 0.5), and the number of channels (48 ± 5 vs. 48 ± 4) were statistically similar in both groups. At 6 months, two class angina relief was similar in both groups (92% vs. 100%, p = 0.4); however, the TMR + PRP group had a lower average angina score (1.3 vs. 0.4, p = 0.07) and more were angina free (23% vs. 78%, p = 0.04) than the TMR-alone group. EF improved in the TMR + PRP group (–2.0% vs. +9.0%, p = 0.07) compared to the TMR-alone group. Two 30-day morbidities occurred in the TMR-alone group (atrial fibrillation and left pleural effusion) and one mortality occurred in the TMR + PRP group. Intramyocardial injection of autologous PRP combined with TMR may be more efficacious at relieving angina and improving myocardial function than TMR alone.
Implantation of a Three-Dimensional Fibroblast Matrix Improves Left Ventricular Function and Blood Flow after Acute Myocardial Infarction
Authors: Thai Hoang M. , Juneman Elizabeth , Lancaster Jordan , Hagerty Tracy , Do Rose , Castellano Lisa , Kellar Robert , Williams Stuart , Sethi Gulshan , Schmelz Monika , Gaballa Mohamed , Goldman Steven
Published: June 2009

This study was designed to determine if a viable biodegradable three-dimensional fibroblast construct (3DFC) patch implanted on the left ventricle after myocardial infarction (MI) improves left ventricular (LV) function and blood flow. We ligated the left coronary artery of adult male Sprague-Dawley rats and implanted the 3DFC at the time of the infarct. Three weeks after MI, the 3DFC improved LV systolic function by increasing ( p < 0.05) ejection fraction (37 ± 3% to 62 ± 5%), increasing regional systolic displacement of the infarcted wall (0.04 ± 0.02 to 0.11 ± 0.03 cm), and shifting the passive LV diastolic pressure volume relationship toward the pressure axis. The 3FDC improved LV remodeling by decreasing ( p < 0.05) LV end-systolic and end-diastolic diameters with no change in LV systolic pressure. The 3DFC did not change LV end-diastolic pressure (LV EDP; 25 ± 2 vs. 23 ± 2 mmHg) but the addition of captopril (2mg/L drinking water) lowered ( p < 0.05) LV EDP to 12.9 ± 2.5 mmHg and shifted the pressure–volume relationship toward the pressure axis and decreased ( p < 0.05) the LV operating end-diastolic volume from 0.49 ± 0.02 to 0.34 ± 0.03 ml. The 3DFC increased myocardial blood flow to the infarcted anterior wall after MI over threefold ( p < 0.05). This biodegradable 3DFC patch improves LV function and myocardial blood flow 3 weeks after MI. This is a potentially new approach to cell-based therapy for heart failure after MI.
Regioselective alkynylation followed by Suzuki coupling of 2,4-dichloroquinoline: Synthesis of 2-alkynyl-4-arylquinolines
Authors: Reddy Ellanki Amarender , Islam Aminul , Mukkanti K , Bandameedi Venkanna , Bhowmik Dipal Ranjan , Pal Manojit
Published: July 2009

A two step synthesis of 2-alkynyl-4-arylquinolines has been accomplished via Pd/C-mediated regioselective C-2 alkynylation of 2,4-dichloroquinoline in water followed by Suzuki coupling at C-4 of the resulting 4-chloro derivative.
Effects of Photosystem‐II‐Interfering Herbicides Atrazine and Bentazon on the Soybean Transcriptome
Authors: Zhu Jin , Patzoldt William L. , Radwan Osman , Tranel Patrick J. , Clough Steven J.
Published: July 2009

Atrazine and bentazon are both photosystem‐II (PSII)–inhibiting herbicides that interfere with photosynthetic electron transport, provoking oxidative stress. While atrazine is lethal to soybean [Glycine max (L.) Merr.], bentazon does not kill soybean because of the capability of soybeans to metabolize the herbicide. Gene expression profiling was conducted using cDNA microarrays to understand the responses of soybeans to PSII interruption and concomitant stress caused by atrazine and bentazon by monitoring expression at 1, 2, 4, and 8 h after treatment (HAT). The microarray study revealed that 6646 genes were differentially expressed with high statistical significance over the experiment, with 88% of them sharing similar expression pattern between the atrazine and bentazon treatments. Many genes related to xenobiotic detoxification and antioxidation, such as cytochrome P450s, glutathione‐S‐transferases, superoxide dismutases, catalases, and tocophero cyclases, were induced by the herbicides. The study also discovered plants treated with bentazon started to recover between 4 and 8 HAT as reflected in the decreased amplitude of fold changes of most genes from 4 to 8 HAT. The 12% of the genes that were differentially expressed between atrazine and bentazon were largely related to cell recovery, such as genes related to ribosomal components.
MODELLING MODAL SHIFT DUE TO THE ENHANCED LEVEL OF BUS SERVICE
Authors: Vedagiri Perumal , Arasan Venkatachalam Thamizh
Published: July 2009

This study is concerned with the estimation of the probable shift of auto‐rickshaw (three wheeled motorized para‐transit vehicle) users to the bus due to an increase in its level of service after providing exclusive bus lanes on Indian city roads carrying heterogeneous traffic. The quantum of an increase in the level of bus service due to the introduction of an exclusive bus lane was determined using a recently developed simulation model of heterogeneous traffic flow. The data on other factors (variables) that might cause modal shift from the auto‐rickshaw to the bus was collected conducting a home‐interview survey based on the stated preference approach. A binary logit model of mode‐choice was then calibrated using the collected data and the model was also validated using a holdout sample. A mode‐choice probability curve to depict the possible shift of auto‐rickshaw users to the bus is developed taking difference in the travel times of two‐modes as the basis to serve as a user friendly tool to analyze the possible modal shift for a wide range of the values of the involved variables.
INVESTIGATION INTO THE STABILITY OF DRIVING AN AUTOMOBILE ON THE ROAD PAVEMENT WITH RUTS
Authors: Vansauskas Vidmantas , Bogdevičius Marijonas
Published: July 2009

The article reviews the stability of an automobile on the road pavement with ruts. The problems of road safety depend on many factors. One of those is the quality of the road surface. Big heavy lorries deform the road surface causing ruts especially in the first line of the road. The article analyzes the influence of the geometry of ruts on the stability of the automobile. A mathematical model of a 3D automobile and the interaction between wheels and the road surface and the geometry of the road surface are presented. The mathematical criterion of the stability of the moving automobile on the road surface with ruts is laid down. The results of mathematical simulation and the criterion of the stability of the moving automobile (60 km/h) on the road surface depending on the depth of ruts are introduced.
Evidence for Genetic Association and Interaction Between the TYK2 and IRF5 Genes in Systemic Lupus Erythematosus
Authors: HELLQUIST ANNA , JÄRVINEN TIINA M. , KOSKENMIES SARI , ZUCCHELLI MARCO , ORSMARK-PIETRAS CHRISTINA , BERGLIND LINDA , PANELIUS JAANA , HASAN TAINA , JULKUNEN HEIKKI , D’AMATO MAURO , SAARIALHO-KERE ULPU , KERE JUHA
Published: July 2009

Objective.

Several candidate genes have been implicated in susceptibility for systemic lupus erythematosus (SLE), a complex autoimmune disease. The proposed genes include members of the type I interferon (IFN) pathway and genes involved in immunological defense functions. Our aim was to systematically replicate 6 such genes, TYK2, IRF5, CTLA4, PDCD1, FCGR2A, and NOD2.

Methods.

Single-nucleotide polymorphisms in TYK2, IRF5, CTLA4, PDCD1, FCGR2A, and NOD2 were genotyped in 277 SLE patients and 356 healthy controls from Finland, giving a power of 42%–70% for different genes at published allele frequencies.

Results.

Significant association was seen for rs2304256 (p = 0.0001) and rs12720270 (p = 0.0031) in TYK2 and rs10954213 (p = 0.0043) in IRF5 in our samples, but not for the other genes. We found evidence for genetic interaction (p = 0.014) between rs2304256 in TYK2 and rs10954213 in IRF5, both members of the type I IFN pathway, strengthening the role of the type I IFN pathway in the pathogenesis of SLE.

Conclusion.

The IFN pathway genes IRF5 and TYK2 may act epistatically in increasing risk for SLE, but our lack of replication does not exclude effects of the other genes studied.
Mechanisms of Opioid-Mediated Inhibition of Human T Cell Receptor Signaling
Authors: Börner Christine , Warnick Beate , Smida Michal , Hartig Roland , Lindquist Jonathan A. , Schraven Burkhart , Höllt Volker , Kraus Jürgen
Published: June 2009

Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid β-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-κB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by μ opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since β-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for β-endorphin in the physiological regulation of T cell activation.
Focal Adhesion Kinase Regulates Pathogen-Killing Capability and Life Span of Neutrophils via Mediating Both Adhesion-Dependent and -Independent Cellular Signals
Authors: Kasorn Anongnard , Alcaide Pilar , Jia Yonghui , Subramanian Kulandayan K. , Sarraj Bara , Li Yitang , Loison Fabien , Hattori Hidenori , Silberstein Leslie E. , Luscinskas William F. , Luo Hongbo R.
Published: June 2009

Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.
The Opposite Effects of Acute and Chronic Alcohol on Lipopolysaccharide-Induced Inflammation Are Linked to IRAK-M in Human Monocytes
Authors: Mandrekar Pranoti , Bala Shashi , Catalano Donna , Kodys Karen , Szabo Gyongyi
Published: June 2009

Impaired host defense after alcohol use is linked to altered cytokine production, however, acute and chronic alcohol differently modulate monocyte/macrophage activation. We hypothesized that in human monocytes, acute alcohol induces hyporesponsiveness to LPS, resulting in decreased TNF-α, whereas chronic alcohol increases TNF-α by sensitization to LPS. We found that acute alcohol increased IL-1R-associated kinase-monocyte (IRAK-M), a negative regulator of IRAK-1, in human monocytes. This was associated with decreased IκBα kinase activity, NFκB DNA binding, and NFκB-driven reporter activity after LPS stimulation. In contrast, chronic alcohol decreased IRAK-M expression but increased IRAK-1 and IKK kinase activities, NFκB DNA binding, and NFκB-reporter activity. Inhibition of IRAK-M in acute alcohol-exposed monocytes using small interfering RNA restored the LPS-induced TNF-α production whereas over-expression of IRAK-M in chronic alcohol macrophages prevented the increase in TNF-α production. Addition of inhibitors of alcohol metabolism did not alter LPS signaling and TNF-α production during chronic alcohol exposure. IRAK-1 activation induces MAPKs that play an important role in TNF-α induction. We determined that acute alcohol decreased but chronic alcohol increased activation of ERK in monocytes and ERK inhibitor, PD98059, prevented the chronic alcohol-induced increase in TNF-α. In summary, inhibition of LPS-induced NFκB and ERK activation by acute alcohol leads to hyporesponsiveness of monocytes to LPS due to increased IRAK-M. In contrast, chronic alcohol sensitizes monocytes to LPS through decreased IRAK-M expression and activation of NFκB and ERK kinases. Our data indicate that IRAK-M is a central player in the opposite regulation of LPS signaling by different lengths of alcohol exposure in monocytes.
Autocrine IL-10 Induces Hallmarks of Alternative Activation in Macrophages and Suppresses Antituberculosis Effector Mechanisms without Compromising T Cell Immunity
Authors: Schreiber Tanja , Ehlers Stefan , Heitmann Lisa , Rausch Alexandra , Mages Jörg , Murray Peter J. , Lang Roland , Hölscher Christoph
Published: June 2009

Elevated IL-10 has been implicated in reactivation tuberculosis (TB). Since macrophages rather than T cells were reported to be the major source of IL-10 in TB, we analyzed the consequences of a macrophage-specific overexpression of IL-10 in transgenic mice (macIL-10-transgenic) after aerosol infection with Mycobacterium tuberculosis (Mtb). MacIL-10 transgenic mice were more susceptible to chronic Mtb infection than nontransgenic littermates, exhibiting higher bacterial loads in the lung after 12 wk of infection and dying significantly earlier than controls. The differentiation, recruitment, and activation of Th1 cells as well as the induction of IFN-γ-dependent effector genes against Mtb were not affected by macrophage-derived IL-10. However, microarray analysis of pulmonary gene expression revealed patterns characteristic of alternative macrophage activation that were overrepresented in Mtb-infected macIL-10 transgenic mice. Importantly, arginase-1 gene expression and activity were strikingly enhanced in transgenic mice accompanied by a reduced production of reactive nitrogen intermediates. Moreover, IL-10-dependent arginase-1 induction diminished antimycobacterial effector mechanisms in macrophages. Taken together, macrophage-derived IL-10 triggers aspects of alternative macrophage activation and promotes Mtb recrudescence independent of overt effects on anti-TB T cell immunity.
Lethal Effect of CD3-Specific Antibody in Mice Deficient in TGF-β1 by Uncontrolled Flu-Like Syndrome
Authors: Perruche Sylvain , Zhang Pin , Maruyama Takashi , Bluestone Jeffrey A. , Saas Philippe , Chen WanJun
Published: June 2009

CD3-specific Ab therapy results in a transient, self-limiting, cytokine-associated, flu-like syndrome in experimental animals and in patients, but the underlying mechanism for this spontaneous resolution remains elusive. By using an in vivo model of CD3-specific Ab-induced flu-like syndrome, we show in this paper that a single injection of sublethal dose of the Ab killed all TGF-β1−/− mice. The death of TGF-β1−/− mice was associated with occurrence of this uncontrolled flu-like syndrome, as demonstrated by a sustained storm of systemic inflammatory TNF and IFN-γ cytokines. We present evidence that deficiency of professional phagocytes to produce TGF-β1 after apoptotic T cell clearance may be responsible, together with hypersensitivity of T cells to both activation and apoptosis, for the uncontrolled inflammation. These findings indicate a key role for TGF-β1 and phagocytes in protecting the recipients from lethal inflammation and resolving the flu-like syndrome after CD3-specific Ab treatment. The study may also provide a novel molecular mechanism explaining the early death in TGF-β1−/− mice.
Heat Shock Protein 60, via MyD88 Innate Signaling, Protects B Cells from Apoptosis, Spontaneous and Induced
Authors: Cohen-Sfady Michal , Pevsner-Fischer Meirav , Margalit Raanan , Cohen Irun R.
Published: June 2009

We recently reported that heat shock protein 60 (HSP60) via TLR4 signaling activates B cells and induces them to proliferate and secrete IL-10. We now report that HSP60 inhibits mouse B cell apoptosis, spontaneous or induced by dexamethasone or anti-IgM activation. Unlike HSP60 enhancement of B cell proliferation and IL-10 secretion, TLR4 signaling was not required for the inhibition of apoptosis by HSP60; nevertheless, MyD88 was essential. Inhibition of apoptosis by HSP60 was associated with up-regulation of the antiapoptotic molecules Bcl-2, Bcl-xL, and survivin, maintenance of the mitochondrial transmembrane potential, and inhibition of caspase-3 activation. Moreover, B cells incubated with HSP60 manifested prolonged survival following transfer into recipient mice. These results extend the varied role of HSP60 in the innate regulation of the adaptive immune response.
Activation of the Akt-NF-κB Pathway by Subtilase Cytotoxin through the ATF6 Branch of the Unfolded Protein Response
Authors: Yamazaki Hiroaki , Hiramatsu Nobuhiko , Hayakawa Kunihiro , Tagawa Yasuhiro , Okamura Maro , Ogata Ryouji , Huang Tao , Nakajima Shotaro , Yao Jian , Paton Adrienne W. , Paton James C. , Kitamura Masanori
Published: June 2009

Shiga toxin has the potential to induce expression of inflammation-associated genes, although the underlying mechanisms are not well understood. We examined the effects of subtilase cytotoxin (SubAB), an AB5 toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-κB. SubAB is known to be a protease which selectively degrades GRP78/Bip. Treatment of NRK-52E cells with SubAB caused rapid cleavage of GRP78. Following the degradation of GRP78, transient activation of NF-κB was observed with a peak at 6–12 h; the activation subsided within 24 h despite the continuous absence of intact GRP78. The activation of NF-κB was preceded by transient phosphorylation of Akt. Treatment of the cells with a selective inhibitor of Akt1/2 or an inhibitor of PI3K attenuated SubAB-induced NF-κB activation, suggesting that activation of Akt is an event upstream of NF-κB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-κB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1α, XBP1, or PERK did not attenuate activation of NF-κB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-κB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-κB through the ATF6 branch of the UPR.
Patent Filarial Infection Modulates Malaria-Specific Type 1 Cytokine Responses in an IL-10-Dependent Manner in a Filaria/Malaria-Coinfected Population
Authors: Metenou Simon , Dembélé Benoit , Konate Siaka , Dolo Housseini , Coulibaly Siaka Y. , Coulibaly Yaya I. , Diallo Abdallah A. , Soumaoro Lamine , Coulibaly Michel E. , Sanogo Dramane , Doumbia Salif S. , Wagner Marissa , Traoré Sekou F. , Klion Amy , Mahanty Siddhartha , Nutman Thomas B.
Published: June 2009

The effect of filarial infections on malaria-specific immune responses was investigated in Malian villages coendemic for filariasis (Fil) and malaria. Cytokines were measured from plasma and Ag-stimulated whole blood from individuals with Wuchereria bancrofti and/or Mansonella perstans infections (Fil+; n = 19) and those without evidence of filarial infection (Fil−; n = 19). Plasma levels of IL-10 (geometric mean [GM], 22.8 vs 10.4) were higher in Fil+ compared with Fil−, whereas levels of IFN-inducible protein (IP)-10 were lower in Fil+ (GM, 66.3 vs 110.0). Fil+ had higher levels of spontaneously secreted IL-10 (GM, 59.3 vs 6.8 pg/ml) and lower levels of IL-2 (1.0 vs 1.2 pg/ml) than did Fil−. Although there were no differences in levels of Staphylococcus aureus enterotoxin B-induced cytokines between the two groups, Fil+ mounted lower IL-12p70 (GM, 1.11 vs 3.83 pg/ml; p = 0.007), IFN-γ (GM, 5.44 vs 23.41 pg/ml; p = 0.009), and IP-10 (GM, 29.43 vs 281.7 pg/ml; p = 0.007) responses following malaria Ag (MalAg) stimulation compared with Fil−. In contrast, Fil+ individuals had a higher MalAg-specific IL-10 response (GM, 7318 pg/ml vs 3029 pg/ml; p = 0.006) compared with those without filarial infection. Neutralizing Ab to IL-10 (but not to TGFβ) reversed the down-regulated MalAg-specific IFN-γ and IP-10 (p < 0.001) responses in Fil+. Together, these data demonstrate that filarial infections modulate the Plasmodium falciparum-specific IL-12p70/IFN-γ secretion pathways known to play a key role in resistance to malaria and that they do so in an IL-10-dependent manner.
T Cell Responses in the Absence of IFN-γ Exacerbate Uterine Infection with Chlamydia trachomatis
Authors: Gondek David C. , Roan Nadia R. , Starnbach Michael N.
Published: June 2009

Infection with the obligate intracellular bacterium Chlamydia trachomatis is controlled primarily by IFN-γ and Th1 immunity. In this study, we used cells from a Chlamydia-specific CD4+ TCR-transgenic mouse to assess the role of IFN-γ in development of Th1 immunity. We show that secretion of host IFN-γ or the ability of host cells to respond to secreted IFN-γ is not required to initiate a Th1 immune response. Additionally, we found that Ag-specific CD4+ cells that were preskewed toward Th1 confer protection, whereas cells preskewed toward Th2 cause a previously unreported exacerbation of disease leading to higher bacterial load. Chlamydia-specific Th1 cells transferred into an IFN-γ−/− recipient mouse demonstrate protective effects, but the same cells exacerbate bacterial burden when transferred into IFN-γR−/− mice. Thus, we demonstrate that the secretion of IFN-γ is necessary for protection against C. trachomatis and that in the absence of host cell IFN-γR expression, both Th1 and Th2 cells lead to increased burden of C. trachomatis.
Role of MAPK Kinase 6 in Arthritis: Distinct Mechanism of Action in Inflammation and Cytokine Expression
Authors: Yoshizawa Toshio , Hammaker Deepa , Boyle David L. , Corr Maripat , Flavell Richard , Davis Roger , Schett Georg , Firestein Gary S.
Published: June 2009

Development of p38α inhibitors for rheumatoid arthritis has been hindered by toxicity and limited efficacy. Therefore, we evaluated whether MKK6, an upstream kinase that regulates multiple p38 isoforms, might be an alternative therapeutic target in inflammatory arthritis. Wild-type (WT), MKK6−/−, and MKK3−/− mice were administered K/B×N serum to induce arthritis. Articular expression of activated kinases and cytokines was determined by Western blot, qPCR, ELISA, and multiplex analysis. Immunoprecipitation and confocal microscopy experiments were performed to determine the subcellular location of MKK6, P-p38, and MAPKAPK2 (MK2). Arthritis scores were significantly lower in MKK6−/− mice compared with WT mice. Joint destruction and osteoclast differentiation were lower in MKK6−/−, as were articular IL-6 and matrix metalloproteinase-3 expression. Phospho-p38 levels were modestly decreased in the joints of arthritic MKK6−/− mice compared with WT but were significantly higher than MKK3−/− mice. P-MK2 was low in MKK6−/− and MKK3−/− mice. Uncoupled p38 and MK2 activation was also observed in cultured, MKK6−/− FLS and confirmed using kinase assays. Immunoprecipitation assays and confocal microscopy showed that P-p38 and MK2 colocalized in activated WT but not MKK6−/− FLS. Distinct patterns of cytokine production were observed in MKK6−/− and MKK3−/− cells. MKK6 deficiency suppresses inflammatory arthritis and joint destruction, suggesting it might be a therapeutic target for inflammation. Although MKK3 and MKK6 activate the p38 pathway, they regulate distinct subsets of proinflammatory cytokines. MKK6 appears mainly to facilitate p38 and MK2 colocalization in the nucleus rather than to phosphorylate p38.
An Activating and Inhibitory Signal from an Inhibitory Receptor LMIR3/CLM-1: LMIR3 Augments Lipopolysaccharide Response through Association with FcRγ in Mast Cells
Authors: Izawa Kumi , Kitaura Jiro , Yamanishi Yoshinori , Matsuoka Takayuki , Kaitani Ayako , Sugiuchi Masahiro , Takahashi Mariko , Maehara Akie , Enomoto Yutaka , Oki Toshihiko , Takai Toshiyuki , Kitamura Toshio
Published: June 2009

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of FcεRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by FcεRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRγ. Analysis of FcRγ-deficient BMMCs demonstrated that both Y276/303 and FcRγ played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRγ and thereby functions as an activating receptor in concert with TLR4 stimulation.
Pathogen-Induced Interleukin-1β Processing and Secretion Is Regulated by a Biphasic Redox Response
Authors: Tassi Sara , Carta Sonia , Vené Roberta , Delfino Laura , Ciriolo Maria Rosa , Rubartelli Anna
Published: June 2009

In this study, we show that IL-1β processing and secretion induced by pathogen-associated molecular pattern (PAMP) molecules in human monocytes is regulated by a biphasic redox event including a prompt oxidative stress and a delayed antioxidant response. Namely, PAMPs induce an early generation of reactive oxygen species (ROS) followed by increase of intracellular thioredoxin and release of reduced cysteine: this antioxidant phase is paralleled by secretion of mature IL-1β. ROS production and antioxidant response are both required, because either inhibitors of NADPH oxidase and of thioredoxin reductase impair IL-1β secretion. These inhibitors also hinder cysteine release and consequently prevent reduction of the extracellular medium: addition of exogenous reducing agents restores IL-1β secretion. Not only silencing of thioredoxin, but also of the ROS scavenger superoxide dismutase 1 results in inhibition of IL-1β secretion. Thus, PAMP-induced ROS trigger an antioxidant response involving intracellular redox enzymes and release of cysteine, ultimately required for IL-1β processing and secretion.
Targeted Liposomal Delivery of TLR9 Ligands Activates Spontaneous Antitumor Immunity in an Autochthonous Cancer Model
Authors: Hamzah Juliana , Altin Joseph G. , Herringson Thomas , Parish Christopher R. , Hämmerling Günter J. , O'Donoghue Helen , Ganss Ruth
Published: June 2009

Accessibility of tumors for highly effective local treatment represents a major challenge for anticancer therapy. Immunostimulatory oligodeoxynucleotides (ODN) with CpG motifs are ligands of TLR9, which prime spontaneous antitumor immunity, but are less effective when applied systemically. We therefore developed a liposome-based agent for selective delivery of CpG-ODN into the tumor environment. A peptide that specifically targets angiogenic endothelial cells in a transgenic tumor model for islet cell carcinogenesis was engrafted into CpG-ODN containing liposomes. Intravenous injection of these liposomes resulted in specific accumulation around tumor vessels, increased uptake by tumor-resident macrophages, and retention over time. In contrast, nontargeted liposomes did not localize to the tumor vasculature. Consequently, only vascular targeting of CpG-ODN liposomes provoked a marked inflammatory response at vessel walls with enhanced CD8+ and CD4+ T cell infiltration and, importantly, activation of spontaneous, tumor-specific cytotoxicity. In a therapeutic setting, 40% of tumor-bearing, transgenic mice survived beyond week 45 after systemic administration of vascular-directed CpG-ODN liposomes. In contrast, control mice survived up to 30 wk. Therapeutic efficacy was further improved by increasing the frequency of tumor-specific effector cells through adoptive transfers. NK cells and CD8+ T cells were major effectors which induced tumor cell death and acted in conjunction with antivascular effects. Thus, tumor homing with CpG-ODN-loaded liposomes is as potent as direct injection of free CpG-ODN and has the potential to overcome some major limitations of conventional CpG-ODN monotherapy.
TLR9 Activation Coupled to IL-10 Deficiency Induces Adverse Pregnancy Outcomes
Authors: Thaxton Jessica E. , Romero Roberto , Sharma Surendra
Published: June 2009

Pregnancy outcome is severely compromised by intrauterine infections and inflammation. Although the pregnant uterine microenvironment is replete with innate immune cells and TLR expression, the mechanisms that facilitate adverse effects of their activation are largely unknown. In this study, we mimic the activation of TLR9 with its pathogenic ligand hypomethylated CpG and demonstrate that IL-10 proficiency protects against CpG-induced pregnancy complications. We show that fetal resorption and preterm birth are rapidly induced in IL-10−/− mice by low doses of CpG (∼25 μg/mouse) when injected i.p. on gestational day 6 or gestational day 14, respectively. In contrast, wild-type mice failed to experience such effects at comparable doses, but pups born at term displayed craniofacial/limb defects in response to higher doses (∼400 μg/mouse). Pregnancy complications in IL-10−/− mice were associated with unexpected and robust TLR9-triggered activation and amplification of uterine neutrophil and macrophage subpopulations followed by their migration to the placental zone. Furthermore, a dramatic increase in serum levels of mouse KC and TNF-α production by uterine F4/80+ cells, but not uterine NK or Gr-1+CD11b+ cells, was observed. Depletion of F4/80+ macrophages or neutralization of TNF-α rescued pregnancy to term. Our results have important implications for IL-10-mediated “uterine tolerance” against CpG-driven innate immune activation.
Immunomodulatory Function of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Type 1 Diabetes
Authors: Fiorina Paolo , Jurewicz Mollie , Augello Andrea , Vergani Andrea , Dada Shirine , La Rosa Stefano , Selig Martin , Godwin Jonathan , Law Kenneth , Placidi Claudia , Smith R. Neal , Capella Carlo , Rodig Scott , Adra Chaker N. , Atkinson Mark , Sayegh Mohamed H. , Abdi Reza
Published: June 2009

Human clinical trials in type 1 diabetes (T1D) patients using mesenchymal stem cells (MSC) are presently underway without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in NOD mice. In comparison to NOD mice, BALB/c-MSC mice were found to express higher levels of the negative costimulatory molecule PD-L1 and to promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e., nonobese resistant mice or BALB/c), but not from NOD mice, delayed the onset of diabetes when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC temporarily resulted in reversal of hyperglycemia in 90% of NOD mice (p = 0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit. We also noted soft tissue and visceral tumors in NOD-MSC-treated mice, which were uniquely observed in this setting (i.e., no tumors were present with BALB/c- or nonobese resistant mice-MSC transfer). The importance of this observation remains to be explored in humans, as inbred mice such as NOD may be more susceptible to tumor formation. These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and, potentially, for other autoimmune disorders.
Treatment with a C5aR Antagonist Decreases Pathology and Enhances Behavioral Performance in Murine Models of Alzheimer’s Disease
Authors: Fonseca Maria I. , Ager Rahasson R. , Chu Shu-Hui , Yazan Ozkan , Sanderson Sam D. , LaFerla Frank M. , Taylor Stephen M. , Woodruff Trent M. , Tenner Andrea J.
Published: June 2009

Alzheimer’s disease (AD) is an age-related dementia, characterized by amyloid plaques, neurofibrillary tangles, neuroinflammation, and neuronal loss in the brain. Components of the complement system, known to produce a local inflammatory reaction, are associated with the plaques and tangles in AD brain, and thus a role for complement-mediated inflammation in the acceleration or progression of disease has been proposed. A complement activation product, C5a, is known to recruit and activate microglia and astrocytes in vitro by activation of a G protein-coupled cell-surface C5aR. Here, oral delivery of a cyclic hexapeptide C5a receptor antagonist (PMX205) for 2–3 mo resulted in substantial reduction of pathological markers such as fibrillar amyloid deposits (49–62%) and activated glia (42–68%) in two mouse models of AD. The reduction in pathology was correlated with improvements in a passive avoidance behavioral task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau (69%). These data provide the first evidence that inhibition of a proinflammatory receptor-mediated function of the complement cascade (i.e., C5aR) can interfere with neuroinflammation and neurodegeneration in AD rodent models, suggesting a novel therapeutic target for reducing pathology and improving cognitive function in human AD patients.
Crystallization and Grain Growth Kinetics for Precipitation‐Based Ceramics: A Case Study on Amorphous Ceria Thin Films from Spray Pyrolysis
Authors: Rupp Jennifer L. M. , Scherrer Barbara , Harvey Ashley S. , Gauckler Ludwig J.
Published: June 2009

The introductory part reviews the impact of thin film fabrication, precipitation versus vacuum‐based methods, on the initial defect state of the material and microstructure evolution to amorphous, biphasic amorphous‐nanocrystalline, and fully nanocrystalline metal oxides. In this study, general rules for the kinetics of nucleation, crystallization, and grain growth of a pure single‐phase metal oxide thin film made by a precipitation‐based technique from a precursor with one single organic solvent are discussed. For this a complete case study on the isothermal and non‐isothermal microstructure evolution of dense amorphous ceria thin films fabricated by spray pyrolysis is conducted. A general model is established and comparison of these thin film microstructure evolution to kinetics of classical glass‐ceramics or metallic glasses is presented. Knowledge on thermal microstructure evolution of originally amorphous precipitation‐based metal oxide thin films allows for their introduction and distinctive microstructure engineering in devices‐based on microelectromechanical (MEMS) technology such as solar cells, capacitors, sensors, micro‐solid oxide fuel cells, or oxygen separation membranes on Si‐chips.
Perfumery quaternary diagrams for engineering perfumes
Authors: Teixeira Miguel A. , Rodríguez Oscar , Mata Vera G. , Rodrigues Alírio E.
Published: June 2009

The Perfumery Ternary Diagram (PTD®) methodology predicts the headspace odor character and intensity of fragrant mixtures, applying the concept of odor value (OV) to multi‐component systems. This methodology is extended here to quaternary and quinary odorant systems through the use of tetrahedric diagrams. To present this new methodology, the effect of different base notes in quaternary systems of the type (limonene + geraniol + base note + ethanol) and its forming ternary subsystems has been studied. Base notes selected were: vanillin, tonalide, ambrox, and galaxolide. The Perfumery Quaternary Diagrams (PQD) of the mixtures studied show the different headspace odor character, with ambrox and galaxolide dominating most of the composition spectrum (OVmax). The methodology was also applied to the quinary mixture (limonene + geraniol + vanillin + tonalide + ethanol), and the effect of different concentrations of tonalide on the headspace is presented. © 2009 American Institute of Chemical Engineers AIChE J, 2009