Mesenchymal stem cells (MSC) represent a heterogeneous population exhibiting stem cell-like properties which are distributed almost ubiquitously among perivascular niches of various human tissues and organs. Organismal requirements such as tissue damage determine interdisciplinary functions of resident MSC including self-renewal, migration and differentiation, whereby MSC support local tissue repair, angiogenesis and concomitant immunomodulation. However, growth of tumor cells and invasion also causes local tissue damage and injury which subsequently activates repair mechanisms and consequently, attracts MSC. Thereby, MSC exhibit a tissue-specific functional biodiversity which is mediated by direct cell-to-cell communication via adhesion molecule signaling and by a tightly regulated exchange of a multifactorial panel of cytokines, exosomes, and micro RNAs. Such interactions determine either tumor-promoting or tumor-inhibitory support by MSC. Moreover, fusion with necrotic/apoptotic tumor cell bodies contributes to re-program MSC into an aberrant phenotype also suggesting that tumor tissue in general represents different types of neoplastic cell populations including tumor-associated stem cell-like cells. The present work summarizes some functional characteristics and biodiversity of MSC and highlights certain controversial interactions with normal and tumorigenic cell populations, including associated modulations within the MSC microenvironment. Mesenchymal stem cells (MSC) can be identified in and isolated from nearly all kinds of human tissues. MSC are often synonymously termed stromal cells while other reports distinguish MSC as a precursor of stromal cells. As firstly described, bone marrow-derived human MSC (BM-hMSC) still represent the most frequently investigated hMSC population to compare their properties to those of other tissue-originating hMSC populations . Among tin vitro senescence of BM-MSC [in vitro results of MSC may dramatically differ from the in vivo situation. In this context, effects of the cellular microenvironment such as oxidative stress and DNA damage clearly influence the proliferative capacity and premature aging of the cells [The hMSC originating from different human tissues or organs are designated as stem/stromal or stem-like cells since they share functional properties such as continuous cell cycle progression and plasticity by the capability to differentiate at least along the mesodermal lineage. However, several tissue-originating MSC display significant differences in their proliferative capacities. According to the tissue-specific microenvironment of the diverse MSC populations, UC-MSC exhibit a higher proliferation potential than BM-MSC -19. Thusf BM-MSC . Howeverhe cells . Indeed,he cells .With respect to differentiation capacity, previous work has demonstrated that MSC can acquire certain functions associated with adipogenic, chondrogenic or osteogenic maturation -27. This- phenotype whereby CD44 expression is acquired in the course of in vitro culture of BM-hMSC [Although hMSC have a common minimal expression marker profile of surface receptors, additional distinct surface molecules and metabolic alterations can be detected between different hMSC populations which may determine their functional diversity within the different tissues. For example, umbilical cord-derived (UC) hMSC and some adipose tissue-derived MSC express significant levels of CD44 in contrast to bone-marrow-derived (BM) hMSC. However, there are controversial reports on CD44 expression in BM-hMSC which suggested that the original BM-hMSC represent the CD44 BM-hMSC ,35. The  BM-hMSC . A simil BM-hMSC -40. Ther+ BM-MSC can inhibit lymphocyte proliferation [+ hematopoietic stem cells and endothelial cells [Further markers are proposed for MSC which characterize at least a certain MSC subpopulation. Such markers include Stro-1 identified in BM-MSC . The Strferation , althougal cells . Moreoveal cells . Furtheral cells  -abundant reticular (CAR) cells contribute to hematopoietic stem cell functions within the endo-osteal and the perivascular niche of the bone marrow. The chemokine SDF-1 CXCL12) which is produced and released by CAR cells represents one of the key players for cell migration in several tissues and for homing of hematopoietic stem cells to the appropriate bone marrow niches . The cap which ismacrophage inflammatory protein 1-alpha), CCL3)), and MIP2α , CXCL2, GROβ (Growth-regulated protein beta)) which attracts pro-inflammatory M1 macrophages via their corresponding chemokine receptors CCR1 and CXCR2, respectively, to exhibit antimicrobial activities [monocyte chemotactic protein-1), CCL2)) and IL-8 which increases the mobility of MSC [2, whereby the MSC-mediated local increase in prostaglandin concentration contributes to silence the macrophage inflammatory activity. Similarly, indoleamine 2,3-dioxygenase which is also inducible in MSC by γ-IFN, catalyzes the conversion from tryptophan to kynurenine and tryptophan depletion in the cellular microenvironment inhibits the immune cell activity [Further MSC activities in addition to the exchange of miRs and other exosome-containing compounds include the mutual release of chemokines and cytokines between MSC and target cells during wound healing. For the repair of damaged tissues, MSC interact with macrophages that infiltrate the wound bed in injured epithelia. During this process MSC can secrete factors such as MIP1α , interleukins , hematopoiesis-stimulating factors , angiogenic growth factors , and tropic growth factors  -82. More2,85 which2. The mol2,87  production of the previously LPS-stimulated immune cells . MoreoveAlthough precise molecular interactions still remain unclear, how the two different actions of MSC mediate immune-modulation, either by direct MSC-to-immune cell attachment (e.g. via ICAM-1 and VCAM-1) or by the release of a large panel of cytokines and micro vesicle-containing miRs, it becomes more and more conceivable that both mechanisms may be mutually combined rather than representing exclusive phenomena. Based on this hypothesis, the direct and timely limited MSC-to-immune cell attachment could trigger and enhance a short-term release of required factors which contribute to immune cell-specific  responses. In addition, the MSC-immune cell attachment establishes a close interpopulation complex which provides a minimized intercellular space for maximal action of the released cytokines and micro vesicles within this microenvironment Figure.Besides their immune-modulatory capacity, hMSC also possess immune-privileged properties which means that these cells do not challenge a response of allogeneic immune cells suggesting an active self-protection . Due to Taken together, hMSC represent a heterogeneous population of stromal/stem cells originating from perivascular niches of nearly all tissues which exhibit altered proliferation and differentiation capacities, and provide distinct levels of immune-modulatory activities . This imAccording to the heterogeneity of MSC functions already observed during MSC interaction with normal cells and particularly immune cells, there are also a variety of controversial reports suggesting both, tumor-inhibitory and tumor-promoting activities of MSC. These conflicting observations may be related in part to the cellular microenvironment whereby hypoxia establishes an extracellular milieu to reduce aging and enrich poorly differentiated tumor cells and mesenchymal stromal/stem cells. Such an undifferentiated hypoxic microenvironment provides essential cellular interactions and environmental signals for the preferential maintenance of MSC and cancer cells ,49,102.The contradictory reports on cellular interactions between MSC and tumors can also be associated with different activation levels of MSC which are switched by intracellular triggers such as different miRs. Indeed, different states of MSC activation are relayed by variable expression levels of certain miR including miR-335. High levels of miR-335 expression contribute to a potentially non-activated (silenced) MSC state of auto-maintenance and low amounts of miR-335 suggest an activated state leading to proliferation, migration and differentiation in MSC  Figure11.Of interest, a functional role in the regulation of a neoplastic development and the formation of metastases has also been attributed to miRs in breast cancer cells, particularly miR-21 and miR-205 for tumor development and miR-126 and miR-335 for breast cancer metastases. Whereas different amounts of miR-335 determine the activation status of MSC, certain expression levels of this miR also contribute to metastasis of breast cancer cells. The constitutive expression of miR-335 targets more than 62 genes  includinTumor-promoting effects are also attributed to an epithelial-to-mesenchymal transition (EMT), whereby MSC play a crucial role. Thus, EMT represents a key event for colorectal cancer progression and MSC are implicated in this process . Likewis2+), inositol phosphates (different IP3 and IP4) and cyclic nucleotides Thus, a derailed GJIC or defects in GJIC can modulate the metastatic potential of the breast cancer cells [Interactions of MSC with breast cancer cells include the formation of gap junctions  The gap er cells . Moreoveer cells .in vitro[Consequently, MSC provide direct cell-cell contact with tumor cells whereby these interactions rather than soluble factors predominantly promote tumor cell proliferation and survival in vitro. Indeed,in vitro. MUC1  attribute to a direct recruitment of CrkL binding via its SH2 domain. The SH2/SH3 adaptor protein CrkL is involved in the regulation of cell migration through the association with guanine nucleotide exchange factors such as Dock180 to catalyze GTPase activation and GDP/GTP exchange. Consequently, the ICAM-1-MUC1-mediated Src-CrkL complex formation further activates the small GTPases Rac1 and Cdc42 which are key effectors of the actin cytoskeleton protrusion machinery and thereby, reorganize the actin cytoskeleton and promote an actin-based cell motility . SupportTogether, these studies suggest that direct cell-to-cell interactions between MSC and tumor cells via gap junctions and the exchange of exosomes or via membrane receptor assembly promote tumor cell growth. In contrast, tumor inhibitory effects of MSC are reported preferentially via the ECM compounds of the microenvironment and soluble factors Figure. Thus, eIn vivo application of MSC into the contralateral hemisphere of a glioma in rats was associated with migration of the MSC towards the tumor through the corpus callosum. Moreover, intratumoral injection of MSC resulted in a significant inhibition of tumor growth and increased the survival of these rats [More detailed studies in a tumor model revealed that UC-MSC possess a significantly elevated migratory potential as compared to BM-MSC. The capacity to migrate towards glioma cells is accompanied by an elevated expression of IL-8 receptors and CXC chemokine receptors-1 and −2 on UC-MSC . Malignaese rats . In the ese rats . Since Mese rats . In addiese rats ,127.In conclusion, these effects suggest that MSC display tumor-inhibitory functions preferably via the ECM and the release of distinct factors into the tumor surrounding microenvironment, whereas tumor-promoting effects are relayed predominantly by direct cell-to-cell interaction and the exchange of membrane vesicles.A variety of further evidences suggest that disease- and cancer-associated stromal cells are distinctly abnormal as compared to MSC from healthy tissues. E.g., multiple myeloma represents a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities and a significant amount of MSC derived from multiple myeloma carry chromosomal abnormalities and defects in gene and protein expression . Likewisin vitro and in an in vivo co-implantation model [The close vicinity of MSC to the tumor entity also enables interactions with biological material obtained from necrotic tumor tissue and dying cancer cells. Such necrotic factors – also termed damage-associated molecular patterns (DAMPs), can activate MSC by enhancing their angiogenic capacity or affect the immune-regulatory potential of MSC by interfering with the expression of indoleamine 2,3-dioxygenase in MSC . Moreoveon model . Likewison model ,137. Furon model . Finallyon model  and someon model ,141. Thion model ,143.Together, MSC can directly interact with a variety of different adjacent normal cell types either via adhesion molecules and gap junctions or via exchange of a large panel of cytokines/chemokines, exosome-containing miRs and ECM components. In case of tumor cells, such interactions with MSC result in the support of tumor-promoting and tumor-inhibiting activities, respectively. Moreover, uptake of DAMPs and fusion with vesicles or apoptotic bodies from tumor cells contribute to alter the MSC functionality into an aberrant phenotype.MSC demonstrate a functional heterogeneity within different tissue origins. This heterogeneity is even displayed within the same population and suggests MSC subpopulations exhibiting specialized functions according to local tissue requirements which may develop from certain precursor cells and still maintain stem cell properties . TherebyWhereas MSC appear to function almost as a cellular all-rounder in support of tissue homeostasis and plasticity, these unique capabilities of biodiversity may also be misused during transformation into aberrant MSC phenotypes.The authors declare that they have no competing interests.AO contributed to Figure


The cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway is activated by nitric oxide (NO), natriuretic peptides , and cGMP-elevating drugs. It regulates important physiological functions such as platelet aggregation, smooth muscle tonus, and cell growth and survival. Recent reports indicate that cGMP might also play a role in tumorigenesis. In the present study we found that cGKI is expressed in melanoma cells of murine and human origin.Treatment of intact mouse B16 melanoma cells with the membrane-permeable cGMP analog 8-Br-cGMP induced phosphorylation of the cGKI substrates, vasodilator-stimulated phosphoprotein and phosphodiesterase 5. ANP and CNP, ligands of the membrane-bound guanylyl cyclase GC-A and GC-B, respectively, activated the endogenous cGMP/cGKI pathway. CNP-induced cGMP signals were detected in cell extracts by ELISA and in living cells by a FRET-based cGMP sensor [We have identified a natriuretic peptide/cGMP/cGKI pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. Pharmacologic inhibition of cGMP signaling may offer a promising strategy for the treatment of melanoma.


Newer chemotherapeutic agents target extracellular signaling, including the mitogen-activated protein kinase kinase (MEK) pathway. We present a case of a 54-year-old female who developed bilateral multifocal central serous-like chorioretinopathy shortly after starting MEK inhibition for metastatic cutaneous melanoma. There was a complete resolution of findings after drug stoppage. After resuming a lower dose of the MEK inhibitor, the findings recurred again but resolved after drug stoppage. Other etiologies were unlikely given the clinical course. The presumed mechanism involves toxicity to the retinal pigment epithelium, with breakdown of the blood-retinal barrier. Recognition of this side effect is important with this new class of chemotherapy. Recently developed chemotherapeutic agents target the mitogen-activated protein kinase kinase (MEK) pathway . CentralA 54-year-old Caucasian female presented for an eye examination prior to initiation of dabrafenib  and trametinib  chemotherapy for cutaneous melanoma with axillary and cervical lymph node metastases. Past medical history was positive for hypertension and past ocular history was negative. Current medications included diltiazem, meloxicam, losartan, and hydrochlorothiazide. Visual acuity (VA) was 20/20 OU with normal exam findings.Three weeks after beginning therapy, she presented with decreased vision of 20/60 OD and 20/50 OS. Fundus examination showed bilateral multifocal neurosensory retinal detachments that were hyperautofluorescent on fundus autofluorescence Figures . SubretiFour months later, her vision gradually deteriorated to 20/30 OU with recurrence of SRF Figures . DabrafeDysregulation of extracellular signaling is an increasingly recognized factor in the development of human cancers. Three kinase enzymes are part of this pathway: mitogen-activated protein kinase (MAPK), MEK, and extracellular signal-regulated kinase (ERK) . InhibitSeveral cases of MEK inhibitor-induced retinopathy have been reported. In a prospective, randomized, phase I/II study of a B-raf/MEK inhibitor, 2% of patients in the higher dose group developed chorioretinopathy . Velez-MMechanisms underlying retinopathy in the setting of MEK inhibition are unknown, but we hypothesize that MEK inhibition results in acute retinal pigment epithelial (RPE) toxicity and dysfunction with breakdown of the blood-retinal barrier. In support of this, the MEK pathway appears to be important in maintaining the blood-retinal barrier and protecting RPE cells against oxidative and light-induced damage. Animal models demonstrate that MEK inhibition induces oxidative stress and inflammation with subsequent endothelial and blood-retinal barrier damage and RPE hyperpermeability , 6, provThe combination of B-raf and MEK inhibition in patients with cutaneous melanoma significantly improves progression-free survival . In addi


Circulating progenitor cells (CPC) are bone marrow-derived elements with reparative properties known to be mobilised in the context of acute coronary syndromes. Controversy persists over the determinants of their release in the acute phase of ST-elevation myocardial infarction (STEMI). We used cardiovascular magnetic resonance (CMR) to investigate this issue.Fifteen STEMI patients undergoing successful primary percutaneous coronary intervention (pPCI) were included. Peripheral vein blood samples were drawn at 48 hours after pPCI to measure CPC (CD34+/ KDR+/CD45-). At 2-7 days after pPCI all patients underwent CMR assessment with SSFP, T2-STIR, first-pass perfusion and late gadolinium-enhancement imaging. Quantitative T2 area-at-risk , infarct size , myocardial salvage  were calculated. Microvascular obstruction (MVO) was assessed both as perfusion defect score on first-pass imaging  or as MVO score in the late enhancement images.CPC levels  were more strongly related to extent of AAR  than to IS , but did not correlate to MS . Moreover, CPC were related to PD score  but not to MVO score .Our data suggest that CPC are mobilized in STEMI patients in response to the total myocardial ischemic insult, as represented by the area-at-risk on T2-STIR. Furthermore, CPC positive correlation with PD suggests that they could be mobilized in attempt to overcome ongoing microvascular dysfunction.


A suspension protein microarray was developed using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles for potential applications in multiplex and high-throughput immunoassays. A simple photopatterning process produced various shapes of hydrogel micropatterns that were weakly bound to poly(dimethylsiloxane) (PDMS)-coated substrates. These micropatterns were easily detached from substrates during the washing process and were collected as non-spherical microparticles. Acrylic acids were incorporated into hydrogels, which could covalently immobilize proteins onto their surfaces due to the presence of carboxyl groups. The amount of immobilized protein increased with the amount of acrylic acid due to more available carboxyl groups. Saturation was reached at 25% v/v of acrylic acid. Immunoassays with IgG and IgM immobilized onto hydrogel microparticles were successfully performed with a linear concentration range from 0 to 500 ng/mL of anti-IgG and anti-IgM, respectively. Finally, a mixture of two different shapes of hydrogel microparticles immobilizing IgG (circle) and IgM (square) was prepared and it was demonstrated that simultaneous detection of two different target proteins was possible without cross-talk using same fluorescence indicator because each immunoassay was easily identified by the shapes of hydrogel microparticles. A numSince the Doyle group developed outstanding methods for generating various shapes of hydrogel microparticles using continuous flow or stop-flow lithography ,19, seveIn this study, we prepared a shape-coded suspension protein microarray capable of multiplexed immunoassays using PEG-based hydrogel microparticles. A photopatterning process fabricated different shapes of hydrogel microparticle. Acrylic acid was incorporated into the hydrogel to provide functional groups that can covalently immobilize probe proteins onto hydrogel surfaces. After confirming that IgG or IgM immobilized onto different hydrogel microparticles could each function in the immunoassay, the possibility of using shape-coded hydrogel microparticles for multiplexed immunoassay was demonstrated by simultaneously detecting two different analytes.2.2.1.W 575), 2-hydroxy-2-methylpropiophenone (HOMPP) photoinitiator, bovine serum albumin conjugated with fluorescein isocyanate (FITC-BSA), 1-ethyl-3(3-dimethylaminoprophyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and acrylic acid were purchased from Sigma-Aldrich . Poly(dimethylsiloxane) (PDMS) elastomer was purchased as Dow Corning Sylgard 184  and is composed of prepolymer and a curing agent. FITC-rabbit anti-mouse immunoglobulin G(FITC-anti-IgG), mouse IgG, FITC-rabbit anti-mouse immunoglobulin M(FITC-anti-IgM), and mouse IgM were purchased from ZYMED Laboratories . Chrome soda lime photomask for the photolithographic patterning of hydrogels was purchased from Advanced Reproductions . Phosphate buffered saline  consisted of 1.1 mM potassium phosphate monobasic, 3 mM sodium phosphate dibasic heptahydrate, and 0.15 M NaCl in deionized water.Poly(ethylene glycol) diacrylate (PEG–DA)  for 1 s through the photomask. UV photopolymerization created hydrogel micropatterns because only precursor solution exposed to UV light underwent free-radical cross-linking and became insoluble in common PEG solvents such as water. The final hydrogel microparticles were obtained by flushing the PDMS-coated glass slide and photomask and collecting the released hydrogel micropatterns. To incorporate carboxyl groups into hydrogel, a given amount of acrylic acid was added to the precursor solution and copolymerized with PEG-DA. Resultant hydrogel microparticles were washed overnight to remove any unreacted or unbound molecules from the hydrogels.Hydrogel microparticles were prepared by UV-initiated free radical polymerization as described in our previous study . The ini2.3.Proteins such as FITC-BSA, IgG, and IgM were covalently immobilized on hydrogel microparticles via an EDC/NHS-mediated reaction. Carboxylated hydrogel microparticles were immersed into a solution containing 2 mM EDC and 5 mM NHS for three hours to convert the carboxyl groups in acrylic acid into reactive intermediates susceptible to attack by amine groups in proteins. The activated hydrogel microparticles were then washed with PBS and immediately reacted with protein solutions  for three hours at 4 °C. The reactions resulted in amide bond formation that covalently attached proteins to the hydrogel surface. After immobilization of proteins, hydrogel microparticles were thoroughly washed with PBS to remove weakly-bound or physically-entrapped proteins.2.4.For immunoassays, IgG or IgM-immobilized hydrogel microparticles were incubated with blocking solution (1 wt% BSA in PBS solution) for two hours and subsequently reacted with different concentration of FITC-anti-IgG or FITC-anti-IgM at 4 °C for two hours. After the reaction, hydrogel microparticles were thoroughly washed to remove physically-adsorbed or hydrogel-entrapped anti-IgG or anti-IgM. Immunoreactions on the hydrogel microparticles were visualized and quantified using fluorescence microscopy. To investigate cross-reactivity, IgG-immobilized hydrogel microparticles were reacted with different concentration of anti-IgM, or IgM-immobilized hydrogel microparticles were reacted with anti-IgG. A Zeiss Axiovert 200 microscope equipped with an integrated color CCD camera  was used to obtain fluorescent and optical images. The fluorescence intensity was quantified using commercially available image analysis software .3.A photopatterning process, described in The feasibility of covalently immobilizing proteins onto hydrogel microparticles was investigated by incubating FITC-BSA with activated carboxylated hydrogel microparticles. Relative amounts of immobilized proteins were determined from the quantitative fluorescence intensity data. Acrylic acid-incorporating hydrogel can achieve different levels of swelling at different pH levels by ionizing carboxyl groups above their pKa (4.7). Because of this, changing the pH was expected to alter the morphology of carboxylated hydrogel microparticles. To investigate the pH-response of hydrogel microparticles, acrylic acid-incorporating hydrogel microparticles were fabricated using a photomask containing arrays of circles or squares with a lateral dimension of 100 μm. The microparticles were then incubated with different pH buffer solutions. As shown in the optical images of After successful covalent immobilization of proteins onto hydrogel microparticles, immunoassays were performed to test the potential application of hydrogel microparticles as suspension array-based biosensors. First, IgM was immobilized onto square-shaped hydrogel microparticles and reacted with FITC-anti-IgM. Fluorescent images demonstrated that anti-IgM bound specifically to IgM-immobilized microparticles and that fluorescence intensity increased with anti-IgM concentration . Figure Finally, we demonstrated simultaneous analysis of different immunoassays using suspension arrays of hydrogel microparticles. As a proof of concept, multiplex analysis was performed with two different shapes of hydrogel microparticles, circular hydrogel immobilizing IgG and square hydrogel immobilizing IgM. The two suspension arrays were prepared separately and combined in a well plate at a 1:1 volume ratio. Each reaction was identified by the hydrogel microparticle shape, generating a shape-coded suspension microarray system. Five different samples (mixtures of FITC-anti-IgG and FITC-anti-IgM) were prepared and reacted with the suspension arrays of shape-coded hydrogel microparticles. When a solution containing either anti-IgG or anti-IgG was introduced, only the hydrogel microparticles immobilizing corresponding probe molecules emitted green florescence without any cross-talk, as shown in 4.This paper has described the preparation of hydrogel microparticles and their potential application to multiplexed immunoassays in a suspension array format. Hydrogel microparticles were fabricated by a simple photopatterning process that cross-linked liquid precursor solution containing acrylic acid, PEG-DA, and photoinitiator to form hydrogel. Because of the carboxyl groups in acrylic acid, resultant hydrogel microparticles were capable of covalently immobilizing proteins via an EDC/NHS-mediated reaction. For the immunoassay, a suspension array of hydrogel microparticles immobilizing IgM and IgG were prepared. The binding events between IgM and anti-IgM and between IgG and anti-IgG were successfully investigated using fluorescence detection. Furthermore, a mixture of two different shapes of hydrogel microparticle-immobilizing IgM and IgG was prepared. This shape-coded suspension array of hydrogel microparticles could simultaneously characterize two different immunoassays without cross-talk, despite using the same fluorescence indicator. Since many unique shapes of hydrogel microparticles can be prepared by changing photomask designs, more than two analytes can be detected by randomly assembling a mixture of desired hydrogel microparticles and incorporating appropriate receptor molecules.


Cardiac involvement in systemic lupus erythematosus (SLE) is a common complication and is associated with a considerable morbidity and mortality in these patients. As affected individuals often present with subclinical or non-specific symptoms the betimes confirmation of cardiac manifestations is challenging. Cardiovascular magnetic resonance (CMR) with late gadolinium enhancement (LGE) is the current gold-standard for non-invasive tissue characterization as well as for the evaluation of systolic and diastolic function. In this study we assessed the cardiac morphology and function in SLE patients with low disease activity.We studied twenty-nine SLE patients  fulfilling the SLE diagnostic criteria of the American College of Rheumatology and compared them to thirty age-matched healthy volunteers. All patients were in a stable clinical condition and only patients with a low disease activity (SLEDAI Index < 5) were included. All of them received an individually optimized medication. CMR images were acquired on a 1.5 T whole-body MRI  using a 32-element cardiac phased-array coil. Short and long axis views were obtained applying a standard clinical steady-state free-precession sequence and LGE CMR imaging was performed  in 27 patients. Left and right ventricular  end-diastolic and end-systolic volumes , stroke volume (SV), ejection fraction (EF) as well as the mitral/tricuspid annular plane systolic excursion (M/TAPSE) and LV wall mass were measured.SLE patients and healthy controls had comparable demographic and clinical characteristics. SLE patients had significantly higher LV-EDV, LV-ESV and LV-EF. LV-SV and MAPSE did not differ significantly between patients and healthy controls. However SLE patients had significantly higher RV-EDV, RV-ESV, RV-SV, RV-EF and TAPSE values. LV mass showed no significant differences. Nineteen of twenty-seven assessed patients (70%) showed LGE CMR as a sign of myocardial involvement.Even in SLE patients with a low disease activity there is a high prevalence of myocardial and functional alterations. Especially the RV function is affected which could be due to early onset pulmonary manifestations of SLE. Regarding the high morbidity and mortality of cardiac complications in SLE patients, CMR may be an important non-invasive method for the early diagnosis of cardiopulmonary involvement.none


Associations between measures of subjective health and mortality risk have previously been shown. We assessed the impact and comparative predictive performance of a multi-biomarker panel on this association.Data from 4,261 individuals aged 20-79 years recruited for the population-based Study of Health in Pomerania was used. During an average 9.7 year follow-up, 456 deaths (10.7%) occurred. Subjective health was assessed by SF-12 derived physical (PCS-12) and mental component summaries (MCS-12), and a single-item self-rated health (SRH) question. We implemented Cox proportional-hazards regression models to investigate the association of subjective health with mortality and to assess the impact of a combination of 10 biomarkers on this association. Variable selection procedures were used to identify a parsimonious set of subjective health measures and biomarkers, whose predictive ability was compared using receiver operating characteristic (ROC) curves, C-statistics, and reclassification methods.In age- and gender-adjusted Cox models, poor SRH (hazard ratio (HR), 2.07; 95% CI, 1.34-3.20) and low PCS-12 scores  were significantly associated with increased risk of all-cause mortality; an association independent of various covariates and biomarkers. Furthermore, selected subjective health measures yielded a significantly higher C-statistic (0.883) compared to the selected biomarker panel (0.872), whereas a combined assessment showed the highest C-statistic (0.887) with a highly significant integrated discrimination improvement of 1.5% (p < 0.01).Adding biomarker information did not affect the association of subjective health measures with mortality, but significantly improved risk stratification. Thus, a combined assessment of self-reported subjective health and measured biomarkers may be useful to identify high-risk individuals for intensified monitoring. Subjective health measures are widely used within clinical and epidemiological research, as well as health policy settings, being easily assessed by a single-item self-rated health (SRH) question or more thoroughly using health related quality of life (HRQoL) instruments. The Short Form Health Survey SF-36) is a well documented and validated HRQoL instrument -3, with 6 is a weIn particular, the relationship between SRH and mortality has been repeatedly reported -8, suggeThe present study aims to investigate the impact and comparative predictive performance of a multi-biomarker panel on the association of subjective health with mortality, analyzing data from the 10-year follow-up of the population-based Study of Health in Pomerania (SHIP).The SHIP is a population-based cohort study in West Pomerania, conducted in the north-eastern area of Germany comprising the cities of Greifswald, Stralsund, Anklam and 29 surrounding communities with a total of 212,157 residents ,26. A reA computer-assisted personal interview assessed socio-demographic information including age, gender, educational level , civil status (cohabitation), and occupational status ; health-related behaviour including physical activity , excessive/high-risk alcohol consumption , smokinLaboratory assessment of the multi-biomarker panel included measurement of 10 biomarkers from distinct biological pathways associated with increased morbidity and mortality including inflammation [high sensitive C-reactive protein (hs-CRP)], hemostasis (fibrinogen), metabolic disturbances , liver disease [gamma glutamyltransferase (GGT)], kidney disease [urine albumin and glomerular filtration rate (GFR)], thyroid status [thyrotropin (TSH)], and hypothalamic-pituitary-adrenal axis activity [insulin-like growth factor-I (IGF-I)]. Biomarkers were measured as follows: hs-CRP determined immunologically on a Behring Nephelometer II with commercially available reagents from Dade Behring ; plasma fibrinogen assayed according to Clauss using an Electra 1600 analyzer ; HbA1c determined by high-performance liquid chromatography ; total cholesterol measured photometrically ; triglyceride determined enzymatically using reagents from Roche Diagnostics ; urine albumin determined on a Behring Nephelometer ; GGT measured photometrically ; creatinine determined with the Jaffé method  and the GFR estimated according to the modified MDRD formula ; TSH meaInformation on vital status were acquired at regular intervals from time of enrollment into the study through December 15, 2009. Individuals were censored at either death or loss to follow-up. The number of months between baseline examination and censoring was used as follow-up length.2 test  or a Mann-Whitney-U test (quantitative data). PCS-12 and MCS-12 were divided into quartiles to calculate crude incidence rates (per 1000 person-years) and to perform multivariable Cox proportional-hazards regression models associating PCS-12, MCS-12, and SRH with all-cause mortality. Kaplan-Meier survival curves were graphed for SRH and compared using the log-rank test. First, we prespecified gender- and age-adjusted Cox models and included alternately socio-demographic factors , behavioral factors , comorbidities , and the multi-biomarker panel . In secondary analyses, we used backward  and forward elimination (p < 0.05 for inclusion) procedures to identify a parsimonious adjustment set among all of the applied standard covariates and the multi-biomarker panel.Data on quantitative and qualitative characteristics are expressed as median (inter-quartile range), or percent, respectively. Intergroup comparisons with regard to vital status were performed using χTo compare the predictive performance of the implemented models, we measured the area under the receiver operating characteristic (ROC) curve, or C-statistic, and tested their differences using STATA's "roccomp" command. The C-statistic ranges from 0.5 (no discrimination) to a theoretical maximum of 1 (perfect discrimination) and is equivalent to the probability that the predicted risk is higher for a case (decedent) than for a non-case (survivor) . FurtherWe assessed the potential effect modification of the investigated associations by age and gender through additional inclusion of multiplicative interaction terms  into the applied multivariable Cox models. To account for the potential impact of changing risk factor patterns over time, we entered the applied adjustment sets as time-varying covariates into the analysis. Further sensitivity analyses were performed by recalculating the applied models stratified by 20-year age-groups and gender, as well as adjusting for possible non-response bias by using inverse probability weights . An elevth, 9.3; 75th, 10.7) of follow-up, 456 individuals (10.7%) died, resulting in an overall death rate of 11.1 deaths per 1000 person-years. Crude incidence rates of all-cause mortality decreased across quartiles of PCS-12 but not MCS-12  compared to subjects in the highest quartile. The inclusion of potentially confounding socio-demographic factors , behavioral factors , comorbidities , as well as the multi-biomarker panel , attenuated the estimates only slightly  and the multi-biomarker panel . The ROC curves depicted in Figure Sensitivity analyses with the inclusion of multiplicative interaction terms did not yield any significant effect modification caused by gender or age (p < 0.05). Furthermore, we found virtually no differences comparing the risk estimates between Cox models with and without time-varying covariates. Stratified analyses revealed that especially middle-aged men (40-59 years) were responsive to the detected associations of low PCS-12  and poor SRH  with all-cause mortality. Finally, the additional inclusion of non-response weights altered the revealed estimates only slightly (data not shown).The present study investigated the associations between subjective health, multiple biomarkers, and mortality risk; offering three principal findings. First, we found that poor SRH and low PCS-12 scores were significantly associated with increased risk of all-cause mortality, independent of a broad spectrum of standard covariates and multiple biomarkers. Second, we found that a risk assessment using subjective health instruments and standard covariates yielded a better mortality risk prediction compared to that of a multi-biomarker panel. Finally, we were able to show that the most accurate mortality risk prediction was obtained from a combined assessment of subjective health and biomarkers.Our presented risk estimates of the association between low PCS-12 and mortality were similar to those previously reported among Taiwanese community-dwelling elderly . We wereOur observed effect sizes between SRH and mortality reflect fairly well the range found in the literature, although the confidence intervals are somewhat wider ,43. Our To the best of our knowledge, this is the first population-based study to systematically assess the impact of a comprehensive multi-biomarker panel on the subjective health-mortality association. Our finding of an association between poor SRH and low PCS-12 scores with increased mortality risk was independent of a broad spectrum of multiple biomarkers from distinct biological pathways. This finding is in line with previous results from a five-year follow-up of 4,065 individuals aged 71 years or older, showing SRH significantly associated with mortality risk after adjustment for various biomarkers including albumin, white blood cell count, hemoglobin, high-density lipoprotein cholesterol, and creatinine . It is iWhen we applied variable selection procedures to answer the question which of the 10 biomarkers were most predictive in terms of mortality risk, we identified fibrinogen, HbA1c, albumin, and GGT as the most predictive biomarkers. These pathophysiological highly plausible mortality risk candidate biomarkers reflect disturbances in hemostasis (fibrinogen), metabolism (HbA1c), kidney disease , and liver disease (GGT). Their predictive ability has been shown to be similar to the full biomarker panel, suggesting improved risk stratification effectiveness using this parsimonious set of biomarkers. But even more interestingly, the predictive performance of these selected four biomarkers was shown to be nearly as good as a risk assessment based on subjective health instruments and standard covariates. While previous studies accumulated evidence that subjective health is a powerful mortality risk predictor, the present findings add to the existing literature indicating that this selected biomarker set plus information about gender and age do about as well. Finally, we were able to show that the combined assessment of subjective health and biomarkers significantly improved the discriminatory ability of the mortality risk prediction model beyond that of each separate panel. There is only one previous study among US veterans that investigated the predictive power of PCS-12 . CompareThe strengths of our study include a prospective population-based sample of adults aged 20-79 years, a completed 10-year follow-up period utilizing valid and reliable mortality data based on a national death register, comprehensive subjective health and covariable assessment, as well as a broad multi-biomarker panel. Nonetheless, the present study has several limitations. First, non-response bias may potentially exist in this sample, because non-response is particular relevant for self-report of mental properties. But when we performed sensitivity analyses accounting for non-response bias, we detected only minimal deviations from our main results. Second, this study sampled a healthy adult population, with only 11% of participants deceased over the follow-up period. But although this proportion is lower than in several previous studies, a comprehensive review of single-item measured SRH and mortality concluded that studies with lower than 10% mortality found similar effect sizes as studies with higher mortality rates . Third, The present results from a large population-based epidemiological study demonstrate that the association of poor SRH and low PCS-12 with mortality is independent of a broad spectrum of standard covariates and multiple biomarkers from distinct biological pathways. But the key finding of the present study is that a small set of biomarkers conjointly with subjective health instruments and socio-demographic standard measures significantly improved the mortality risk prediction above and beyond a separate assessment.While the first finding is more confirmative in nature, the latter holds important implications from epidemiological and public health perspectives. Incorporating both subjective health assessment and a small set of biomarkers into routine data collection could be used to monitor population health, especially to identify subpopulations particularly at risk. Doing so, the improved identification of high-risk individuals could increase this efficacy of disease prevention strategies. However, further research must be conducted to elucidate how subjective health and biomarkers are interrelated and interdependent. Carrying the present results forward to cause-specific mortality and morbidity will lead to a better understanding of these relationships.The authors declare that they have no competing interests.RH; JM, TK, and HW contributed to the study design and ideas for the data analysis. HV, MD, MN, and HW organized the sample collection and data preparation. Statistical analyses were performed by RH. RH, YSF, JM, HW, and TK contributed to the interpretation of the results and the discussion. RH drafted the manuscript and wrote the final version together with all other co-authors. All authors read and approved the final manuscript.The authors have nothing to disclose.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/103/prepubAdjusted hazard ratios (95% CI) for PCS-12, MCS-12, and self-rated health associated with all-cause mortality. Complete regression results of all Cox proportional-hazards analyses.Click here for file


Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterise the feline homologue of tetherin and assess its effects on the replication of feline immunodeficiency virus (FIV).A feline homologue of tetherin was amplified from IL2-dependent T cell cDNA. Real-time PCR analyses revealed that feline tetherin transcripts were expressed in many feline cell lines and were induced by interferons . Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release, however, this activity resisted antagonism by either HIV-1 VPU, or the FIV Env and "OrfA" proteins. Further, as over-expression of a complete FIV genome in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. When expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV, indeed, syncytia formation was significantly enhanced in tetherin-expressing cells infected with CD134-independent strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Confocal microscopy revealed co-localisation of tetherin and FIV envelope glycoprotein (Env) in regions at the periphery of syncytia. Moreover, while treatment of feline cells with ínterferon-ω suppressed viral protein production and release , syncytia formation was enhanced in cells treated with interferon-ω either pre- or post- infection with a CD134-independent strain of FIV.in vivo may promote the selective expansion of viral variants capable of more efficient cell to cell spread; contributing to the emergence of CD134-independent strains of FIV.While feline tetherin prevents the release of nascent viral particles, cell to cell spread remains efficient in the presence of abundant viral receptors and interferon-induced up-regulation of tetherin expression may enhance syncytia formation. Accordingly, tetherin expression


Currently, there is much debate on whether epithelial thinning from hormonal contraceptives can increase HIV acquisition. Previously, we illustrated that HIV can penetrate to depths in squamous epithelium where it can interact with target cells, such as CD4+ T-cells and macrophages. Using a similar approach, we show that epithelial thinning affects virus penetration, along with target cell and cellular junction distribution.To investigate how genital epithelial thickness may affect HIV penetration, ten female rhesus macaques were pre-treated with 30mg depo-medroxyprogesterone acetate (Depo-provera®) 4-5 weeks prior to vaginal photoactivatable (PA-GFP) HIV exposure. Additionally, eight female pigtail macaques were exposed to PA-GFP HIV at various menstrual cycle stages. Genital tracts were removed 4 hours post-exposure and immediately dissected and snap frozen in optimal cutting temperature (OCT) compound. Comparison of pre- and post-photoactivation image z-stacks revealed the presence of virus, accounting for background.Within 4 hours, PA-GFP virions were observed between squamous epithelial cells penetrating up to depths of 50μm. This is within the reach of target cell populations. Furthermore, current analysis illustrated epithelial thickness to be inversely proportional to the number of penetrating virions and target cells, independent of thinning mechanism. Also, cellular junction distribution in pigtail macaques with thinned squamous epithelia mirrored those results of progesterone-treated rhesus macaques.Our current results suggest that HIV acquisition in women may be influenced by menstrual cycle and hormonal contraceptives. CD4+ T-cells and CD68+ macrophage distribution and virus penetration were dependent on epithelial thickness, suggesting HIV interactions with female genital epithelia may differ in the luteal and follicular stages of the menstrual cycle. Our results also suggest that progestin-based contraceptives may alter the barrier properties of the stratified squamous epithelium, possibly increasing the risk of HIV acquisition in women.


Abacavir use has been associated with an increased risk of ischemic cardiovascular events in several cohort studies, but the pathogenic mechanisms are still unknown. Recently Martinez et al. observed no differences in markers of inflammation, endothelial dysfunction, insulin resistance or hypercoagulability in HIV infected persons treated with either abacavir or tenofovir. While several studies have shown endothelial dysfunction in HIV-infected patients, only a few data are available on the involvement of platelets. Aim of our study was to evaluate markers of platelet activation and endothelial dysfunction in HIV infection comparing patients treated with abacavir or tenofovir.In a retrospective, case-control study, the time course of some endothelial  and platelet activation markers  was examined in 62 HIV-infected patients, before starting HAART and after 6-12 months of therapy with either Abacavir (n=31) or Tenofovir (n=31). Data were compared with those from 20 untreated HIV-infected patients at diagnosis and after 6 months and 10 healthy matched controls.Soluble P-selectin (sP-sel), sPLA2, soluble vascular cell adhesion molecule-1 (sVCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in HIV-infected patients than in healthy controls. During 6-12 months of follow-up, we found no significant differences between abacavir and tenofovir-treatment for endothelial markers, while sPLA2  and sPsel  increased significantly in the abacavir group as compared with the tenofovir-treated group. sGPV, the platelet glycoprotein V major fragment released upon thrombin cleavage, was increased after 6-12 months as compared to baseline in both treatment groups . In naïve patients, not treated with HAART, significantly increased plasma markers of endothelial dysfunction sPsel and sPLA2 were confirmed at diagnosis, as compared with healthy controls, with no changes upon follow-up..Our results confirm that chronic HIV infection induces endothelial dysfunction but indicate that a short-term treatment with abacavir enhances also some parameters of platelet activation, suggesting a role of platelets too in the increased incidence of ischemic cardiovascular events in HIV-infected patients treated with abacavir


Objective. Presence of anti-HLA antibodies has a well-known impact on kidney grafts survival; however their role in liver transplantation has not been fully elucidated. We conducted a 7-year prospective study to show correlation between presence of anti-HLA and anti-MICA antibodies and liver graft survival. Methods. Blood samples from 123 liver transplant recipients were collected during patients routine visits. Time from transplantation to blood sample collection was different for each patient. Blood samples were tested for anti-HLA (separately class I and II) and MICA antibodies using Luminex assays. Results. There were 32 (26%) patients with positive anti-HLA and 37 (30%) with positive anti-MICA antibodies. Graft loss occurred in 7 cases (23%) in anti-HLA positive group compared to 20 (22%) in anti-HLA negative group (P = ns) and in 8 cases (22%) in anti-MICA positive group but 19 (23%) in anti-MICA negative group (P = ns). No correlations were detected between presence of antibodies and acute graft rejection (AGR). Presence of any antibodies (anti-HLA or anti-MICA antibodies) correlated with late graft rejection (P = 0.04). Conclusion. Presence of anti-HLA or anti-MICA had no impact on long-term liver graft survival; however, detection of any antibodies was correlated with episodes of late graft rejection. Presence of donor specific anti-HLA antibodies (DSA) is a negative predictor of graft survival in kidney transplantation. Moreover, the presence of any anti-HLA antibodies without accessing their specificity increases the risk of kidney graft failure . TransplThere are conflicting data regarding impact of anti-MICA antibodies on acute rejection episodes and kidney graft survival , 8. The The 14th International HLA and Immunogenetics Workshop Prospective Chronic Rejection Project was an international collaborative study of 45 transplant centers to assess impact of anti-HLA and anti-MICA antibodies detected after transplant on chronic graft failure. The 1-year follow-up data published in 2007 showed significant impact of anti-HLA antibodies on kidney graft survival  and P = 0.005, OR = 5.26 [1.63–17.03], resp.). HCV infection was a significant negative predictor of all AGR  but not biopsy proven early AGR cases. Early AGR was not significantly correlated with patients age and sex, primary liver disease , HBV infection, nor number or type of immunosuppressive drugs. Late biopsy proven AGR was diagnosed in 13 cases and histopathological findings of concomitant chronic rejection were found in 4 (31%) cases. Late AGR did not significantly correlate with patients age and sex, time since liver transplantation to blood collection, primary liver disease , HBV and HCV infection, induction with basiliximab, nor number or type of immunosuppressive drugs.Biopsy-proven early AGR were diagnosed in 23 cases and early AGR was clinically suspected in 24 cases based on retrospective data. Basiliximab induction was a negative predictor of both all and biopsy-proven cases of early AGR .No correlations were detected between presence of anti-HLA antibodies  or anti-MICA antibodies (neither positive nor only strong positive cases) with early or late AGR. In contrast, presence of any antibodies (anti-HLA or anti-MICA antibodies) correlated with late AGR (P = 0.02) than that in patients with no AGR.Liver function test was performed every 3 months during patients' routine visits in Outpatients Department. The anti-HLA positive versus anti-HLA negative groups did not differ in serum bilirubin concentration, alanine aminotransferase (ALAT), or gamma-glutamyl transpeptidase (GGTP) activity at the end of the 7-year study. Similarly, these variables did not differ between the anti-MICA positive and anti-MICA negative groups. However, mean bilirubin serum concentration was significantly higher in a group of patients with late acute graft rejection  tend to disappear after liver transplantation in most cases but if persist complement activation in the liver allograft could be found in biopsy but it did not impact graft function at 1-year after transplantation . Our datP = 0.003) and that patients with chronic rejection had a higher mean fluorescence intensity (MFI) of DSA than comparator patients [In our analyses, no significant correlations were detected between presence of anti-HLA/anti-MICA antibodies and factors which could potentially activate humoral immune system response, like primary autoimmune liver diseases, minimalization of immunosuppression (HCV and HBV infection or HCC in explanted liver), or type and number of immunosuppressive drugs. The presence of anti-HLA  and anti-MICA antibodies did not significantly correlate with 7-year graft survival or acute graft rejection in either early or late posttransplant period. In contrast, the presence of any antibodies (anti-HLA or anti-MICA antibodies) significantly correlated with late graft rejection. This observation suggests that increased activation of humoral immune system could be involved in late rejection episodes and agrees with results of a prospective study showing that DSA were found significantly more in liver transplant recipients with biopsy proven chronic rejection (36 of 39 (96%)) than in patients without rejection (24 of 39 (61%); patients . In anotpatients . MonitorLimitations of this study included that antibodies were screened only once and at different time points after-transplantation for each patient in the study group; however it was caused by the methodology of the Workshop. Secondly, the presence of anti-HLA antibodies generally, but not DSA, was tested; however, availability of single-antigen Luminex assays was limited when the study was started. We are planning to reevaluate remaining patients sera for the presence of DSA.Presence of anti-HLA or anti-MICA antibodies did not significantly correlate with inferior graft survival nor with increased incidence of acute graft rejection. In contrast, higher humoral immune system activity defined as presence of any antibodies (anti-HLA or anti-MICA antibodies) was a significant predictor of late graft rejection.


The neuroinflammatory response to West Nile virus (WNV) infection can be either protective or pathological depending on the context. Although several studies have examined chemokine profiles within brains of WNV-infected mice, little is known about how various cell types within the central nervous system (CNS) contribute to chemokine expression. Here, we assessed chemokine expression in brain microvascular endothelial cells and astrocytes, which comprise the major components of the blood–brain barrier (BBB), in response to a non-pathogenic (WNV-MAD78) and a highly pathogenic (WNV-NY) strain of WNV. Higher levels of the chemokine CCL5 were detected in WNV-MAD78-infected brain endothelial monolayers compared with WNV-NY-infected cells. However, the opposite profile was observed in WNV-infected astrocytes, indicating that pathogenic and non-pathogenic strains of WNV provoke different CCL5 profiles at the BBB. Thus, cells comprising the BBB may contribute to a dynamic pro-inflammatory response within the CNS that evolves as WNV infection progresses. Flaviviridae. Prior to the 1990s, WNV infections were typically asymptomatic or associated with a self-limiting mild febrile illness known as West Nile fever; however, recent outbreaks of WNV infection in the Western hemisphere and Europe have been associated with an increased incidence of neuroinvasive disease, including meningitis, encephalitis and acute flaccid paralysis (http://www.cdc.gov/ncidod/dvbid/westnile/index.htm) (West Nile virus (WNV) is a positive-strand RNA virus in the family dex.htm) .One of the hallmarks of WNV neuroinvasion is the recruitment of inflammatory leukocytes into the central nervous system (CNS)  . Consistm ImageJ , WNV infm ImageJ . Thus, WP<0.005). However, the level of secreted and cell-associated CCL5 was significantly higher in WNV-MAD78-infected cultures compared with WNV-NY-infected cultures [P<0.01) and f ], suggesting that CCL5 expression is regulated at both the transcriptional and translational level within these cells. Moreover, these findings suggest that pathogenic and non-pathogenic strains of WNV differentially induce CCL5 expression in HBMECs.Unlike CCL2, CCL5 mRNA expression was upregulated ~6000-fold in both WNV-NY- and WNV-MAD78-infected HBMECs compared with mock-infected HBMECs , P<0.005P<0.005). Together, these findings suggest that the chemokine profiles induced by pathogenic and non-pathogenic strains of WNV vary as the infection progresses within the BBB from endothelial cells into astrocytes. The higher levels of CCL5 detected in WNV-NY-infected HBCA cultures were probably due to the enhanced ability of WNV-NY to spread from cell to cell within the HBCA monolayer compared with WNV-MAD78 . Approximately 70 % of the FITC–dextran was detected in the lower chamber of control wells lacking cells or containing HBMEC monolayers treated with EDTA to disrupt tight junctions. In contrast, only 5–10 % of the FITC–dextran was detected in the lower chamber of mock-, WNV-NY- and WNV-MAD78-infected cultures, confirming that WNV infection of the brain endothelial cells is not sufficient to induce disruption of the monolayer  to abluminal supernatants and total fluorescence determined using a FLUOstar Omega fluorescence microtitre plate reader (−1), WNV infection did not significantly enhance THP-1 migration compared with mock treated co-cultures. This suggests that the chemokine profiles induced in response to WNV infection of HBMECs and HBCAs were not sufficient to mediate monocyte transmigration across the endothelial cell monolayer.In many cases, viral disruption of the BBB is the result of breakdown of the endothelial tight junctions by matrix metalloproteinases secreted from infected astrocytes (tom well . HBMECs tom well . Thus, te reader . Whilst in vitro model of the BBB containing both endothelial cells and astrocytes. Other cell types within the CNS, such as neurons or activated microglia, may therefore be responsible for the establishment of the chemokine gradients that attract immune cells into the CNS during WNV infection. Likewise, WNV replication did not affect the integrity of the HBMEC monolayer, suggesting that additional host factors are required for the breakdown of the BBB that occurs in vivo late in infection (In conclusion, our data suggest that pathogenic and non-pathogenic strains of WNV differentially induce the pro-inflammatory chemokine CCL5 at the BBB, although neither virus substantially induced CCL2. During initial infection of brain endothelial cells, the enhanced chemokine expression in response to WNV-MAD78 may stimulate the rapid recruitment of circulating leukocytes to the BBB. However, the induction of CCL5 in response to WNV infection was not sufficient to promote leukocyte transmigration across the endothelial layer in an nfection . Nonethe


A peer-driven intervention (PDI) for female sex workers (FSWs) was implemented in harm reduction projects in two Ukrainian cities. The goal was to recruit and interview 500 FSWs in 6 months who had never received services before, and to measure how well FSW-recruiters educated FSWs in a body of fresh HIV prevention information. Recruiters were rewarded by earning tickets to win special prizes in a weekly lottery.A PDI relies on respondent educating and recruiting peers for services. All recruits also get to serve as peer-educator/recruiters. Each recruit’s score on an 8-item knowledge test, measuring how well the recruiter educated her, determined how many lottery tickets the recruiter earned. A weekly lottery was held each week offering prizes to 4 lucky winners.The two PDIs in 6 months recruited 532 and 437 FSWs never seen before, 2-3 times more new FWSW-recruits than the number recruited 6 months prior to the PDIs’ start-up. Both projects held 23 lotteries with an average attendance of 8 FSWs. Very significant differences in levels of drug- and sex-related risk behaviors were found between FSW drug-users and non-users, heavily shaped by education, knowledge, and other social variables, suggesting more targeted types of intervention.The FSW-respondents were eager to serve as peer-educators/recruiters. The lottery proved to be a cost-saving and effective reward system that was highly motivating. The PDI offers harm reduction projects an entirely new model for accessing and educating FSWs, as well as a new method for targeting special sub-populations of FSWs.


Isolated tuberculosis of the pancreas is rare even in developing countries where abdominal tuberculosis continues to be prevalent. We present a case of pancreatic tuberculosis in an immunocompetent male with confounding imaging findings and non-contributory clinical details. Mycobacterium tuberculosis DNA. The patient was initiated on multi-drug anti-tuberculous regimen and remained well 6-months post treatment.A 48-year-old male presented with progressive abdominal discomfort and weight loss of 3 months duration. Laboratory investigations revealed total bilirubin of 3.3 mg/dL, aspartate aminotransferase 141 IU, alanine aminotransferase 100 IU, alkaline phosphatase 436 IU, and erythrocyte sedimentation rate of 78 mm the first hour. Computed tomography revealed a well-marginated cystic lesion in the head of the pancreas with upstream biliary dilatation . The remM. tuberculosis is extremely rare, presumably because of the resistance offered by the pancreatic enzymes2Pancreatic involvement by


Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery (MCAO) or occlusion of the distal segment of middle cerebral artery (dMCAO) on day 14 of vehicle or GCV treatment and mice were killed 24 hr or 12 weeks later. Increased infarct volume or brain atrophy was found in GCV- compared to vehicle-treated middle-aged DCX-TK(+) mice, both 24 hr after MCAO and 12 weeks after dMCAO. More severe motor deficits were also observed in GCV-treated, middle-aged DCX-TK(+) transgenic mice at both time points. Our results indicate that ischemia-induced newborn neurons contribute to anatomical and functional outcome after experimental stroke in middle-aged mice.Depletion of neurogenesis worsens functional outcome in young-adult mice after focal cerebral ischemia, but whether a similar effect occurs in older mice is unknown. Using middle-aged (12-month-old) transgenic (DCX-TK Aging is associated with a striking increase in the incidence of stroke and neurodegenerative diseases, which are major causes of disability among those aged 70 years and older In the adult brain, neurogenesis occurs in the subventricular zone (SVZ) of the lateral ventricles, from where newly generated cells migrate into the olfactory bulb, and the subgranular zone (SGZ) of the dentate gyrus (DG), where new granule cells become integrated into the local neuronal network. Neural stem cells (NSCs) in these regions proliferate in response to focal ischemia (+) middle-aged mice resulted in both increased infarct volume (acute phase) or brain atrophy (chronic phase) and more severe neurologic deficits, compared with vehicle-treated DCX-TK(+) middle-aged mice and either GCV- or vehicle-treated DCX-TK(−) mice. Our results indicate that ischemia-induced new-born neurons also contribute functional recovery in middle-aged mice after experimental stroke.In the current study, we conditionally depleted newly generated neurons in middle-aged mouse and assessed outcome after focal cerebral ischemia. We found that depletion of DCX-expressing cells in GCV-treated DCX-TK(+)) that express HSV-1 TK under control of the DCX promoter and DCX-TK(−) mice were generated at Buck Institute for Research on Aging as described in our previous publication Transgenic CD1 mice (DCX-TK(+) or DCX-TK(−) mice were anesthetized with 4% isoflurane in 70% N2O/30% O2, implanted with osmotic minipumps , and infused continuously for 14 days with 0.25 μl/hr of either 20 mM ganciclovir (GCV)  or vehicle (PBS). Focal ischemia was induced on day 14 of GCV administration.Middle-aged (12-month-old) male DCX-TK5-bromo-2-deoxyuridine  was dissolved in saline and injected intraperitoneally twice daily at 24 hr prior to termination of GCV administration or on the last day after dMCAO and animals were killed 24 h later.(+) and DCX-TK(+)) were anesthetized with 2.0% isoflurane in 70% N2O/30% O2 using a vaporizer. Permanent intraluminal suture occlusion of the middle cerebral artery (MCAO) and distal middle cerebral artery occlusion (dMCAO) were performed for short-term and long-term outcome assessment respectively. MCAO was induced by intraluminal occlusion with a nylon monofilament suture as described previously Middle-aged (12-month-old) male mice  for 2 hr at room temperature, before being treated with mouse monoclonal anti-BrdU overnight at 4°C. Immunohistochemistry was conducted using a standard protocol with antigen retrieval , according to the manufacturer's instructions. The primary antibodies used were mouse monoclonal anti-BrdU  and affinity-purified goat anti-DCX . The second antibodies were biotinylated donkey anti-goat or biotinylated horse anti-mouse antibody . Sections were examined with a Nikon E800 epifluorescence microscope. Controls included omitting the primary or secondary antibody.Immunohistochemistry was performed as described previously Double-label immunohistochemistry (5 mice/group) was performed as described elsewhere BrdU- and DCX-positive cells in the SVZ and DG were counted by an observer blind to experimental condition using a Zeiss microscope in bright field mode and a 40 x objective. A two-photon confocal microscope was used to count double-labeled cells. In the SVZ, DCX- and BrdU-labeled cells were counted along the lateral walls of the lateral ventricles, corresponding to coronal coordinates interaural 8.7 to 10.2 mm, bregma −0.30 to bregma −1.2 mm, for a total of 6-µm coronal sections  per mouse (6 mice per group for single immunostaining and 5 mice per group for double immunostaining). In the DG, DCX- and BrdU-labeled cells within two cell diameters from the inner edge of the granule cell layer (GCL) of the dente gyrus  were included in the analysis.(+) or DCX-TK(−) mice (n = 4) were anesthetized and decapitated 24 hr after MCAO or 12 weeks after dMCAO. Brains were removed and 50-µm coronal sections were prepared and stained with H&E. Infarct area (24 hr after MCAO) or brain atrophy area (12 weeks after dMCAO), left hemisphere area, and total cross-sectional brain area were measured by a blinded observer using the NIH Image program, and areas were multiplied by the distance between sections to obtain the respective volumes. Infarct volume or volume loss (brain atrophy) was calculated as a percentage of the volume of the contralateral hemisphere, as described previously DCX-TKThe beam-walking test was used to assess deficits in coordination and integration of motor movement, especially in the hindlimb 3. The two boards were gradually moved to enclose the mouse from both sides and encourage the mouse to enter into a 30° corner with a small opening along the joint between the two boards. When the mouse entered deep into the corner, both the vibrissae on both sides were stimulated by the boards. The mouse then reared forward and upward, and then turned back to face the open end. Twenty trials were performed for each mouse and the percentage of left turns was calculated. Only turns involving full rearing along either board were recorded.The corner test, used to test integrated sensorimotor function, was performed according to a previous protocol The elevated body swing test was used to test asymmetric motor behavior. Mice (n = 12 per group) held by the base of the tail were raised ≈10 cm above the testing surface. The initial direction of body swing, constituting a turn of the upper body of >10° to either side, was recorded in three sets of 10 trials, performed over 5 min. The number of turns in each (left or right) direction was recorded, and the percentage of turns made to the side contralateral to the ischemic hemisphere (percent left-biased swing) was calculated. For each mouse, average scores were determined.Limb-placing, a test of lateralized post-ischemic sensorimotor dysfunction Mice (12 mice/group) were trained on an accelerating (5 to 40 rpm) rotating rod (rotarod) for 3 days before focal ischemia; only those mice able to remain on the rod for 20 s at 40 rpm were subjected to focal ischemia post hoc multiple comparison tests (Fisher PLSD or Student's paired t test with the Bonferroni correction). Brain atrophy data were analyzed by one-way ANOVA followed by Fisher PLSD post hoc tests. P values <0.05 were considered significant.Quantitative data were expressed as mean ± SEM from the indicated number of experiments. Behavioral data were analyzed by two-way analysis of variance (ANOVA) with repeated measures, followed by (+) and DCX-TK(−) mice were treated with GCV or vehicle for 14 days and received behavioral training, and then underwent MCAO or dMACO. Behavioral testing was conducted for 12 weeks after dMCAO ((+) transgenic mice treated with vehicle. After treatment with GCV for 14 days, DCX- and BrdU-positive cells in SVZ and SGZ were barely detectable middle-aged DCX-TK(+) transgenic, compared to DCX-TK(−) mice. Double immunostaining shows that most BrdU-positive cells expressed DCX (Middle-aged DCX-TK(+) transgenic mice treated with GCV, compared to vehicle-treated DCX-TK(+) mice and either vehicle- or GCV- treated middle-aged DCX-TK(−) mice, suggesting that NSCs in middle-aged brain influence acute histological outcome after MCAO.To determine the role of neurogenesis in outcome from focal ischemia in middle-aged mice, we first induced focal ischemia by permanent intraluminal suture occlusion of MCA after administration of GCV or vehicle for 14 days, and mice were killed 24 hr later. As shown in (+) mice was significantly higher than that in vehicle-treated DCX-TK(+) and vehicle- or GCV-treated DCX-TK(−) mice.To determine if depletion of DCX-positive cells could also affect long-term histological outcome, we induced focal cerebral ischemia by permanent dMCAO in middle-aged mice treated with GCV or vehicle for 14 days. dMCAO was used in these experiments because it produces stroke mortality is generally lower in older rats (+) mice. We found that deficits in beam-walking ((+) transgenic mice tested up to 4 weeks after dMCAO, compared to vehicle-treated DCX-TK(+) and vehicle- or GCV-treated DCX-TK(−) mice, suggesting involvement of newly generated cells in functional outcome after focal ischemia.Next we measured neurological deficits in middle-aged DCX-TK(−) mice treated with GCV or vehicle incorporated into BrdU, but only less than 5% DCX-positive cells in the SGZ of dentate gyrus incorporated into BrdU ((+)mice treated with GCV. Collectively, these results indicate that neurogenesis in middle-aged brain can be slowly restored to normal baseline at certain recovery period even after severely depletion.Finally, we asked whether NSCs in the SVZ and SGZ of dentate gyrus were able to restore newborn neurons in middle-aged mice after nearly complete ablation of DXC-positive cells following focal ischemia. As shown in In the current study, we found that the NSC depletion causes larger infarct volumes or volume loss and increases neurological deficits in middle-aged mice after focal cerebral ischemia. This suggests that ischemia-induced neurogenesis exerts a beneficial effect on short-term and long-term outcome not only in young-adult (25), but also in middle-aged mice.(+) transgenic mice, in which neurogenesis could be selectively ablated after administration of GCV. Using this approach, we found that depletion of newborn cells in young-adult mice results in worsened functional outcome after experiment stroke. As stroke predominantly occurs in more aged populations Brain damage, such as stroke, triggers reparative mechanisms, among which neurogenesis may be one. Experimental (4) and clinical (23) stroke are both associated with increased neurogenesis, and the time course over which occurs coincides with the most active period of functional recovery. Although this does not necessarily imply a causal relationship between neurogenesis and recovery, inhibition of neurogenesis by antimitotic drugs is associated with worsened outcome after cerebral ischemia Although basal neurogenesis in the SVZ and SGZ of dentate gyrus declines with age, we found previously that SVZ neurogenesis is still increased after focal ischemia, albeit to a lesser extent, in older rats, who therefore appear to retain the capacity for stroke-induced neurogenesis Neurogenesis in the aging brain can be manipulated by pharmaceutical tools, such as growth factors. A recent study showed that granulocyte-colony stimulating factor (G-CSF), a cytokine, exerts a robust and sustained beneficial effect on survival, motor function, and working memory after focal ischemia, and stimulates the proliferation of NSCs cells in the ipsilateral SVZ


In recent decades Chinese medicine has been used worldwide as a complementary and alternative medicine to treat cancer. Plenty of studies have shown that microRNAs (miRNAs) play fundamental roles in many pathological processes, including cancer, while the anti-cancer mechanisms of Chinese medicinal herbs targeting miRNAs also have been extensively explored. Our previous studies and those of others on Chinese medicinal herbs and miRNAs in various cancer models have provided a possibility of new cancer therapies, for example, up-regulating the expression of miR-23a may activate the positive regulatory network of p53 and miR-23a involved in the mechanism underlying the anti-tumor effect of berberine in hepatocellular carcinoma (HCC). In this review, we survey the role of Chinese medicinal herbal products in regulating miRNAs in cancer and the use of mediating miRNAs for cancer treatment. In addition, the controversial roles of herb-derived exogenous miRNAs in cancer treatment are also discussed. It is expected that targeting miRNAs would provide a novel therapeutic approach in cancer therapy by improving overall response and survival outcomes in cancer treatment, especially when combined with conventional therapeutics and Chinese medicinal herbal products. With the rapid development of the Human Genome Project, the non-coding sequence that accounts for 99% of the total human genome is attracting more and more attention by researchers. So far, in recent decades several kinds of non-coding RNAs have been discovered and identified, including Piwi-interacting RNAs (piRNAs), short interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs) and microRNAs (miRNAs) ,2,3. AmoAs miRNAs are involved in many physiological functions of human cells, the dysregulation of miRNAs is associated with many kinds of human disorders such as heart and nervous system diseases, obesity and cancer ,11,12. ITraditional Chinese medicine (TCM) is one of the most widely used adjunctive therapies for cancer patients worldwide. TCM plays an important role in cancer treatment by suppressing tumor proliferation, preventing complications, improving quality of life and reducing the side effects of conventional treatments in cancer patients ,22,23. Sin vivo experiment results also confirmed that curcumin could suppress tumor growth by miR-21-mediated tumor cell apoptosis in a xenograft mice model [Curcumin is the principal curcuminoid of turmeric, which belongs to the ginger family. Curcumin treatment could result in a >80% knockdown of miR-21 expression in prostate cancer cells, thus increasing expression of several downstream target genes, including PTEN and PDCD4, and inducing apoptosis in prostate cancer cells. The ce model .Coptis chinensis. A recent study has proved that berberine could induce apoptosis in multiple myeloma cells by decreasing the expression of miR-99a, miR-17 and miR-106 and regulating the TP53, Erb and MAPK signaling pathways [Camptotheca acuminata could induce apoptosis in cancer cells by down-regulating miR-125b and activation of the mitochondrial apoptosis pathways by increasing the expression of the Bak1, Mcl1, and p53 genes [Berberine is a famous anti-cancer herbal product from the root of pathways . A studypathways . A Chine53 genes . MiR-125Polygonum cuspidatum and some fruit skins. Recent studies have found that resveratrol could induce apoptosis by up-regulating miR-137 levels and subsequently reducing EZH2 expression in neuroblastoma. Decreasing EZH2 expression could further induce H3K27me3 down-regulation and activation of the tumor suppressor genes CLU and NGFR [Resveratrol is a popular natural anti-cancer agent isolated from and NGFR . In anotand NGFR . Terminalia chebula. The pro-apoptosis effects of luteolin in gastric cancer cells are related to down-regulation of Bcl-2 expression by decreasing miR-34a expression. Further studies showed that anti-miR-34a oligonucleotides could block the luteolin-induced Bcl-2 suppression in gastric cancer cells [Luteolin is a natural flavone from the leaves of er cells . Genistein is a kind of isoflavone which has shown activity inducing apoptosis in prostate cancer cells. MiR-1260b was down-regulated by more than 50 percent after genistein treatment in prostate cancer cells. Knocking down miR-1260b caused inhibition of two target genes, sFRP1 and Smad4. The expression of these two genes induced apoptosis and reduced survival of prostate cancer cells .Sophora flavescens. The pro-apoptotic effect of matrine has been identified by many studies. It was found that matrine in combination with sorafenib could induce apoptosis in HCC cells, and the mechanism study showed that inhibition of miR-21 and the subsequent increased of PTEN expression contributed to the pro-apoptosis effect of matrine and sorafenib [Matrine is one of the major bioactive ingredients isolated from orafenib .in vitro as well as lung metastasis in vivo by inhibiting MMP-2 activation. Further mechanistic studies showed that this MMP-2 inhibition was mediated by miR-328 up-regulation, treatment of osteosarcoma 143B and U2OS cells with resveratrol (RESV) for 6 h can up-regulate miR-328 expression by approximately 2-fold in both cell lines [in vivo studies, one recent research report has found that resveratrol can inhibit tumor metastasis in an immunodeficient mice model. Further mechanistic studies confirmed that resveratrol can inhibit the mesenchymal-epithelial transition process by suppressing the expression of FOXC2 through regulation of miRNA-520h-mediated signal cascade in lung cancer cells [The epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells transform into mesenchymal type ones and become more invasive and migratable. Many studies have confirmed the relationship between EMT and cancer metastasis ,36. Recell lines . Isoflavll lines . Panax Nll lines ,42. For Coptidisrhizoma have been extensively elaborated in our lab. Recently, we have demonstrated that berberine could up-regulate miR-23a expression by 5-fold compared to the control group and mediate the up-regulation of the tumor suppressive genes p21/GADD45α and induce G2/M cell cycle arrest in HCC cells [Coptidisrhizome is also effective in reducing cancer cell proliferation by targeting miRNAs. After treating HCC cells with Coptidisrhizoma aqueous extract, both miR-21 and miR-23a were obviously up-regulated. The downstream target genes of these miRNAs still need further exploration in future studies [The anti-cancer effects of berberine and CC cells . Accordi studies . Ginseno studies . Ginseno studies . Ganoderma fruiting body has been used as a precious herbal medicine for thousands years in some Asian countries, but Ganoderma spore lipid was only discovered and applied in cancer adjunctive therapy several decades ago. Recent studies have demonstrated that Ganoderma spore lipid inhibited cell proliferation of lung adenocarcinoma in a dose-dependent manner. Mechanistic studies revealed that Ganoderma spore lipid could inhibit the expression of miR-21 and obviously increase the expression of PTEN and PDCD4 [As an anti-cancer alkaloid agent, matrine has been used as effective treatment against breast cancer with approval by the public health authorities in China. Recent research has shown that matrine could induce cell cycle arrest at G1/S phase in human breast cancer cells. Further mechanistic studies demonstrated that matrine could increase PTEN expression by decreasing miR-21 levels which subsequently led to dephosphorylated Akt, and further induced accumulation of Bad, p27(/KIP1) and p21(/WAF1/CIP1) . The Chind PDCD4 . in vitro and in vivo. miR-135a and miR-135b were down-regulated by approximately 80% after mistletoe lectin-I treatment via degradation of their precursors. Western blot analysis showed that levels of their their target gene adenomatous polyposis coli (APC) were increased, which contributed to the anti-proliferative effect displayed by mistletoe lectin-I [The herb mistletoe is widely used in gynecological and obstetrical diseases as well as a complementary cancer therapy in TCM. The major active component of mistletoe called mistletoe lectin-I have been shown to display significant proliferation inhibition towards colorectal cancer cells both lectin-I ,57.in vivo tumorigenicity of cancer stem-like MHCC97L cells obviously decreased and the proliferation and invasion ability of these cells were also suppressed. Further studies showed that miR-122 regulation may participate in the inhibition of cancer-stem like cells by hydroxycamptochecin, along with suppression of albumin, α-fetoprotein and hepatocyte nuclear factor-4 expression [A novel analog of curcumin has shown inhibitory effect on cancer stem-like cells in chemoresistant colon cancer cells. A mechanistic study demonstrated that this agent could down-regulate miR-21 expression with up-regulation of PTEN and inhibition of Akt phosphorylation. As the PTEN-Akt pathway plays an important role in chemoresistance in cancer stem-like cells, this finding may provide a novel therapeutic agent targeting cancer stem-like cells . Hydroxypression . Anotherpression . Magnolia. Because of its special chemical properties and high oral bioavailability, honokiol has been used as a promising herbal agent for cancer treatment. A study demonstrated that honokiol could increase miR-141 expression and further down-regulate the target gene zinc finger E-box binding homeobox 2 (ZEB2) in renal cancer cells. As ZEB2 is associated with cancer stem cell properties and EMT, honokiol could inhibit metastasis as well as the stem-like properties of cancer cells at least partly through the miR-141/ZEB2 pathway [Honokiol is a bioactive ingredient isolated from a number of species of  pathway .et al. According to their studies, plant-derived miRNAs could transfer into animal’s blood and tissues from food sources. More surprisingly, their further studies showed that the plant-derived exogenous miR-168 could down-regulate the expression level of low-density lipoprotein receptor-associated protein 1 in the liver of mice [et al., attributed Zhang’s results to an underestimated effect of contamination [In 2012, an interesting hypothesis on exogenous miRNAs was presented by Zhang  of mice . Several of mice ,64,65. T of mice ,67. Howemination . These omination ,71. In fmination . HoweverAccording to the previous studies, miRNAs target more than 5000 human genes, which represent probably 30% of the human genome . These sin vitro and in vivo studies, have shown encouraging safety profiles and significant anti-cancer effect via targeting specific miRNAs. Chinese herbal medicines and miRNAs may provide a new possibility for alternative therapies of cancer. We hope this review on Chinese herbal medicines targeting miRNAs will contribute to a novel understanding of anti-cancer Chinese herbal medicines and shed light on their future clinical applications.In conclusion, these Chinese medical herbal products, which have been tested extensively by


Donor Lymphocyte infusion (DLI) is a therapy for the patients with relapsed hematological malignancy after allogeneic hematopoetic stem cell transplantation. However, the development of Graft-Versus-Host Disease (GVHD) is a serious adverse event and the efficacy is limited when one needs to control the GVHD. To inhibit the development of GVHD with increased tumor-specificity of transferred allogeneic lymphocytes, here we demonstrate the development of DLI with lymphocytes engineered to express tumor-specific T cell receptor (TCR) in combination with decreased GVHD-inducing potential utilizing the novel retrovirus vector (siTCR vector) that specifically silences endogenous TCR in gene-engineered T cells.3H uptake proliferation assay. Immunodeficient NOG mice were inoculated with a NY-ESO-1-expressing human melanoma cell line NW-MEL-38, received TCR gene-transduced T cells, and monitored for tumor growth and the development of GVHD.Human PBMC were transduced with a high affinity TCR specific to a cancer/testis antigen, NY-ESO-1, by the retrovirus vector with siRNA specific to the endogenous TCR. Resulting TCR gene-transduced T cells were examined for their reactivity to allogeneic LCL by Human lymphocytes that were genetically engineered to express a high affinity NY-ESO-1-specific TCR with siTCR vector showed reduced expression of endogenous TCR associated with the dramatically diminished reactivity to allogeneic lymphocytes. When administrated into NOG mice, these TCR gene-transduced T cells induced tumor regression without the development of GVHD.The results here suggest that the allogeneic T cells transduced with a tumor-specific TCR by siTCR vector showed diminished GVHD potential. These T cells will be applicable to the donor lymphocytes infusion therapy after allogeneic stem cell transplantation for the treatment of hematological malignancy, providing diminished GVHD and increased tumor eradication.


Reactivation of KLK5 suppressed key EMT genes. Unexpectedly, we identified altered expression of genes encoding enzymes of the mevalonate pathway typical of those observed upon cholesterol starvation. Consistently, we found that SREBF1, the master regulator of the mevalonate pathway was induced. KLK5 re-expression leads to reduced cellular cholesterol and fatty acid synthesis and enhanced uptake of LDL-cholesterol. Suppression of the mevalonate pathway in KLK5 transfectants was further shown by reduced synthesis of isoprenoids. Indeed, we found diminished levels of active RhoA, a signaling oncoprotein that requires prenylation for activation. We propose that reduced RhoA activation plays a dominant role in suppression of malignancy by KLK5, since geranylgeranyl pyrophosphate restored active RhoA in KLK5-reverted cells resulting in increased malignancy. For the first time, we suggest that a protease may suppress breast cancer by modulating the mevalonate pathway.Kallikrein-related peptidase 5 (KLK5) displays aberrant expression in cancer. However, any functional association is missing. Here, we show that reconstitution of KLK5 expression in non-expressing MDA-MB-231 breast cancer cells suppresses malignancy  In vitro studies indicated that KLK5 can activate the protease-activated receptors (PARs) -acetate into fatty acids was quantified by autoradiography and visualized with phosphomolybdic acid.The assay is based on the incorporation of the [1-ynthesis . Cells w


Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disease caused by the autoantibody against granulocyte-macrophage colony stimulating factor (GM-CSF). The clinical course of aPAP is variable; in severe cases, patients develop lethal respiratory failure due to pulmonary fibrosis. However, the pathogenesis of pulmonary fibrosis in aPAP has never been delineated.Here, we describe a rare case of aPAP that was subsequently complicated by microscopic polyangiitis-related pulmonary fibrosis. The patient was a 75-year-old Japanese man diagnosed with aPAP based on the crazy-paving appearance on high-resolution computed tomography (HRCT), “milky” appearance of broncho-alveolar lavage fluid (BALF), and elevated serum levels of the anti-GM-CSF antibody. The patient was followed-up without aPAP-specific treatment for 3 years. During this period, both hematuria and proteinuria appeared; in addition, serum myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA) turned positive and increased markedly. The second BAL performed one year after the diagnosis, showed that the “milky” appearance had resolved. The HRCT showed that fibrotic changes had developed and that the crazy-paving appearance had disappeared. These data suggest an association between pulmonary fibrosis that developed during the natural course of aPAP and ANCA-related systemic vasculitis.This is the first case report that suggests the existence of a pathogenetic relationship between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis. The link between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis requires further investigation. Autoimmune pulmonary alveolar proteinosis (aPAP), which causes 90% of all PAP cases, is an autoimmune disease caused by the presence of an autoantibody against granulocyte-macrophage colony stimulating factor (GM-CSF) . The supThe pathogenesis of pulmonary fibrosis in aPAP is unknown. It has been hypothesized that the retention of lipoproteinaceous material in the alveoli, silica exposure, and/or superimposed pulmonary infections induces damage to cells lining the alveoli and causes pulmonary fibrosis in aPAP patients . In ratsAnti-neutrophil cytoplasmic antibody (ANCA) is a sensitive and specific marker for ANCA-associated systemic vasculitis, as observed in granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), “idiopathic” necrotizing crescentic glomerulonephritis and allergic granulomatous angiitis . In vitr5) showed a typical “milky” appearance. The total cell count in BALF was 320/μL. Differential cell count revealed 50% alveolar macrophages, 36% lymphocytes, 12% neutrophils, 2% eosinophils. Pathological examination of a transbronchial lung biopsy sample revealed periodic-acid-Schiff stain-positive material along the alveolar wall. After the serum anti-GM-CSF autoantibody concentration  was confirmed and the comorbidity of hematological diseases, such as chronic myelocytic leukemia, myelodysplastic syndrome, or monoclonal gammopathy of undetermined significance, were excluded, the patient was diagnosed with aPAP.A 75-year-old Japanese man, who self-identified as a chronic smoker (30 pack-years), was referred to our hospital because of a cough, which had been continued for 2 months, and an abnormal chest X-ray. He had no significant past medical history. He was an owner of a liquor shop and had no prior exposure to harmful dust. The physical examination revealed fine crackles bilaterally over the lower lung. The laboratory findings showed elevated levels of lactate dehydrogenase , Krebs von den Lungen-6 , surfactant protein-D , carcinoembryonic antigen , and CYFRA21.1 (cytokeratin-19 fragments)  in the serum. Serum antinuclear antibody titer was 1:40 , and myeloperoxidase (MPO)-ANCA testing was negative. The chest X-ray showed diffuse bilateral ground-glass opacities, and high-resolution computed tomography (HRCT) showed a crazy-paving appearance  as grade 2. The chest HRCT obtained at this visit showed that the crazy-paving pattern had been replaced by fibrotic changes, i.e., subpleural honeycombing and traction bronchiectasis Figure A. In conMany studies have shown that ANCA production is key to the pathogenesis of MPA. ANCA is known to activate neutrophils and allow their accumulation at the endothelial portion of a vasculitic lesion . ANCA isThere are several reports of pulmonary fibrosis that developed in patients with aPAP. In these cases, as in ours, the features of PAP disappeared as pulmonary fibrosis progressed , 7. As LIt is generally thought that autoimmune diseases are induced by dysfunctional immunotolerance to self-antigens due to genetic as well as environmental factors . ClinicaPrevious research has not succeeded in explaining why pulmonary fibrosis occurs in patients with aPAP, or how we should treat this complication, which is often associated with a poor prognosis. This is the first case report to suggest a pathogenetic relationship between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis. The link between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis must be explored with further research.Because the patient passed away due to the progression of respiratory failure, written informed consent was obtained from the patient’s wife for publication of this case report and any accompanying images. A copy is available for review by the Editor of this journal.