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1.  Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix 
Journal of microscopy  2013;251(3):212-223.
Summary
Mammary branching morphogenesis occurs over a period of weeks deep inside an adipocyte-rich stroma. The adipocytes contain light-scattering lipid droplets that limit the depth of penetration of visible light. Organotypic culture methods were developed to enable high-resolution optical monitoring of branching morphogenesis ex vivo. A challenge has been to identify the best culture conditions to model specific developmental events. We recently demonstrated that collagen I induces protrusive invasion in both normal and neoplastic mammary epithelium. In this study, we observed that the abundance of collagen I fibrils correlated strongly with invasive behaviour, even when the collagen I concentration was identical. We found that the extent of fibril assembly was experimentally manipulable by varying the incubation time at 4°C following pH neutralization. We next tested the capacity of collagen I fibrils to induce invasive behaviour when presented in combination with basement membrane proteins (Matrigel). We found that epithelial organoids in mixed gels of collagen I and basement membrane proteins exhibited more extensive branching morphogenesis but did not initiate protrusions into the matrix. Organoids in pure Matrigel produced many small epithelial buds that were bare of myoepithelial cells. Surprisingly, organoids in mixed gels of collagen I and Matrigel produced fewer epithelial buds, the buds elongated further, and the elongating buds remained covered by myoepithelial cells. Our mixed gels therefore provide a more physiologically accurate model of mammary branching morphogenesis. Our results also suggest that changes in the composition of the extracellular matrix could induce migration of epithelial cells past myoepithelial coverage.
doi:10.1111/jmi.12017
PMCID: PMC3978143  PMID: 23432616
Basement membrane; collagen I; extracellular matrix; mammary branching morphogenesis
2.  Retardation of arsenic transport through a Pleistocene aquifer 
Nature  2013;501(7466):204-207.
Groundwater drawn daily from shallow alluvial sands by millions of wells over large areas of South and Southeast Asia exposes an estimated population of over 100 million to toxic levels of arsenic (1). Holocene aquifers are the source of widespread arsenic poisoning across the region (2, 3). In contrast, Pleistocene sands deposited in this region more than ~12,000 years ago mostly do not host groundwater with high levels of arsenic. Pleistocene aquifers are increasingly used as a safe source of drinking water (4) and it is therefore important to understand under what conditions low levels of arsenic can be maintained. Here we reconstruct the initial phase of contamination of a Pleistocene aquifer near Hanoi, Vietnam. We demonstrate that changes in groundwater flow conditions and the redox state of the aquifer sands induced by groundwater pumping caused the lateral intrusion of arsenic contamination over 120 m from Holocene aquifer into a previously uncontaminated Pleistocene aquifer. We also find that arsenic adsorbs onto the aquifer sands and that there is a 16–20 fold retardation in the extent of the contamination relative to the reconstructed lateral movement of groundwater over the same period. Our findings suggest that arsenic contamination of Pleistocene aquifers in South and Southeast Asia as a consequence of increasing levels of groundwater pumping have been delayed by the retardation of arsenic transport.
doi:10.1038/nature12444
PMCID: PMC3772538  PMID: 24025840
3.  Diabetes Mellitus Accelerates Cartilaginous Metaplasia and Calcification in Atherosclerotic Vessels of LDLr Mutant Mice 
Background
Vascular calcification is highly prevalent in patients with type II diabetes mellitus (T2DM). Little is known about whether T2DM is causative.
Methods
Low density lipoprotein receptor mutant (LDLr−/−) mice were fed with customized diabetogenic and/or procalcific diets to induce atherosclerosis, cartilaginous metaplasia and calcification, along with obesity, hyperglycemia, hyperinsulinemia, and hypercholesterolemia at various levels, and euthanized for study after 18–24 weeks on diet.
Results
We found that T2DM accelerated cartilaginous and calcific lesion development by ~3- and 13-folds as determined by incidence of vascular cartilaginous metaplasia and calcification in LDLr−/− mice. Lowering dietary fat from ~60% to ~40% kcal reduced body weight and serum glucose and insulin levels, leading to a 2-fold decrease in aortic calcium content. Correlation analysis of calcium content with a calculated insulin resistance index, HOMA-IR, showed a positive correlation of insulin resistance with vascular calcification. Finally, we used genetic fate mapping strategy to trace cells of SM origin in these animals. Vascular SMCs were found to be a major cell source contributing to osteochondrogenic differentiation and calcification. Receptor for advanced glycation end-products (RAGE) was up-regulated, co-localizing with osteochondrogenic SMCs.
Conclusions
Through quantitative measure of aortic calcium content, we provided experimental findings that LDLr−/− mice, like T2DM patients, are predisposed to vascular calcification. Our study is also the first to establish a distinct role of hyperglycemia and hypercholesterolemia in osteochondrogenic differentiation of SMCs and determined these cells as a major source contributing to cartilaginous and calcifying lesions of T2DM blood vessels, possibly mediated by RAGE.
doi:10.1016/j.carpath.2012.06.007
PMCID: PMC3481013  PMID: 22818582
atherosclerosis; receptor for glycation end-products; smooth muscle cells; type 2 diabetes mellitus; vascular calcification
4.  Patellar dislocation due to iatrogenic quadriceps fibrosis: results of operative treatment in 54 cases 
Objective
To evaluate the clinical and functional results of a surgical treatment of patellar dislocation whose etiology was iatrogenic quadriceps fibrosis in children.
Materials and methods
A prospective study was undertaken from February 2004 to December 2009. The study included 54 pediatric patients (56 knees) that had developed dislocation of the patella after repeated intramuscular injections of antibiotic(s) into the quadriceps muscle. There were 11 males (20.4 %) and 43 females (79.6 %). The patients’ mean age at surgery was 7 years, 9 months (range 6 years, 4 months to 12 years, 6 months). A complete history of each patient was recorded. The affected knees were evaluated preoperatively and postoperatively on the basis of the symptoms, signs, and roentgenographic findings. Patellar dislocation was classified according Bensahel’s criteria. All patients had a three-part surgical procedure that combined capsulorrhaphy, quadricepsplasty, and transfer of the vastus medialis oblique to the superior border of the patella.
Results
There has been no poor postsurgical result or recurrence so far; we have noted an ugly scar in nine knees (16.1 %), limitation of the knee flexion in five knees (8.9 %), and loss of extension of 5 °–20 ° in four knees (7.1 %). Overall, we attained excellent results in 39 knees (69.7 %), good results in 13 knees (23.2 %), and fair results in four knees (7.1 %).
Conclusion
In our cases of pediatric dislocation of the patella caused by iatrogenic quadriceps fibrosis, the introduced three-part surgical procedure has shown great success in restoring the realignment mechanism of the patella. The technique is simple, safe, and effective in skeletally immature children.
doi:10.1007/s11832-014-0564-5
PMCID: PMC3935028  PMID: 24510420
Patellar instability; Patellar dislocation; Iatrogenic quadriceps fibrosis; Surgical treatment; Quadricepsplasty; Capsulorrhaphy; Subluxation of the patella; Developmental dysplasia of the patella (DDP); Malformative dislocation
5.  Draft Genome Sequence of Pseudomonas nitroreducens Strain TX1, Which Degrades Nonionic Surfactants and Estrogen-Like Alkylphenols 
Genome Announcements  2014;2(1):e01262-13.
Pseudomonas nitroreducens TX1 ATCC PTA-6168 was isolated from rice field drainage in Taiwan. The bacterium is of special interest because of its capability to use nonionic surfactants (alkylphenol polyethoxylates) and estrogen-like compounds (4-t-octylphenol and 4-nonylphenol) as a sole carbon source. This is the first report on the genome sequence of P. nitroreducens.
doi:10.1128/genomeA.01262-13
PMCID: PMC3907738  PMID: 24482523
6.  Randomised Pharmacokinetic Trial of Rifabutin with Lopinavir/Ritonavir-Antiretroviral Therapy in Patients with HIV-Associated Tuberculosis in Vietnam 
PLoS ONE  2014;9(1):e84866.
Background
Rifampicin and protease inhibitors are difficult to use concomitantly in patients with HIV-associated tuberculosis because of drug-drug interactions. Rifabutin has been proposed as an alternative rifamycin, but there is concern that the current recommended dose is suboptimal. The principal aim of this study was to compare bioavailability of two doses of rifabutin (150 mg three times per week and 150 mg daily) in patients with HIV-associated tuberculosis who initiated lopinavir/ritonavir-based antiretroviral therapy in Vietnam. Concentrations of lopinavir/ritonavir were also measured.
Methods
This was a randomized, open-label, multi-dose, two-arm, cross-over trial, conducted in Vietnamese adults with HIV-associated tuberculosis in Ho Chi Minh City (Clinical trial registry number NCT00651066). Rifabutin pharmacokinetics were evaluated before and after the introduction of lopinavir/ritonavir -based antiretroviral therapy using patient randomization lists. Serial rifabutin and 25-O-desacetyl rifabutin concentrations were measured during a dose interval after 2 weeks of rifabutin 300 mg daily, after 3 weeks of rifabutin 150 mg daily with lopinavir/ritonavir and after 3 weeks of rifabutin 150 mg three times per week with lopinavir/ritonavir.
Results
Sixteen and seventeen patients were respectively randomized to the two arms, and pharmacokinetic analysis carried out in 12 and 13 respectively. Rifabutin 150 mg daily with lopinavir/ritonavir was associated with a 32% mean increase in rifabutin average steady state concentration compared with rifabutin 300 mg alone. In contrast, the rifabutin average steady state concentration decreased by 44% when rifabutin was given at 150 mg three times per week with lopinavir/ritonavir. With both dosing regimens, 2 – 5 fold increases of the 25-O-desacetyl- rifabutin metabolite were observed when rifabutin was given with lopinavir/ritonavir compared with rifabutin alone. The different doses of rifabutin had no significant effect on lopinavir/ritonavir plasma concentrations.
Conclusions
Based on these findings, rifabutin 150 mg daily may be preferred when co-administered with lopinavir/ritonavir in patients with HIV-associated tuberculosis.
Trial Registration
ClinicalTrials.gov NCT00651066
doi:10.1371/journal.pone.0084866
PMCID: PMC3898920  PMID: 24465443
7.  Targeting Cancer Cells with Reactive Oxygen and Nitrogen Species Generated by Atmospheric-Pressure Air Plasma 
PLoS ONE  2014;9(1):e86173.
The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H2O2, Ox, OH−, •O2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH2-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells.
doi:10.1371/journal.pone.0086173
PMCID: PMC3897664  PMID: 24465942
8.  Genetics and Epigenetics of Recurrent Hydatidiform Moles: Basic Science and Genetic Counselling 
Gestational trophoblastic disease (GTD) is a group of conditions that originate from the abnormal hyperproliferation of trophoblastic cells, which derive from the trophectoderm, the outer layer of the blastocyst that would normally develop into the placenta during pregnancy. GTDs encompass hydatidiform mole (HM) (complete and partial), invasive mole, gestational choriocarcinoma, placental-site trophoblastic tumor, and epithelioid trophoblastic tumor. Of these, the most common is HM, and it is the only one that has been reported to recur in the same patients from independent pregnancies, which indicates the patients’ genetic predisposition. In addition, HM is the only GTD that segregates in families according to Mendel’s laws of heredity, which made it possible to use rare familial cases of recurrent HMs (RHMs) to identify two maternal-effect genes, NLRP7 and KHDC3L, responsible for this condition. Here, we recapitulate current knowledge about RHMs and conclude with the role and benefits of testing patients for mutations in the known genes.
doi:10.1007/s13669-013-0076-1
PMCID: PMC3920063  PMID: 24533231
NLRP7; KHDC3L; Recurrent hydatidiform moles; Genetics; Epigenetics; DNA methylation; GTD; Live birth; Recurrent HMs (RHMs); Gestational choriocarcinoma; Gestational trophoblastic disease; Management of gestational trophoblastic diseases
9.  High Calcium Bioglass Enhances Differentiation and Survival of Endothelial Progenitor Cells, Inducing Early Vascularization in Critical Size Bone Defects 
PLoS ONE  2013;8(11):e79058.
Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects.
doi:10.1371/journal.pone.0079058
PMCID: PMC3828289  PMID: 24244419
19.  Gene Set Signature of Reversal Reaction Type I in Leprosy Patients 
PLoS Genetics  2013;9(7):e1003624.
Leprosy reversal reactions type 1 (T1R) are acute immune episodes that affect a subset of leprosy patients and remain a major cause of nerve damage. Little is known about the relative importance of innate versus environmental factors in the pathogenesis of T1R. In a retrospective design, we evaluated innate differences in response to Mycobacterium leprae between healthy individuals and former leprosy patients affected or free of T1R by analyzing the transcriptome response of whole blood to M. leprae sonicate. Validation of results was conducted in a subsequent prospective study. We observed the differential expression of 581 genes upon exposure of whole blood to M. leprae sonicate in the retrospective study. We defined a 44 T1R gene set signature of differentially regulated genes. The majority of the T1R set genes were represented by three functional groups: i) pro-inflammatory regulators; ii) arachidonic acid metabolism mediators; and iii) regulators of anti-inflammation. The validity of the T1R gene set signature was replicated in the prospective arm of the study. The T1R genetic signature encompasses genes encoding pro- and anti-inflammatory mediators of innate immunity. This suggests an innate defect in the regulation of the inflammatory response to M. leprae antigens. The identified T1R gene set represents a critical first step towards a genetic profile of leprosy patients who are at increased risk of T1R and concomitant nerve damage.
Author Summary
Leprosy type 1 reversal reactions (T1R) are an important cause of nerve damage in leprosy patients and accurate prediction of patients at increased risk of T1R is a major challenge of current leprosy control. The incidence of T1R differs widely from 6% to 67% of leprosy patients in different leprosy endemic settings. Whether or not this reflects the impact of unknown environmental triggers or differences in the genetic background across ethnicities is not known. We performed a comparative transcriptome analysis between leprosy patients affected and free of T1R in response to M. leprae antigens. As the discovery sample we enrolled cured leprosy patients who had been diagnosed with T1R at the time of leprosy diagnosis and leprosy patients who had never undergone T1R (retrospective arm). Whole genome transcriptome analysis after stimulation of blood with M. leprae antigen resulted in the definition of a T1R signature gene set. We validated the T1R gene set in RNA samples obtained from T1R-free patients at the time of leprosy diagnosis and followed for 3 years for development of T1R (prospective arm). These results confirm the role of innate factors in T1R and are a first step towards a predictive genetic T1R signature.
doi:10.1371/journal.pgen.1003624
PMCID: PMC3708838  PMID: 23874223
20.  Congenital constriction ring in children: sine plasty combined with removal of fibrous groove and fasciotomy 
Objective
To evaluate the clinical and functional results of a technical procedure used in the surgical treatment of congenital constriction ring (CCR) in children.
Materials and methods
This was a retrospective study undertaken to evaluate the results of surgical techniques performed from January 1995 to December 2005 on 95 patients with 134 congenital constriction bands. Due to the drop-out of nine patients during follow-up, data on 86 patients (121 congenital constriction rings; average age at surgery 1 year 2 months) were analyzed. The extent of the constrictions was classified by according to the Patterson criteria. All patients were treated by two-stage sine plasty combined with removal of the fibrous groove and fasciotomy, with one-half of the ring removed during the first stage and the other half removed 1 week later during the second state. The surgical outcomes were assess according to the Moses criteria.
Results
Three types of CCR (Patterson criteria) were identified among the 86 patients (121 constriction rings): types I (5 patients, 4.1 %), II (107, 88.5 %), III (9, 7.4 %). Of the 121 constriction rings, good results were attained in 73.6 % and fair results in 26.4 %. Sensory deficits were seen in six patients immediately after the surgery but all six had improved to a normal condition at the final follow-up examination. There were no skin necrosis or wound healing problems.
Conclusion
The combined sine plasty/removal of fibrous groove and fasciotomy method reported here is a simple and safe surgical technique for treating CCR in children.
doi:10.1007/s11832-012-0420-4
PMCID: PMC3399999  PMID: 23814619
Congenital constriction band syndrome; Dermofat flap; Amniotic bands; Direct closure; Z-plasty
21.  New strategy toward dioxin risk reduction for local residents surrounding severe dioxin hotspots in Vietnam 
Global Health Action  2013;6:10.3402/gha.v6i0.21105.
Background
A public health intervention program with active involvement of local related stakeholders was piloted in the Bien Hoa dioxin hotspot (2007–2009), and then expanded to the Da Nang dioxin hotspot in Vietnam (2009–2011). It aimed to reduce the risk of dioxin exposure of local residents through foods. This article presents the results of the intervention in Da Nang.
Methodology
To assess the results of this intervention program, pre- and post-intervention knowledge, attitude, and practice (KAP) surveys were implemented in 400 households, randomly selected from four wards surrounding the Da Nang Airbase in 2009 and 2011, respectively.
Results
After the intervention, the knowledge on the existence of dioxin in food, dioxin exposure pathways, potential high-risk foods, and preventive measures significantly increased (P<0.05). Ninety-eight percent were willing to follow advice on preventing dioxin exposure. Practices to reduce the risk of dioxin exposure also significantly improved (P<0.05). After intervention, 60.4% of households undertook exposure preventive measures, significantly higher than that of the pre-intervention survey (39.6%; χ2=40.15, P<0.001). High-risk foods had quite low rates of daily consumption (from 0 to 2.5%) and were significantly reduced (P<0.05).
Conclusions
This is seen as an effective intervention strategy toward reducing the risk of human exposure to dioxin at dioxin hotspots. While greater efforts are needed for remediating dioxin-polluted areas inside airbases, there is also evidence to suggest that, during the past four decades, pollution has expanded to the surrounding areas. For this reason, this model should be quickly expanded to the remaining dioxin hotspots in Vietnam to further reduce the exposure risks in other areas.
doi:10.3402/gha.v6i0.21105
PMCID: PMC3689901  PMID: 23791241
dioxin hotspots; intervention program; dioxin exposure through foods; risk communication; dioxin risk reduction; Vietnam
22.  Sources of cells that contribute to atherosclerotic intimal calcification: an in vivo genetic fate mapping study 
Cardiovascular Research  2012;94(3):545-554.
Aims
Vascular cartilaginous metaplasia and calcification are common in patients with atherosclerosis. However, sources of cells contributing to the development of this complication are currently unknown. In this study, we ascertained the origin of cells that give rise to cartilaginous and bony elements in atherosclerotic vessels.
Methods and results
We utilized genetic fate mapping strategies to trace cells of smooth muscle (SM) origin via SM22α-Cre recombinase and Rosa26-LacZ Cre reporter alleles. In animals expressing both transgenes, co-existence within a single cell of β-galactosidase [marking cells originally derived from SM cells (SMCs)] with osteochondrogenic (Runx2/Cbfa1) or chondrocytic (Sox9, type II collagen) markers, along with simultaneous loss of SM lineage proteins, provides a strong evidence supporting reprogramming of SMCs towards osteochondrogenic or chondrocytic differentiation. Using this technique, we found that vascular SMCs accounted for ∼80% of Runx2/Cbfa1-positive cells and almost all of type II collagen-positive cells (∼98%) in atherosclerotic vessels of LDLr−/− and ApoE−/− mice. We also assessed contribution from bone marrow (BM)-derived cells via analysing vessels dissected from chimerical ApoE−/− mice transplanted with green fluorescence protein-expressing BM. Marrow-derived cells were found to account for ∼20% of Runx2/Cbfa1-positive cells in calcified atherosclerotic vessels of ApoE−/− mice.
Conclusion
Our results are the first to definitively identify cell sources attributable to atherosclerotic intimal calcification. SMCs were found to be a major contributor that reprogrammed its lineage towards osteochondrogenesis. Marrow-derived cells from the circulation also contributed significantly to the early osteochondrogenic differentiation in atherosclerotic vessels.
doi:10.1093/cvr/cvs126
PMCID: PMC3353803  PMID: 22436847
Atherosclerosis; Circulating progenitors; Osteochondrogenic differentiation; Smooth muscle cells; Vascular calcification
23.  Pediatric Psychosomatic Medicine: Creating a Template for Training 
Psychosomatics  2012;53(6):532-540.
There is a critical public health problem in the United States today, the problem of childhood psychiatric disorders in youngsters with physical illnesses. Currently there is a pressing need for well-trained pediatric psychosomatic medicine practitioners as well as advanced training in the field. Yet, this training does not currently exist. This article will present the innovative Montefiore Medical Center/Albert Einstein College of Medicine (MMC/AECOM) program as a model for a training curriculum, clinical training experience, and clinical research training setting in this important and rapidly expanding area of need in pediatric mental health.
doi:10.1016/j.psym.2012.01.008
PMCID: PMC3525319  PMID: 22658325
24.  Ascorbic Acid Improves the Developmental Competence of Porcine Oocytes After Parthenogenetic Activation and Somatic Cell Nuclear Transplantation 
Abstract
In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.
doi:10.1262/jrd.2012-114
PMCID: PMC3943238  PMID: 23154385
Culture system; Embryo development; Handmade cloning; Parthenote; Vitamin C
25.  PARK2 Mediates Interleukin 6 and Monocyte Chemoattractant Protein 1 Production by Human Macrophages 
Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D3 (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors.
Author Summary
Leprosy is an infectious disease with a strong host genetic component. The identification of host genetic lesions predisposing to disease is a powerful approach for mapping key junctions in the host pathogen interplay. Genetic variants located in the promoter region of the PARK2 gene are replicated leprosy susceptibility factors. To better understand a possible contribution of PARK2 to host effector mechanisms in leprosy patients, we developed a cellular model to test the contribution of the PARK2 encoded parkin protein to host responses to mycobacterial antigens. We observed that parkin was a mediator of IL-6 production in response to mycobacterial antigen in both THP-1 macrophages and human Schwann cells while human monocyte-derived macrophages needed to be pre-activated with VitD to show the same impact. Parkin also impacted on the constitutive production of MCP-1. The regulatory activity of parkin on cytokine production was found to be independent of the canonical TLR-NFκB signalling pathway. We also tested association of IL6 and CCL2 gene expression levels in whole blood assays with PARK2 polymorphisms. For both cytokines, we found significant associations with those PARK2 variants that were established leprosy susceptibility factors. Hence, our results show that genetic PARK2 variants that are correlated with leprosy susceptibility are also correlated with production of these cytokines following stimulation with M. leprae sonicate.
doi:10.1371/journal.pntd.0002015
PMCID: PMC3547867  PMID: 23350010

Results 1-25 (67)