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1.  [No title available] 
PMCID: PMC3932334  PMID: 24239751
2.  [No title available] 
PMCID: PMC3944912  PMID: 24220607
3.  Mechanisms of IFNγ regulation of autoimmune myocarditis 
A protective effect of interferon-gamma (IFNγ) has been described in a number of models of autoimmune disease, including experimental autoimmune myocarditis (EAM). Some reports have suggested that regulation of apoptosis in autoreactive lymphocytes mediate these protective functions. We examined the potential of IFNγ to regulate apoptotic mechanisms in detail, both in vitro and in vivo in EAM. We observed multiple apoptotic defects in caspase activity, and the expression of TNF superfamily members on CD4+ T cells. In addition, we observed selective defects in CD4+ T cell activation in response to antigenic stimulation. These activation and apoptotic defects were CD4+ cell autonomous, independent of the genotype of APCs. Inhibition of nitric oxide production in vivo did not reproduce the severe form of EAM of IFNγ-deficient mice, indicating that this pathway does not mediate the protective effect of IFNγ. Crosswise adoptive transfer of wild type, IFNγ−/−, and IFNγR−/− EAM demonstrated that IFNγ signaling was critical in CD4+ cells, but that non-CD4+ sources of IFNγ production were also involved in the control of disease. Together, these data indicate multiple mechanisms of autonomous and non-autonomous CD4+ T cell regulation mediated by IFNγ in the control of autoimmune heart disease.
PMCID: PMC4266481  PMID: 20599938
myocarditis; interferon-gamma; apoptosis; caspase 8; nitric oxide; autoimmune disease
4.  Deep Sequencing and Circos Analyses of Antibody Libraries Reveal Antigen-driven Selection of Ig VH Genes during HIV-1 Infection 
The vast diversity of antibody repertoires is largely attributed to heavy chain (VH) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some VH families are selected and preferentially expressed after long-term infection with HIV-1. We also demonstrated that the immune system responds to HIV-1 chronic infection by selectively expanding certain HV families in an attempt to combat infection. Our findings may have implications for understanding immune responses in pathology as well as for development of new therapeutics and vaccines.
PMCID: PMC3889869  PMID: 24158018
454 sequencing; antibodyome; antibody repertoire; HIV-1; human monoclonal antibody; VDJ analysis
5.  Extracellular Transport of Cell-Size Particles and Tumor Cells by Dendritic Cells in Culture 
Many particulate materials of sizes approximating that of a cell disseminate after being introduced into the body. While some move about within phagocytic inflammatory cells, others appear to move about outside of, but in contact with, such cells. In this report, we provide unequivocal photomicroscopic evidence that cultured, mature, human dendritic cells can transportin extracellular fashion over significant distances both polymeric beads and tumor cells. At least in the case of polymeric beads, both fibrinogen and the β2-integrin subunit, CD18, appear to play important roles in the transport process. These discoveries may yield insight into a host of disease-related phenomena, including and especially tumor cell invasion and metastasis.
PMCID: PMC3953141  PMID: 24145002
In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade®), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly down regulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to down regulate the enzymes 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were down regulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly down regulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then down regulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP down regulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in down regulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used.
PMCID: PMC4197193  PMID: 22445925
Fatty acid; Triacylglycerol and Cholesterol Synthesis; Proteasome inhibitor
7.  Adiponectin receptor 1 C-terminus interacts with PDZ-domain proteins such as syntrophins 
Experimental and molecular pathology  2013;95(2):10.1016/j.yexmp.2013.07.002.
Adiponectin receptor 1 (AdipoR1) is one of the two signalling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to β2-syntrophin (SNTB2). Hybridization of a PDZ domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including βl-and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C-and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2.
PMCID: PMC3841378  PMID: 23860432
Adiponectin; PDZ-protein; hepatocyte; syntrophin
8.  Active Systemic Lupus Erythematosus is associated with decreased blood conventional dendritic cells 
To determine the frequency and functionality of blood conventional dendritic cells (cDC) in relation to disease activity in Systemic Lupus Erythematosus.
Blood cDC were enumerated for 34 SLE patients, defined as “active” (SLEDAI≥4) or “inactive” (SLEDAI<4), 26 RA and 8 healthy subjects by FACS. cDC activation was measured by IL-12p40/70 staining following resiquimod stimulation.
The frequency of blood cDC were significantly lower in active compared to inactive patients, however, with comparable cDC functionality.
cDC frequency in active SLE is decreased with no perturbation in cDC function, possibly due to enhanced turnover and/or tissue-specific migration.
PMCID: PMC3963522  PMID: 23773850
Systemic Lupus Erythematosus; dendritic cells; disease activity; autoimmunity
10.  Lysosome-dependent p300/FOXP3 degradation and limits Treg cell functions and enhances targeted therapy against cancers 
p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our recent studies have defined a complex set of histone acetyltransferase interactions which can lead to enhanced or repressed changes in FOXP3 function. We have explored the use of a natural p300 inhibitor, Garcinol, as a tool to understand mechanisms by which p300 regulates FOXP3 acetylation. In the presence of Garcinol, p300 appears to become disassociated from the FOXP3 complex and undergoes lysosome-dependent degradation. As a consequence of p300's physical absence, FOXP3 becomes less acetylated and eventually degraded, a process that cannot be rescued by the proteasome inhibitor MG132. p300 plays a complex role in FOXP3 acetylation, as it could also acetylate a subset of four Lys residues that repressively regulate total FOXP3 acetylation. Garcinol acts as a degradation device to reduce the suppressive activity of regulatory T cells (Treg) and to enhance the in vivo anti-tumor activity of a targeted therapeutic anti-p185her2/neu (ERBB2) antibody in MMTV-neu transgenics implanted with neu transformed breast tumor cells. Our studies provide the rationale for molecules that disrupt p300 stability to limit Treg functions in targeted therapies for cancers.
PMCID: PMC3963828  PMID: 23644046
Acetyltransferase; Lysosome degradation; FOXP3; Garcinol; p300; Regulatory T cells; Treg
11.  Over expression of miR-10a and miR-375 and down regulation of YAP1 in medullary thyroid carcinoma 
MicroRNAs are a primordial mechanism of gene expression control that appear to be crucial to cellular development and may play an important role in tumor development. Much is known about the genetics of medullary thyroid carcinomas, as approximately 25% are hereditary and harbor germ line activating mutations in the RET gene. Somatic RET mutations are also seen in roughly 50% of sporadic medullary thyroid carcinomas. Few studies, however, have evaluated the role of microRNA expression in these tumors. DNA and RNA were extracted from formalin-fixed paraffin-embedded tissue blocks of 15 medullary thyroid carcinomas [10 with RET mutations (3 hereditary) and 5 without RET mutations] and 5 non-tumor thyroid glands. miRNA expression of 754 targets was quantitated by real time PCR using the ABI OpenArray miRNA assay. Three miRNAs showed significant differential expression and were validated in a larger cohort of 59 cases by real-time PCR. Expression of potential downstream targets and upstream regulators were also investigated by real-time PCR. miR-375 and miR-10a were significantly overexpressed, while miR-455 was underexpressed in medullary thyroid carcinomas. Expression of all 3 miRNAs were validated in the larger cohort of cases (miR-375, p = 3.3×10−26; miR-10a, p = 5.6×10−14; miR-455, p = 2.4×10−4). No significant differences in miRNA expression were found between RET mutation positive and negative tumors nor between sporadic and hereditary tumors. Expression of the potential downstream targets of miR-375, YAP1 (a growth inhibitor) and SLC16a2 (a transporter of thyroid hormone), was downregulated in the tumors suggesting that miR-375 is a negative regulator of the expression of these genes. Thus, differential expression of miR-375, miR-10a and miR-455 may be important for tumor development and/or the reflect c-cell lineage of medullary thyroid carcinoma. Furthermore, the growth inhibitor YAP1 is identified as a potential important downstream target of miR-375.
PMCID: PMC3712278  PMID: 23685355
medullary thyroid carcinoma; microRNA; miR-375; miR-10a; miR-455; YAP1
12.  Heterogeneity of Colorectal Cancer (CRC) in reference to KRAS proto-oncogene utilizing WAVE technology 
New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10–20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS.
In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (High performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for presence of KRAS proto-oncogene mutations at codon 12 and 13.
Initially, sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations.
In these studies the analysis of three patients’ colorectal cancer tissue were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.
PMCID: PMC4015467  PMID: 23528430
13.  Perivascular, but not Parenchymal, Cerebral Engraftment of Donor Cells after Non-Myeloablative Bone Marrow Transplantation 
Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2 Gy irradiation, and 5.5 Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5 Gy irradiation resulted in significant engraftment of BMT-derived cells in brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5 Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms MyA, but not NMyA, is able to overcome.
PMCID: PMC3706527  PMID: 23567123
14.  Calcineurin and Akt expression in hypertrophied bladder in STZ-induced diabetic rat 
Diabetes causes significant increases in bladder weight but the natural history and underlying mechanisms are not known. In this study, we observed the temporal changes of detrusor muscle cells (DMC) and the calcineurin (Cn) and Akt expressions in detrusor muscle in the diabetic rat. Male Sprague–Dawley rats were divided into 3 groups: streptozotocin-induced diabetics, 5% sucrose-induced diuretics, and age-matched controls. The bladders were removed 1, 2, or 9 weeks after disease induction and the extent of hypertrophy was examined by bladder weights and cross sectional area of DMC. Cn and Akt expression were evaluated by immunoblotting. Both diabetes and diuresis caused significant increases in bladder weight. The mean cross sectional areas of DMC were increased in both diabetic and diuretic animals 1, 2, or 9 weeks after disease induction. The expression levels of both the catalytic A (CnA) and regulatory B (CnB) subunits of Cn were increased at 1 and 2 weeks, but not at 9 weeks. Expression of Akt was similar among control, diabetic, and diuretic rat bladder at all time points. In conclusion, diabetes and diuresis induce similar hypertrophy of detrusor muscle during the first 9 weeks, indicating that bladder hypertrophy in the early stage of diabetes is in response to the presence of increased urine output in diabetes. Our results suggest that the Cn, but not the Akt signaling pathway may be involved in the development of bladder hypertrophy.
PMCID: PMC4097027  PMID: 22305959
Calcineurin; Akt; Bladder; Hypertrophy diabetes
15.  Transplantation of Bone Marrow-derived MSCs Improves Cisplatinum-induced Renal Injury through Paracrine Mechanisms 
Mesenchymal stem cells (MSCs) have been reported to preserve renal function in various models of acute kidney injury (AKI). Different routes were used to transplant MSCs but the role of cell transplantation routes in directing outcomes has been unknown. In the present study, we evaluated organ bio-distributions of transplanted MSCs, and correlated survival of transplanted cells with outcomes in mice with cisplatinum-induced AKI. We found that after intravenous administration MSCs were largely localized in pulmonary capillaries and only a minute fraction of MSCs entered kidneys and the cells survived only transiently. Therefore, we also transplanted MSCs via intraperitoneal and renal subcapsular routes. Transplanted MSCs survived longer in peritoneal cavity and renal subcapsular space. Interestingly, when MSCs transplantation was followed by cisplatinum-induced AKI, renal morphology and renal functions were better preserved, irrespective of the cell transplantation route. As transplanted MSCs did not migrate to kidneys from either peritoneal cavity or renal subcapsular space, this finding suggested that migration of cells was not required for the beneficial response. The possibility of indirect mechanisms was confirmed when administration of the conditioned medium from MSCs also protected renal tubular cells from cisplatinum-induced cytotoxicity. We identified presence of over forty regulatory cytokines in the conditioned medium obtained from MSCs. Since paracrine factors released by transplanted cells accounted for improvements, it appears that the route of cell transplantation is not critical for realizing benefits in AKI of cell therapy with MSCs. Studies of specific cytokines secreted by MSCs will help to obtain new therapeutic mechanisms for renal protection.
PMCID: PMC3647016  PMID: 23534987
16.  Deficit of p66ShcA Restores Redox-sensitive Stress Response Program in Cisplatin-induced Acute Kidney Injury 
Overwhelming oxidative stress and compromised tubular cell antioxidant response have been incriminated for cisplatin (Cis) -induced acute kidney injury (AKI). We hypothesized that Cis-induced KI was the outcome of the deactivated redox-sensitive stress response program (RSSRP). Wild (WT) and heterozygous p66ShcA+/− mice in groups of six were administered either normal saline (WT) or Cis (12.5 mg/kg, intraperitoneal, Cis/WT). Renal biomarkers were collected and kidneys were harvested for renal histology. Cis/WT showed elevated blood urea nitrogen levels and enhanced tubular cell apoptosis, necrosis, and dilated tubules filled with casts when compared to Cis/p66+/−. Cis/p66+/− developed only a clinically occult ARF (normal blood urea levels and only microscopic alterations). Immunoblots from the lysates of renal tissues of Cis/WT displayed enhanced expression of phospho-p66ShcA, and phospho-Foxo3A but attenuated expression of MnSOD and catalase; conversely, p66 deficit prevented these alterations in Cis milieu. In in vitro studies, Cis treated mouse proximal tubular cells (MPTCs) displayed enhanced phosphorylation of p66ShcA and no increase in tubular cell expression of MnSOD. In addition, renal tissues of Cis/WT and Cis-treated MPTCs displayed enhanced phosphorylation of p53 and Bax expression. However, MPTC partially silenced for p66ShcA displayed partial inhibition of Cis-induced tubular cell apoptosis as well as necrosis. These findings indicated that Cis-induced AKI was the outcome of the deactivated RSSRP (attenuated anti-oxidant response) and activation of pro-apoptotic (p53-induced Bax expression) pathway.
PMCID: PMC3720132  PMID: 23506954
17.  The cholangiocyte marker, BD. 1, forms a stable complex with CLIP170 and shares an identity with eIF3a, a multifunctional subunit of the eIF3 initiation complex 
We have previously described the generation of a monoclonal antibody recognizing a novel cholangiocyte marker, designated BD.1, that is expressed by fetal and adult rat cholangiocytes but not hepatocytes or the hepatic progenitor cells known as oval cells. In the present report, we have undertaken a comprehensive examination of BD.1 expressed by long-term cultures of bile duct epithelial cells (BDEC) and prostate epithelial cells (PEC). We show that with continued passage, the levels of BD.1 expressed by BDEC and PEC drop significantly, a decrease that is temporally associated with transition from a diploid to an aneuploid karyotype. Cell cycle analysis revealed cell cycle dependent expression of BD.1 characterized by decreased BD.1 levels within the first 10 h after release from serum starvation followed by reacquisition as cells entered S phase. MAb BD.1 recognized a 170 kDa protein in Western blots and showed strong reactivity with a 170 kDa band in blots prepared from phosphoproteins isolated by metal affinity chromatography. Analysis by mass spectrometry of tryptic peptides generated from BD.1 purified by continuous elution electrophoresis identified the plus end microtubule-binding protein, CLIP170, in the fraction reactive with MAb BD.1. Double immunofluorescence with MAb BD.1 and a MAb specific for CLIP170 showed that both were reactive with intrahepatic bile ducts. However, overexpression or siRNA knockdown of CLIP170 in 293T cells did not significantly alter BD.1 levels, indicating that CLIP170 and BD.1 were distinct, co-migrating proteins. Immunoprecipitation analysis with MAb BD.1 and anti-CLIP170 antibodies showed that under microtubule depolymerizing conditions the two proteins could be co-precipitated with both antibodies, leading us to conclude they were capable of forming stable complexes. Two different protocols were devised to enrich for the CLIP170 binding protein recognized by MAb BD.1. Analysis of tryptic peptides by LC-ESI-MS/MS identified BD.1 as eIF3a, the largest subunit of the elongation initiation factor 3 (eIF3) complex. This identity was confirmed by the simultaneous knockdown of both BD.1 and eIF3a by eIF3a-specific siRNAs and by the strong reactivity of MAb BD.1 with the 170 kDa protein immunoprecipitated with the anti-eIF3a antibody, 5H10. Based on these findings, we concluded that the BD.1 antigen was identical to eIF3a, a multifunctional subunit of the eIf3 complex shown here to associate with microtubules through its interactions with CLIP170.
PMCID: PMC4035227  PMID: 22613460
eIF3a; p170; CLIP170; Cholangiocytes; Prostate epithelial cells; Neoplastic conversion
18.  Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu 
Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p<30), and onset of aneuploidy. At high passage (p>85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.
PMCID: PMC4012332  PMID: 20655306
spontaneous transformation; cholangiocyte; cholangiocarcinoma; ErbB-2/Neu
19.  Generation of large numbers of highly purified dendritic cells from bone marrow progenitor cells after co-culture with syngeneic murine splenocytes 
Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes. From a total of 20×106 BM cells and 120×106splenocytes, 3 × 106 BM cells along with 20×106splenocytes were co-cultured in petri dishes for DC generation; 120×106 splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. The control group were BM cells cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8α, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA.
The number of DCs derived from co-culture of BM and splenocytes (DCsTME) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2 % and 77.2%, respectively; p<0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCsTME compared to BM-derived DCs in the absence of splenocytes (193 pg/ml and 630 pg/ml, respectively; p<0.05).
The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCsTME could be more advantageous for DC-based immunotherapy and vaccination techniques.
PMCID: PMC3602144  PMID: 23269574
Dendritic cells; bone marrow; spleen; co-culture; purity; large number
20.  Ablation of the Microglial Protein DOCK2 Reduces Amyloid Burden in a Mouse Model of Alzheimer’s Disease 
Alzheimer’s disease (AD) neuropathology is characterized by innate immune activation primarily through prostaglandin E2 (PGE2) signaling. Dedicator of cytokinesis 2 (DOCK2) is a guanyl nucleotide exchange factor expressed exclusively in microglia in the brain and is regulated by PGE2 receptor EP2. DOCK2 modulates microglia cytokine secretion, phagocytosis, and paracrine neurotoxicity. EP2 ablation in experimental AD results in reduced oxidative damage and amyloid beta (Aβ) burden. This discovery led us to hypothesize that genetic ablation of DOCK2 would replicate the anti-Aβ effects of loss of EP2 in experimental AD. To test this hypothesis, we crossed mice that lacked DOCK2 (DOCK2−/−), were hemizygous for DOCK2 (DOCK2+/−), or that expressed two DOCK2 genes (DOCK2+/+) with APPswe-PS1Δe9 mice (a model of AD). While we found no DOCK2-dependent differences in cortex or in hippocampal microglia density or morphology in APPswe-PS1Δe9 mice, cerebral cortical and hippocampal Aβ plaque area and size were significantly reduced in 10-month-old APPswe-PS1Δe9/DOCK2−/− mice compared with APPswe-PS1Δe9/DOCK2+/+ controls. DOCK2 hemizygous APPswe-PS1Δe9 mice had intermediate Aβ plaque levels. Interestingly, soluble Aβ42 was not significantly different among the three genotypes, suggesting the effects were mediated specifically in fibrillar Aβ. In combination with earlier cell culture results, our in vivo results presented here suggest DOCK2 contributes to Aβ plaque burden via regulation of microglial innate immune function and may represent a novel therapeutic target for AD.
PMCID: PMC3602334  PMID: 23318649
innate immunity; microglia; amyloid beta; PGE2 receptor EP2; Alzheimer’s disease
21.  Acute endotoxemia is associated with upregulation of lipocalin 24p3/Lcn2 in lung and liver 
Acute endotoxemia is associated with production of acute phase proteins which regulate inflammatory responses to tissue injury. Consistent with DNA microarray experiments, we found that acute endotoxemia, induced by administration of lipopolysaccharide (LPS) to mice (1 mg/kg) or rats (5 mg/kg), resulted in increased expression of the hepatic acute phase protein, lipocalin 24p3, which was evident within 4 h and persisted for 24–48 h. Increases in 24p3 expression were also observed in the lung after LPS administration, as well as in isolated liver and lung macrophages, and Type II alveolar epithelial cells. The actions of LPS are dependent, in part, on Toll-like receptor (TLR) proteins. Macrophages from C3H/HeJ mice, which possess a nonfunctional TLR-4, expressed low levels of 24p3 mRNA when compared to cells from control C3H/OuJ mice. Whereas LPS administration increased 24p3 expression in lung and liver macrophages from control C3H/OuJ mice, minimal effects were observed in TLR-4 mutant mice demonstrating that TLR-4 is important in regulating 24p3 expression during acute endotoxemia. Promoters for genes encoding lipocalin proteins including 24p3 contain consensus sequences for transcription factors including NF-κB, and C/EBP. Acute endotoxemia resulted in NF-κB nuclear binding activity in both alveolar macrophages and Type II cells. In contrast, C/EBP activation was evident only in Type II cells, suggesting differential effects of LPS on these cell types. These data suggest that the acute phase response to acute endotoxemia involves induction of 24p3 in both the lung and liver. This protein may be important in restoring tissue homeostasis following LPS-induced injury.
PMCID: PMC3954125  PMID: 17490638
Lcn2; 24p3; LPS; Alveolar macrophage; Type II cell
22.  The liver is populated by a broad spectrum of markers for macrophages. In alcoholic hepatitis the macrophages are M1 and M2☆ 
Liver cell injury in alcoholic hepatitis (AH) is in part, due to macrophage generated proinflammatory cytokines i.e., M1, M2a, M2b, and M2c might be involved in ALD. The T cell response to chemokines and cytokines differs not only when M1 and M2 macrophages are compared but even when individual M2 subtypes are profiled.
In AH, M1 monocytes in the blood show increased sensitivity in the TNF-α response to LPS. Immunohistochemistry (IHC) studies showed that the liver sinusoids in ALD are abundantly populated by CD163 expressing type 2 macrophages. In this report, we profile many of the molecules associated with M1 and M2 macrophages in livers with AH using IHC.
Using immunofluorescent antibody-labeling, we profiled the receptors, cytokines and chemokines observed in M1, M2a, M2b, and M2c macrophages in liver biopsies from patients with AH.
The increased CD 163 expression found in previous studies was confirmed as well an additional macrophage phenotypic marker CD206, suggesting that AH pathogenesis at least partially involves M2a and M2c macrophages. TGF-β was found to be robustly over expressed by liver sinusoidal macrophages. Macrophage expression of the phenotypic markers TLR-2, TLR-4 and TLR-8 – found in both M1 and M2 macrophages – as well as the chemokines CCL-1 and CCL-18 was found. However, IRF-4, which is related to IL-4 production and M2a polarization as well as the cytokines CCL-1 and Il-1β and the chemokine CXCL-1 were also observed, suggesting that M2a and M2b also play a role in AH pathogenesis.
Livers with AH show robust macrophage over expression of TGF-β, a growth factor more commonly associated with M2 type macrophages and mostly known for its fibrogenetic properties. However, our immunoprofiling of macrophage over expression also shows that AH is driven by receptors, interferons, and cytokines that are commonly associated not just with M2 macrophages, but with M1 as well. Thus, a complex interplay between different types of macrophages expressing a diverse array of molecules and receptors is involved in AH.
PMCID: PMC3944995  PMID: 24145004
Alcoholic hepatitis; Macrophages; CD163; TLR-4
23.  LMP2, a novel immunohistochemical marker to distinguish renal oncocytoma from the eosinophilic variant of chromophobe renal cell carcinoma 
LMP2 is a subunit of the immunoproteasome that is overexpressed in oncocytic lesions of the thyroid gland. This study was designed to assess the expression profile and diagnostic utility of LMP2 in two renal oncocytic tumors that share similar morphologic features but have different clinical outcomes: renal oncocytoma (RO) and the eosinophilic variant of chromophobe renal cell carcinoma (CHRCC-EO). A total of 56 RO), 38 classic CHRCC, and 7 CHRCC-EO cases, as well 84 normal kidney controls, were selected from the Johns Hopkins surgical pathology archive and stained for LMP2 using a standard immunohistochemical protocol. Sections were scored for cellular location (nuclear versus cytosolic), intensity (from 0 to 3), and percent of area involved (from 0 to 100%), and an H score was calculated multiplying the intensity by the extent of the staining signal. The cytoplasmic expression of LMP2 was similar among the renal lesions, being present in 44 of 56 (79%) ROs, 27 of 38 (71%) CHRCCs, and 7 of 7 (100%) CHRCC-EO cases. The nuclear expression of LMP2, however, was more informative. All CHRCC-EO cases (7 of 7, 100%) strongly showed nuclear LMP2 staining, as opposed to only 2 of 56 (4%, P<0.0001) ROs and 9 of 38 (24%, P=0.0001) classic CHRCCs. These results suggest that the nuclear LMP2 expression can be used in clinical scenarios where histological distinction between RO and CHRCC-EO remains challenging.
PMCID: PMC3459143  PMID: 22705098
LMP2; renal oncocytomas; chromophobe renal cell carcinoma
24.  Nef interaction with actin compromises human podocyte actin cytoskeletal integrity 
The HIV-1 accessory protein Nef is considered to play an important role in the development of podocyte phenotype in HIV-1 associated nephropathy. We hypothesized that Nef may be altering podocyte phenotype both structurally and functionally. To elucidate the involved mechanisms, podocyte proteins interacting with Nef were identified using GST pull down assay and yeast two hybrid assay. The GST pull down assay on protein extracts made from stable colonies of conditionally immortalized human podocytes expressing Nef (Nef/CIHP) displayed a band at 45 kD, which was identified as actin by mass spectrometry. Yeast two hybrid assay identified the following Nef-interacting proteins: syntrophin, filamin B, syntaxin, translational elongation factor 1, and zyxin. The Nef-actin and Nef- zyxin interactions were confirmed by co-localization studies on Nef/CIHP stable cell lines. The co-localization studies also showed that Nef/CIHP stable cell lines had decreased number of actin filaments (stress fibers), displayed formation of lamellipodia, and increased number of podocyte projectons (filopodia). Nef/CIHP displayed enhanced cortical F-actin score index (P<0.001) and thus indicating reorganization of F-actin in the cortical regions. Microarray analysis showed that Nef enhanced the expression of Rac1, syndecan-4, Rif, and CDC42 and attenuated the expression of syndecan-3 and syntenin. In addition, Nef/CIHPs displayed diminished sphingomyelinase (ASMase) activity. Functionally, Nef/CIHPs displayed diminished attachment and enhanced detachment to their substrate. These findings indicate that Nef interaction with actin compromises podocyte cytoskeleton integrity.
PMCID: PMC3463768  PMID: 22721673
25.  Sex differences in TLR2 and TLR4 expression and their effect on coxsackievirus-induced autoimmune myocarditis 
Coxsackievirus B3 (CVB3) infection of C57Bl/6 mice shows a sex bias with males developing more severe cardiac inflammation than females because males develop a Th1 inflammatory response, whereas females develop a Th2 response. Since their discovery, Toll-like receptors have been shown to play an important role in the development of the immune response against harmful pathogens. To assess the role of TLRs in coxsackievirus-induced myocarditis wild type and Toll-like Receptor 2 −/− male and female mice were infected and assessed for viral replication, myocarditis, helper T-cell generation, and regulatory T-cell generation. TLR2−/− mice show reduced Th1 expression compared to controls. Treatment of wild type mice with either Pam3CSK4 (TLR2) or LPS (TLR4) specific TLR agonists resulted in increased Th1 expression in male and female mice and a decrease in FoxP3+ regulatory T-cells in male mice. The suppression of Tregulatory cells by TLR signaling in males but not females correlates with the increased myocarditis susceptibility of the males.
PMCID: PMC3485413  PMID: 22750431
Coxsackievirus B3; Toll-like receptors; myocarditis

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