Selective gene delivery to cancer cells secreting matrix metalloproteinases using a gelatin/polyethylenimine/DNA complex

We developed a gene delivery strategy targeting metastatic tumors by exploiting the specific matrix metalloproteinases (MMPs) secreting properties of metastatic tumor cells. A ternary polyplex has been formed by coating polyethylenimine/DNA (PD) complex with an excessive amount of negatively charged gelatin B (GPDB). We show that GPD-B’s gene delivery activity could be targeted to cancer cells via the MMP-mediated proteolytic process, while GPD-A, made from positively charged gelatin A, was not successful in exhibiting such activity. The 1,10-Phenanthroline, an MMP2 inhibitor, abrogated the MMP-dependent transfection activity of GPD-B. GPD-B carried much less positive surface charges than PD, and thus exhibited significantly reduced interactions with erythrocytes. However, MMP2 elevated the positiveness in GPDB’s surface charge and, thus, its interaction with erythrocytes. These results suggest that the anionic gelatin coating may confer improved stabilities on GPD-B in the surrounding medium, while MMP2-mediated disintegration of the gelatin coat enhances the gene delivery to metastatic cancer cells via increasing the likelihood of local chargemediated interactions between the polyplex and cancer cell membrane.     

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8⁺ T cell epitope, NY-ESO-1_88–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1_157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1_88–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1_157–165. On the other hand, NY-ESO-1_157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A_26–35; whereas NY-ESO-1_88–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1_88–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18⁺ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1_88–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8⁺ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.     

Paradoxical Impact of Two Folate Receptors,FRα and RFC,in Ovarian Cancer: Effect on Cell Proliferation,Invasion and Clinical Outcome

Despite being an essential vitamin, folate has been implicated to enhance tumor growth, as evidenced by reports on overexpression of folate receptor alpha (FRα) in carcinomas. The role of another folate transporter, reduced folate carrier (RFC), is largely unknown. This study investigated the roles of folate, FRα and RFC in ovarian cancers. We demonstrated FRα mRNA and protein overexpression and reduced RFC expression in association with FRα gene amplification and RFC promoter hypermethylation, respectively. FRα overexpression was associated with tumor progression while RFC expression incurred a favorable clinical outcome. Such reciprocal expression pattern was also observed in ovarian cancer cell lines. Folate was shown to promote cancer cell proliferation, migration and invasion in vitro, and down-regulate E-cadherin expression. This effect was blocked after either stable knockdown of FRα or ectopic overexpression of RFC. This hitherto unreported phenomenon suggests that, RFC can serve as a balancing partner of FRα and confer a protective effect in patients with high FRα-expressing ovarian carcinomas, as evidenced by their prolonged overall and disease-free survivals. In conclusion, we report on the paradoxical impact of FRα (putative oncogenic) and RFC (putative tumor suppressive) in human malignancies. FRα and RFC may potentially be explored as therapeutic target or prognostic marker respectively. We recommend caution and additional research on folate supplements in cancer patients.     

Segregation of LIPG, CETP, and GALNT2 Mutations in Caucasian Families with Extremely High HDL Cholesterol

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.     

Male Microchimerism in the Human Female Brain

In humans, naturally acquired microchimerism has been observed in many tissues and organs. Fetal microchimerism, however, has not been investigated in the human brain. Microchimerism of fetal as well as maternal origin has recently been reported in the mouse brain. In this study, we quantified male DNA in the human female brain as a marker for microchimerism of fetal origin (i.e. acquisition of male DNA by a woman while bearing a male fetus). Targeting the Y-chromosome-specific DYS14 gene, we performed real-time quantitative PCR in autopsied brain from women without clinical or pathologic evidence of neurologic disease (n = 26), or women who had Alzheimer’s disease (n = 33). We report that 63% of the females (37 of 59) tested harbored male microchimerism in the brain. Male microchimerism was present in multiple brain regions. Results also suggested lower prevalence (p = 0.03) and concentration (p = 0.06) of male microchimerism in the brains of women with Alzheimer’s disease than the brains of women without neurologic disease. In conclusion, male microchimerism is frequent and widely distributed in the human female brain.     

The Role of Interferon-γ Inducible Protein-10 in a Mouse Model of Acute Liver Injury Post Induced Pluripotent Stem Cells Transplantation

Background

Liver injuries are important medical problems that require effective therapy. Stem cell or hepatocyte transplantation has the potential to restore function of the damaged liver and ameliorate injury. However, the regulatory factors crucial for the repair and regeneration after cell transplantation have not been fully characterized. Our study investigated the effects and the expression of the regulatory factors in mouse models of acute liver injury either transplanted with the induced pluripotent stem cells (iPS) or the hepatocytes that differentiated from iPS cells (iHL).

Methods/Principal Findings

Mice received CCl₄ injection and were randomized to receive vehicle, iPS, or iHL transfusions vial tail veins and were observed for 24, 48 or 72 hours. The group of mice with iPS transplantation performed better than the group of mice receiving iHL in reducing the serum alanine aminotransferase, aspartate aminotransferase, and liver necrosis areas at 24 hours after CCl₄ injury. Moreover, iPS significantly increased the numbers of proliferating hepatocytes at 48 hours. Cytokine array identified that chemokine IP-10 could be the potential regulatory factor that ameliorates liver injury. Further studies revealed that iPS secreted IP-10 in vitro and transfusion of iPS increased IP-10 protein and mRNA expressions in the injured livers in vivo. The primary hepatocytes and non-parenchyma cells were isolated from normal and injured livers. Hepatocytes from injured livers that received iPS treatment expressed more IP-10 mRNA than their non-hepatocyte counter-parts. In addition, animal studies revealed that administration of recombinant IP-10 (rIP-10) effectively reduced liver injuries while IP-10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rIP-10 significantly reduced the 72-hour mortality rate in mice that received multiple CCl₄-injuries.

Conclusions/Significance

These findings suggested that IP-10 may have an important regulatory role in facilitating the repair and regeneration of injured liver after iPS transplantation.     

Lipocalin 2 Regulates Inflammation during Pulmonary Mycobacterial Infections

Pulmonary tuberculosis (TB), caused by the intracellular bacteria Mycobacterium tuberculosis, is a worldwide disease that continues to kill more than 1.5 million people every year worldwide. The accumulation of lymphocytes mediates the formation of the tubercle granuloma in the lung and is crucial for host protection against M.tuberculosis infection. However, paradoxically the tubercle granuloma is also the basis for the immunopathology associated with the disease and very little is known about the regulatory mechanisms that constrain the inflammation associated with the granulomas. Lipocalin 2 (Lcn2) is a member of the lipocalin family of proteins and binds to bacterial siderophores thereby sequestering iron required for bacterial growth. Thus far, it is not known whether Lcn2 plays a role in the inflammatory response to mycobacterial pulmonary infections. In the present study, using models of acute and chronic mycobacterial pulmonary infections, we reveal a novel role for Lcn2 in constraining T cell lymphocytic accumulation and inflammation by inhibiting inflammatory chemokines, such as CXCL9. In contrast, Lcn2 promotes neutrophil recruitment during mycobacterial pulmonary infection, by inducing G-CSF and KC in alveolar macrophages. Importantly, despite a common role for Lcn2 in regulating chemokines during mycobacterial pulmonary infections, Lcn2 deficient mice are more susceptible to acute M.bovis BCG, but not low dose M.tuberculosis pulmonary infection.     

Changes in inflammatory response after endovascular treatment for type B aortic dissection

This present study aims to investigate the changes in the inflammatory markers after elective endovascular treatment of Type B aortic dissection with aneurysm, as related to different anatomical features of the dissection flap in the paravisceral perfusion. Consecutive patients with type B aortic dissections with elective endovascular stent graft repair were recruited and categorized into different groups. Serial plasma levels of cytokines (Interleukin-1β, -6, -8, -10, TNF-α), chemokines (MCP-1), and serum creatinine were monitored at pre-, peri- and post-operative stages. The length of stent graft employed in each surgery was retrieved and correlated with the change of all studied biochemical parameters. A control group of aortic dissected patients with conventional medication management was recruited for comparing the baseline biochemical parameters. In total, 22 endovascular treated and 16 aortic dissected patients with surveillance were recruited. The endovascular treated patients had comparable baseline levels as the non-surgical patients. There was no immediate or thirty day-mortality, and none of the surgical patients developed post-operative mesenteric ischaemia or clinically significant renal impairment. All surgical patients had detectable pro-inflammatory mediators, but none of the them showed any statistical significant surge in the peri-operative period except IL-1β and IL-6. Similar results were obtained when categorized into different groups. IL-1β and IL-6 showed maximal levels within hours of the endovascular procedure (range, 3.93 to 27.3 higher than baseline; p?=?0.001), but returned to baseline 1 day post-operatively. The change of IL-1β and IL-6 at the stent graft deployment was statistically greater in longer stent graft (p>0.05). No significant changes were observed in the serum creatinine levels. In conclusion, elective endovascular repair of type B aortic dissection associated with insignificant changes in inflammatory mediators and creatinine. All levels fell toward basal levels post-operatively suggesting that thoracic endovascular aortic repair is rather less aggressive with insignificant inflammatory modulation.