Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease. Y.S. and A.S are the incumbents of the Tillie and Charles Lubin Professorial Chair in Biochemical Endocrinology, and the Robert and Yaddele Sklare Professorial Chair in Biochemistry, respectively. S.J. is the incumbent of the Pauline Recanati Career Development Chair. D.P. in partial fulfillment of her PhD Thesis requirements at the Feinberg graduate school of the Weizmann Institute of Science.     

Imaging endothelin ET(B) receptors using [18F]-BQ3020: in vitro characterization and positron emission tomography (microPET)

The endothelin (ET) receptor system has been shown to play a role in a number of vascular diseases. We have synthesized 18F-and 11C-labeled radioligands to enable in vivo imaging of the fundamental processes involved in ET receptor pharmacology in normal and diseased tissue using positron emission tomography (PET). One aim is to elucidate the proposed role of the ET(B) subtype as clearing receptor, removing ET-1 from the circulation, and whether this is an important mechanism to limit the detrimental effects caused by upregulated ET-1 in disease. To image ET(B) receptors we have labeled the selective agonist BQ3020 with 18F. In vitro characterization verified that [18F]-BQ3020 bound with a single subnanomolar affinity (K(D) = 0.34 +/- 0.10 nM, B(max) = 9.23 +/- 3.70 fmol/mg protein) to human left ventricle. Binding of [18F]-BQ3020 to human kidney was inhibited by ET-1 and unlabeled BQ3020 but not by the ET(A) selective antagonist FR139317, confirming that selectivity for the ET(B) receptor was retained. In vitro autoradiography revealed, as expected, high levels of ET(B) receptor densities in lung and kidney medulla, whereas kidney cortex and heart showed lower levels of ET(B) receptor densities. Furthermore, a high level of [18F]-BQ3020 binding was found to colocalize to macrophages in atherosclerotic coronary arteries. MicroPET studies demonstrated high uptake of [18F]-BQ3020 in ET(B) receptor-rich tissue, including lung, liver and kidney. The in vivo biodistribution of [18F]-BQ3020 was comparable to that previously obtained for [18F]-ET-1, supporting our hypothesis that the ET(B) receptor plays a significant role in the uptake of ET-1. In conclusion, [18F]-BQ3020 has retained high affinity and selectivity, allowing imaging of ET(B) receptor distributions in vitro and in vivo in human and animal tissue. Furthermore, in vitro data suggest that [18F]-BQ3020 potentially can be used to image atherosclerotic lesions in vivo using PET.     

Analysis of Spliceosomal Proteins in Trypanosomatids Reveals Novel Functions in mRNA Processing

In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions.     

Quantitative trait loci analysis of seed‐specialized metabolites reveals seed‐specific flavonols and differential regulation of glycoalkaloid content in tomato

Given the potential health benefits (and adverse effects), of polyphenolic and steroidal glycoalkaloids in the diet there is a growing interest in fully elucidating the genetic control of their levels in foodstuffs. Here we carried out profiling of the specialized metabolites in the seeds of the Solanum pennellii introgression lines identifying 338 putative metabolite quantitative trait loci (mQTL) for flavonoids, steroidal glycoalkaloids and further specialized metabolites. Two putative mQTL for flavonols and one for steroidal glycoalkaloids were cross‐validated by evaluation of the metabolite content of recombinants harboring smaller introgression in the corresponding QTL interval or by analysis of lines from an independently derived backcross inbred line population. The steroidal glycoalkaloid mQTL was localized to a chromosomal region spanning 14 genes, including a previously defined steroidal glycoalkaloid gene cluster. The flavonoid mQTL was further validated via the use of transient and stable overexpression of the Solyc12g098600 and Solyc12g096870 genes, which encode seed‐specific uridine 5′‐diphosphate‐glycosyltransferases. The results are discussed in the context of our understanding of the accumulation of polyphenols and steroidal glycoalkaloids, and how this knowledge may be incorporated into breeding strategies aimed at improving nutritional aspects of plants as well as in fortifying them against abiotic stress.     

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.     

Innervation patterns of autonomic axons in the human endocrine pancreas

The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, thus impacting glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are unknown, particularly in human. Here we demonstrate that the innervation of human islets is different from that of mouse islets and does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, unlike mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet, and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.     

Involvement of host stroma cells and tissue fibrosis in pancreatic tumor development in transgenic mice

Introduction

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.

Methods

Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.

Results

Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.

Conclusions

The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.     

Insights into interstitial flow,shear stress,and mass transport effects on ECM heterogeneity in bioreactor-cultivated engineered cartilage hydrogels

Interstitial flow in articular cartilage is secondary to compressive and shear deformations during joint motion and has been linked with the well-characterized heterogeneity in structure and composition of its extracellular matrix. In this study, we investigated the effects of introducing gradients of interstitial flow on the evolution of compositional heterogeneity in engineered cartilage. Using a parallel-plate bioreactor, we observed that Poiseuille flow stimulation of chondrocyte-seeded agarose hydrogels led to an increase in glycosaminoglycan and type II collagen deposition in the surface region of the hydrogel exposed to flow. Experimental measurements of the interstitial flow fields based on the fluorescence recovery after photobleaching technique suggested that the observed heterogeneity in composition is associated with gradients in interstitial flow in a boundary layer at the hydrogel surface. Interestingly, the interstitial flow velocity profiles were nonlinearly influenced by flow rate, which upon closer examination led us to the original observation that the apparent hydrogel permeability decreased exponentially with increased interfacial shear stress. We also observed that interstitial flow enhances convective mass transport irrespective of molecular size within the boundary layer near the hydrogel surface and that the convective contribution to transport diminishes with depth in association with interstitial flow gradients. The implications of the nonlinearly inverse relationship between the interfacial shear stress and the interstitial flux and permeability and its consequences for convective transport are important for tissue engineering, since porous scaffolds comprise networks of Poiseuille channels (pores) through which interstitial flow must navigate under mechanical stimulation or direct perfusion.