Synthesis of 68Ga-Labeled Biopolymer-based Nanoparticle Imaging Agents for Positron-emission Tomography.

AIM: The purpose of this study was to develop a folate receptor-targeted 68Ga-labeled agent for the detection of cancer cells in mouse models of ovarian cancer by dual positron-emission tomography (PET) and magnetic resonance imaging (MRI). Moreover, we aimed to develop a controlled biopolymer-based chemistry that enables linking metal-binding (here Ga-68) chelators. MATERIALS AND METHODS: The nanoparticle (NP) agent was created by self-assembling of folic acid-modified polyglutamic acid and chelator-modified chitosan followed by radiolabeling with 68Ga (III) ions (68Ga-NODAGA-FA). The structure of modified biopolymers was characterized by spectroscopy. Particle size and mobility were determined. RESULTS: Significant selective binding of NPs was established in vitro using folate receptor-positive KB and - negative MDA-MB-231 cell lines. In vivo tumor uptake of folate-targeted 68Ga3+-radiolabeled NPs was tested using subcutaneous tumor-bearing CB17 SCID mice models. PET/MR dual modalities showed high tumor uptake with 6.5 tumor-to-muscle ratio and NP localization. CONCLUSION: In vivo results supporting the preliminary in vitro tests demonstrated considerably higher 68Ga-NODAGA-FA nanoparticle accumulation in KB tumors than in MDA-MB-231 tumors, thereby confirming the folate receptor-mediated uptake of this novel potential PET imaging agent.

First comparative results about the direct effect of traditional cigarette and e-cigarette smoking on lung alveolocapillary membrane using dynamic ventilation scintigraphy.

INTRODUCTION: Dynamic inhalation scintigraphy (DIS) with technetium-99m-diethylenetriamine-pentaacetate aerosol is a useful nuclear medicine procedure for staging and monitoring the damage of alveolocapillary membrane. The e-cigarette is a new popular smoking device producing vapor from the nicotine solution. Many studies have shown that e-cigarettes appear to be safer than smoking, but there are still debates to what extent e-cigarettes are less harmful than smoking.In this prospective, self-controlled study, we compared DIS results among volunteers smoking an e-cigarette and their results after they returned to traditional cigarette smoking for a week. PARTICIPANTS AND METHODS: We included 24 healthy volunteers into this study who regularly used e-cigarette containing at least 10 mg nicotine/ml. We performed baseline DIS study in volunteers with e-cigarette smoking and then we asked them to return to traditional cigarette smoking for a week. Conventional respiration tests were also measured. We statistically analyzed the effect of traditional cigarette on clinical parameters and pulmonary clearance of the radiopharmacon. RESULTS: There was no significant change in the parameters of peak expiratory flow rate and Tiffeneau-Pinelli index respiration tests; forced vital capacity and forced expiratory volume in 1 s slightly decreased (P<0.05), whereas the exhaled CO and COHb levels were significantly higher at traditional cigarette use (P<0.0001), and increased in every case. The pulmonary clearance was significantly faster at traditional cigarette smoking compared with e-cigarette use (P<0.0001). CONCLUSION: On the basis of our results, we suppose that e-cigarette smoking is less harmful to the lung function than a traditional cigarette, and it can be recommended to heavy smokers who are unable to stop smoking.

Characterization of luteinizing hormone-releasing hormone receptor type I (LH-RH-I) as a potential molecular target in OCM-1 and OCM-3 human uveal melanoma cell lines.

INTRODUCTION: Uveal melanoma (UM) is the most common primary intraocular malignancy with very poor prognosis. Conventional chemotherapy only rarely prolongs the survival, therefore patients require novel treatment modalities. The discovery of specific receptors for hypothalamic hormones on cancer cells has led to the development of radiolabeled and cytotoxic hormone analogs. MATERIALS AND METHODS: In the present study, our aim was to investigate the expression of mRNA for receptors of luteinizing hormone-releasing hormone type I (LH-RH-I) and LH-RH ligand in OCM-1 and OCM-3 human uveal melanoma cell lines. The presence and binding characteristics of LH-RH-I receptor protein was further studied by Western blot, immunocytochemistry and ligand competition assay. The expression of mRNA and protein for LH-RH-I receptors has been also studied using tumor samples originating from nude mice xenografted with OCM-1 or OCM-3 cells. RESULTS: The mRNA for LH-RH-I receptor has been detected in OCM-1 and OCM-3 cell lines and was found markedly higher in OCM-3 cells. The mRNA for LH-RH-I receptors was also observed in both UM xenograft models in vivo with higher levels in OCM-3. The presence of LH-RH-I receptor protein was found in both cell lines in vitro by immunocytochemistry and Western blot, and also in tumor tissue samples grown in nude mice by Western blot. Both human uveal melanoma models investigated showed specific high affinity receptors for LH-RH-I using ligand competition assay. The mRNA for LH-RH ligand has also been detected in OCM-1 and OCM-3 cell lines and cancer tissues. CONCLUSION: The demonstration of the expression of LH-RH-I receptors in OCM-1 and OCM-3 human UM cell lines suggests that they could serve as potential molecular target for therapy. Our findings support the development of new therapeutic approaches based on cytotoxic LH-RH analogs or modern powerful antagonistic analogs of LH-RH targeting LH-RH-I receptors in UM.

Effects of non-toxic zinc exposure on human epidermal keratinocytes.

Zinc is an essential microelement; its importance to the skin is highlighted by the severe skin symptoms in hereditary or acquired zinc deficiency, by the improvement of several skin conditions using systemic or topical zinc preparations and by the induced intracellular zinc release upon UVB exposure, which is the main harmful environmental factor to the skin. Understanding the molecular background of the role of zinc in skin may help gain insight into the pathology of skin disorders and provide evidence for the therapeutic usefulness of zinc supplementation. Herein, we studied the effects of zinc chloride (ZnCl2) exposure on the function of HaCaT keratinocytes, and the results showed that a non-toxic elevation in the concentration of extracellular zinc (100 µM) facilitated cell proliferation and induced significant alterations in the mRNA expression of NOTCH1, IL8, and cyclooxygenase-2. In addition, increased heme oxygenase-1 (HMOX1) expression and non-toxic generation of superoxide were detected in the first 4 h. Regarding the effects on the UVB-induced toxicity, although the level of cyclobutane pyrimidine dimers in the keratinocytes pre-treated with zinc for 24 h was reduced 3 h after UVB irradiation, significantly enhanced superoxide generation was observed 10 h after UVB exposure in the zinc pre-exposed cells. The overall survival was unaffected; however, there was a decrease in the percentage of early apoptotic cells and an increase in the percentage of late apoptotic plus necrotic cells. These results suggest that the exposure of human keratinocytes to non-toxic concentrations of ZnCl2 impacts gene expression, cell proliferation and the responses to environmental stress in the skin. It would be important to further examine the role of zinc in skin and further clarify whether this issue can affect our thinking regarding the pathogenesis of skin diseases.

Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.

UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine.

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons.

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.

Lymphatic spread of mesenchymal renal tumor to metastatic parathymic lymph nodes in rat.

Rat mesenchymal renal tumor cells (NeDe) transplanted under the kidney capsule of F344 rats resulted in metastases in the parathymic lymph nodes. Tumor cells were isolated from these tumor-bearing lymph nodes and 106 cells were implanted under the kidney capsule. Tumor growth after this implantation could be traced within six days. India ink was implanted to prove that there is a connection between the lymphatic vessels of the kidney capsule and the parathymic lymph nodes. The distribution of the radioligand 18FDG in different organs also provided evidence that the parathymic lymph nodes are the primary sites of metastatic tumor growth. Tumor growth was followed after staining sections of biopsies of normal, tumorous kidneys and parathymic lymph nodes embedded in paraffin. The progression of tumor formation was seen as a frontline between the healthy and tumor bearing tissue. This demarcation line was sharp at the beginning of the invasion and at the peripheral regions of the tumor, while the central region infiltrated into the healthy kidney tissue. The initial invasion gradually turned to an infiltration resulting in the disruption of the renal tissue, especially at the periphery. Accumulation of lipids and flow of blood to the lymphatic vessels was due to the lack of angiogenesis, leading to an increased pressure of the interstitial fluid. Interstitial damage ultimately led to the appearance of blood and the growth of tumor cells in parathymic lymph nodes. The kidney capsule-parathymic lymph node complex is proposed as a suitable metastatic model for the isolated in vivo examination of tumor development and for the analysis of secondary tumors.

Gamma irradiation induced apoptotic changes in the chromatin structure of human erythroleukemia K562 cells.

Exponentially growing human erythroleukemia K562 cells were synchronized by centrifugal elutriation prior to and after Co60 gamma-irradiation (4 Gy). Forward scatter flow cytometry used for size analysis revealed the increase of an early apoptotic cell population ranging from lower (0.05 C-value) to higher DNA content (approximately 1 C) as the cells progressed through the S phase. The increase in cellular DNA content expressed in C-values correlated with apoptotic chromatin changes manifested as many small apoptotic bodies in early S phase and larger but less numerous disintegrated apoptotic bodies in late S phase. Most significant changes after exposure to gamma-irradiation took place in early S phase resulting in an increase of nuclear size by more than 50%. Cell fractions containing irradiated cells showed enhanced growth arrest at 2.4 C-value, which was accompanied by apoptosis. Apoptotic cell cycle arrest near to the G1/G0 checkpoint and apoptotic changes indicate that the radiation resistance of K562 cells is related to the bypass of the early stage of the p53 apoptotic pathway. Apoptotic changes in chromatin structure induced by gamma-irradiation indicate that these injury-specific changes can be identified and distinguished from chromatin changes induced by UV radiation or heavy metals.