[Albumin-based Drug Delivery System Targeting Mannose Receptors and Its Application to Medical Treatments].

The onset and progression of liver diseases and cancer have shown to be affected by over-active macrophages and fibroblasts. Therefore, developing methods to suppress the activation of these cells has become an urgent task. Prior to this study, a mannosylated-albumin (Man-HSA) that targets mannose receptors expressed in hepatic macrophages (Kupffer cells) or fibroblasts was created. Here, we report on the development of medical treatments based on Man-HSA. To target the reactive oxygen species or inflammation derived from Kupffer cells, we developed a nano-antioxidant, i.e., polythiolated (SH)-Man-HSA, by introducing thiol groups into Man-HSA, or a nano-anti-inflammatory drug, i.e., Man-HSA-IFNα2b, by fusing Man-HSA and IFNα2b. SH-Man-HSA or Man-HSA-IFNα2b attenuated Kupffer cell-derived oxidative stress or inflammation, respectively, resulting in the suppression of liver damage and overall improvement of the survival rate in mice with acute and chronic liver injuries. Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF), both of which are present in the stroma of intractable cancers, also express mannose receptors. Thus, mono-polyethylene glycol modified Man-HSA (monoPEG-Man-HSA) was synthesized as a novel drug delivery carrier targeting TAM/CAF. A complex of monoPEG-Man-HSA with paclitaxel suppressed tumor growth by decreasing the number of TAM/CAF and the stroma area. For the present study, we focused on the mannose receptors expressed in macrophages and fibroblasts, and developed drug delivery carriers that target these cells. Considering the excellent drug-carrying capacity and high biocompatibility of HSA, it is expected that this research will pave the way for innovative pharmacotherapy to treat unmet medical needs, i.e., intractable liver diseases and cancer.

Albumin-based drug carrier targeting urokinase receptor for cancer therapy.

Urokinase plasminogen activator receptor (uPAR) is a key participant in extracellular proteolysis, tissue remodeling and cell motility. uPAR overexpresses in most solid tumors and several hematologic malignancies, but has low levels on normal tissues, thus is advocated as a molecular target for cancer therapy. One of the obstacles for the evaluation of uPAR targeting agents in preclinical study is the species specificity, where targeting agents for human uPAR  usually not bind to murine uPAR. Here, we develop a targeting agent that binds to both murine and human uPAR. This targeting agent is genetically fused to human serum albumin, a commonly used drug carrier, and the final construct is named as uPAR targeting carrier (uPARTC). uPARTC binds specifically to uPAR-overexpressing 293T/huPAR and 293T/muPAR as demonstrated by flow cytometry. A cytotoxic compound, celastrol, is embedded into uPARTC non-covalently. The resulting macromolecular complex show effective proliferation inhibition on both murine and human uPAR overexpressing cells, and exhibit potent antitumor efficacy on hepatoma H22-bearing mice. This work demonstrates that uPARTC is a promising tumor targeting drug carrier, which address the species-specificity challenge of uPAR targeting agents and can be used to load other cytotoxic compounds.

The New Delivery Strategy of Albumin Carrier Utilizing the Interaction with Albumin Receptors.

Albumin, the most abundant protein in human serum, is applied to various diseases as a drug delivery carrier because of its superior blood retention, high biocompatibility, and a wide variety of drug binding abilities. Albumin is known to distribute widely in the blood and various interstitial fluids and organs. Different albumin receptors skillfully regulate the distribution characteristics of albumin in the body. Albumin receptors are a group of diverse proteins, such as FcRn, gp60, gp18, megalin, cubilin, SPARC, and CD36. Their tissue distributions in vivo are unique, with different albumin's recognition sites. Therefore, the distribution of albumin in vivo is ingeniously controlled by these multiple albumin receptors. Reevaluation of these albumin receptors opens up new possibilities for applying albumin as a drug delivery carrier. If the tissue distributions of albumin receptors were known and the albumin recognition site of the receptor was identified, organ-specific active targeting would be possible. In this review, we would like to scrutinize what is currently known and share information to develop next-generation albumin carriers that focus on interactions with albumin receptors.

Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver.

Lipid-like nanoparticles (LNPs) have potential as non-viral delivery systems for mRNA therapies. However, repeated administrations of LNPs may lead to accumulation of delivery materials and associated toxicity. To address this challenge, we have developed biodegradable lipids which improve LNPs clearance and reduce toxicity. We modify the backbone structure of Dlin-MC3-DMA by introducing alkyne and ester groups into the lipid tails. We evaluate the performance of these lipids when co-formulated with other amine containing lipid-like materials. We demonstrate that these formulations synergistically facilitate robust mRNA delivery with improved tolerability after single and repeated administrations. We further identify albumin-associated macropinocytosis and endocytosis as an ApoE-independent LNP cellular uptake pathway in the liver. Separately, the inclusion of alkyne lipids significantly increases membrane fusion to enhance mRNA release, leading to synergistic improvement of mRNA delivery. We believe that the rational design of LNPs with multiple amine-lipids increases the material space for mRNA delivery.

HcTTR: a novel antagonist against goat interleukin 4 derived from the excretory and secretory products of Haemonchus contortus.

Haemonchus contortus (H. contortus) has evolved sophisticated evasion mechanisms to ensure their survival, including generating excretion and secretion products (ESPs) to regulate the secretion of host cytokines. Interleukin 4 (IL4) is a classic T-helper cell type 2 (Th2)-type cytokine that plays an irreplaceable role against nematode infection. In this study, three proteins, glutathione S-transferase domain containing protein (HcGST), transthyretin domain containing protein (HcTTR) and calponin actin-binding domain containing protein (HcCab), were identified to bind to goat IL4 by co-immunoprecipitation (Co-IP) assays and yeast two-hybrid screening. Additionally, cell proliferation analysis showed that HcTTR blocked the IL4-induced proliferation of peripheral blood mononuclear cells in goats, while HcGST and HcCab did not. In addition, HcTTR could also downregulate the transcription of candidate genes in the IL4-induced JAK/STAT pathway. These results indicated that HcTTR is a novel antagonist against goat IL4 from HcESPs, and this information could improve our understanding of the relationship between host cytokines and parasite infections.

Mir-382 Promotes Differentiation of Rat Liver Progenitor Cell WB-F344 by Targeting Ezh2.

BACKGROUND/AIMS: Liver progenitor cells (LPCs) were considered as a promising hepatocyte source of cell therapy for liver disease due to their self-renewal and differentiation capacities, while little is known about the mechanism of LPC differentiate into hepatocytes. This study aims to explore the effect of miR-382, a member of Dlk1-Dio3 microRNA cluster, during hepatic differentiation from LPCs. METHODS: In this study, we used rat liver progenitor cell WB-F344 as LPC cell model and HGF as inducer to simulate the process of LPCs hepatic differentiation, then microRNAs were quantified by qPCR. Next, WB-F344 cell was transfected with miR-382 mimics, then hepatocyte cell trait was characterized by multiple experiments, including that periodic acid schiff staining and cellular uptake and excretion of indocyanine green to evaluate the hepatocellular function, qPCR and Western Blotting analysis to detect the hepatocyte-specific markers (ALB, Ttr, Apo E and AFP) and transmission electron microscopy to observe the hepatocellular morphology. Moreover, Luciferase reporter assay was used to determine whether Ezh2 is the direct target of miR-382. RESULTS: We found that miR-382 increased gradually and was inversely correlated with the potential target, Ezh2, during WB-F344 hepatic differentiation. In addition, functional studies indicated that miR-382 increased the level of hepatocyte-specific genes. CONCLUSIONS: This study demonstrates that miR-382 may be a novel regulator of LPCs differentiation by targeting Ezh2.

Albumin uptake in human podocytes: a possible role for the cubilin-amnionless (CUBAM) complex.

Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes.

Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modification on siRNA.

Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. In addition, the GNA-T nucleobase adopts a rotated conformation in which the 5-methyl group points into the minor groove, rather than the major groove as in a normal Watson-Crick base pair. This observation of reverse Watson-Crick base pairing is further supported by thermal melting analysis of GNA-C and GNA-G containing duplexes where it was demonstrated that a higher thermal stability was associated with isoguanine and isocytosine base pairing, respectively, over the canonical nucleobases. Furthermore, it was also shown that GNA nucleotide or dinucleotide incorporation increases resistance against snake venom phosphodiesterase. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc conjugate duplex targeting mouse transthyretin (TTR) indicated that GNA is well tolerated in the seed region of both strands in vitro, resulting in an approximate 2-fold improvement in potency. Finally, these conjugate duplexes modified with GNA were capable of maintaining in vivo potency when subcutaneously injected into mice.

Expression of thyroid hormone regulator genes in the yolk sac membrane of the developing chicken embryo.

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo.

Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify transthyretin amyloid deposits in carpal tunnel syndrome.

Transthyretin-derived (ATTR) amyloidosis is a frequent finding in carpal tunnel syndrome. We tested the following hypotheses: the novel fluorescent amyloid ligand heptameric formic thiophene acetic acid (h-FTAA) has a superior sensitivity for the detection of amyloid compared with Congo red-staining; Amyloid load correlates with patient gender and/or patient age. We retrieved 208 resection specimens obtained from 184 patients with ATTR amyloid in the carpal tunnel. Serial sections were stained with Congo red, h-FTAA and an antibody directed against transthyretin (TTR). Stained sections were digitalized and forwarded to computational analyses. The amount of amyloid was correlated with patient demographics. Amyloid stained intensely with h-FTAA and an anti-TTR-antibody. Congo red-staining combined with fluorescence microscopy was significantly less sensitive than h-FTAA-fluorescence and TTR-immunostaining: the highest percentage area was found in TTR-immunostained sections, followed by h-FTAA and Congo red. The Pearson correlation coefficient was .8 (Congo red vs. h-FTAA) and .9 (TTR vs. h-FTAA). Amyloid load correlated with patient gender, anatomical site and patient age. h-FTAA is a highly sensitive method to detect even small amounts of ATTR amyloid in the carpal tunnel. The staining protocol is easy and h-FTAA may be a much more sensitive procedure to detect amyloid at an earlier stage.

Vitreous Amyloidosis: Ocular, Systemic, and Genetic Insights.

PURPOSE: To report the unique clinical and surgical characteristics encountered in eyes with vitreous amyloidosis. Systemic evaluation and visual outcome after vitrectomy are discussed. A novel mutation in the transthyretin gene (TTR) in Indian patients with familial amyloid polyneuropathy (FAP) is described. DESIGN: Retrospective, observational study. PARTICIPANTS: Ten eyes of 5 patients from 2 pedigrees with a diagnosis of vitreous amyloidosis. METHODS: Detailed history, pedigree charting, systemic and ocular examination of 10 eyes (5 patients from 2 pedigrees) were carried out. Tests were performed to rule out vitreitis, retinal vasculitis, vitreous hemorrhage, and systemic amyloidosis. Genetic analysis to identify the mutation was performed in 1 patient. Vitreous biopsy, followed by 25-gauge pars plana vitrectomy, was performed in the same sitting in all cases. Samples were sent for Congo red staining and polarized microscopy. Patients were followed up on days 1, 7, and 28 and then every 2 months. Visual acuity assessment, intraocular pressure measurement, and fundus examination were performed each time. MAIN OUTCOME MEASURES: Mutations in TTR and postoperative visual acuity. RESULTS: Mean age at presentation was 32 years, with a 3:2 male-to-female distribution. Family history was positive in all patients. Nine eyes had pseudopodia lentis, whereas all 10 had glass wool-like vitreous. Glaucoma developed in 1 patient (2 eyes). Waxy paper-like vitreous with firm vitreous adhesions beyond major arcades and along retinal vessels was noted during surgery in all eyes. Congo red staining and apple green birefringence demonstrated vitreous amyloidosis. The mean preoperative best-corrected visual acuity (BCVA) was 1.39±0.64 logarithm of the minimum angle of resolution (logMAR), whereas the postoperative BCVA improved to 0.17±0.07 logMAR (P = 0.004). Gene sequencing revealed a phenylalanine→isoleucine mutation in the 33rd position of exon 2 of TTR in 1 patient of 1 pedigree, confirming the diagnosis of FAP. Two patients subsequently were found to have sensorimotor autonomic neuropathy, whereas 2 others had subclinical autonomic dysfunction. CONCLUSIONS: The clinical clues, management strategy, surgical characteristics, vitrectomy outcomes, and significance of systemic evaluation in vitreous amyloidosis are highlighted. A novel single mutation (Phe33Ile) in a case of FAP with vitreous amyloidosis from India is reported.

siRNA carrying an (E)-vinylphosphonate moiety at the 5΄ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2.

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5΄- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5΄- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5΄-(E)-vinylphosphonate (5΄-E-VP) guide RNA at 2.5-Šresolution. The structure demonstrates how the 5΄ binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5΄-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5΄-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.

Familial prion protein mutants inhibit Hrd1-mediated retrotranslocation of misfolded proteins by depleting misfolded protein sensor BiP.

Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases.

TTR kinetic stabilizers and TTR gene silencing: a new era in therapy for familial amyloidotic polyneuropathies.

INTRODUCTION: Transthyretin Familial Amyloid Polyneuropathy (TTR-FAP) is a rare disease with autosomal dominant transmission due to a point mutation of the TTR gene. By removing the main source of systemic mutant TTR, liver transplantation (LT) has become the reference therapy of this severe and fatal polyneuropathy of adult-onset, stopping disease progression in subgroup of patients. Recently, new therapeutic strategies have emerged, which intend to stabilize TTR or to silence the TTR gene. Amongst them, the TTR kinetic stabilizer tafamidis is the first drug approved in the EU. AREAS COVERED: We shall review the natural history of TTR-FAP and the best indications for LT. Data on the efficacy, safety and tolerability of the TTR kinetic stabilizers, tafamidis and diflunisal, have been reviewed, from the pivotal Phase III clinical trials published in PubMed medical journals or presented at international meetings. We will review the ongoing phase III clinical trials of TTR gene silencing with RNAi therapeutics and ASO published in clinicaltrialgov. EXPERT OPINION: Due to the data on efficacy, tolerability, safety, tafamidis and diflunisal became the first line anti-amyloid treatment in stage 1 TTR-FAP. Both drugs slow progression of the disease. Only tafamidis got marketing authorization. We are waiting for results of the 2 phase III clinical trials of TTR gene silencing in varied stages of the disease.

Multisystemic Disease Modeling of Liver-Derived Protein Folding Disorders Using Induced Pluripotent Stem Cells (iPSCs).

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.

The role of albumin receptors in regulation of albumin homeostasis: Implications for drug delivery.

Albumin is the most abundant protein in blood and acts as a molecular taxi for a plethora of small insoluble substances such as nutrients, hormones, metals and toxins. In addition, it binds a range of medical drugs. It has an unusually long serum half-life of almost 3weeks, and although the structure and function of albumin has been studied for decades, a biological explanation for the long half-life has been lacking. Now, recent research has unravelled that albumin-binding cellular receptors play key roles in the homeostatic regulation of albumin. Here, we review our current understanding of albumin homeostasis with a particular focus on the impact of the cellular receptors, namely the neonatal Fc receptor (FcRn) and the cubilin-megalin complex, and we discuss their importance on uses of albumin in drug delivery.

In vivo detection of nerve injury in familial amyloid polyneuropathy by magnetic resonance neurography.

Transthyretin familial amyloid polyneuropathy is a rare, autosomal-dominant inherited multisystem disorder usually manifesting with a rapidly progressive, axonal, distally-symmetric polyneuropathy. The detection of nerve injury by nerve conduction studies is limited, due to preferential involvement of small-fibres in early stages. We investigated whether lower limb nerve-injury can be detected, localized and quantified in vivo by high-resolution magnetic resonance neurography. We prospectively included 20 patients (12 male and eight female patients, mean age 47.9 years, range 26-66) with confirmed mutation in the transthyretin gene: 13 with symptomatic polyneuropathy and seven asymptomatic gene carriers. A large age- and sex-matched cohort of healthy volunteers served as controls (20 male and 20 female, mean age 48.1 years, range 30-73). All patients received detailed neurological and electrophysiological examinations and were scored using the Neuropathy Impairment Score-Lower Limbs, Neuropathy Deficit and Neuropathy Symptom Score. Magnetic resonance neurography (3 T) was performed with large longitudinal coverage from proximal thigh to ankle-level and separately for each leg (140 axial slices/leg) by using axial T2-weighted (repetition time/echo time = 5970/55 ms) and dual echo (repetition time 5210 ms, echo times 12 and 73 ms) turbo spin echo 2D sequences with spectral fat saturation. A 3D T2-weighted inversion-recovery sequence (repetition time/echo time 3000/202 ms) was acquired for imaging of the spinal nerves and lumbar plexus (50 axial slice reformations). Precise manual segmentation of the spinal/sciatic/tibial/common peroneal nerves was performed on each slice. Histogram-based normalization of nerve-voxel signal intensities was performed using the age- and sex-matched control group as normative reference. Nerve-voxels were subsequently classified as lesion-voxels if a threshold of >1.2 (normalized signal-intensity) was exceeded. At distal thigh level, where a predominant nerve-lesion-voxel burden was observed, signal quantification was performed by calculating proton spin density and T2-relaxation time as microstructural markers of nerve tissue integrity. The total number of nerve-lesion voxels (cumulated from proximal-to-distal) was significantly higher in symptomatic patients (20 405 ± 1586) versus asymptomatic gene carriers (12 294 ± 3199; P = 0.036) and versus controls (6536 ± 467; P < 0.0001). It was also higher in asymptomatic carriers compared to controls (P = 0.043). The number of nerve-lesion voxels was significantly higher at thigh level compared to more distal levels (lower leg/ankle) of the lower extremities (f-value = 279.22, P < 0.0001). Further signal-quantification at this proximal site (thigh level) revealed a significant increase of proton-density (P < 0.0001) and T2-relaxation-time (P = 0.0011) in symptomatic patients, whereas asymptomatic gene-carriers presented with a significant increase of proton-density only. Lower limb nerve injury could be detected and quantified in vivo on microstructural level by magnetic resonance neurography in symptomatic familial amyloid polyneuropathy, and also in yet asymptomatic gene carriers, in whom imaging detection precedes clinical and electrophysiological manifestation. Although symptoms start and prevail distally, the focus of predominant nerve injury and injury progression was found proximally at thigh level with strong and unambiguous lesion-contrast. Imaging of proximal nerve lesions, which are difficult to detect by nerve conduction studies, may have future implications also for other distally-symmetric polyneuropathies.

A novel predictive equation for potential diagnosis of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second most common-primary liver cancer. The difficulties in diagnosis limit successful treatment of CCA. At present, histological investigation is the standard diagnosis for CCA. However, there are some poor-defined tumor tissues which cannot be definitively diagnosed by general histopathology. As molecular signatures can define molecular phenotypes related to diagnosis, prognosis, or treatment outcome, and CCA is the second most common cancer found after hepatocellularcarcinoma (HCC), the aim of this study was to develop a predictive model which differentiates CCA from HCC and normal liver tissues. An in-house PCR array containing 176 putative CCA marker genes was tested with the training set tissues of 20 CCA and 10 HCC cases. The molecular signature of CCA revealed the prominent expression of genes involved in cell adhesion and cell movement, whereas HCC showed elevated expression of genes related to cell proliferation/differentiation and metabolisms. A total of 69 genes differentially expressed in CCA and HCC were optimized statistically to formulate a diagnostic equation which distinguished CCA cases from HCC cases. Finally, a four-gene diagnostic equation (CLDN4, HOXB7, TMSB4 and TTR) was formulated and then successfully validated using real-time PCR in an independent testing set of 68 CCA samples and 77 non-CCA controls. Discrimination analysis showed that a combination of these genes could be used as a diagnostic marker for CCA with better diagnostic parameters with high sensitivity and specificity than using a single gene marker or the usual serum markers (CA19-9 and CEA). This new combination marker may help physicians to identify CCA in liver tissues when the histopathology is uncertain.

Avaliação do uso "precoce" de albumina em crianças com queimadura extensa: um ensaio clínico randomizado controlado / Evaluation of the "early" use of albumin in children with extensive burns: a clinical randomized controlled trial

Introdução: A reanimação fluídica da criança queimada é um desafio devido à intolerância à insuficiente ou excessiva oferta de líquidos. Há dúvidas em relação à utilização de solução coloide na ressuscitação volêmica e também quanto ao melhor momento a ser administrada. O momento ideal para a administração de albumina permanece em foco de debate, se deveria ser utilizada como estratégia de resgate, quando o volume de cristaloide infundido se torna excessivo, ou rotineiramente, como intervenção primária em pacientes com queimaduras extensas. Objetivos: Avaliar e comparar quanto a evolução clínica de crianças com lesões térmicas que receberam abordagem de infusão precoce (entre 8 e 12 horas do acidente) de solução coloide natural versus crianças que receberam abordagem de infusão tardia (após 24 horas do acidente) da mesma solução para reanimação na fase aguda. Metodologia: Ensaio Clínico Randomizado Controlado, realizado no Centro de Tratamento de Queimados do Hospital Universitário de Londrina. Foram estudadas 46 crianças (1 a 12 anos), apresentando entre 15% e 45% de Superfície Corporal Queimada, admitidas até a 12a hora após o acidente. Intervenção: Para a ressuscitação hídrica dos pacientes, foi utilizada solução cristaloide baseada na Fórmula de Parkland modificada, ajustada de acordo com o débito urinário. O Grupo Intervenção (23 pacientes) foi randomizado para receber solução de albumina entre 8 e 12 horas do acidente, e o Grupo Controle (23 pacientes) recebeu a mesma solução após 24 horas do acidente. Resultados: Houve possibilidade de redução de infusão de solução cristaloide durante o período de ressuscitação dos pacientes. O grupo Intervenção recebeu um volume de solução cristaloide com uma mediana de -31,99% (P=0,025) no 1º dia, -19,37% (P=0,002) no 2º dia e -45,3% (P=0,002) no 3º dia de ressuscitação. Não foram observadas diferenças significantes entre os grupos em relação à diurese. A incidência acumulada de fluid creep na população...

Introduction: The fluidic resuscitation of burned children is a challenge due to intolerance to insufficient or excessive supply of liquids. There are questions regarding the use of colloids in fluid resuscitation solution as well as the timing of the administration. The ideal time for the administration of albumin, in other words whether it should be used as a rescue strategy when the volume of crystalloid infused becomes excessive, or routinely, as primary intervention for patients with extensive burns, remains in the focus of discussion. Objectives: Evaluate the clinical outcomes of children with burn injuries who have received early infusion treatment (between 8 and 12 hours after the accident) with natural colloids solution compared to the children who have received late infusion treatment (24 hours after the accident) with the same solution, in order to resuscitate them in the acute phase. Methods: Randomized Controlled Trial carried out at the Burn Treatment Center, State University of Londrina. Forty-six children (1 to 12 year olds) who had between 15% and 45% total body surface area and were admitted up to 12 hours after the accident were studied. Intervention: For the fluid resuscitation of patients, a crystalloid solution based on modified Parkland Formula was adjusted according to urine output. The Intervention Group (n= 23) were randomized to receive albumin solution between 8 and 12 hours after the accident and the Control Group (n= 23) received the same solution later than 24 hours after the burn injury. Results: During the resuscitation of patients, it was possible to reduce the infusion of crystalloid solution. The Intervention Group required a volume of crystalloid solution with a median of -31.99% (P = 0.025) on day 1, - 19.37% (P = 0.002) on day 2 and -45.3% (P = 0.002) on day 3 of resuscitation. No significant differences were observed in the groups in relation to diuresis. The cumulative incidence of fluid creep in the population...

[Effect of Hanfangji compound on proliferation of hepatic stellate cells and expressions of TTR, ITIH1 and SERPINF2].

OBJECTIVE: To observe and compare the effects of Hanfangji Compound and IFN-gamma on expressions of transthyretin (TTR) , inter-alpha inhibitor H1 (ITIH1) and serpin peptidase inhibitor clade F member 2 (SERPINF2) of hepatic stellate cells (HSC-T6). METHOD: Hanfangji Compound and IFN-gammaof different concentrations were used in hepatic stellate cell-T6 (HSC-T6) for 48 h. Flow cytometer was used to detect the effects of Hanfangji Compound and IFN-gamma on HSC proliferation. RT-PCR method was adopted to detect mRNA expressions of TFR, ITIH1 and SERPINF2. TTR, ITIH1 and SERPINF2 secretions were detected by ELISA. The protein localizations of TTR, ITIH1 and SERPINF2 were examined by immune fluorescence. The protein expression of TfR and ITIHI were determined by Western blot. RESULT: After Hanfangji Compound and IFN-gamma were adopted in HSC-T6, compared with the control group, the cell proliferation was inhibited obviously (P < 0. 05) , protein expressions of TTR, ITIH1 and SERPINF2 and mRNA expression increased significantly, with certain correlation with concentrations of Hanfangji Compound. The 2. 5 g L-I Hanfangji Compound group was superior to the IFN-gamma group (P <0. 05). CONCLUSION: Hanfangji Compound can inhibit HSC proliferation, upregulated TTR, ITIH1 and SERPINF2 proteins and mRNA expression, which may be one of mechanisms of anti-hepatic fibrosis of Hanfangji Compound.