Cationic Glucan Dendrimer Gel-Mediated Local Delivery of Anti-OC-STAMP-siRNA for Treatment of Pathogenic Bone Resorption.

Osteoclast stimulatory transmembrane protein (OC-STAMP) plays a pivotal role in the promotion of cell fusion during osteoclast differentiation (osteoclastogenesis) in the context of pathogenic bone resorption. Thus, it is plausible that the suppression of OC-STAMP through a bioengineering approach could lead to the development of an effective treatment for inflammatory bone resorptive diseases with minimum side effects. Here, we synthesized two types of spermine-bearing (Spe) cationic glucan dendrimer (GD) gels (with or without C12) as carriers of short interfering RNA (siRNA) to silence OC-STAMP. The results showed that amphiphilic C12-GD-Spe gel was more efficient in silencing OC-STAMP than GD-Spe gel and that the mixture of anti-OC-STAMP siRNA/C12-GD-Spe significantly downregulated RANKL-induced osteoclastogenesis. Also, local injection of anti-OC-STAMP-siRNA/C12-GD-Spe could attenuate bone resorption induced in a mouse model of periodontitis. These results suggest that OC-STAMP is a promising target for the development of a novel bone regenerative therapy and that C12-GD-Spe gel provides a new nanocarrier platform of gene therapies for osteolytic disease.

Nanoparticle approaches for the renin-angiotensin system.

The renin-angiotensin system (RAS) is a hormonal cascade that contributes to several disorders: systemic hypertension, heart failure, kidney disease, and neurodegenerative disease. Activation of the RAS can promote inflammation and fibrosis. Drugs that target the RAS can be classified into 3 categories, AT1 angiotensin receptor blockers (ARBs), angiotensin-converting enzyme (ACE) inhibitors, and renin inhibitors. The therapeutic efficacy of current RAS-inhibiting drugs is limited by poor penetration across the blood-brain barrier, low bioavailability, and to some extent, short half-lives. Nanoparticle-mediated drug delivery systems (DDSs) are possible emerging alternatives to overcome such limitations. Nanoparticles are ideally 1-100 nm in size and are considered efficient DDSs mainly due to their unique characteristics, including water dispersity, prolonged half-life in blood circulation, smaller size, and biocompatibility. Nano-scale DDSs can reduce the drug dosage frequency and acute toxicity of drugs while enhancing therapeutic success. Different types of nanoparticles, such as chitosan, polymeric, and nanofibers, have been examined in RAS-related studies, especially in hypertension, cardiovascular disease, and COVID-19. In this review article, we summarize the physical and chemical characteristics of each nanoparticle to elaborate on their potential use in RAS-related nano-drug delivery research and clinical application.

Productive Performance and Cecum Microbiota Analysis of Broiler Chickens Supplemented with ß-Mannanases and Bacteriophages-A Pilot Study.

This study was conducted to evaluate the productive performance, intestinal health, and description of the cecum microbiota in broilers supplemented with ß-mannanases (MNs) and bacteriophages (BPs). Six hundred one-day-old broilers were divided into four groups and fed one of the following diets: CON-corn-soybean meal + 10 ppm enramycin (ENR); MN: CON + 500 ppm MN; BP: CON + 500 ppm BP; MN + BP: BP + 500 ppm MN. The BP and MN factors showed similar performances to ENR. MN improved the concentration of IgA in the jejunum at 35 days of age. The morphometric index (IM) of the thymus increased by adding MN, while BP increased the liver and thymus IM. The histological analysis showed that BP and MN improved the intestinal morphology. MN + BP showed a tendency to decrease the abundance of Proteobacteria and increase the abundance of Bacteroidetes, indicating better microbiota function. In conclusion, our results demonstrate that the combination of MN + BP has potential in poultry nutrition; however, we highly recommend further experiments to confirm this hypothesis.

Enhanced control of bioactivity of tissue plasminogen activator (tPA) through domain-directed enzymatic oxidation of terminal galactose.

Introduction: In targeted enzyme prodrug constructs, it is critical to control the bioactivity of the drug in its prodrug form. The preparation of such constructs often involves conjugation reactions directed to functional groups on amino acid side chains of the protein, which result in random conjugation and incomplete control of bioactivity of a prodrug, which may result in significant nontarget effect. Thus, more specific method of modification is desired. If the drug is a glycoprotein, enzymatic oxidation may offer an alternative approach for therapeutic glycoproteins. Methods: Tissue plasminogen activator (tPA), a model glycoprotein enzyme, was treated with galactose oxidase (GO) and horseradish peroxidase, followed by thiolation reaction and conjugation with low molecular weight heparin (LMWH). The LMWH-tPA conjugate was isolated by ion-exchange chromatography followed by centrifugal filtration. The conjugate was characterized for its fibrinolytic activity and for its plasminogen activation through an indirect amidolytic assay with a plasmin-specific substrate S-2251 when LMWH-tPA conjugate is complexed with protamine-albumin conjugate, followed by triggered activation in the presence of heparin. Results: LMWH-tPA conjugate prepared via enzymatic oxidation retained ~95% of its fibrinolytic activity with respect to native tPA. Upon complexation with protamine-albumin conjugate, the activity of LMWH-tPA was effectively inhibited (~90%) whereas the LMWH-tPA prepared by random thiolation exhibited ~55% inhibition. Addition of heparin fully generated the activities of both conjugates. Conclusion: The tPA was successfully modified via enzymatic oxidation by GO, resulting in enhanced control of its activity in the prodrug construct. This approach can be applied to other therapeutic glycoproteins.

Targeted delivery of thrombolytic enzymes.

Although thrombolytic agents have been used for several decades in the treatment of thromboembolic conditions, there is an unmet need for the development of safer thrombolytic agents. The development of new molecules themselves may not be sufficient. This has sparked a growing interest in the design of novel nanoscale drug carrier systems for the delivery of thrombolytic enzymes in an effort to address its fatal side effects. There are numerous proof-of-concept reports on such nanoscale systems that seek to capitalize on the pathophysiologic signatures of thrombosis as well as external biochemical/physical triggers. Although there may be a long road ahead before we have such new nanoscale therapeutics on the bedside, hopes remain high.

Pluronic® F127-mediated control of insulin release rates from NPH microcrystals and blood glucose depression in STZ-induced diabetic rats.

Introduction: Neutral protamine Hagedorn (NPH) insulin is an intermediate-acting basal insulin with a long history of clinical use, consisting native human insulin. Its rather undesirable action profile, characterized by a peak release within a few hours, followed by insufficient insulin delivery upon a single subcutaneous (s.c.) dose, is well-documented. This may have been caused by the inherent microcrystal structure involving the basic peptide protamine, as well as the presence of tissue enzyme activities that readily act on protamine at the injection site. This issue may be circumvented by utilizing thermosensitive, erodible Pluronic F127 (PF127) to modulate the kinetics of insulin release from NPH over a period of 24 hours in which the hydrogel is completely eroded. Methods: Previously, we have shown that insulin release rates in vitro from NPH/PF127 formulations (0-25% PF127) markedly reduced the initial insulin release, especially in the presence of enzyme activity that selectively degraded protamine at 1-5 U/mL. Insulin release over the course of 20 hours was better modulated in the presence of increasing PF127 content. In this study, the insulin formulations (0, 20, and 25% PF127) were administered s.c. (4 U/kg) to streptozotocin (STZ)-induced diabetic rats and blood glucose levels were monitored over 24 hours. Results: In vivo blood glucose depression profiles in STZ-induced diabetic rats exhibited a similar pattern of control to in vitro data at the single s.c. dose of 4 U/kg, apparently extending the duration of action of NPH over a 24-hour period in the presence of PF127. Conclusion: Our findings suggest that the undesirable kinetics of insulin release from NPH is significantly influenced by tissue enzyme activity and that the presence of PF127 provided a timely modulation of insulin release from NPH microcrystals in the STZ-induced diabetic rat model.

Institutional reductions in opioid prescribing do not change patient satisfaction on Press Ganey surveys after total shoulder arthroplasty.

BACKGROUND: With an ongoing opioid epidemic in the United States, it is important to examine if decreased opioid prescribing can affect patient experience, namely satisfaction with pain control. PURPOSE: The purpose of this study was to investigate what effect, if any, decreased opioid prescribing after total shoulder arthroplasty had on Press Ganey satisfaction surveys. METHODS: A retrospective review was conducted on patients who underwent primary anatomic or reverse total shoulder arthroplasty between October 2014 and October 2019. Patients with complete Press Ganey survey information and no history of trauma, fracture, connective tissue disease, or prior shoulder arthroplasty surgery were included in the analysis. Patients were segregated into 2 groups, pre-protocol and post-protocol, based on the date of surgery relative to implementation of an institutional opioid reduction protocol, which occurred in October 2018. Prescriptions were converted to morphine milligram equivalents (MME) for direct comparison between different opioid medications. RESULTS: A total of 201 patients met inclusion criteria, and there were 110 reverse total shoulder arthroplasties and 91 anatomic total shoulder arthroplasties. Average opioids prescribed on discharge for the pre-protocol group were 426.3 ± 295 MME (equivalent to 56.8 tablets of oxycodone 5 mg), whereas after the initiation of the protocol, they were 193.8 ± 199 MME (equivalent to 25.8 tablets of oxycodone 5 mg); P < .0001. Average satisfaction with pain control did not change significantly between pre-protocol and post-protocol (4.71 ± 0.65 pre-protocol and 4.74 ± 0.44 post-protocol, P = .82). CONCLUSION: A reduction in opioids prescribed after a total shoulder replacement is not associated with any negative effects on patient satisfaction, as measured by the Press Ganey survey.

Effects of Housing Types on Cecal Microbiota of Two Different Strains of Laying Hens During the Late Production Phase.

Due to animal welfare issues, European Union has banned the use of conventional cages (CC) and non-EU countries including the US are also under constant public pressure to restrict their use in egg production. Very limited information is available on the composition of the microbial community of hens raised in different housing environments. This study was conducted to determine the effects of CC and enriched colony cages (EC) on cecal microbiota of two commercial laying hen strains, Hy-Line W36 (W36) and Hy-Line Brown (HB) during the late production stage (53, 58, 67, and 72 weeks of age). Cecal microbiota was studied by analyzing 16S rRNA gene sequences with Quantitative Insights Into Microbial Ecology (QIIME) 2 ver. 2018.8. Differentially abundant taxa were identified by Linear discriminant analysis Effect Size (LEfSe) analysis (P < 0.05, LDA score > 2.0). At phylum level, Actinobacteria was significantly enriched in W36 at all time points while Synergistetes (53 weeks), Spirochaetes (58 weeks), and Synergistetes and Spirochaetes (67 weeks) were significantly higher in HB. At genus level, Bifidobacterium (at all time points) and butyric acid producing genera such as Butyricicoccus and Subdoligranulum (58 and 72 weeks) were significantly higher in W36 as compared to HB. Moreover, Proteobacteria (72 weeks) and its associated genus Campylobacter (67 and 72 weeks) were significantly enriched in EC as compared to CC. Alpha diversity was significantly higher in HB (at all time points) and in EC (67 weeks) as compared to W36 and CC, respectively. Similarly, there was a significant difference in community structure (beta diversity) between W36 and HB (all time points) as well as between EC and CC (67 weeks). The effect of housing and strains was not only seen at the bacterial composition and structure but also reflected at their functional level. Notably, KEGG metabolic pathways predicted to be involved in carbohydrates degradation and amino acids biosynthesis by PICRUSt analysis were significantly different between W36 and HB housed at CC and EC. In sum, cecal microbiota composition, diversities, and their functional pathways were affected by housing type which further varied between two commercial laying hen strains, HB and W36. This suggests that both housing and genetic strains of laying hens should be considered for selection of the alternative housing systems such as enriched colony cage.

Evaluation of the Epithelial Barrier Function and Ileal Microbiome in an Established Necrotic Enteritis Challenge Model in Broiler Chickens.

Necrotic enteritis (NE) is a recognized multifactorial disease that cost annually to the poultry industry around $2 billion. However, diverse aspects related to its presentation are not completely understood, requiring further studies using known induction experimental models. Therefore, the purpose of this study was to measure the changes occurring in performance, intestinal integrity and ileal microbiome using a previously established NE-challenge model. Chickens were assigned to a negative control group (NC) or a positive control group (PC). In the PC, broilers were orally gavaged with Salmonella Typhimurium (ST) (1 × 107 cfu/chick) at day 1, Eimeria maxima (EM) (2.5 × 104 oocyst/chick) at day 18 and Clostridium perfringens (CP) (1 × 108 cfu/chick/day) at 23-24 days of age. Weekly, body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) were evaluated. Morbidity and mortality were determined throughout the study, and NE lesion scores were recorded at day 25. Additionally, blood and liver samples were collected to measure gut permeability as determined by levels of serum fluorescein isothiocyanate-dextran (FITC-d) and bacterial translocation (BT). Ileal contents were processed for 16S rRNA gene-based microbiome analysis. Performance parameters and intestinal permeability measurements were negatively impacted in the PC resulting in elevated serum FITC-d and BT with a -6.4% difference in BWG. The NE lesion score in PC (1.97 vs. 0.00) was significantly higher in comparison to NC, although there was no difference in mortality. The microbiome analysis showed a dramatic shift of ileal microbiomes in PC groups as compared to NC (ANOSIM: R = 0.76, P = 0.001). The shift was characterized by reduced abundance of the phylum Actinobacteria (P < 0.01), and increased abundance of the genera Butyrivibrio, Lactobacillus, Prevotella and Ruminococcus in PC compared to NC (P < 0.05). Expectedly, Clostridium was found higher in PC (2.98 ± 0.71%) as compared to NC (1.84 ± 0.36%), yet the difference was not significant. In conclusion, results of the present study showed the different intestinal epithelial and microbiological alterations occurring in an established NE-challenge model that considers paratyphoid Salmonella infections in young chicks as an important predisposing factor for presentation of NE.

Global Screening of Salmonella enterica Serovar Typhimurium Genes for Desiccation Survival.

Salmonella spp., one of the most common foodborne bacterial pathogens, has the ability to survive under desiccation conditions in foods and food processing facilities for years. This raises the concerns of Salmonella infection in humans associated with low water activity foods. Salmonella responds to desiccation stress via complex pathways involving immediate physiological actions as well as coordinated genetic responses. However, the exact mechanisms of Salmonella to resist desiccation stress remain to be fully elucidated. In this study, we screened a genome-saturating transposon (Tn5) library of Salmonella Typhimurium (S. Typhimurium) 14028s under the in vitro desiccation stress using transposon sequencing (Tn-seq). We identified 61 genes and 6 intergenic regions required to overcome desiccation stress. Salmonella desiccation resistance genes were mostly related to energy production and conversion; cell wall/membrane/envelope biogenesis; inorganic ion transport and metabolism; regulation of biological process; DNA metabolic process; ABC transporters; and two component system. More than 20% of the Salmonella desiccation resistance genes encode either putative or hypothetical proteins. Phenotypic evaluation of 12 single gene knockout mutants showed 3 mutants (atpH, atpG, and corA) had significantly (p < 0.02) reduced survival as compared to the wild type during desiccation survival. Thus, our study provided new insights into the molecular mechanisms utilized by Salmonella for survival against desiccation stress. The findings might be further exploited to develop effective control strategies against Salmonella contamination in low water activity foods and food processing facilities.

Characterization of the Culturable Subpopulations of Lactobacillus in the Chicken Intestinal Tract as a Resource for Probiotic Development.

To gain better understanding of the distributions of the culturable Lactobacillus species in the chicken intestinal tract, we collected ceca, and distal ileum from 10 3-weeks-old broiler chickens. Lactobacillus strains from cecal lumen contents (M-CL), and those associated with mucosa of ceca (M-CM) and ileum (M-IM) were recovered on de Man, Rogosa and Sharpe (MRS) agar plates, and used for microbiota analysis. The total cecal content (T-CL) was also used directly for microbiota analysis. We purposefully focused on MRS-recovered populations to gain understanding of the culturable subpopulations of Lactobacillus, since the culturability is an important phenotype in order to exploit the chicken gut microbiota as a resource for development of probiotics. The V1-V3 regions of 16S rRNA gene was amplified from genomic DNA samples, and the pooled amplicons were analyzed by MiSeq sequencing with paired-end read 300 cycle option. Among MRS groups, Firmicutes were significantly higher in M-IM and M-CL as compared to M-CM, whereas Proteobacteria were significantly higher in M-CM as compared to M-IM and M-CL at p < 0.05. Among Lactobacillus, L. salivarius (36%) and L. johnsonii (21%) were higher in M-IM as compared to M-CL (L. salivarius, 28%; L. johnsonii, 15%), and M-CM (L. salivarius, 20%; L. johnsonii, 11%). L. crispatus was found significantly higher in M-CL as compared to M-IM (p < 0.01) whereas L. gasseri was found significantly higher in M-IM as compared to M-CM (p < 0.05). L. aviarius, and L. fornicalis were only observed in T-CL. In summary, Lactobacillus populations recovered on MRS vary with different regions and locations in chicken GIT, which might indicate their distinct functional roles in different gastrointestinal tract (GIT) niches, and some species of Lactobacillus are not culturable on MRS agar media. This study is the first attempt to define culturable Lactobacillus subpopulations in the chicken intestinal tract comprehensively using 16S rRNA gene profiling, and the findings of this study will be used as a platform to develop a new strategy for isolation of effective Lactobacillus probiotic candidates based on comparative analyses of chicken gut microbiota.

Bio-responsive delivery of tissue plasminogen activator for localized thrombolysis.

In this study, we have developed an albumin-camouflaged/thrombin-triggered delivery system for site-specific delivery of tissue plasminogen activator (tPA). The camouflaged construct is expected to suppress tPA's enzymatic activity in the systemic circulation but regenerate its thrombolytic action upon contact with thrombin present on the thrombus. tPA was camouflaged with human serum albumin (HSA) via a thrombin-cleavable peptide (GFPRGFPAGGCtPA). The surface of the albumin molecule was decorated with a homing peptide (CQQHHLGGAKQAGDV) that binds with GPIIb/IIIa expressed on activated platelets. To avoid non-specific interactions, the unpaired cysteine-34 of HSA was permanently blocked by iodoacetamide and the primary amines were temporarily masked with citraconic anhydride. Shielding with HSA suppressed 75% of tPA's activity which, upon contact with 25 nM thrombin, was regenerated to ~90% of that of native tPA. The fibrin agar plate assay further confirmed the thrombin-mediated release of tPA from the camouflaged construct. The integrity of camouflaged construct was maintained in human plasma or blood. The fluorescence microscopic studies confirmed the binding affinity of camouflaged tPA with the activated platelets. Furthermore, when evaluated in a rat thrombosis model, the thrombolytic activity of camouflaged tPA was similar to that of native tPA. However, the degradation of circulating fibrinogen was reduced by 2-fold with HSA-decorated tPA compared with that of native tPA, which is an indication of reduced risk of hemorrhagic incidence. This proof-of-principle study suggests that the activity of tPA can be suppressed by HSA and regenerated by thrombin present at the thrombus.

Serum albumin-protamine conjugate for biocompatible platform for targeted delivery of therapeutic macromolecules.

A well-defined, one-to-one conjugate between human serum albumin (HSA) and protamine was synthesized and characterized as a biocompatible carrier for macromolecules. In circulation, the conjugate will camouflage drug molecules upon complex formation, while liberating free drug at the desired location using a triggering mechanism. The N-terminus of protamine was thiolated and conjugated with the unpaired Cysteine-34 of HSA, and was purified by ion-exchange chromatography. The molecular weight of the conjugate was 70.8 kDa, confirming one-to-one conjugation between HSA (66.6 KDa) and protamine (4200 Da). Superimposed fluorescence spectra of native HSA and HSA-protamine conjugate indicated no conformational change around the Trp-214. The conjugate had marked reduction in hemolytic and cytotoxic properties compared to protamine. When therapeutic potential was tested using tissue plasminogen activator as a model drug, HSA-protamine conjugate suppressed the enzymatic activity by 65%, which was fully recovered by a triggering agent, heparin. The construct showed binding characteristics with activated platelets upon conjugation with a targeting peptide, demonstrating flexibility to introduce suitable homing moiety on the surface. The camouflaged construct retained triggered release property in human plasma condition. Overall, the conjugate has a good potential to serve as a biocompatible platform for macromolecular drugs.

Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1.

Aptamers are artificial oligonucleotides (DNA or RNA) that can bind to a broad range of targets. In diagnostic and detection assays, aptamers represent an alternative to antibodies as recognition agents. The objective of this study was to select and characterize DNA aptamers that can specifically bind to avian influenza virus (AIV) H5N1 based on Systematic Evolution of Ligands by EXponential enrichment (SELEX) and surface plasmon resonance (SPR). The selection was started with an ssDNA (single-stranded DNA) library of 10¹4 molecules randomized at central 74 t. For the first four selection cycles, purified hemagglutinin (HA) from AIV H5N1 was used as the target protein, and starting from the fifth cycle, entire H5N1 virus was applied in order to improve the specificity. After 13 rounds of selection, DNA aptamers that bind to the H5N1 were isolated and three aptamer sequences were characterized further by sequencing and affinity binding. Dot blot analysis was employed for monitoring the SELEX process and conducting the preliminary tests on the affinity and specificity of aptamers. With the increasing number of selection cycles, a steady increase in the color density was observed, indicating that the aptamers with good binding affinity to the target were enriched. The best aptamer candidate had a dissociation constant (KD) of 4.65 M as determined by SPR, showing a strong binding between the HA and the selected aptamer. The specificity was determined by testing non-target AIV H5N2, H5N3, H5N9, H9N2 and H7N2. Negligible cross-reactivity confirmed the high specificity of selected aptamers. The developed aptamer was then applied for detection of AIV H5N1 in spiked poultry swab samples. The obtained aptamers could open up possibilities for the development of aptamer-based medical diagnostics and detection assays for AIV H5N1. (The H5N1 used in this study was inactivated virus.).

Preparation and characterization of anionic oligopeptide-modified tissue plasminogen activator for triggered delivery: an approach for localized thrombolysis.

PURPOSE: The study sought to synthesize anionic peptide-conjugated tissue plasminogen activator (tPA) for its targeted/triggered delivery, where tPA's activity would be masked in the circulation and regenerated at the thrombus site by a commonly used anticoagulant, heparin, to minimize tPA associated bleeding complications. METHODS: tPA was conjugated to Polyglutamate, and the activity of oligoanion-modified tPA was tested by fibrinolytic assay. Separately human serum albumin (HSA) was conjugated to protamine and the formation of its electrostatic complex with anionic peptide was monitored by Förster Resonance Energy Transfer (FRET). The masking of tPA-activity via steric hindrance created by albumin, and subsequent regeneration with therapeutic dose of heparin was tested by enzymatic assay. Stability of 'camouflaged-tPA' was determined in human plasma. Using fluorescence microscope, binding of camouflaged-tPA with activated platelets was monitored. Heparin modulated clot-lysis was evaluated in human blood clot. RESULTS: The anionic tPA retained ~97% activity of the unmodified-tPA. FRET experiments confirmed the electrostatic interaction between polyglutamate and protamine which was subsequently reversed by heparin. Complexation with HSA-protamine masked ~60% of tPA activity which was fully regenerated by heparin. The complex retained its prodrug character in human plasma after incubation at 37°C. Fluorescence microscopic study confirmed binding of the construct with activated platelets. In lysing human clot, the camouflage could mask tPA-activity until it was triggered at a heparin level of 0.4U/mL. CONCLUSION: Oligoanion-modified tPA could be used for targeted/triggered delivery where its enzymatic activity could be masked by HSA-protamine conjugate and successfully regenerated by therapeutic dose of heparin.

Heparin-triggered release of camouflaged tissue plasminogen activator for targeted thrombolysis.

A targetable, heparin-triggered release system for tissue plasminogen activator (tPA) was designed to prevent the excessive 'lytic' state associated with the current tPA therapy for acute thrombotic conditions, such as myocardial infarction (MI). The strategy is, upon target accumulation, to trigger tPA release from a prodrug construct by a usual heparin dose. A relatively inactive form of tPA was constructed by conjugating tPA with low-molecular weight heparin followed by complexation with albumin-protamine conjugate, termed 'camouflage'. The modified tPA was ~97% as active as native tPA. The prodrug construct of tPA significantly masked the enzymatic activity, which was fully recovered upon heparin addition. The camouflaged tPA was stable in human blood for at least 30min and was able to trigger enzyme activation in vitro at heparin level of 0.4U/mL. In vivo studies on jugular vein rat thrombosis model showed that the clot lysis of the heparin-triggered camouflaged tPA group was equivalent to the tPA+heparin group without prolongation of activated partial thromboplastin time (aPTT) before and after the treatment. This proof-of-principle study suggests that the activity of the tPA prodrug construct can be triggered at the thrombus site at therapeutic heparin concentration conjunctively used for MI with reduced bleeding risk.

Coexpression of carcinoembryonic antigen and E-cadherin in colorectal adenocarcinoma with liver metastasis.

Carcinoembryonic antigen (CEA) has been suggested as a metastatic activator in colorectal carcinoma, whereas the E-cadherin expression is downregulated in a variety of carcinomas. CEA and E-cadherin expressions were simultaneously assessed with regard to tumor progression in the various sites of colorectal carcinomas with liver metastasis. Twenty-six consecutive patients who had colorectal carcinoma with liver metastasis underwent curative surgery for primary tumor and liver metastasis. CEA and E-cadherin expression were identified on immunohistochemical staining using the labeled streptavidin-biotin method. Their mRNA expression was also detected by RT in situ PCR using one-step reverse transcription-polymerase chain reaction (RT-PCR). CEA and E-cadherin expression scores in the tumor center were greater than those in the tumor margin in both primary tumor and liver metastasis (P<0.001 to 0.006). CEA expression scores were closely associated with E-cadherin expression scores on the corresponding tumor site (P<0.001 to 0.017). CEA and E-cadherin mRNA expression was greatest in the hepatocytes adjacent to liver metastasis, next greatest in the primary tumor, and least in the liver metastasis (P<0.001 to 0.002). CEA mRNA expression was also closely correlated with E-cadherin mRNA expression in the primary tumor (P<0.001) and in the adjacent hepatocytes of the liver metastasis (P=0.018). Patients with a lesser CEA expression score in the liver metastasis margin appeared to have a longer disease-free survival period than did those with a greater CEA expression score. Expression of CEA and E-cadherin was closely correlated with the mRNA levels. Furthermore, these correlations may be implicated in the tumor progression of colorectal carcinoma considering their biological properties.