Impact of oral function on regaining oral intake and adjusting diet forms for acute stroke patients.

BACKGROUND: Outcome prediction for dysphagia recovery is essential for rehabilitation treatment planning. Several studies have reported various predictors for resuming oral feeding after post-stroke dysphagia. However, evidence on oral health and function, a crucial part of feeding, has rarely been reported. Therefore, the goal of this study was to identify the oral status-related factors that could predict oral intake resumption in acute stroke patients. METHODS: 80 acute stroke patients with dysphagia were included. Clinical data, including the changes of general condition, oral and swallowing functions, were collected once a week until discharge. Patients were divided into two groups based on the outcome of the food intake level scale at discharge, and data were compared between the groups. RESULTS: 60 patients had regained complete oral intake before discharge. Multiple logistic regression showed that posterior tongue pressure could significantly predict complete oral intake recovery. Tongue pressure and modified water swallowing test score also significantly influenced diet forms. In addition, Spearman correlation analysis showed that improvement of other oral status-related factors, such as oral moisture and dentition status, also indicated the improvement of diet forms and swallowing function during the hospital stay. CONCLUSION: Tongue pressure measurement could be a useful oral status-related indicator for predicting complete oral intake and adjusting diet forms for acute stroke patients during hospitalization. Acute stroke patients should receive proper oral status evaluation and implementation to enhance functional recovery.

Biocompatible Polymers Modified with d-Octaarginine as an Absorption Enhancer for Nasal Peptide Delivery.

Peptide and protein drugs, which are categorized as biologics, exhibit poor membrane permeability. This pharmacokinetic disadvantage has largely restricted the development of noninvasive dosage forms of biologics that deliver into systemic circulation. We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel absorption enhancer to overcome this challenge. Since our previous study revealed that biocompatible poly( N-vinylacetamide- co-acrylic acid) modified with d-octaarginine, a typical cell-penetrating peptide, enhanced in vitro permeation of biomolecules such as plasmid DNA and bovine serum albumin through cell membranes, the present study evaluated whether the polymers enhanced in vivo absorption of biologics applied on the mucosa. Mouse experiments demonstrated that d-octaarginine-linked polymers drastically enhanced nasal absorption of exendin-4, whose injection is clinically used. The mean bioavailability was 20% relative to subcutaneous administration, even though it fell short of 1% when exendin-4 alone was administered nasally. The absorption-enhancing function of the polymers was superior to that of sodium caprate and sodium N-(8-(2-hydroxybenzoyl)amino) caprylate, which have been used for humans as an absorption enhancer. In vitro experiments using several biologics with different characteristics revealed that biologics interacted with d-octaarginine-linked polymers and were taken up into cells when incubated with the polymers. The interaction and cellular uptake were enhanced as molecular weights of the biologics increased; however, their charge-dependent in vitro performance was not clearly observed. The current data suggested that biologics formulated with our polymers became an alternative to their conventional invasive parenteral formulations.

Evaluation of serum carbohydrate-deficient transferrin by HPLC and MALDI-TOF MS.

BACKGROUND: The percentage of carbohydrate-deficient transferrin (%CDT) in serum is a marker of habitual alcohol intake that can be determined by antibody detection of abnormal disialo sugar chains at D432 and D630. However, this approach lacks specificity for alcoholic liver disease. To decrease the false-positive rate in patients with non-alcoholic liver diseases, we developed a screening method using the disialo sugar chain at D630 alone. METHODS: Serum was obtained from 12 patients with alcoholic liver disease, 12 with type C chronic liver disease, 6 with non-alcoholic steatohepatitis (NASH), and 12 healthy non-alcohol drinkers. Transferrin with two sialic acids (disialotransferrin) was fractionated from serum using HPLC, digested with trypsin, and evaluated using MALDI-TOF MS. RESULTS: An abnormal sugar chain at D630 of transferrin was not detected in healthy subjects or in patients with chronic liver disease or NASH, but was detected in 9 patients (75%) with alcoholic liver disease. Positive results were found in 3 samples that were negative using an N-Latex CDT kit and in one sample negative for γ-glutamylaminotransferase and CDT. CONCLUSIONS: Detection of CDT by HPLC/MALDI-TOF MS based on an abnormal sugar chain at D630 may permit identification of habitual alcohol drinkers when used in combination with current markers.

Overexpression of hydroxymethylglutaryl CoA synthase 2 and 2,4-dienoyl-CoA reductase in rat pancreas following chronic alcohol consumption.

OBJECTIVES: The mechanism of alcohol-induced pancreatic damage is unclear. The aim of this study was to clarify the effects of chronic alcohol intake on the pancreatic proteome. METHODS: Rats were fed an alcohol-containing Lieber-DeCarli liquid diet, and the pancreatic proteome was compared with that of pair-fed control rats using agarose 2-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. RESULTS: The expression of 3 proteins was consistently altered in alcohol-fed rats: 1 protein was down-regulated, and 2 proteins were up-regulated. The 2 up-regulated proteins were identified as 2,4-dienoyl-CoA reductase and hydroxymethylglutaryl-CoA synthase (HMGCS2). The combined concentration of malondialdehyde and 4-hydroxyalkenals was significantly greater in alcohol-fed rats. It is noteworthy that the reactivity of anti-4-hydroxy-2-nonenal antibody was significantly higher toward HMGCS2 isolated from alcohol-fed rats. The activity of HMGCS2 was higher in alcohol-fed rats, but the relative increase in enzyme activity in alcohol-fed rats was less than the relative increase in HMGCS2 expression. CONCLUSIONS: Chronic alcohol consumption results in distinct alterations in the expression of 3 pancreatic proteins. The reactivity of 4-hydroxy-2-nonenal toward one of the up-regulated proteins, HMGCS2, increased markedly following chronic alcohol intake, suggesting that up-regulation of HMGCS2 is connected with alterations of lipid peroxidation induced by alcohol.

Combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats.

BACKGROUND: Proteomic approaches may provide new insights into pathological conditions associated with alcoholism. The aim of this study was to conduct a proteomic analysis of liver tissue and serum in chronically alcohol-fed rats using agarose 2-dimensional gel electrophoresis (2-DE) and 3-step serum proteome analysis. METHODS: A total of 12 rats were pair-fed nutritionally adequate liquid diet containing ethanol as 36% of the total energy or an isocaloric control diet for 2 months. Rat liver homogenates and cytosol fractions were subjected to agarose 2-DE. Serum samples were subjected to 3-step serum proteome analysis involving immunodepletion of abundant proteins followed by fractionation using reverse-phase high-performance liquid chromatography and 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Candidate proteins were digested with trypsin and identified using mass spectrometry. Observed differences in protein expression levels were confirmed using Western blotting. RESULTS: A total of 46 protein spots were found to be differentially expressed in the liver homogenates and cytosol fractions of alcohol-fed rats relative to pair-fed controls. The most notable change was down-regulation of a 29-kDa protein, which was subsequently identified as carbonic anhydrase III (CA III). Down-regulation of this protein in alcohol-fed rats was confirmed by Western blotting. The messenger RNA level of CA III was decreased as well. In rat serum, a total of 41 proteins were differentially expressed. Of these proteins, only betaine-homocysteine methyltransferase (BHMT) was also found to be differentially expressed in the liver. CONCLUSIONS: A combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats revealed that the expression of CA III is significantly down-regulated in the liver of alcohol-fed rats. Our results also showed that BHMT expression is up-regulated in both the liver and serum of alcohol-fed rats.

The measurement of a fibrinogen α C-chain 5.9 kDa fragment (FIC 5.9) using MALDI-TOF MS and a stable isotope-labeled peptide standard dilution.

BACKGROUND: We previously identified a 5.9 kDa peptide fragment of fibrinogen α C-chain (FIC 5.9) as a novel biomarker candidate for heavy drinking. In an effort to improve FIC 5.9 measurement for potential use in clinical diagnostics, we combined the ClinProt System and a stable isotope-labeled peptide standard dilution as a simple and reproducible system for measuring FIC 5.9. METHODS: We analyzed 104 serum samples that were obtained from patients with alcohol dependency, from patients with chronic hepatitis C, and from healthy volunteers. Serum FIC 5.9 levels were measured using the ClinProt system with and without a stable isotope-labeled synthetic FIC 5.9 as an internal standard. RESULTS: The within-day and between-day CVs were significantly smaller with stable isotope dilution mass spectrometry (SID-MS) than with conventional MALDI-TOF MS. Of the two different MALDI-TOF MS platforms, we obtained concordant results with SID-MS. Furthermore, only SID-MS detected a small but significant difference between the serum FIC 5.9 levels in the chronic hepatitis C group and the controls. CONCLUSIONS: MALDI-TOF MS with a stable isotope-labeled peptide spike can determine serum FIC 5.9 levels more precisely than conventional MS. This will make inter-laboratory FIC 5.9 comparisons possible.

Effect of various forms of the C1 esterase inhibitor (C1-INH) and DAF on complement mediated xenogeneic cell lysis.

The purpose of the present study was to assess the effect of various forms of the surface-bound form of the C1 esterase inhibitor (C1-INH-PI) and decay accelerating factor (DAF) on xenogenic cells. cDNAs of various deletion mutants of the C1-INH-PI, such as delta-1-99 amino acid (AA), delta-108-183AA loop, delta-whole loop, delta-exon5, delta-exon6 + 7, and delta-exon5 + 6 + 7, and that of DAF, the delta-short consensus repeat (SCR) 1-DAF were established. While all deletion mutants of C1-INH-PI except the delta-1-99AA were expressed in the cytoplasm but not on the cell surface, the delta-1-99AA was clearly expressed on the xenogeneic cell surface. Amelioration of complement-mediated xenogeneic cell lysis by delta-1-99AA was next tested, and compared with delta-SCR1 DAF. Both molecules blocked human complement-mediated cell lysis by approximately 57 to 90 and 93 to 98%, respectively, in Chinese hamster ovarian tumor (CHO) cells and pig endothelial cells (PECs). The CHO cell transfectants were incubated with 20% normal human serum, and the amounts of C4 and C3 deposition on the cell surface were analysed by flow cytometry. The DAF transfectant showed a large amount of C4-deposition and much less C3-deposition than the controls (approximately 85% suppression), whereas the delta-1-99AA showed approximately a 40% suppression in both C4- and C3-deposition. Consequently, both the delta-1-99AA C1-INH-PI and delta-SCR1 DAF molecules are quite effective in down-regulating the xenogeneic cell lysis, but accomplished this in different manners.

Modulation of the leader peptide sequence of the HLA-E gene up-regulates its expression and down-regulates natural killer cell-mediated swine endothelial cell lysis.

BACKGROUND: The inhibitory function of HLA class I molecules, HLA-G1 and HLA-E, on natural killer (NK) cell-mediated cytolysis has previously been reported. In this study, we report on a study of the effects of the co-expression of these molecules on the inhibition of NK cell-mediated cytolysis, using a newly constructed gene. METHODS: Complementary DNA (cDNA) of HLA-G (G1 and G3), HLA-E, and human beta2-microglobulin (hbeta2m) were prepared and transfected into swine endothelial cell (SEC) and Chinese hamster ovarian tumor (CHO) cell. The leader peptide sequences of HLA-G1 and HLA -E genes were changed to VMAPRTLFL or VMAPRTLVL, which corresponds to the original HLA-G1 and HLA-A2. The cell surface expression of the modified genes was evaluated by flow cytometry, and NK cell-mediated cytolysis by human peripheral blood mononuclear cells (PBMC) was assessed. RESULTS: The transfectant with the hbeta2m and HLA-G1 genes showed a clear expression of the HLA-G1 molecule and had an inhibitory effect on NK cell-mediated SEC lysis. Whereas neither the transfectant with the hbeta2m and HLA-E genes, nor that with the hbeta2m and HLA-G3 genes, expressed the HLA molecule on SEC, the transfectant with triple genes, hbeta2m, HLA-E, and HLA-G3, expressed the HLA-E molecule and also inhibited NK-mediated SEC lysis. Conversely, the modification of the leader sequence of the HLA-E gene successfully induced the expression of the HLA-E molecule on the SEC surface. Furthermore, the transfectant expressed both HLA-G1 and HLA-E molecules, thus efficiently enhancing the inhibition of NK-mediated SEC lysis. CONCLUSION: The co-expression of HLA-G1 and HLA-E molecules with the modified genes has potential for use in preventing xenograft rejection, as mediated by human NK cells.

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter.

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.