Pig Red Blood Cells Research Articles

Effect of dietary boron on immune function in growing beef steers*

Thirty-six Angus and Angus x Simmental cross steers (initial BW 269.5 +/- 22.3 kg) were used to determine the effects of dietary boron (B) on performance and immune function. Steers were fed on one of the three dietary treatments: (i) control (no supplemental B; 7.2 mg B/kg DM), (ii) 5 mg supplemental B/kg DM and (iii) 50 mg supplemental B/kg DM, from sodium borate for 78 days. Supplementation of dietary B had no effect on body weight (BW) gain, feed intake or gain:feed during the study. Jugular blood samples were collected prior to feeding on days 28, 63 and 77 for plasma-B analysis. Supplementation of dietary B increased (p < 0.001) plasma B-concentration in a dose-responsive manner. Furthermore, plasma B-concentration was correlated (p < 0.001; R(2) = 0. 95) to daily B-intake (mg B/day). Jugular blood was also collected, from an equal number of steers from each treatment, on day 42 or 44 for determination of in vitro production of interferon-gamma and tumour necrosis factor-alpha from isolated monocytes and assessment of lymphocyte proliferation. Dietary B did not affect T- or B-lymphocyte proliferation or in vitro cytokine production from monocytes. On day 49 of the study, the humoral immune response was assessed by i.m. injection of a 25% pig red blood cell (PRBC) solution for determination of anti-PRBC IgG and IgM titre responses. Boron-supplemented steers had greater (p = 0.035) anti-PRBC IgG titres than controls on day 7 but not on day 14 or 21 post-injection. Anti-PRBC IgM titres did not differ throughout the sampling period. Results from this study indicate that supplemental B had minimal effects on immune function and did not affect performance of growing steers.

Comparison of human and guinea pig acetylcholinesterase sequences and rates of oxime-assisted reactivation

Poisoning via organophosphorus (OP) nerve agents occurs when the OP binds and inhibits the enzyme acetylcholinesterase (AChE). This enzyme is responsible for the metabolism of the neurotransmitter acetylcholine (ACh) which transmits signals between nerves and several key somatic regions. When AChE is inhibited, the signal initiated by ACh is not properly terminated. Excessive levels of ACh result in a cholinergic crisis, and in severe cases can lead to death. Current treatments for OP poisoning involve the administration of atropine, which blocks ACh receptors, and oximes, which reactivate AChE after inhibition. Efforts to improve the safety, efficacy, and broad spectrum utility of these treatments are ongoing and usually require the use of appropriate animal model systems. For OP poisoning, the guinea pig ( Cavia porcellus) is a commonly used animal model because guinea pigs more closely mirror primate susceptibility to OP poisoning than do other animals such as rats and mice. This is most likely because among rodents and other small mammals, guinea pigs have a very low relative concentration of serum carboxylesterase, an enzyme known to bind OPs in vitro and to act as an endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE is substantially more resistant to oxime-mediated reactivation than human AChE. To examine the molecular basis for this difference, we reverse transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of guinea pig AChE were then compared to the human version. Several amino acid differences were noted, and the predicted locations of these differences were mapped onto a structural model of human AChE. To examine directly how these differences affect oxime-mediated reactivation of AChE after inhibition by OPs, human and guinea pig red blood cell ghosts were prepared and used as sources of AChE, and the relative capacity of several different oximes to reactivate each OP-inhibited AChE were determined. The differences we report between human and guinea pig AChE raise additional concerns about the suitability of the guinea pig as an appropriate small animal model to approximate human responses to OP poisoning and therapies.

Brief report: a new profile of terminal N-acetyllactosamines glycans on pig red blood cells and different expression of α-galactose on Sika deer red blood cells and nucleated cells

It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galbeta1-4GlcNAc-R) and alpha-Gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galbeta1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack alpha-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that alpha-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-alpha-Gal epitopes. Whereas, the expression of the alpha-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution.

5,6,3′,4′-Tetrahydroxy-7-methoxyflavone as a Novel Potential Proteasome Inhibitor

Proteasome inhibition is one of the new pharmaceutical approaches to study antitumor activity. Although the active components are not yet identified for either treating or preventing cancer, the low toxicity plant Anisomeles ovata R. Br. has been used in traditional herbal medicine for more than 1000 years. In this study, the methanol extract from Anisomeles ovata showed potent inhibition of proteasome activity. Twenty compounds, two macrocycylic diterpenoids, six aromatics, seven flavonoids, and five phenylethanoids were isolated from Fang Feng Cao the dried aerial parts of A. ovata. Their structures have been established on the basis of spectral evidence. Using a proteolysis assay for inhibition of 26S proteasome from pig red blood cells, we found that 5,6,3',4'-tetrahydroxy-7-methoxyflavone inhibited 90.5 %, 85.4 % and 73.1 % the chymotrypsin-like, caspase-like and trypsin-like activities of 26S proteasome with IC (50) values of 14.0, 5.4 and 24.1 microM, respectively, when Suc-LLVY-AMC, Z-LLE-AMC and Ac-RLR-AMC were used as substrates. 5,6,3',4'-Tetrahydroxy-7-methoxyflavone had a higher inhibitory at 15 minutes. A combination of 5,6,3',4'-tetrahydroxy-7-methoxyflavone and 5,6,4'-trihydroxy-7,3'-dimethoxyflavone increased the inhibition ability on 26S enzymatic activity. This combination appears to be a potentially attractive chemotherapy approach. We have found that 5,6,3',4'-tetrahydroxy-7-methoxyflavone has the highest inhibitory effects on 26S proteasome activities when compared to the other 11 flavonoids. These results suggest that both the 6-hydroxy and 7-methoxy positions of the flavone may play an important role in targeting 26S proteasome activity.

Significant neutralizing activity of human immunoglobulin preparations against pandemic 2009 H1N1

The influenza-like illness that began in the Unites States and Mexico was first reported by the World Health Organization (WHO) on 24 April, 2009, and declared a phase 6 pandemic on 11 June. As of 6 July 2009, over 90 000 cases and more than 400 deaths in some 120 countries had been confirmed (WHO, 2009). Importantly, on July 8th, the WHO announced that oseltamivir (Tamiflu)-resistant viruses had been identified in Denmark, Japan and Hong Kong (WHO, 2009). The pandemic virus 2009 H1N1 was a triple reassortant of human-, swine- and avian-derived influenza A virus segments and the HA gene was classified as being of swine-origin (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, (2009). Evidence is accumulating that specific IgG antibodies against this virus are present in certain populations, especially the elderly (Itoh et al, 2009). However, Katz et al (2009) reported that cross reactive IgG against a pandemic influenza virus (A/California/04/2009) was found in no serum specimens of children aged 6 months–9 years old, 8% of samples from 5- to 9-year olds, 9% of samples from 18- to 64-year olds, 6% of samples of 18- to 40-year olds and 33% of samples of those over 60 years old, suggesting that immunoglobulin preparations derived from pooled plasma from over 10 000 healthy donors could contain such cross reactive IgG. The present study evaluated haemagglutinin-inhibition (HI) and virus neutralization (VN) activities against 2009 H1N1 and seasonal H1N1 as a positive control in intravenous human immunoglobulin (IVIG) preparations manufactured in 1999 and 2008. An influenza A/H1N1 vaccine strain (A/New Caledonia/20/99), a clinical isolate of A/H1N1 (A/Osaka/16/2008), a classical swine isolate of A/H1N1 (A/Swine/Hokkaido/2/1981) and a pandemic influenza isolate of A/H1N1 (A/Osaka/168/2009 H1N1 pdm) were used in this study. Three lots (Lot. A, B and C) of IVIG derived from pooled plasma collected in Japan and manufactured in 2008 (IVIG2008JP, ‘Kenketsu Venoglobulin®-IH Yoshitomi; Benesis Corp., Osaka, Japan’) were also used. In addition, two lots of IVIG that were manufactured in 1999, derived from plasma pooled collected in Japan and the USA (IVIG1999JP ‘Kenketsu Venoglobulin®-IH’, IVIG1999US ‘Venoglobulin®-IH; Yoshitomi Pharmaceutical Industries, Ltd. at the time, currently Benesis Corp.’), were used. The viruses were propagated in Madin-Darby canine kidney (MDCK) cells or in the allantoic cavity of chicken embryonated eggs. The culture media and the allantoic fluids were stored at −80°C prior to use. Infectivity, as infectious focus-forming units (FFU) per ml, was titrated in MDCK cells using peroxidase and an anti-peroxidase (PAP) staining technique (Okuno et al, 1990). The haemagglutinin-inhibition (HI) test using 0·75% guinea pig red blood cells was carried out as described previously (Okuno et al, 1993). The results were expressed as the reciprocal of the highest dilution of the culture medium to show inhibition. The virus neutralization (VN) test was carried out as described (Okuno et al, 1990). Briefly, IVIG was diluted twofold with serum-free medium. The diluted IVIG (50 μl) was mixed with 100 FFU (50 μl) of virus, then applied to MDCK cells in a 96-well microplate. After culturing, the cells were fixed with ethanol and stained by PAP as above. The results were expressed as the reciprocal of the dilution giving 50% neutralization. Intravenous human immunoglobulins were manufactured using plasma pooled from over 10 000 healthy donors. The HI and VN activities of IVIGs were titrated against pandemic, seasonal human and swine influenza A viruses (Table I). Of note, both the 1999 and 2008 IVIGs were shown to have anti pandemic and classical swine influenza A/H1N1 virus titres with HI (×4–×8) and VN (×32–×64). The 2008 IVIGs showed titres against the vaccine strain A/New Caledonia/20/99, which was isolated in 1999, with HI (×160–×320) and VN (×640–×1280), while the 1999 IVIGs showed titres with HI (×10–×40) and VN (×32–×128). These results suggested that the IVIG derived from the pooled plasma contained a certain amount of functional IgG, including IgG against pandemic or classical swine influenza A/H1N1. Of note, such IgG titres were slightly higher in the IVIG2008JP products compared with IVIG1999JP. However, the titres were slightly higher in IVIG1999US than in IVIG1999JP. Higher titres against the vaccine and clinical strains were observed in IVIG1999US than IVIG1999JP. Interestingly, the difference in the increase in titres against the vaccine strain was much greater between the products manufactured in 2008 and 1999 than between the others. This difference seems to be an outcome of vaccination. Our preliminary results showed a HI titre >×40 in 1·2% (7/580), ×20 in 3·1% (18/580) and ×10 in 4·3% (25/580), indicating the possible production of hyperimmune globulin with these sources of plasma collected in 2008, Japan. Table I Cross reactivity of several lots of IVIG against pandemic 2009, classical swine and seasonal H1N1 viruses.

English

This study determined the haemolytic, haemagglutinating and antibiotic susceptibility activities of Plesiomonas shigelloides isolated from stool samples of patients attending different health centers in the Venda region of South Africa. P.shigelloides was isolated and identified using the API 20E, API 20NE systems. Antibiotic susceptibility profiles of the isolates were determined using the disc diffusion method and analyzed according to NCCLS standards. The hemolytic and hemagglutination activities of the isolates on human, sheep, pig and chicken red blood cells were determined using the plate and slide methods. A total of 89 (13%) P. shigelloide were isolated from 660 samples. The hemolytic activities of the isolates were variable with no heamolysis on sheep red cells. 33 (37%) of isolates were beta lactamase producers. There was a high level of resistance to the penicilllins with 100% resistance to Penicillin G, Amoxicillin and Ampicillin. This study has demonstrated multiple resistance to different antibiotics and production of beta lactamase. Most of the isolates showed evidence of pathogenicity as demonstrated by hemolytic and haemagglutinating activities.

Inhibitory Effect of Flavonoids on 26S Proteasome Activity

Inhibiting proteasomal degradation has been shown to induce apoptosis in tumor cells. Utilization of proteasome inhibition is therefore one approach to anticancer therapy. Some of the flavonoids can induce cell apoptosis via inhibiting proteasome 26S activity. In this study, the inhibition of 26S proteasome from pig red blood cells was analyzed on 12 flavones, 5 flavanones, and 9 isoflavones by using a proteolysis assay. Several flavonoids such as apigenin-6-hydroxy-7-O-beta-D-glucoside, quercetin, rutin, 6-hydroxyapigenin, 5,6,4'-trihydroxy-7,3'-dimethoxyflavone, 5,6,3',4'-tetrahydroxy-7-methoxyflavone, glycitecin, and 6,7,4'-trihydroxyisoflavone inhibited the chymotrypsin-like, caspase-like, or trypsin-like activity of 26S proteasome when Suc-LLVY-AMC, Z-LLE-AMC, and Ac-RLR-AMC were used as substrates. Three peptidase activities of flavonoids were found to be significantly correlated with one another. Flavones had significantly stronger inhibitory effects on chymotrypsin-like and caspase-like activities than flavanones and isoflavones. 5,6,3',4'-Tetrahydroxy-7-methoxyflavone, 5,6,4'-trihydroxy-7,3'-dimethoxyflavone, and quercetin displayed a mixed type inhibition of 26S by Lineweaver-Burk plots analysis. Furthermore, 5,6,3',4'-tetrahydroxy-7-methoxyflavone is found to have a higher inhibitory effect on 26S proteasome activities and is the only flavonoid to inhibit all three peptidase activities, whereas the inhibition of flavonoids was not affected by ubiquitin-induced stimulation of the three peptidase activities of 26S proteasome; 5,6,3',4'-tetrahydroxy-7-methoxyflavone inhibited 75% casein degradation. These results suggest that both the 6-hydroxy and 7-methoxy positions of the flavone may play an important role in targeting 26S activity.

Genetically engineered pig red blood cells for clinical transfusion: initial in vitro studies

Pigs are a potential source of red blood cells (RBCs) and could resolve the shortage of human blood for transfusion. This study investigated in vitro the compatibility of genetically engineered pig RBCs (pRBCs) with the human innate immune response. Human volunteers of all ABO blood types were sources of sera and those of O blood type were sources of circulating monocytes/macrophages. RBCs from ABO-compatible (ABO-C) and ABO-incompatible (ABO-I) humans and wild-type (WT) and alpha-1,3-galactosyltransferase gene-knockout (GTKO) pigs were tested for hemagglutination, immunoglobulin (Ig)M/IgG antibody binding, and complement-dependent cytotoxicity (CDC) using human sera. Phagocytosis of RBCs by human monocyte-derived macrophages was measured by coculture in the absence or presence of pooled human O serum. RBCs showed significant differences (p < 0.01) with regard to hemagglutination, IgM and IgG binding, and CDC (ABO-C < GTKO < ABO-I < WT). In the absence of pooled human O serum (antibodies), there was no phagocytosis of any RBCs; in the presence of serum (antibodies), phagocytosis of ABO-I RBCs was greater than of WT (p < 0.01), which in turn was greater than of GTKO RBCs (p < 0.05). GTKO RBCs were significantly more compatible than ABO-I and WT RBCs, but were not comparable to ABO-C combinations. In the presence of antibody, human monocyte-derived macrophages phagocytosed ABO-I RBC/sera combinations more efficiently than pRBCs. These observations contribute to our ultimate goal of using genetically engineered pRBCs for clinical blood transfusion. However, pigs will require other modifications or manipulations if they are to become suitable for human transfusion.

In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds

Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.

CD1d-Mediated Interaction Between Activated T Cells and B Cells Is Essential to B-Cell Proliferation and Anti-α-Gal Antibody Production

Antibody-mediated rejection is central to ABO-incompatible transplantation as well as to xenotransplantation. The carbohydrate structure of xenoantigen α-Gal is highly analogous to the human blood group antigens. Both require memory B-cell activation for antibody production. We hypothesized that B cells, reactive to the α-Gal xenoantigen, required the presence of fully activated T cells to survive and proliferate in vitro. This hypothesis was contrary to the traditional theory that the response of B cells to carbohydrate antigens is T cell independent (Wong and Arsequell: Immunobiology of Carbohydrates. New York: Kluwer; 2003). When we compared the capacity of B cells to proliferate, we observed that activated T cells were necessary for B-cell proliferation. However, this proliferation was independent of the presence of antigen. A relevant question was also to investigate the role of the specific class of T cells: the CD1d-restricted iNKT (iNKT) cells in the activation of α-Gal-reactive B cells. The iNKT cells are reactive to glycolipids and capable of producing both Th1 and Th2 cytokine responses. We therefore wanted to determine the role of the iNKT cells as mediators of a Th2-type response when B cells were exposed to a glycolipid antigen extracted from pig red blood cells, which express blockade of the α-Gal epitope. We observed that the interaction between B cells and iNKT cells prevents B-cell proliferation and anti-α-Gal antibody production.

Assessment of antioxidant activity of natural extracts

The aim of this study was investigate and compare the antioxidant activity of extracts from Labiatae and Oleaceae cell cultures and a natural Verbenaceae extract on pig whole blood. The antioxidant activitiy was assessed using the KRL biological test. Extract obtained from Ajuga reptans (Labiatae) cell cultures and titrated at 50% of phenylpropanoids expressed as teupolioside (T), extract obtained from Siringa vulgaris (Oleaceae) cell cultures and titrated at 50% of phenylpropanoids expressed as verbascoside (V), V and T 50/50 mixture and a natural Verbenaceae extract (NE) titrated at 5% of phenylpropanoids, expressed as verbascoside, were tested for their ability to protects pig red blood cells from 2,2’azobis (2-amidinopropane) dihydrochloride (AAPH) induced hemolysis. The protective effect of the compounds was dose-dependent at concentrations of 0.1 to 1g/L. The active principle in the NE shows an antioxidant activity 2.27 times higher (P<0.01) than the cell culture extract V and 5.11 times higher (P<0.01) than the cell culture extract T. Taking into account the total phenylpropanoids content of cell cultures and natural extract, these results clearly indicate a greater antioxidant activity for the natural Verbaneceae extract.

Oxygen consumption and blood flow distribution in perfused skeletal muscle of chinook salmon

An isolated, perfused salmon tail preparation showed oxyconformance at low oxygen delivery rates. Addition of pig red blood cells to the perfusing solution at a haematocrit of 5 or 10% allowed the tail tissues to oxyregulate. Below ca. 60 ml O(2) kg(-1) h(-1) of oxygen delivery (DO(2)), VO(2) was delivery dependent. Above this value additional oxygen delivery did not increase VO(2) of resting muscle above ca. 35 ml O(2) kg(-1) h(-1). Following electrical stimulation, VO(2) increased to ca. 65 ml O(2) kg(-1) h(-1), with a critical DO(2) of ca. 150 ml O(2) kg(-1) h(-1). Dorsal aortic pressure fell to 69% of the pre-stimulation value after 5 min of stimulation and to 54% after 10 min. Microspheres were used to determine blood flow distribution (BFD) to red (RM) and white muscle (WM) within the perfused myotome. Mass specific BFD ratio at rest was found to be 4.03 +/- 0.49 (RM:WM). After 5 min of electrical stimulation the ratio did not change. Perfusion with saline containing the tetrazolium salt 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) revealed significantly more mitochondrial activity in RM. Formazan production from MTT was directly proportional to time of perfusion in both red and WM. The mitochondrial activity ratio (RM:WM) did not change over 90 min of perfusion.

Role of Ginsenoside Rd in Inhibiting 26S Proteasome Activity

Drugs targeting 26S proteasome as antitumor agents are considered to be important for cancer therapy. Although the active components are yet to be identified, for more than 1000 years, the low-toxicity Panax ginseng has been used in traditional herbal medicine for either treating or preventing cancer. Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 are distinct components that can be isolated from P. ginseng C.A. Meyer. In this study 26S proteasome was purified from pig red blood cells, and the activity of the seven isolated ginsenosides was analyzed by proteolysis assay. It was found that ginsenoside Rd inhibited 52.9% the chymotrypsin-like activity of 26S proteasome with an IC(50) value of 107.5 microM when Suc-LLVY-AMC was used as a substrate. Ginsenoside Rd displayed a mixed type inhibition of 26S proteasome when analyzed by Lineweaver-Burk plots of the inhibition kinetics. Unlike ginsenoside Rd, the other ginsenosides showed low inhibitory effect of the chymotrypsin-like activity of 26S proteasome. Seven ginsenosides did not inhibit the trypsin-like and caspase-like activities of 26S when Ac-RLR-AMC or Z-LLE-AMC was used as substrate. These results suggest that ginsenoside Rd is a potential drug for cancer prevention due to its specific 26S proteasome inhibitory effect and known low toxicity. Furthermore, both 3-O-Glc(2)-Glc and 20-O-beta-Glc positions of the ginsenoside may play a role in the inhibitory property of the chymotrypsin-like activity in 26S proteasome.

MDCK-SIAT1 Cells Show Improved Isolation Rates for Recent Human Influenza Viruses Compared to Conventional MDCK Cells

The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. The suitability of MDCK-SIAT1 cells, engineered to express increased levels of alpha-2,6-linked sialic acid receptors, as an alternative to conventional MDCK cells for isolation of circulating influenza virus was assessed. A greater number of influenza A (H1N1 and H3N2) and B viruses from stored human clinical specimens collected between 2005 and 2007 were isolated following inoculation in MDCK-SIAT1 cells than in MDCK cells. In addition, a higher titer of virus was recovered following culture in MDCK-SIAT1 cells. All A(H1N1) viruses recovered from MDCK-SIAT1 cells were able to agglutinate both turkey and guinea pig red blood cells (RBC), while half of the A(H3N2) viruses recovered after passage in MDCK-SIAT1 cells lost the ability to agglutinate turkey RBC. Importantly, the HA-1 domain of the hemagglutinin gene was genetically stable after passaging in MDCK-SIAT1 cells, a feature not always seen following MDCK cell or embryonated chicken egg passage of human influenza virus. These data indicate that the MDCK-SIAT1 cell line is superior to conventional MDCK cells for isolation of human influenza virus from clinical specimens and may be used routinely for the isolation and propagation of current human influenza viruses for surveillance, diagnostic, and research purposes.

Prevalence, pathogenesis, antibiotic susceptibility profiles, and in-vitro activity of selected medicinal plants against Aeromonas isolates from stool samples of patients in the Venda region of South Africa.

The prevalence, pathogenic indices, such as haemolytic and haemagglutinating activities, antibiograms, and in-vitro activities of local medicinal plants against Aeromonas isolates in Vhembe district of Limpopo province, South Africa, were studied using standard microbiological methods. In total, 309 diarrhoeic stool samples were collected from patients attending five health centres in the region during December 2004-May 2005. Aeromonas species were identified using the API 20E system. The haemagglutinating and haemolytic activities of isolates on human, sheep, pig and chicken red blood cells were investigated. Antibiotic susceptibility profiles of the isolates to several antibiotics and in-vitro activity of local medicinal plants were also ascertained using previously-reported schemes. Results showed that 104 (33.6%) of the 309 samples were positive for Aeromonas species, of which 89 (85.6%) were Aeromonas hydrophila, 12 (11.5%) A. sobria, and three (2.9%) A. caviae. All strains of A. hydrophila and A. caviae produced haemolysis on sheep blood, while eight of the 12 A. sobria strains were haemolytic on sheep blood. The haemolytic activities of the isolates were variable on other red blood cells tested. High level of resistance was observed to amoxicillin and ampicillin, followed by cefuroxime (79%), chloramphenicol (74%), and erythromycin (65%). The carbapenems were the most active drugs with only 7% resistance to meropenem and 11% to imipenem. About 12% of the isolates were resistant to ciprofloxacin. The extracts of three of seven medicinal plants tested showed inhibitory activity against all Aeromonas isolates; these included acetone and hexane extracts of Pterocarpus angolensis, Syzygium cordatum, and Zornia milneana. The results suggest a high prevalence of Aeromonas species in the region. The isolates demonstrated multiple resistant profiles to different antibiotics tested. Some local medicinal plants were inhibitory to Aeromonas isolates, indicating a potential role in the management of Aeromonas-related infections. Structural elucidation of the active components may pave the way for the discovery of candidate templates for eventual drug design. Most isolates possessed important virulence characteristics based on their haemolytic and haemagglutinating ability. However, the genetic characterization of the isolates will further confirm their pathogenicity and the origin of multiple antibiotic resistance.

Effects of Trace Mineral Supplementation, Source, and Growth Implants on Immune Response of Growing and Finishing Feedlot Steers

<h2>ABSTRACT</h2> To determine the effects of growth implants and trace mineral (TM) supplementation and source on immune response, 375 steers (approximately 7 mo of age and 250.0 ± 11.0 kg), stratified by initial body weight, were randomly assigned to 1 of 36 pens that were then randomly assigned to treatments. Treatments consisted of 1) control (no supplemental Cu, Zn, Mn, and Co), 2) inorganic TM (CuSO₄, ZnSO₄, MnSO₄, and CoCO₃), and 3) organic TM (iso-amounts of organic Cu, Zn, Mn, and Co). On d 28 post-arrival to the feedlot, steers from 6 pens per treatment received a growth implant containing 200mg progesterone and 20mg estradiol benzoate, whereas the other half of the steers within the same treatment (6 pens) did not receive a growth implant. Steers were fed a corn silage-based growing diet for 56 d and were then gradually switched toa high-concentrate finishing diet. At the beginning of the finishing phase, only steers receiving growth implants during the growing phase were reimplanted with 80mg of trenbolone acetate and 16mg of estradiol. During the finishing phase, all minerals excluding Zn were fed at NRC recommended concentrations in inorganic form. Treatments during the finishing phase consisted of 1) control (no supplemental Zn), 2) inorganic Zn (30mg Zn/kg DM from ZnSO₄), and 3) organic Zn (iso-amounts of organic Zn). At the end of the growing phase, steers receiving organic TM had greater (P < 0.02) total IgM concentrations than inorganic-supplemented steers, and implanted steers tended (P < 0.07) to have greater IgG antibody titer concentrations specific for pig red blood cells than nonimplanted steers. During the finishing phase, steers supplemented with organic Zn had greater (P = 0.04) ovalbumin antibody titer concentrations than steers supplemented with inorganic TM.Superoxide dismutase activity, interferon gamma concentrations, and antibody titers specific to pig red blood cells and infectious bovine rhinotracheitis virus were unaffectedby TM treatment or growth implants.

A Novel Antiproliferative and Antifungal Lectin from Amaranthus viridis Linn Seeds

A lectin from the seeds of Amaranthus viridis Linn has been purified by affinity chromatography on asialofetuin-linked amino activated silica. Amaranthus viridis lectin (AVL) has a native molecular mass of 67 kDa. It is a homodimer composed of two 36.6 kDa subunits. The lectin gave a single band in non-denaturing PAGE at pH 4.5 and pH 8.3 and a single peak on HPLC size exclusion and cation exchange columns. The purified lectin was specific for both T-antigen and N-acetyl-D-lactosamine, markers for various carcinomas, in addition to N-acetyl-D-galactosamine, asialofetuin and fetuin. This lectin reacted strongly with red blood cells (RBCs) from human ABO blood groups and rat. It also reacted with rabbit, sheep, goat and guinea pig RBCs. The lectin is a glycoprotein having no metal ion requirement for its activity. Denaturing agents such as urea, thiourea and guanidine-HCl had no effect on its activity when treated for 15 minutes. AVL showed significant antiproliferative activity towards HB98 and P388D1 murine cancer cell lines. It also exerted antifungal activity against phytopathogenic fungi Botrytis cincerea and Fusarium oxysporum but not against Rhizoctonia solani, Trichoderma reesei, Alternaria solani and Fusarium graminearum.

Identification of peptide mimetics of xenoreactive α-Gal antigenic epitope by phage display

The prevention of hyperacute rejection (HAR) triggered by interaction between the human natural antibody and xenoreactive antigenic epitope (Gal-α1, 3Gal) present on pig cells is the key to success in pig-to-human xenotransplantation. The phage display technology offers an effective strategy for screening peptides which can interact with the anti-Gal antibody to block α-Gal antigen binding site. Two peptide libraries, linear 7 peptide library and C7C library, were panned on the anti-B monoclonal antibody which has the characteristic of binding to the α-Gal antigenic epitope. After four rounds of panning, 22 positive phage clones were selected. Highly homologous sequence PT and STL existed among these selected peptides. Stachyose competitive ELISAs revealed that these peptides specifically bound to α-Gal antigen binding site. Eight peptide mimics of α-Gal antigenic epitope could inhibit the agglutination of pig red blood cells mediated by human sera in a dose-dependent manner. These results demonstrated that the selected peptides can mimic the conformational structure of α-Gal antigenic epitope and have the therapeutic potential in xenotransplantation.

Species- and cell type-specific interactions between CD47 and human SIRPα

AbstractCD47 on red blood cells (RBCs) reportedly signals “self” by binding SIRPα on phagocytes, at least in mice. Such interactions across and within species, from mouse to human, are not yet clear and neither is the relation to cell adhesion. Using human SIRPα1 as a probe, antibody-inhibitable binding to CD47 was found only with human and pig RBCs (not mouse, rat, or cow). In addition, CD47-mediated adhesion of human and pig RBCs to SIRPα1 surfaces resists sustained forces in centrifugation (as confirmed by atomic force microscopy) but only at SIRPα-coating densities far above those measurable on human neutrophils, monocytes, and THP-1 macrophages. While interactions strengthen with deglycosylation of SIRPα1, low copy numbers explain the absence of RBC adhesion to phagocytes under physiologic conditions and imply that the interaction being studied is not responsible for red cell clearance in humans. Evidence of clustering nonetheless suggests mechanisms of avidity enhancement. Finally, using the same CD47 antibodies and soluble SIRPα1, bone marrow-derived mesenchymal stem cells were assayed and found to display CD47 but not bind SIRPα1 significantly. The results thus demonstrate that SIRPα-CD47 interactions, which reportedly define self, exhibit cell type specificity and limited cross-species reactivity. (Blood. 2006;107:2548-2556)

Pathogenicity of Vietnamese Enterotoxigenic Escherichia coli Strains in Colostrum-deprived One-day-old Piglets

Preweaning colibacillosis is a major cause of economic loss to the swine industry in Vietnam. The aim of this study was to examine the enteropathogenicity of representative enterotoxigenic Escherichia coli (ETEC) strains obtained during an earlier epidemiologic survey conducted in five provinces in North Vietnam. This included isolates belonging to serotype O8 that produced heat-stable and heat-labile enterotoxins but did not produce any of the recognized fimbriae (F4, F5, F6, F41, F18). In vitro hemagglutination (unique mannose-resistant hemagglutination activity with guinea pig, sheep, human, and chicken red blood cells at 37 degrees C, but not at 18 degrees C) and enterocyte brush border attachment assays suggested that the F- ETEC strains produced an unidentified colonization factor that promoted adherence to the intestinal epithelium. Colostrum-deprived 1-day-old piglets challenged with an F- strain (1-2 x 10(9) bacteria) developed acute watery diarrhea within 4 hours of inoculation and suffered up to 20% weight loss, with comparable severity to piglets challenged with conventional F4 and F5 strains. At necropsy, viable counts and histopathologic examination of intestinal sections demonstrated colonization of the duodenum, jejunum, and ileum by F4-positive strains. In comparison, the F- and F5-positive strains attached exclusively to the ileum. Transmission electron micrographs of negatively stained F- cells grown at 37 degrees C demonstrated the presence of fimbriae. These results confirm the presence of a potentially new pathogenic ETEC fimbrial type in piggeries in Vietnam, with a unique hemagglutination property and attachment characteristics similar to ETEC bearing F5 fimbriae.