Development and composition of lymphoid lesions in the spleens of Marek's disease virus-infected chickens: Association with virus spread and the pathogenesis of Marek's disease

Changes in lymphocyte distribution in spleens of Marek's disease virus (MDV) infected White Leghorn chickens of line 72 (MD susceptible) and line 61 (MD resistant) were studied by immunocytochemistry. Lymphocytes expressing the MDV antigen pp38 (predominantly B cells) were detected from 4 to 6 days post-inoculation (d.p.i.) but not at or after 8 d.p.i., and were more numerous in line 72. In line 61, infection resulted in depletion of B lymphocytes and an increase in T lymphocytes from 3 to 6 d.p.i., but no change in distribution of these cells. From 8 d.p.i., the B-dependent tissue began to recover and the T cells decreased in number. In line 72, infection caused a dramatic change in lymphocyte distribution, with formation of 'lymphoid lesions'. Diffuse, irregular patches of B lymphocytes, around the capillaries, became surrounded by large aggregates of TCRαβ1⁺ CD8⁺ and CD4⁺ lymphocytes, bordered by a band of TCRγδ⁺ lymphocytes. From 8 d.p.i., the B-dependent areas partially recovered, while TCRαβ1⁺ CD4⁺ and CD8⁺ lymphocytes, potentially transformed, became extensively scattered throughout the spleen. We conclude that in line 72, replication and spread of MDV is more efficient and T cell responses in early infection are greater, favouring the tumour stage of the disease.     

The Effect of l‐Arginine on Porphyromonas gingivalis‐Induced Phagocytosis of a Murine Macrophage‐like Raw 264.7 Cell Line

The aim of this study was to determine the effect of l‐arginine on Porphyromonas gingivalis‐induced phagocytosis by RAW 264.7 cells. The cells were pretreated with l‐arginine or d‐arginine prior to incubation with either unopsonized or opsonized P. gingivalis. In other experiments, the cells were pretreated with l‐arginine and various concentrations of NMLA (N^G‐monomethyl‐l‐arginine) prior to incubation with the bacteria. The phagocytosis was microscopically assessed and determined by the phagocytic index. The results showed that l‐arginine, but not d‐arginine enhances the ability of RAW264.7 cells to engulf the bacteria. The upregulatory effect of l‐arginine on P. gingivalis‐induced phagocytosis was abolished by NMLA. The results of the present study suggest that l‐arginine may upregulate the P. gingivalis‐induced phagocytic activity of RAW264.7 cells, perhaps, via modulation of nitric oxide synthase.     

Granulocyte macrophage colony–stimulating factor is required for cytokine induction by a highly 6-branched 1,3-β-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes

We have previously obtained and elucidated the precise structure of a highly branched 1,3-β-d-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented β-d-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony–stimulating factor (GM-CSF); this is similar way to be a typical 1,3-β-d-glucan from Sparassis crispa (SCG), which is a 1,3-β-d-glucopyranosyl backbone with single 1,6-β-d-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of β-d-glucan but will also aid in the design and development of more effective β-d-glucan agents.     

Cardiac index,oxygen delivery,and tissue oxygen extraction in slow and fast growing chickens,and in chickens with heart failure and ascites: A comparative study

The present study examines the differences in blood gas parameters, cardiac output, cardiac index, oxygen delivery and tissue oxygen extraction in slow growing chickens (leghorn and feed restricted broilers), fast growing chickens (broilers fed ad libitum) and chickens with fulminant heart failure and ascites. In comparison to leghorns, broiler chickens had lower pO2 and O2 saturation levels in venous blood (P < 0.001). At the age of 35 days, broilers had arterial and venous pO2 significantly lower than 7-day-old broilers (P < 0.05). Overall, blood pO2 and O2 saturation tended to decline, and CO2 content tended to increase with age. Chickens developing ascites had lower blood pO2 and O2 saturation levels, and higher blood CO2 content in comparison to normal chickens (P < 0.05). In comparison to other chickens, ascitic chickens had the lowest pO2 and O2 saturation, and highest CO2 content in both venous and arterial blood (all P < 0.001). Broilers at 35 days of age had higher arterial O2 content than leghorn chicks, and there were only minor differences between normal and ascitic chickens. However, ascitic chickens had the lowest venous O2 content (P < 0.001), but the highest tissue O2 extraction index (P < 0.001). Cardiac index was higher in leghorn chicks than in broilers (P 0.001). Ascitic birds had the lowest cardiac index (P < 0.001). Oxygen delivery was higher in leghorns than in broilers (P < 0.001). Ascitic birds had the lowest oxygen delivery index. The present study has identified significant differences in previously unexamined performance indicators of the cardiovascular system between slow growing chickens, fast growing chickens and chickens with heart failure. Low cardiac index in broiler chickens appears to be the key haemodynamic problem leading to hypoxaemia and ultimately cardiovascular failure in fast growing broilers.     

A critical review on properties and applications of microbial l-asparaginases

Abstract

l-Asparaginase is one of the main drugs used in the treatment of acute lymphoblastic leukemia (ALL), a commonly diagnosed pediatric cancer. Although several microorganisms are found to produce l-asparaginase, only the purified enzymes from E. coli and Erwinia chrysanthemi are employed in the clinical and therapeutic applications in humans. However, their therapeutic response seldom occurs without some evidence of hypersensitivity and other toxic side effects. l-Asparaginase is also of prospective use in food industry to reduce the formation of acrylamide in fried, roasted or baked food products. This review is an attempt to compile information on the properties of l-asparaginases obtained from different microorganisms. The complications involved with the therapeutic use of the currently available l-asparaginases, and the enzyme’s potential application as a food processing aid to mitigate acrylamide formation have also been reviewed. Further, avenues for searching alternate sources of l-asparaginase have been discussed, highlighting the prospects of endophytic microorganisms as a possible source of l-asparaginases with varied biochemical and pharmacological properties.     

Subject Index to Volume 21

ABSTRACT

A novel amperometric immunosensor, based on graphite paste (graphite powder and paraffin oil), has been constructed for the assay of l-T₄. The graphite paste is impregnated with mouse monoclonal anti-(+)-3,3′,5,5′-tetraiodo-l-thyronine (anti-l-T₄). The immunosensor can be reliably used for the assay of l-T₄ in thyroid and in drugs, using chronoamperometry technique, at ppt up to ppb concentration levels. The potential used for l-T₄ assay was +450 mV vs. Ag/AgCl electrode. The surface of the immunosensor can be regenerated by simply polishing, obtaining fresh immunocomposite ready to be used in a new assay.     

Oral Newcastle disease vaccination trials in Ethiopia

Vaccination experiments were carried out in Ethiopia to study the efficacy of the NDV-I2 vaccine against challenge with an Ethiopian velogenic strain of NDV. In experiment A, which comprised 300 broiler chicks, the efficacy of the ocular/drinking water application of the HB1/La Sota vaccine was compared with the ocular/drinking water and the feed application of the NDV-I2 vaccine on untreated barley and sorghum. The NDV-I2 vaccine applied by eye-drop or drinking-water protected the chickens against challenge as efficiently as combined HB1/La Sota vaccination but untreated barley and sorghum were unsuitable vaccine carriers. The vaccine virus could not be recovered and chickens neither seroconverted nor were they protected. In experiment B, 120 broiler chicks were divided into 6 treatment groups. One group each received NDV-I2 vaccine mixed with untreated barley or sorghum which was applied immediately, or 14h after mixing and standing at ambient temperature. The fifth group was vaccinated intraocularly and via the drinking water with the NDV-I2 vaccine. The sixth group remained untreated. Experiment B confirmed the results of experiment A. In experiment C, 100 chicks were divided into 5 groups of 20 chickens each. One group each received the NDV-I2 vaccine on parboiled barley or sorghum as vaccine carriers 0 and 6h after mixing. The last group remained untreated. Parboiled barley given 0 or 6 h and parboiled sorghum given 0 h after mixing with the vaccine led to seroconversion and protection of the chickens. Parboiled sorghum given 6h after mixing with the vaccine did not. It is concluded that the thermostable NDV-I2 vaccine may be a suitable vaccine for oral application under Ethiopian conditions.     

Maximum aerobic power and physical dimensions of children

Age, height, mass, fat-free mass and vital capacity were used as predictors of maximum aerobic power (V˙o_{2 max}). The variables were cast in linear form by logarithmic transformation and submitted to multiple regression analysis. Results indicate V˙o_{2 max} as a power function of age, height and mass in 50 untrained boys aged 7 to 13 years. In this group the relationship between V˙o_{2 max} and body mass may be expressed by the equation Y=0·076X^0·88 (r=0·92, P<0·01). Age, height and mass together accounted for 89 per cent of the variance in V˙o_{2 max} (R=0·94, P<0·01). In 30 girl swimmers and in 14 young boys during 22 months of running training, V˙o_{2 max} was proportional to body mass and indicated greater maximum aerobic power for their size and age. In normally growing children, V˙o_{2 max} appears to increase more slowly than body mass. Children subjected to aerobic training evidently maintain V˙o_{2 max} in proportion to their increasing mass throughout adolescence.     

β-d-Xylosides Stimulate GAG Synthesis in Chondrocyte Cultures Due to Elevation of the Extracellular GAG Domains,Accompanied by the Depletion of the Intra–pericellular GAG Pools,with Alterations in the GAG Profiles

The familial disease of hereditary multiple exostoses is characterized by abnormal skeletal deformities requiring extensive surgical procedures. In hereditary multiple exostoses patients there is a shortage in the pericellular glycosaminoglycan (GAG) of heparan sulfate (HS), related to defective activity of HS glycosyltransferases, mainly in the pericellular regions of chondrocytes. This study searched for a novel approach employing xylosides with different aglycone groups priming a variety of GAG chains, in attempting to alter the GAG compositional profile. Cell cultures of patients with osteochondroma responded to p-nitrophenyl β-d-xyloside by a significant increase in total GAG synthesis, expressed mainly in the extracellular domains, limited to chondroitin sulfate). The different β-d-xylosides, in addition to increasing the synthesis of extracellular GAGs, led to a significant depletion of the intracellular GAG domains. In mouse chondrocyte cultures, β-d-xylosides with different aglycones created a unique distribution of the GAG pools. Of special interest was the finding that the naphthalene methanol β-d-xyloside showed the highest absolute levels of HS-GAGs in both extracellular and intra–pericellular moieties compared with other β-d-xylosides and with controls without xyloside. In summary, β-d-xylosides can be utilized in chondrocyte cultures to modify the distribution of GAGs between the extracellular and intracellular compartments. In addition, xylosides may alter the profile of specific GAG chains in each moiety.     

HB-EGF stimulates eNOS expression and nitric oxide production and promotes eNOS dependent angiogenesis

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family of ligands that is expressed by many cell types including endothelial cells. We have previously shown that HB-EGF stimulates angiogenesis in vitro in human umbilical vein endothelial cells (HUVEC). Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is an important regulator of angiogenesis. However, the role of HB-EGF in regulation of eNOS has not yet been investigated. Whether HB-EGF-induced endothelial cell migration and vascular network formation are mediated via production of NO from eNOS is also unknown. To address these questions, we stimulated HUVEC with HB-EGF and evaluated the expression of eNOS at the mRNA and protein levels. HB-EGF significantly upregulated expression of eNOS mRNA, stimulated eNOS protein production, and increased NO release from HUVEC. HB-EGF phosphorylated eNOS in a phosphatidylinositol 3-kinase (PI3K) dependent fashion, and stimulated in vitro angiogenesis. eNOS siRNA inhibited HB-EGF-stimulated HUVEC migration in a scratch assay. NG-nitro-L-arginine-methyl-ester (L-NAME) and L-N5-(1-lminoethyl)ornithine,dihydochloride (L-NIO) (specific inhibitors of eNOS) also abolished HB-EGF-induced HUVEC migration and angiogenesis. More importantly, we found that HB-EGF also promotes angiogenesis in vivo in the Marigel plug assay. Lastly, inhibition of the p38 MAPK pathway enhanced HB-EGF-induced EC migration and angiogenesis. We conclude that HB-EGF, through its interaction with EGF receptors (EGFR), stimulates eNOS activation and NO production via a PI3K-dependent pathway. Thus, activation of eNOS appears to be one of the key signaling pathways necessary for HB-EGF mediated angiogenesis. These novel findings highlight an important role for HB-EGF as a regulator of endothelial cell function.     

Toxic effects of d-galactose on thymus and spleen that resemble aging

Continuous low-dose injection of d-galactose induces changes in mice that resemble accelerated aging. As such, these mice have been used as models to study mechanisms of aging. Here, we examined whether repeated (daily, for 60 days) subcutaneous injections (at 50?mg d-galactose/kg) into young adult (i.e., 2-month-old) mice induced changes in key immune system organs that were on par with those associated with aging. The results showed that galactose-treated mice develop histologic changes in their thymic cortical and medullary regions; immunohistochemical analysis revealed unorganized distributions of keratin-5 and keratin-8 proteins in the thymus of these hosts. These histological changes in the thymus of d-galactose-treated mice were also observed in the organs of aged (i.e., 24-month-old control mice); however, in this latter group, these changes were accompanied by a strong infiltration of adipose cells. Galactose-treated mice also evinced alterations within their splenic white and red pulp. Further, ultrastructural analyses of the thymus and spleen of the treated mice revealed increases in irregularly shaped lymphocytes bearing visible pyknosis. It was also seen that levels of autophagy within thymic epithelial cells were greatly decreased in the tissues of the galactose-treated mice, an outcome also seen in aged mice. Lastly, the level of memory T-lymphocytes and percentage of IgM-B220-B-lymphocytes in spleens of the galactose-treated mice were both increased (albeit insignificantly so) relative to values among splenocytes of age-matched control; however, these levels were not clealy as elevated as would be expected in “elderly” mice. Taken together, our results strongly suggest that d-galactose treatment can induce structural changes in the thymus and spleen, and some changes in organ-associated cell phenotypes, that are similar to several effects seen with aging. However, the fact that many endpoints do not appear to be truly reflective of what should be seen in immune system organs/cells of “elderly” mice now calls into question the appropriateness of the use of d-galactose (i.e., is it histologically/immunotoxicologically-proper?) to create age-mimicry in mice.     

Preclinical and pharmacotoxicology evaluation of α-l-guluronic acid (G2013) as a non-steroidal anti-inflammatory drug with immunomodulatory property

Context: Therapeutic effects of α-l-guluronic acid with the greatest tolerability and efficacy (G2013) have been shown in experimental model of multiple sclerosis and other in vitro and in vivo examinations regarding α-l-guluronic acid; there are no toxicological researches on its safety although the pharmacological impacts have been recorded.

Objective: This study was designed to determine the acute and sub chronic toxicity of α-l-guluronic acid in healthy male and female BALB/c mice.

Materials and methods: For the acute toxicity study, the animals orally received five different single doses of α-l-guluronic acid and were kept under observation for 14?d. In the sub-chronic study, 24 male and female BALB/c mice were divided into four groups and treated daily with test substance preparation at dose levels of 0, 50, 250, and 1250?mg/kg body weight for at least 90 consecutive days. The mortality, body weight changes, clinical signs, hematological and biochemical parameters, gross findings, histopathological, and organs weight determinants were monitored during this study.

Results: The results of acute toxicity indicated that the LD50 of α-l-guluronic acid is 4.8?g/kg. We found no mortality or abnormality in clinical signs, body weight, relative organs weight, or necropsy in any of the animals in the subchronic study. Additionally, the results showed no significant difference in hematological, biochemical, and histopathological parameters in rats.

Conclusions: Our results suggest that α-l-guluronic acid has high safety when administered orally in animals.     

Detection of infectious bronchitis virus in experimentally infected chickens by an antigen-competitive ELISA

An antigen-competitive enzyme-linked immunosorbent assay (Ag-C-ELISA) was developed for the detection of infectious bronchitis virus (IBV) antigens, M41 strain, in tissues from experimentally infected chickens, or in allantoic fluid harvested from inoculated embryonated eggs. The detection limit of IBV in the Ag-C-ELISA was 10^4.1 median embryo infective doses (EID50)/well. Tracheal and lung samples from chickens vaccinated with 10^2.5 EID50 of live attenuated infectious bronchitis (H120) vaccine were negative in the direct detection Ag-C-ELISA. The results indicate that the Ag-C-ELISA has the potential to detect IBV, either directly in tissue samples or when combined with the passage of material in embryonated eggs, thereby constituting an alternative method for the diagnosis of IBV.     

The effects of molecular structure on sol-to-gel transition of biodegradable poly(depsipeptide-co-lactide)-g-PEG copolymers

We report on the effects of number and length of PEG chains in poly(depsipeptide-co-dl-lactide)-g-poly(ethylene glycol) (P(DG-dl-LA)-g-PEG) copolymers on their sol-to-gel transition behavior. The graft-type copolymer is suitable for the systematic study of the effects of molecular structure and hydrophilic/hydrophobic balance on its sol-to-gel transition. We prepared various P(DG-dl-LA)-g-PEG copolymers through coupling reactions between the pendant carboxylic acid groups of P(GD-dl-LA) and the end hydroxyl group of MeO-PEG having various molecular weights. Temperature-responsive sol-to-gel transition of the obtained copolymer solution in phosphate-buffered solution (pH 7.4, ionic strength = 0.14) was investigated by the test tube inverting method and rheological measurements. P(GD-dl-LA)-g-PEG copolymer prepared from higher molecular weight PEG showed higher sol-to-gel transition temperatures compared with the copolymers prepared from lower molecular weight PEG, although these copolymers have similar weight content of PEG (23–24?wt.%). Similar trends were observed for groups of copolymers whose PEG contents were 27 or 30?wt.%. These results are informative for providing strategies on rational design of thermo-gelling polymers.     

Pathology of the thymus,spleen and bursa of Fabricius in zinc-deficient ducklings

The weight and growth index of bursa of Fabricius, thymus and spleen were significantly reduced (P < 0.05 or P < 0.01) in zinc (Zn)-deficient ducks (Zn 22.9 mglkg diet) when compared with normal ducks. The G0/Gl phase of the cell cycle of the bursa of Fabricius, thymus and spleen was much higher, and the S and G2+M phases lower in Zn-deficient ducks than in the controls. Histopathologically, there was lymphocyte degeneration and depletion of lymphoid organs, and the reticular cells of thymus were also degenerate or necrotic in the Zndeficient group. The results demonstrated that Zn deficiency seriously inhibited the growth of lymphoid organs and caused marked pathology in the lymphoid organs. The results also showed that the effect of Zn deficiency on the primary lymphoid organs occurred earlier than on the secondary lymphoid organs. The effect of Zn deficiency was greatest on the bursa of Fabricius, followed by the thymus, and then the spleen.     

Zinc L-carnosine suppresses inflammatory responses in lipopolysaccharide-induced RAW 264.7 murine macrophages cell line via activation of Nrf2/HO-1 signaling pathway

Context: Zinc L-carnosine (ZnC) is a chelate of Zn and L-carnosine and is used clinically in the treatment of peptic ulcer.

Objective: In this study, we aim to investigate the involvement of heme oxygenase-1 (HO-1) in the anti-inflammatory effects of ZnC in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages.

Materials and methods: We used immunoblotting analysis to evaluate the involvement of HO-1 in the anti-inflammatory effects of ZnC and the signaling pathway involved was measured using Dual luciferase reporter assay.

Results: Results from immunoblotting analysis demonstrated that pretreatment of cells with ZnC enhanced the expression of HO-1 in RAW 264.7 cells. Pretreatment of cells with HO-1 inhibitor (tin protoporphyrin IX dichloride) significantly attenuated the inhibitory effects of ZnC on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and NF-κB activation in LPS-induced RAW 264.7 cells, suggesting that HO-1 play an important role in the suppression of inflammatory responses induced by ZnC. Furthermore, results from co-immunoprecipitation of Nrf2 and Keap1 and dual luciferase reporter assay showed that pretreatment of ZnC was able to activate the Nrf2 signaling pathway. Treatment of cells with p38 inhibitor (SB203580), c-Jun N-terminal kinase inhibitor (SP600125), and MEK 1/2 inhibitor (U0126) did not significantly suppress the induction of HO-1 by ZnC. Moreover, our present findings suggest that the effects of ZnC on NO production, HO-1 expression, and Nrf2 activation were attributed to its Zn subcomponent, but not l-carnosine.

Conclusion: Pretreatment with ZnC was able to activate Nrf2/HO-1 signaling pathway, thus suppressing the expression of inflammatory mediators, such as NO and iNOS in LPS-induced RAW 264.7 cells.     

The Anti-Tumor Effect of Interleukin-12 is Enhanced by Mild (Fever-Range) Thermal Therapy

The cytokine interleukin 12 (IL-12) has resulted in notable anti-tumor activity in animal models and in patients and as a result there is considerable interest in learning how to maximize its therapeutic potential while at the same time reducing its known toxic side effects. Strategies which could maintain its effectiveness while permitting reduced dosage could be especially valuable. In this study we used BALB/c mice bearing CT26 tumors as a model for testing whether combining murine IL-12 with a mild (fever range) whole body hyperthermia protocol could result in such a strategy. Our data revealed that 100 ng of IL-12/mouse/day used in combination with FR-WBH was as effective as one in which 300 ng of IL-12/mouse/day was used alone. Importantly, the mice receiving the combination treatment exhibited fewer treatment related toxicities compared to those that received high dose IL-12 alone. Initiation of the IL-12 treatment immediately after FR-WBH induced the greatest anti-tumor effect. This effect does not appear to depend on differences in IL-12-induced IFN-γ, but may involve production of nitric oxide (NO), since treatment of mice with a NOS inhibitor, N^G-monomethyl-l-arginine (l-NMA), abolishes the additive anti-tumor effect of the combination treatment. Collectively, these data suggest that modification of physiological parameters in the host by mild fever-like thermal stimuli may be an effective and feasible adjuvant for cytokine-based immunotherapeutic strategies.     

Search for novel cell surface markers of apoptotic cells

Surface markers of apoptotic cells are of great interest as potential targets for non-destructive detection and study of these cells. They are also important for apoptotic cell recognition and subsequent clearance by cells of the immune system. Recently, it was found that apoptosis is accompanied by not only the loss of plasma membrane asymmetry detected by Annexin V, but also by changes in cell surface glycoconjugates. These novel markers of apoptosis are α-d-mannose and β-d-galactose-specific plasma membrane glycoproteins whose expression is substantially increased after induction of apoptosis. The glyconeoepitopes described in this article are proposed to be useful for both, the detection of apoptotic cells and the isolation of the latter, from mixed populations.     

Preparation and structure of drug-carrying biodegradable microspheres designed for transarterial chemoembolization therapy

Biodegradable poly(D,L-lactic acid) drug-eluting microspheres containing anti-tumor drugs, cisplatin, and sorafenib tosylate have been prepared by the emulsion solvent evaporation method with diameter between 200 and 400 μm. Scanning electron microscopy showed that cisplatin microspheres had smooth surfaces, while sorafenib tosylate microspheres and cisplatin + sorafenib tosylate microspheres were porous at the surface and the pits of the latter were larger than those of the former. Notably, cisplatin + sorafenib tosylate microspheres had a fast drug release rate compared with microspheres containing one drug alone. In vitro cytotoxicity experiments and classical matrigel endothelial tube assay certificated the maintaining bioactivity of cisplatin and sorafenib tosylate released from the microspheres, respectively. This work provides a useful approach for the fabrication of drug-eluting beads used in transarterial chemoembolization.