Immune Function in Critically Ill Dogs

Background

People with critical illness (CI) commonly develop various forms of immune dysfunction, however, there is limited information concerning immune dysfunction in dogs with CI.

Hypothesis

The immune response in CI dogs differs from that of healthy dogs.

Animals

Immunologic variables were compared between 14 dogs with CI, defined as APPLE_fast score of >20 points, admitted to the University of Missouri Veterinary Health Center Small Animal Clinic Intensive Care Unit and healthy controls (n = 15).

Methods

Cohort study evaluating constitutive and lipopolysaccharide (LPS)‐stimulated TNF‐α, IL‐6, and IL‐10 production, phagocytosis of opsonized E. coli and respiratory burst capacity after opsonized E. coli or phorbol 12‐myristate 13‐acetate (PMA) stimulation, peripheral blood lymphocyte phenotype, and monocyte expressions of HLA‐DR and TLR‐4.

Results

Lipopolysaccharide‐stimulated leukocyte TNF‐α (median, Q1, Q3; CI, 49, 49, 120; control, 655, 446, 1174 pg/mL; P = < 0.001), IL‐6 (median, Q1, Q3; CI, 49, 49, 64; control, 100, 49, 166 pg/mL; P = 0.029), and IL‐10 (CI, 49, 49, 56; control, 96, 49, 203 pg/mL; P = 0.014) production and both E. coli (median, Q1, Q3; CI, 60.5, 43, 88.5; control, 86.6, 81, 89.2%; P = 0.047) and PMA (CI, 40, 11.7, 70; control, 93, 83, 97.6%; P = < 0.001)‐stimulated respiratory burst capacity significantly decreased in CI dogs. Percentage of monocytes expressing TLR‐4 greater in the CI dogs (median, Q1, Q3; CI, 46.9, 24.3, 64.2; control, 16.4, 9.4, 26.2%; P = 0.005).

Conclusion

These findings suggest dogs with CI develop immune system alterations that result in reduced respiratory burst function and cytokine production despite upregulation of TLR‐4.     

Morphological, structural, and functional characterization of the haemocytes of the scallop, Argopecten irradians

Argopecten irradians is one of the most important commercial species of Pectinidae family in China. The internal defense system of mollusks consists of circulating haemocytes. In order to characterize the haemocytes of the scallop A. irradians, light and electron microscopical studies were carried out. Four types of haemocytes were recognized: type I small hyalinocytes (2.38 ± 0.08 μm, 30-35%), type II large hyalinocytes (4.41 ± 0.33 μm, 15-20%), type III small granulocytes (4.15 ± 0.26 μm, 20-25%), and type IV large granulocytes (8.26 ± 0.52 μm, 25-30%). Granulocyte types showed smaller N/C ratios than hyalinocytes. The mean haemocyte concentration was about (3.75 ± 0.65) × 10⁷ cells ml^− 1 of haemolymph. Among haemocytes, 44.7% are granular and 55.3% are agranular. These gave a relatively systematic classification scheme for haemocytes of A. irradians. Three types of granules were identified: type I, with high electron-density; type II, with low electron-density; and type III, with a middle level of electron-density, based on TEM studies. Different haemocyte types were not separated with DDGC of Percoll in this study. Both granulocytes and hyalinocytes showed a phagocytic response to the two strains of bacteria, Escherichia coli and RLOs. The phagocytic ability of granulocyte was significantly higher (41-48%) than that of hyalinocyte (9.2-11.2%).     

中药参芪肺宝对小鼠免疫功能的影响

[目的]初步探讨参芪肺宝对小鼠免疫功能的影响.[方法]采用碳廓清率法、血清素测定法进行分析,观察参芪肺宝对正常小鼠单核巨噬细胞吞噬功能、溶血素抗体生成的影响.[结果]参芪肺宝可激活网状内皮系统并增加小鼠血清溶血素含量.[结论]参芪肺宝具有一定增强机体免疫力的作用.     

Neutrophil function in septic dogs

BACKGROUND: Leukocytes appear to enter a hypo-inflammatory state in human patients with a severe bacterial infection, whereas, in swine, intra-abdominal sepsis produces an initial increase and subsequent decrease in neutrophil Fc receptor mediated phagocytosis. HYPOTHESIS: Impaired neutrophil function (hypo-inflammatory state) develops in dogs with sepsis. ANIMALS: Thirteen adult client-owned dogs that developed clinical signs consistent with sepsis were assessed for evidence of neutrophil dysfunction. These results were compared with those of 12 healthy dogs. METHODS: Flow cytometry combined with the appropriate fluorescent markers allowed for quantification of the phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis of immune complexes, neutrophil phagocytosis of opsonized Escherichia coli, and the intracellular concentration of reduced glutathione as a measure of oxidative stress. RESULTS: The phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis was significantly lower in neutrophils from septic dogs (mean fluorescence intensity +/- standard deviation; 118 +/- 13 and 165 +/- 27 for septic and control dogs, respectively, p = 0.001), although the phagocytosis of opsonized E. coli was significantly increased (155 +/- 74 and 77 +/- 44 for septic and control dogs, respectively, p = 0.004). Intracellular reduced glutathione was not significantly different in neutrophils from septic and healthy control dogs. CONCLUSIONS: An important component of neutrophil function is decreased in septic dogs. The diminished oxidative burst after Fc receptor-mediated phagocytosis in neutrophils from septic dogs could hinder the ability of the innate immune system to clear bacterial infections or it might help protect these patients from the systemic consequences of infection.     

Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens

The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens.     

Leukocyte response and phagocytic activity in Nile tilapia experimentally infected with <Emphasis Type="Italic">Enterococcus</Emphasis> sp.

This study evaluated the total and differential leukocyte counting and the phagocytic activity in Nile tilapia Oreochromis niloticus experimentally injected with Enterococcus sp. in the swim bladder. Fish were distributed in four treatments in triplicates of non-injected fish, fish injected with 1 ml of sterile saline solution 0.65%, and fish injected with 1 × 10³ and 1 × 10⁶ colony-forming units (CFU) of Enterococcus diluted in 1 ml sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected for white blood cell (WBC) counts, differential counting of WBC, and phagocytic activity of blood leukocytes. The increased numbers of WBC and lymphocytes were followed by decreased number of monocyte after infection. The percentages of phagocytic activities in the blood were 55.3 and 55.9%, respectively, in tilapia injected with 1 × 10³ and 1 × 10⁶ CFU/ml.     

粉纹夜蛾(Trichoplusia ni)培养细胞对家蚕微孢子虫(Nosema bombycis)的吞噬过程观察

观察微孢子虫在非天然宿主细胞系的生物行为过程,有助于探究微孢子虫的侵染特异性机制。选用鳞翅目昆虫粉纹夜蛾(Trichoplusia ni,Tn)细胞系为材料,接种家蚕微孢子虫(Nosema bombycis,Nb)孢子后观察其吞噬过程。结果发现:经氢氧化钾发芽预处理的Nb孢子虽然可以在Tn培养细胞液中发芽,但未见发芽孢子的孢原质进入Tn培养细胞;在接种Nb孢子浓度为2.0×105mL-1以上时,观察到孢子通过内吞作用进入了Tn培养细胞,但未见孢子的增殖;随着时间的延长和进入Tn培养细胞的Nb孢子的增加,可引起Tn培养细胞的超微结构变化,并导致细胞的裂解。以上结果说明,虽未见Nb以发芽形式感染Tn培养细胞,但Nb能够被吞噬进入Tn培养细胞并破坏其结构。     

Trans-10, cis-12 conjugated linoleic acid modulates phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to Clostridium difficile toxin B

Trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) has been reported to enhance phagocyte function. Clostridium difficile toxin B (TcdB) has been known to inhibit Ras-homologous (Rho) guanosine triphosphatases (GTPases) which play essential roles in neutrophil immune functions. Here, we examined whether in vitro treatment with t10c12-CLA modulates the filamentous actin (F-actin) polymerization, phagocytic capacity, and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs) exposed to TcdB. Treatment with t10c12-CLA, but not linoleic acid, enhanced PMN F-actin polymerization, phagocytic capacity, and OBA, while TcdB suppressed these functions. t10c12-CLA reversed the suppressive effects of TcdB on these PMN functions. t10c12-CLA stimulated F-actin polymerization regardless of whether phagocytosis was stimulated by microspheres but only elevated OBA when microspheres were added. We asked whether the effects of t10c12-CLA were associated with changes in the activation of the Rho GTPase Cdc42. Treatment with t10c12-CLA augmented Cdc42 activity in both TcdB-treated and TcdB-naive PMNs during phagocytosis. Thus, t10c12-CLA up-regulates PMN phagocytic responses attenuated by TcdB. This effect is associated with an increase in actin polymerization and may involve the activation of Cdc42.     

粉纹夜蛾培养细胞对家蚕微孢子虫的吞噬及与孢壁蛋白的关系

解明家蚕微孢子虫(Nosema bombycis,Nb)入侵宿主细胞的相关因素,有助于深入研究Nb对家蚕的侵染机制。利用激光共聚焦显微镜观察Nb孢子接种粉纹夜蛾(Trichoplusiani,Tn)培养细胞48h后孢子的分布状态和培养细胞的生长情况,并对接种Nb孢子后的Tn培养细胞培养物总蛋白进行SDS-PAGE分析,证实经0.2mol/LKOH预处理的Nb孢子可以被Tn培养细胞所吞噬。对Nb孢子孢壁蛋白的SDS-PAGE分析显示,Nb孢子经KOH预处理后,大小为80kD和12kD的孢壁蛋白带缺失或明显减弱,30kD的孢壁蛋白带丰度降低。推测KOH预处理使Nb孢子部分孢壁蛋白被分解(部分或完全)或使蛋白结构发生变化,由此影响了Nb孢子与Tn培养细胞的相互作用关系,有利于Tn培养细胞对Nb孢子的吞噬。     

Effect of Mesdc2 on phagocytosis of retinal pigment epithelial cells

AIM: To observe the effect of mesoderm development candidate 2 (Mesdc2) on phagocytosis of retinal pigment epithelial (RPE) cells. METHODS: The purified Mesdc2 protein was used to incubate with mouse primary RPE cells or D407 cells. The bound and ingested photoreceptor outer segments (POSs) by RPE cells were observed by confocal microscopy and flow cytometry. RESULTS: The Mesdc2 protein was expressed throughout the retina, and predominantly in POSs. Internalized POSs were co-localized with phagosome biomarker Rab7, suggesting that Mesdc2-mediated engulfment was involved in a phagocytosis pathway. Mesdc2 stimulated phagocytosis of POS vesicles by D407 RPE cells in a dose-dependent manner. The effect of Mesdc2 on phagocytosis decreased after the saturation concentration. Mesdc2 bound to both D407 RPE cells and shed POS vesicles. CONCLUSION: Mesdc2 stimulates the phagocytosis of retinal pigment epithelial cells.