May-Hegglin anomaly in a dog

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 μM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.     

猪脾转移因子对小鼠吞噬细胞吞噬功能的影响

成年健康小鼠分别口服或注射不同剂量的转移因子 ( Transfer factor,TF) ,第 8d检测小鼠外周血中性粒细胞对葡萄球菌的吞噬率 ,第 1 1 d检测小鼠腹腔巨噬细胞对鸡红细胞的吞噬率。结果表明 ,注射 TF的 、 、 组小鼠中性粒细胞吞噬率分别为( 38.5 6± 3.82 0 ,( 4 4.6 4± 2 .48) ,( 5 1 .0 7±3.84) ,而对照组的相应值为 ( 2 9.81± 2 .5 3) ;注射 TF的 、 、 组小鼠巨噬细胞吞噬率分别为 ( 5 8.0 4± 2 .1 9) ,( 6 7.8± 3.38) ,( 75 .82± 1 .73) ,对照组为 ( 4 6 .1 6± 1 .6 7) ;口服 TF的 、 、 组小鼠中性粒细胞吞噬率分别为 ( 4 0 .6 4± 4.85 ) ,( 5 3.45±5 .39) ,( 5 1 .0 7± 3.84) ,对照组为 ( 2 9.81±2 .5 3) ;口服 TF的 、 、 组小鼠巨噬细胞吞噬率分别为 ( 5 7.71± 2 .80 ) ,( 6 7.8±3.38) ,( 80 .39± 2 .30 ) ,对照组为 ( 4 6 .1 6±2 .6 8)。注射、口服 TF组小鼠脾脏重量与对照组比较 ,无明显差异 ( P >0 .0 5 )     

Failure of Lipopolysaccharides to Directly Trigger the Chemiluminescence Response of Isolated Equine Polymorphonuclear Leukocytes

Divergent results have been reported on the effects of lipopolysaccharides (LPS) on the activation of equine polymorphonuclear leukocytes (PMN). We therefore attempted to determine whether LPS alone can stimulate equine PMN or whether plasma factors are necessary. PMN were isolated from citrated blood on a discontinuous density gradient of Percoll. The luminol (10-3 mol/L)-enhanced chemiluminescence (CL) of 1.25 x 106 cells was measured after addition of Escherichia coli LPS (0.001-10 µg/ml) alone or after incubation in autologous plasma (1 h, 37°C). After direct stimulation with LPS, there were random variations of CL in 16 horses that were not reproducible from one sample to the next for the same horse. LPS which had been incubated in plasma gave a dose-dependent stimulation of the CL of the PMN, which did not occur if the plasma had been heat inactivated (1 h, 56°C). These results indicated a role for plasma factors, which were unlikely to be cytokines, as there were no monocytes or lymphocytes in the plasma incubated with LPS, but might have been complement fragments or LPS ligands, such as LPS binding protein. Studies using specific antibodies against these factors are needed to clarify this question.     

Evaluation of immunomodulatory effect of recombinant human granulocyte‐macrophage colony‐stimulating factor on polymorphonuclear cell from dogs with cancer in vitro

The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre‐clinical model to evaluate the immunomodulatory effects of rhGM‐CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer‐related PMN dysfunction in dogs with cancer were restored following incubation with rhGM‐CSF including PMN phagocytosis, respiratory burst and LPS‐induced TNF‐α production. In addition, rhGM‐CSF increased surface HLA‐DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM‐CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM‐CSF as an immunomodulatory therapy in dogs with cancer.     

Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO.     

Bacterial cell wall components of Staphylococcus aureus induce endophthalmitis

AIM: To compare the responses of intraocular inflammation induced by heat-inactivated Staphylococcus aureus or its bacterial cell wall components in SD rats. METHODS: The rats were randomly divided into heat-inactivated bacteria (HIB) group (96 rats were injected with 10 μg of HIB), heat-inactivated bacteria fragments (HIBF) group (96 rats were injected with 10 μg of HIBF), peptidoglycan (PGN) group (96 rats were injected with 10 μg of PGN) and control group (96 rats were injected with sterile saline equivalent). At time points of 6 h, 12 h, 24 h, 48 h, 72 h and 5 d after vitreous injection of the pathogens, the ocular inflammation scores were determined under slit lamp microscope. The infiltration of white blood cells were counted in histological sections. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and cytokine induced neutrophil chemoattractant-1 (CINC-1) in serum and vitreous body were detected by ELISA, and protein concentration in aqueous humor were also measured. RESULTS: Severe ocular inflammation was observed in the animals of HIB, HIBF and PGN groups 6-72 h after injection. Five days after injection, the endophthalmitis subsided. The peak of intraocular white blood cell infiltration was observed 24 h after exposure to the bacteria and the components in each group and the cell infiltration rapidly declined after 3 days. The concentrations of TNF-α and IL-1β peaked at 24 h in each group, maintained to 48 h, then decreased rapidly, and returned to baseline level after 5 days. The concentration of CINC-1 peaked at 12 h in each group, and maintained to 24 h, then decreased rapidly, and returned to the normal level after 3 days. Significantly higher protein levels in aqueous humor were detected in the experimental groups at all time points as compared to that in control group (P<0.01). CONCLUSION: Staphylococcus aureus cells and its components induce typical experimental endophthalmitis in SD rats. Massive leukocyte infiltration and high levels of TNF-α, IL-1β and CINC-1 are the main pathological features in the experimental model. PGN and the bacterial cell wall fragments induce stronger intraocular inflammations than the whole heat-inactivated S. aureus.     

The effects of supplemental threonine on performance,carcass characteristics,immune response and gut health of broilers in subtropics during pre‐starter and starter period

Three hundred thirty‐day‐old unsexed commercial broiler chicks (Vencobb‐400) with initial average body weight of 44.04 ± 0.42 g were allocated into five experimental groups, in a completely randomized design (CRD) with 21‐day experiment. Groups were formed according to dose of supplemental L‐threonine in various rations i.e., 100% NRC specification, 100% threonine of Vencobb‐400 strain specification, 110% threonine of Vencobb‐400 strain specification, 120% of threonine of Vencobb‐400 strain specification and 130% threonine of Vencobb‐400 strain specification. Average daily feed intake (ADFI), average daily body weight gain (ADG), cumulative feed conversion ratio (CFCR), carcass characteristics, immune response, intestinal morphometry and biochemical profile were studied. The ADFI and ADG increased linearly and quadratically as dietary threonine levels were increased. However, the CFCR did not differ (p ? 0.05) among the groups. Though the carcass weight and drumstick yield did not differ (p ? 0.05) among the groups, the relative breast yield increased linearly (p = 0.007). The relative dressing yield and relative thigh weight increased linearly (p = 0.05 and p = 0.03, respectively). The relative weight of immune organs like bursa and thymus increased linearly. The mean total serum immunoglobulin, ND‐ELISA titre and the mean lymphocyte proliferation response index increased linearly, whereas mean phagocytic activity index of neutrophil increased linearly (p < 0.001) and quadratically (p = 0.001). The mean villus height (VH), crypt depth (CD), villus surface area and mean goblet cell number/villus increased linearly and quadratically, whereas the villus width (VW) and goblet cell density increased quadratically. The serum glucose increased linearly (p = 0.001), whereas serum total protein concentration and serum globulin level increased both linearly and quadratically. The albumin: globulin ratio tended to decrease linearly. There was a significant decrease (p < 0.05) in serum cholesterol and VLDL cholesterol level. However, a linear increment (p = 0.04) in the blood serum HDL cholesterol level with a linear reduction (p = 0.01) in the blood serum LDL cholesterol was noticed.     

Killing of Streptococcus uberis by bovine neutrophils following growth in chemically defined media

Following growth in a chemically defined medium (CDM), five strains of Streptococcus uberis were tested for their ability to survive killing by bovine neutrophils. Strains 0140J, ST10, EF20 and C221 were easily killed, whereas strain C197C was highly resistant. The ability of strain 0140J to resist phagocytosis and killing was increased by supplementation of the growth medium with milk whey, casaminoacids, casein, or, to a lesser extent, bovine serum. Supplementation of the growth medium with yeast extract or bovine serum albumin did not affect the resistance of this strain. Following growth in CDM supplemented with casein, strains ST10 and C221, like strain 0140J, were significantly more resistant to killing by neutrophils. The resistance of strains EF20 and C197C was unaffected by the addition of casein to the medium; strain EF20 remained susceptible and strain C197C highly resistant to killing. The effect of supplementing the growth media with components other than casein was only studied for strain 0140J. Decapsulation of strains C197C, ST10 and 0140J, grown in CDM + casein, with type-X hyaluronidase did not significantly affect their ability to survive in the presence of bovine neutrophils.     

Effects of ovariohysterectomy on canine blood neutrophil respiratory burst: a chemiluminescence study

OBJECTIVE: To examine blood neutrophil counts and luminol-enhanced chemiluminescence (CL) responses in dogs undergoing ovariohysterectomy (OH), premedicated with 2 different drugs. STUDY DESIGN: Randomized clinical study. ANIMALS: Forty-two healthy client-owned bitches. METHODS: Dogs had OH under isoflurane anesthesia with either acepromazine or medetomidine, both in combination with butorphanol, administered as preanesthetic medication. Blood samples were collected when the dog was admitted, at the end of surgery, and the next day (approximately 20 hours after surgery). Blood neutrophils were counted automatically, and neutrophil oxidative activity was assessed by measuring blood CL responses (induced by opsonized zymosan and enhanced by luminol) at 37 degrees C for 40 minutes. RESULTS: Number of circulating neutrophils was significantly increased the day after surgery reflected by enhanced blood CL responses. Neutrophil CL, however, was not significantly altered. No significant differences were detected for perioperative Polymorphonuclear neutrophil (PMN) characteristics between the 2 preanesthetic regimens. CONCLUSIONS: In conclusion, despite clearly increasing the number of circulating neutrophils, OH did not significantly affect neutrophil respiratory burst, as measured by whole-blood CL responses. CLINICAL RELEVANCE: Surgical operation of moderate intensity (e.g., OH) did not significantly alter one of the important immune functions, neutrophil oxidative activity. Further studies are warranted to confirm the significance of this finding, and to assess the value of following this variable in different animal patient populations.     

Combined phagocytosis-nitroblue tetrazolium reduction test in bovine neutrophils

An assay was developed for the simultaneous evaluation of phagocytosis and oxidative metabolism of bovine blood neutrophils. Phagocytosis was evaluated by using opsonized zymosan, and oxidative metabolism was evaluated by nitroblue tetrazolium (NBT) reduction. Normal bovine neutrophils exhibited moderate variation in ability to phagocytize zymosan, but little variation in ability to reduce NBT. The subcellular location of NBT reduction to formazan was determined by electron microscopy. Electron dense formazan precipitate was observed along the inner membrane of phagosomes enclosing zymosan particles and radiating from the membrane toward the center of the phagosome.