Effects of human recombinant granulocyte colony stimulating factor (HR-GCSF) on the hemogram of lactating dairy cattle

Mastitis commonly occurs in the dairy cow and results in an influx of granulocytes into the mammary gland. Presently, colony stimulating factors have been isolated. One factor, granulocyte colony stimulating factor (GCSF), has been identified and reproduced using recombinant DNA technologies. In an experiment, lactating bovine were given a bacterially-synthesized human recombinant granulocyctic colony stimulating factor (Hr-GCSF) at a specified dose rate to monitor and characterize their hematological responses. Doses of HR-GCSF were administered subcutaneously and blood samples collected from the tail vein into vacutainer tubes. Results of the study indicated a toleration by the bovine for the HR-GCSF for the tested period, and that the HR-GCSF can stimulate a sustained elevation of circulating neutrophils.     

鸡粒细胞巨噬细胞集落刺激因子-猪干扰素α1融合基因在大肠埃希菌中的可溶性表达及其生物学活性研究

为可溶性表达重组鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF)与猪干扰素α1(PoIFNα1)融合蛋白,并研究其生物学活性,本研究分别提取鸡、猪肝脏细胞总RNA,采用RT-PCR方法扩增ChGM-CSF和PoIFNα1基因,经linker连接上述两种基因后将其克隆于pET-32a原核表达载体,转化E.coliBL21(DE3)菌株进行诱导表达,SDS-PAGE和western blot检测融合蛋白表达产物。在ST细胞/VSV病毒测定系统以细胞病变抑制法滴定该融合蛋白的抗病毒活性;同时用MTT法检测其对鸡淋巴细胞增殖活性的促进作用。结果显示,PCR扩增并融合后的ChGM-CSF和PoIFNα1融合基因约为1000bp,构建重组表达载体后,诱导表达的rChGM-CSF-PoIFNα1融合蛋白分子量约55ku,主要存在于破碎菌体的上清中,表达量较高。Westernblot检测结果显示,该融合蛋白分别能够与ChGM-CSF多抗和PoIFNα单克隆抗体特异性结合,其抗病毒比活性约为1.1×10^6IU/mg,并且具有促进鸡淋巴细胞增殖的活性。本研究为rChGM-CSF-PoIFNα1重组融合蛋白的研制及其相关活性的测定提供实验依据。     

Hemogram changes in lactating dairy cows given human recombinant granulocyte colony stimulating factor (r-MethuG-CSF).

As a prelude to mammary gland challenge experiments, this investigation was implemented to assess the hematologic changes in lactating dairy cattle induced by two dosage regimes of human recombinant colony stimulating factor (Hr-GCSF). This study documents the capability of the human recombinant colony stimulating factor to produce hematologic changes in both a time and dose dependent manner when administered to the adult lactating bovine. A screening dose of 1 microgram/kg of Hr-GCSF administered to three study subjects produced a three- to four-fold increase in peripheral blood mature neutrophil counts (P less than 0.043) by day 12 of the trial. The priming dose treatment group of four lactating cows (3 micrograms/kg of Hr-GCSF) exhibited a three- to five-fold increase in peripheral blood mature neutrophil counts (P less than 0.05) and two- to three-fold increases in white blood cell counts by day 5 of the trial. Hematologic examinations of the control group (n = 4; no Hr-GCSF administration) did not detect significant changes in their neutrophil counts over baseline values. The milk somatic cell counts did not statistically shift over baseline values in any of the control or Hr-GCSF treatment groups. When attempting to alter the course of infectious disease processes, potential applications of colony stimulating factors provide interesting speculations about new therapeutic modalities.     

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.     

Expression and purification of canine granulocyte colony-stimulating factor (cG-CSF)

Canine granulocyte colony-stimulating factor (cG-CSF) with modification of cysteine at position 17 to serine was expressed in Brevibacillus choshinensis HPD31. cG-CSF secreted into the culture medium was purified by ammonium sulfate precipitation and consecutive column chromatography, using butyl sepharose and DEAE sepharose. Biological activity of the recombinant cG-CSF was 8.0 × 10⁶ U/mg protein, as determined by its stimulatory effect on NFS-60 cell proliferation. Purified cG-CSF was subcutaneously administered once a day for two successive days to dogs (1, 5, 25, or 125 μg). Neutrophil count increased the following day in all dogs except those administered the lowest dose (1 μg). No severe side effects were observed in dogs after administration of cG-CSF.     

The effect of neutrophils, tumor necrosis factor, and granulocyte macrophage/colony stimulating factor on Babesia bovis and Babesia bigemina in culture.

Bovine neutrophils, human recombinant tumor necrosis factor-alpha (TNF), and bovine recombinant granulocyte macrophage/colony stimulating factor (GM/CSF) were added to microaerophilic cultures of Babesia bovis and Babesia bigemina to determine if those substances could inhibit growth. Incorporation of [3H]hypoxanthine by the Babesia spp. was utilized as an indirect measure of parasite growth. When neutrophils were added to cultures of B. bovis and B. bigemina, the highest percentage inhibition of growth was attained. There was no significant enhancement of neutrophil killing when TNF or GM/CSF or both were added to either Babesia spp. Addition of TNF or GM/CSF or both substances (without neutrophils) resulted in an increase in growth of B. bovis and B. bigemina. For B. bovis, the group that contained neutrophils only and the group that contained neutrophils and TNF resulted in significantly higher growth inhibitions than the treatment group which contained neutrophils and GM/CSF or the group that contained neutrophils, TNF, and GM/CSF. No significant differences in inhibition were observed for the same treatment groups between B. bovis and B. bigemina.     

Hematologic effects of subcutaneous administration of recombinant human granulocyte colony-stimulating factor (filgrastim) in healthy alpacas

OBJECTIVE: To determine the effects of SC administration of filgrastim on cell counts in venous blood and bone marrow of healthy adult alpacas. ANIMALS: 10 healthy alpacas. PROCEDURES: Alpacas were randomly assigned to receive treatment with filgrastim (5 microg/kg, SC; n=5) or an equivalent volume of physiologic saline (0.9% NaCl) solution (5) once a day for 3 days. Blood samples were obtained via jugular venipuncture 1 day prior to treatment and once a day for 5 days commencing 24 hours after the first dose was administered. Complete blood counts were performed for each blood sample. Bone marrow aspirates were obtained from the sternum of each alpaca 48 hours before the first treatment was administered and 72 hours after the third treatment was administered. Myeloid-to-erythroid cell (M:E) ratio was determined via cytologic evaluation of bone marrow aspirates. RESULTS: In filgrastim-treated alpacas, substantial increases in counts of WBCs and neutrophils were detected within 24 hours after the first dose was administered. Band cell count and percentage significantly increased 24 hours after the second dose. Counts of WBCs, neutrophils, and band cells remained high 48 hours after the third dose. Red blood cell counts and PCV were unaffected. The M:E ratio also increased significantly after treatment with filgrastim. CONCLUSIONS AND CLINICAL RELEVANCE: Filgrastim induced rapid and substantial increases in numbers of circulating neutrophils and M:E ratios of bone marrow in healthy alpacas. Therefore, filgrastim may be useful in the treatment of camelids with impaired bone marrow function.     

Regulation of endometrial prostaglandin F(2alpha) synthesis during luteolysis and early pregnancy in cattle

Luteal regression is caused by a pulsatile release of prostaglandin (PG) F(2alpha) from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF(2alpha) secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF(2alpha), the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF(2alpha) secretion. It has been recently demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulates PGF(2alpha) output from bovine endometrial tissue not only during the follicular phase but also during the late luteal phase, suggesting that TNF-alpha is a factor in the initiation of luteolysis in cattle. Furthermore, our recent study has shown that IFN-tau suppresses the action of TNF-alpha on PGF(2alpha) synthesis by the bovine endometrium in vitro, suggesting that IFN-tau plays a luteoprotective role by inhibiting TNF-alpha-induced PGF(2alpha) production in early pregnancy. On the other hand, factors other than oxytocin or TNF-alpha have also been suggested to be involved in the regulation of PGF(2alpha) synthesis by bovine endometrium. The purpose of this review is to summarize our current understanding of the endocrine mechanisms that regulate the timing and pattern of uterine PGF(2alpha) secretion during the estrous cycle and early pregnancy.     

Regulation of adipose tissue metabolism during pregnancy and lactation in the ewe: the role of insulin

This study was conducted to examine the effect of insulin on lipid metabolism of adipocytes during pregnancy and lactation in ewes. During the first 3 mo of pregnancy, metabolism of adipocytes from omental adipose tissue was characterized by a high rate of de novo lipogenesis (90 to 125 nmol of acetate incorporated into lipids.2 h-1.10(6) cells-1) and a 38% reduction in response to beta-lipolytic stimulus (isoproterenol 10(-6) M). Simultaneously, there was a rise in the number of high-affinity insulin receptors (Kd = .2 nM), and insulin binding characteristics showed a decrease in the negative cooperativity phenomenon. Moreover, lipogenesis stimulated by insulin (1 mU/ml) increased in comparison with observations in nonpregnant ewes. The last third of pregnancy and early lactation were characterized by a marked fall in lipogenesis and a simultaneous increase in isoproterenol-stimulated lipolysis. During lactation, the number of total insulin receptors was decreased by 62% and insulin stimulation of lipogenesis became inefficient. Results suggest that insulin plays a direct role in adipose tissue metabolism during pregnancy.     

Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3×10⁶ U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.     

Genetic (co)variance components for ewe productivity traits in Katahdin sheep

The objective of this study was to estimate genetic parameters, in Katahdin sheep, for total weight of litter weaned per ewe lambing (TW) and its components, number of lambs born (NB), number of lambs weaned (NW), and average weight of lambs weaned (AW) measured as traits of the ewe. Weaning weights of lambs (WW) were adjusted to 60 d of age and for effects of ewe age, lamb sex, and type of birth and rearing and averaged over all lambs in the litter to obtain AW. The 60-d age-adjusted WW were adjusted for ewe age and lamb sex and summed over all lambs in the litter to obtain TW. A total of 2,995 NB and NW records, 2,622 AW, and 2,714 TW records were available from 1,549 ewes (progeny of 235 sires) over 4 yr. Heritabilities were initially estimated for each trait from univariate REML analyses. Estimates of genetic correlations were obtained from bi- and trivariate analyses. Models for NB, NW, AW, and TW included random ewe additive and permanent environmental effects. A random service sire effect was also fit for AW and TW. Heritabilities of TW, NB, NW, and AW from univariate analyses were 0.12, 0.12, 0.09, and 0.13 (all P < 0.01), respectively. Permanent environmental effects were significant (P < 0.01) for TW and AW. Genetic correlations of TW with NB, NW, and AW ranged from 0.27 to 0.33, 0.88 to 0.91, and 0.72 to 0.76, respectively; those of NB with NW and AW ranged from 0.70 to 0.75 and -0.01 to 0.02, respectively; and that between NW and AW ranged from 0.40 to 0.55. Genetic parameters were also obtained for lamb survival to weaning (LS) and WW measured as traits of the lamb, and the relationships between WW of the ewe as a lamb and her subsequent records for NB and NW were also estimated. A total of 5,107 LS and 5,444 WW records were available. Models for WW and LS included random animal and maternal genetic, maternal permanent environmental, and litter effects. Heritability of WW ranged from 0.15 to 0.20. There was no evidence of genetic effects on LS. Direct genetic correlations of WW with NB and NW were not significantly different from zero. The correlation between maternal genetic effects on WW, and animal genetic effects on NW, averaged 0.35. Results of this study indicate that there are no major antagonisms among TW and its components, so that selection for TW would not have adverse effects on any component traits and vice versa. Maternally superior ewes for WW appear to also be somewhat superior for NW.     

Epidermal tissue-derived T-cell growth factor, colony stimulating factor and nerve growth factor in chickens.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9-to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).     

Plasma granulocyte colony-stimulating factor concentrations in neutropenic, parvoviral enteritis-infected puppies

We evaluated the temporal relationship between neutrophil numbers and plasma granulocyte colony-stimulating factor (G-CSF) concentrations in dogs infected with canine parvovirus, a common infectious cause of neutropenia. G-CSF is produced in response to neutropenia, infection, or inflammation, and results in the production and release of neutrophils from the bone marrow. Adequate numbers of functional neutrophils are necessary for protection from infection, and the timely production of G-CSF is a crucial response to certain diseases. The relationship between peripheral neutrophil numbers and plasma G-CSF concentrations during the course of an infectious disease characterized by neutropenia has not been described previously in dogs. Eight mixed-breed puppies were given an oronasal challenge with canine parvovirus, and peripheral neutrophil numbers as well as plasma G-CSF concentrations were measured daily. G-CSF was not detectable in plasma of any dog before the onset of neutropenia, but G-CSF became detectable just after the onset of neutropenia in the 7 dogs that developed clinical illness. Neutropenia persisted or worsened for at least 2 days after plasma G-CSF became detectable in all 7 dogs. Neutrophil nadir, the highest plasma G-CSF concentrations, and the most severe clinical illness occurred concurrently in most dogs. Although 1 dog died while still neutropenic, plasma G-CSF concentrations declined before resolution of neutropenia in the other 6 dogs, and were again below the limits of detection in 5 of the 6 dogs at the time of resolution.     

猪粒细胞-巨噬细胞集落刺激因子的原核表达和纯化

粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)是一种具有多种生物学活性的细胞因子。本试验根据GenBank发表的猪的GM-CSF基因序列设计特异性引物,对GM-CSF基因片段进行扩增,扩增后的PCR产物经酶切后克隆入pET-28a载体。重组质粒转化入大肠杆菌杆菌BL21中经IPTG诱导表达,SDS-PAGE结果证明,重组蛋白获得了成功表达,并且主要以包涵体的形式存在。Western blot结果证明重组蛋白具有良好免疫反应性。以上结果为进一步应用重组GM-CSF作为血液干细胞动员剂提供了基础。