Bovine Granulocyte Colony-stimulating Factor Research Articles

Stabilization of proteins by recombinant human gelatins

Porcine gelatins have been widely used as stabilizers of macromolecular based pharmaceuticals but the mechanism by which they stabilize has not been precisely established. Their variability and immunogenicity, however, make them less than ideal excipients. In this work, we take advantage of the availability of recombinant human gelatins (rhGs) to explore the mechanism by which they may stabilize proteins. Three model recombinant proteins, human serum albumin (HSA), bovine granulocyte colony stimulating factor (bGCSF), and human fibroblast growth factor-20 (FGF-20) that display a range of isoionic points have been selected for this study. The interaction of these model proteins with four different molecular weight rhGs and porcine gelatin was studied using a variety of biophysical techniques including fluorescence, CD and second derivative UV spectroscopy to monitor tertiary and secondary structure as a function of temperature. The 8.5, 25, and 100 kDa rhGs had the greatest effect on conformational and colloidal stability of HSA. The 8.5, 25, and 50 kDa rhGs also increased the T(0) of aggregation of bGCSF and FGF-20. Experiments to probe the mechanism of interaction of model proteins with rhGs suggest that the rhGs might interact with the partially unfolded states of target proteins through a combination of electrostatic and other intermolecular mechanisms to inhibit aggregation.

Nonnative Protein Polymers: Structure, Morphology, and Relation to Nucleation and Growth

Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼10 3–10 5 kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight ( M w). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation ( τ n) and growth ( τ g), as well as the stoichiometry of the nucleus ( x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τ n, τ g, and x for soluble (chain polymer) aggregates, as well as the relationship between τ n/ τ g and aggregate M w.

Effect of Bovine Granulocyte Colony-Stimulating Factor on the Development of Pneumonia Caused by Mannheimia haemolytica

The role of recruited neutrophils in Mannheimia haemolytica infection is controversial. We hypothesized that the neutrophilia induced by recombinant bovine granulocyte colony-stimulating factor (GCSF) would lead to rapid bacterial clearance and less severe lesions after infection with M. haemolytica. Two experiments (A and B) were conducted in which four calves per experiment were treated daily with 5 microg/kg GCSF and four calves per experiment were treated with saline. All 16 calves were challenged with 5 x 10(9) colony-forming units (cfu)/ml (experiment A) or 4.5 x 10(8) cfu/ml (experiment B) of M. haemolytica bacteria, into the right bronchus by bronchoscope-placed catheter. The mean maximal blood neutrophil counts in non-GCSF-treated and GCSF-treated calves before bacterial challenge were 5.6 +/- 0.7 x 10(9)/liter and 25.4 +/- 2.7 x 10(9)/liter, respectively. Two untreated calves became neutropenic and were euthanatized 2 days after infection because of severe respiratory distress. GCSF-treated calves had a 37% reduction in lung lesions compared with nontreated calves, and this difference was significant (P=0.04) when the effect of previous antibody titre to leukotoxin was considered. The effect of GCSF treatment on the severity of clinical signs seemed to be influenced by the antibody titre to M. haemolytica leukotoxin, although this effect could not be conclusively addressed. In conclusion, GCSF induced neutrophilia and partially protected calves against experimental infection with M. haemolytica. These results imply that increased numbers of neutrophils may, under some circumstances, protect against severe pneumonia caused by M. haemolytica.

Structural Characterization of Bovine Granulocyte Colony Stimulating Factor: Effect of Temperature and pH

The protein bovine granulocyte colony stimulating factor (bGCSF) was studied in solution as a function of pH (2–7) and temperature (10°–90°C) using fluorescence, circular dichroism, and Fourier transform infrared spectroscopies, as well as differential scanning calorimetry and optical density as a measurement of aggregation. bGCSF possesses significant conformational lability under the solution conditions examined. Under all pH conditions examined, a major conformational change is observed as a function of temperature at 50°–60°C, although the magnitude and precise temperature at which this occurs varies with pH. Three major conformations are adopted with changing pH. One is observed at pH 2 and 3, a second at pH 4, and a third at pH 5–7. At low pH (2–3), bGCSF adopts a molten globule-like conformation at moderate temperatures (25°–45°C), whereas at pH 4 the protein appears to form a non-molten globule extended conformation. The use of this type of study as complementary data for protein phase diagram development as well as the relationship between the conformational lability demonstrated by bGCSF and that observed for recombinant human granulocyte colony stimulating factor and other similar cytokines is discussed. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association.

Derivative Absorbance Spectroscopy and Protein Phase Diagrams as Tools for Comprehensive Protein Characterization: A bGCSF Case Study

In protein and macromolecule pharmaceutical formulation development, the amount of information initially gathered about a drug's physical and chemical properties under different conditions is often quite limited. This generally requires more intensive studies using a variety of techniques if problems arise later in the development process. We propose a supplementary approach involving a comprehensive examination of a protein by derivative absorbance spectroscopy in conjunction with other methods and the subsequent construction of a phase diagram that permits the determination of optimal formulation conditions. Using bovine granulocyte colony stimulating factor (bGCSF) as a model protein, a thorough characterization is performed using high-resolution second-derivative absorbance spectroscopy. Derivative ultraviolet absorbance data are used to construct an empirical phase diagram for bGCSF using a multidimensional phase space approach. Between pH 2 and 7, and from 10° to 90°C, bGCSF is found to adopt more than six distinct structural phases. Surprisingly, the combination of the phase diagram approach with derivative absorbance data identifies phase boundaries that are not apparent upon initial examination of complementary biophysical data (previous article in this issue). The simplicity and pharmaceutical utility of this approach are discussed. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association.

High level expression, purification, and in vivo activity of bovine granulocyte-colony stimulating factor produced using a baculovirus system

A bovine granulocyte-colony stimulating factor (bG-CSF) cDNA clone bearing a C-terminal poly-His-tag (bG-CSFHis) was constructed and expressed by the baculovirus expression system. The bG-CSFHis was expressed as an approximately 19 kDa protein in the culture supernatants and was purified using a nickel chelate column. The purified bG-CSFHis had bioactivity in vitro in the NFS-60 bioassay. In order to evaluate activity in vivo, purified bG-CSFHis was administered to cattle as single or multiple dosages. The bG-CSFHis increased neutrophil counts in peripheral blood and modulated the phagocytic activity of the neutrophils. The data indicates that the recombinant protein had activity in vivo.

Irreversible aggregation of recombinant bovine granulocyte-colony stimulating factor (bG-CSF) and implications for predicting protein shelf life

The kinetics of irreversible aggregation of bovine Granulocyte-Colony Stimulating Factor (bG-CSF) in solution were investigated as a function of temperature (T), concentration, and pH, and analyzed in terms of an Extended Lumry-Eyring model of protein aggregation proceeding via a non-native conformational state. In the spirit of classic Lumry-Eyring models, the observed kinetics are separated into contributions from thermodynamic or conformational stability of unaggregated native and non-native states, and the intrinsic aggregation kinetics of non-native molecules. It is found that a detailed treatment of the intrinsic kinetics coupled with a two-state approximation of the reversible unfolding transition is sufficient to allow quantitative prediction of low-T stability from high-T data despite highly non-Arrhenius kinetics. Accounting for shifts in conformational equilibrium quantitatively captures the non-Arrhenius T dependence, without requiring the assumption of a change in the rate-determining step with T. From a more general perspective, the observed aggregation behavior of bG-CSF is consistent with the rate-determining step being aggregation at T below a crossover temperature Tx that is inversely related to initial protein concentration. Above Tx, irreversible unfolding is presumably the rate-determining step. The results illustrate that protein aggregation kinetics can, in principle, be predicted quantitatively from so-called accelerated data provided the thermodynamic and kinetic components can be separately extrapolated to longer term storage conditions.

Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening.

Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by high-dose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25-34 residues were completed, corresponding to 10(21)-10(28) sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10-14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13 degrees C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool.

Aggregation of recombinant bovine granulocyte colony stimulating factor in solution.

Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50 degrees C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50 degrees C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.

Expression, purification, and in vitro biological activities of recombinant bovine granulocyte-colony stimulating factor

Neutrophils are essential components of the innate immune system and they play a critical role in the defense of host against bacterial and fungal infections. The colony stimulating factors are a class of glycoproteins that are required for proliferation, differentiation, and functional activation of hematopoietic progenitor cells. Granulocyte-colony stimulating factor (G-CSF) is a member of this regulatory family of cytokines that specifically stimulates proliferation and maturation of precursor cells in the bone marrow into fully differentiated and functional neutrophils. G-CSF also modulates the biological activities of mature neutrophils in circulation. A bovine G-CSF (bG-CSF) cDNA clone (previously isolated and sequenced in our laboratory) was expressed in Escherichia coli and the biological activities of the solubilized protein from purified inclusion bodies were examined. Flow cytometric analysis of membrane antigen density of neutrophils activated with bG-CSF revealed an upregulation in the expression of CD11a (>114%), CD11b (>148%), CD11c (>87%), and CD18 (>109%). Expression of l-selectin was decreased by more than 43%. There was no change, however, in the expression of CD14. These findings indicate that recombinant bG-CSF (rbG-CSF) expressed in E. coli is biologically active and exerts the same type of effects on neutrophils in vitro as those of human G-CSF (hG-CSF).

Sustained In Vivo Activity of Recombinant Bovine Granulocyte Colony Stimulating Factor (rbG-CSF) Using HEPES Buffer

The purpose of this study was to develop a long-acting injectable formulation of bG-CSF for veterinary use. However, in order to achieve sustained in vivo activity it was first necessary to stabilize the protein at the injection site. Preformulation studies, as well as literature, suggest that bG-CSF aggregates at neutral pH ranges (i.e., pH 6–8) and at temperatures of ∼40°C. Therefore, bG-CSF will not retain its activity for an extended period of time at the injection site. During this study we determined that HEPES buffer has a very significant impact on protein stability as well as on biological performance.Recombinant bovine granulocyte colony stimulating factor (rbG-CSF) was formulated in 1 M HEPES buffer for subcutaneous injection into cows. bG-CSF formulated in 1 M HEPES buffer resulted in sustained in vivo activity of bG-CSF compared to the “control” formulation (control formulation: 5% mannitol, 10 mM acetate buffer, 0.004% tween-80, pH 4). White blood cell (WBC) count was used as a marker to evaluate in vivo activity of the formulation. WBC numbers remained above a threshold value for only 24–30 h for the control formula. However, when bG-CSF was formulated in 1 M HEPES, the WBC remained above threshold for 3 days or 72 h. Formulating bG-CSF in 1 M HEPES at pH 7.5 also resulted in greater solution stability. This was surprising since bG-CSF is intrinsically not stable at neutral pH. The effect of 1 M HEPES on the TM (temperature at maximum heat flow on calorimetry scan) of bG-CSF was determined by microcalorimetry. In the absence of 1 M HEPES buffer the TM was 48°C (onset ∼40°C), while bG-CSF formulated in 1 M HEPES buffer has a TM of 59°C (onset ∼50°C). Similar organic buffers, such as MOPS, HEPPS, TES, and tricine, also resulted in improved solution stability as well as in sustained in vivo activity. The dramatic effect of these buffers on stability and biological performance of bG-CSF is not well understood. One hypothesis is that the electrostatic interaction between the zwitterionic form of these buffers and bG-CSF provides stabilization against denaturation.

Cloning, sequencing, and analysis of cDNA encoding bovine granulocyte-colony stimulating factor

Neutrophils play a critical role in defending against bacterial infections. Hematopoietic growth factors are a class of regulatory cytokines that are required for stimulation, proliferation, and differentiation of blood cells. Granulocyte colony stimulating factor (G-CSF) is a cytokine that induces proliferation and maturation of precursor myeloid cells in the bone marrow into fully differentiated neutrophils. G-CSF also modulates the functional activity of mature neutrophils. Treatment with G-CSF significantly enhances neutrophil phagocytic activity and killing of bacteria and fungi. We have isolated and sequenced a cDNA clone encoding bovine G-CSF (bG-CSF) from an endothelial cell cDNA library using primers designed from ovine G-CSF. The full length cDNA is 1460 nucleotides with 585 nucleotides comprising the open reading frame. Sequence analysis shows 95% identity with ovine, 89% with porcine, 85% with human, and 76% with murine G-CSF. The deduced G-CSF protein consists of 174 amino acids with 95% identity to ovine, 86% to porcine, 81% to human, and 71% to murine. The signal peptide of G-CSF is 21 amino acids long which is nine amino acids shorter than that of human and murine G-CSF. RT-PCR analysis shows that neither freshly isolated nor ConA stimulated neutrophils express G-CSF mRNA. Mononuclear cells, however, expressed G-CSF mRNA after 48 h incubation with or without ConA stimulation.

1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling.

Interleukin 6 (IL-6) has many biological activities in vivo, and deregulation has been implicated in many disease processes. IL-6, a 185 amino acid polypeptide was refolded, purified and crystallized. The crystals diffracted to beyond 1.9 A and the structure was solved using single isomorphous replacement. The X-ray structure of IL-6 is composed of a four helix bundle linked by loops and an additional mini-helix. 157 out of 185 residues are well defined in the final structure, with 18 N-terminal and 8 A-B loop amino acids displaying no interpretable electron density. The three-dimensional structure has been used to construct a model of IL-6 interacting with the IL-6 receptor (alpha-chain) and gp130 (beta-chain) that gives new insight into the process of molecular recognition and signaling. Based on this model, we predict a fourth binding site on IL-6, a low affinity IL-6-IL-6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL-6 receptor complex.

Linkage analysis assignment of bovine granulocyte colony stimulating factor receptor (G-CSFR) to chromosome 3.

Linkage analysis assignment of bovine granulocyte colony stimulating factor receptor (G-CSFR) to chromosome 3.

Monomeric bovine IgG2 is a potent stimulus for bovine neutrophils

Bovine plasma contains factors that can stimulate bovine neutrophils. Bovine plasma at 1:1,000,000-1:1,000 dilution induced a dose-dependent superoxide production in bovine neutrophils. When bovine plasma was fractionated with a protein G column, only the IgG fraction contained induction activity. At similar concentrations purified IgG induced a much stronger response than that of plasma IgG. Purified monomeric bovine IgG induced a dose-dependent increase in superoxide production. The maximum induction can be achieved at 100 micrograms/ml of bovine IgG. When subclasses of bovine IgG were examined, monomeric bovine IgG2 potently stimulated bovine polymorphonuclear leukocytes. In contrast, bovine IgG 1 failed to induce a response at similar concentrations, and neither bovine IgG F(ab')2 and Fc were effective. Both recombinant bovine granulocyte-macrophage colony-stimulating factor (r-BoGM-CSF) and recombinant bovine granulocyte colony-stimulating factor (r-BoG-CSF) primed bovine neutrophils for superoxide production induced by bovine IgG. The above results suggest that: (1) bovine plasma contain factors that can activate bovine neutrophils; (2) bovine plasma IgG is the major component that is responsible for bovine neutrophil activation; (3) bovine plasma contains factors that can inhibit the effect of bovine IgG; (4) monomeric bovine IgG2, but not IgG1, can activate bovine neutrophils directly without Fc receptor cross-linkage; (5) the integrity of bovine IgG is important in bovine polymorphonuclear leukocyte activation; and (6) bovine neutrophil activation induced by bovine IgG can be primed by r-BoGM-CSF or r-BoG-CSF.

Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation.

Interleukin-6 (IL-6) drives the sequential assembly of a receptor complex formed by the IL-6 receptor (IL-6R alpha) and the signal transducing subunit, gp130. A model of human IL-6 (hIL-6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL-6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL-6 and gp130 and therefore essential for IL-6 signal transduction, were subjected to site-directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL-6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild-type affinity towards hIL-6-R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild-type IL-6.

Crystal Structure of Canine and Bovine Granulocyte-Colony Stimulating Factor (G-CSF)

The crystal structures of recombinant canine and bovine granulocyte colony stimulating factor (G-CSF) have been determined by X-ray crystallography, using molecular replacement with recombinant human G-CSF as a model. G-CSF is a member of the cytokine family of glycoproteins that stimulate the differentiation and proliferation of blood cells. Human, bovine and canine G-CSF all have a molecular mass of about 19 kDa and share an amino acid sequence identity of about 80%. Two crystal forms of canine G-CSF have been solved. Form I recombinant canine G-CSF (rcG-CSFI; space group C2) contains one molecule in the asymmetric unit while form II canine G-CSF (rcG-CSFII; space group P21) has two molecules in the asymmetric unit and bovine G-CSF (rbG-CSF; space group P 212121) contains one molecule in the asymmetric unit. rcG-CSFI has been refined to an R factor of 20·7% with data to 2·3 Å resolution and rcG-CSFII has been refined to an R factor of 19·3% with data to 2·2 Å resolution. rbG-CSF has been refined to an R factor of 21·3% with data to 1·7 Å resolution. The structure of human, canine and bovine G-CSF is an antiparallel 4-α-helical bundle with up-up-down-down connectivity. With the exception of one highly exposed loop (residues 66 to 74), the human, canine and bovine structures are very similar to each other. Using our series of G-CSF crystal structures we developed a function that describes the probability that a particular residue position (i) contributes to a G-CSF receptor binding site based on two principles, (1) high sequence conservation in the primary sequence of human, bovine, canine and murine G-CSF and (2) conservation of high solvent accessibility in the human, bovine and canine crystal structures. On the basis of this probability function as well as a comparison of G-CSF to the crystal structure of human growth hormone (hGH) complexed with the extracellular domain of the human growth hormone receptor (hGHbp), residues that contribute to potential G-CSF receptor binding sites are identified.

Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors.

Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid:erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.

Granulocyte Colony-Stimulating Factor Effects on Lymphocytes and Immunoglobulin Concentrations in Periparturient Cows

Immunomodulatory effects of recombinant bovine granulocyte colony-stimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony-stimulating factor (5μg/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG1, conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bovine granulocyte colony-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to alleviate immunosuppression in periparturient cows.

Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period

To determine if periparturient immunosuppression in dairy cattle might be due to an alteration in total numbers or percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks prepartum through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCD8, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4+ cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period.