"Prostate cancer is far more hidden ": Perceptions of stigma, social isolation and help-seeking among men with prostate cancer.

The purpose of this study was to provide in-depth insight into men's experiences of prostate cancer, specifically: perceived stigma and self-blame, social isolation, unmet need and help-seeking. A qualitative descriptive approach was used. Semi-structured interviews were undertaken with 20 men diagnosed with prostate cancer, and thematic analysis was undertaken. Some participants perceived a stigma associated with prostate cancer and cancer in general, which sometimes acted as a barrier to disclosure. Self-blame and internalisation of cause was not a prominent issue. Participants' descriptions of emotional distress, social isolation and anxiety demonstrated the impact of prostate cancer. Social isolation was most commonly reported as a physical consequence of treatment and/or side effects. Participants felt both support and ongoing care were limited at post-treatment. Most did not seek or receive help for emotional or psychosocial problems from a formal source due to anticipated awkwardness, autonomous coping, not burdening others, unwanted sympathy and retaining privacy. Prostate cancer can cause considerable emotional and social burden for some men, and many are unlikely to seek or receive help. Men, and their support networks, require active encouragement throughout diagnosis, treatment and follow-up to overcome barriers and access additional support, particularly for sexual, emotional and psychosocial issues.

A novel, validated method to quantify breast cancer-related lymphedema (BCRL) following bilateral breast surgery.

We sought to develop a formula to quantify breast cancer-related lymphedema (BCRL) after bilateral breast surgery, which functions independently of the contralateral arm and accounts for fluctuations in patient weight. Perometer arm measurements from 265 unilateral breast surgery patients were analyzed. We assessed the relationship between change in patient weight and contralateral arm volume and developed a weight-adjusted volume change formula (WAC). The WAC formula and previously-established RVC formula were compared for classification of BCRL (> or = 10% volume increase) in unilateral breast surgery patients. We then evaluated BCRL incidence using the WAC formula in 225 bilateral mastectomy patients. Change in patient weight and contralateral arm volume demonstrated an approximately linear relationship. Weight-adjusted arm volume change (WAC) was therefore calculated as WAC = (A2*W1)/(W2*A1) - 1 where A1 is pre-operative and A2 is post-operative arm volume, and W1, W2 are the patient's corresponding weights. In the unilateral analysis, there was no significant difference in number of patients classified as having BCRL using the RVC and WAC formulas (p = 0.65). In bilateral mastectomy patients 11.1% (25/225) developed BCRL, defined as > or = 10% WAC. Independent risk factors for lymphedema included axillary lymph node dissection (ALND) and higher pre-operative BMI (p<0.05). Use of this weight-adjusted arm volume change formula should be of value for quantification of BCRL after bilateral breast surgery.

Expression of nogo-a is decreased with increasing gestational age in the human fetal brain.

Nogo is a member of the reticulon family. Our understanding of the physiological functions of the Nogo-A protein has grown over the last few years, and this molecule is now recognized as one of the most important axonal regrowth inhibitors present in central nervous system (CNS) myelin. Nogo-A plays other important roles in nervous system development, epilepsy, vascular physiology, muscle pathology, stroke, inflammation, and CNS tumors. Since the exact role of Nogo-A protein in human brain development is still poorly understood, we studied its cellular and regional distribution by immunohistochemistry in the frontal lobe of 30 human fetal brains. Nogo-A was expressed in the following cortical zones: ependyma, ventricular zone, subventricular zone, intermediate zone, subplate, cortical plate, and marginal zone. The number of positive cells decreased significantly with increasing gestational age in the subplate and marginal zone. Using different antibodies, changes in isoform expression and dimerization states could be shown between various cortical zones. The results demonstrate a significant change in the expression of Nogo-A during the development of the human brain. The effects of its time- and region-specific regulation have to be further studied in detail.

Comparison of perometry-based volumetric arm measurements and bioimpedance spectroscopy for early identification of lymphedema in a prospectively-screened cohort of breast cancer patients.

Breast cancer-related lymphedema (BCRL) affects more than one in five women treated for breast cancer, and women remain at lifelong risk. Screening for BCRL is recommended by several national and international organizations for women at risk of BCRL, and multiple methods of objective screening measurement exist. The goal of this study was to compare the use of perometry and bioimpedance spectroscopy (BIS) for early identification of BCRL in a cohort of 138 prospectivelyscreened patients. At each screening visit, a patient's relative volume change (RVC) from perometer measurements and change in L-Dex from baseline (ΔL-Dex) using BIS was calculated. There was a negligible correlation between RVC and ΔL-Dex (r=0.195). Multiple thresholds of BCRL were examined: RVC ≥5% and ≥10% as well as and ΔL-Dex ≥6.5 and ≥10. While some patients developed an elevated RVC and ΔL-Dex, many demonstrated elevations in only one threshold category. Moreover, the majority of patients with RVC ≥5%, ΔL-Dex ≥6.5, or ΔL-Dex ≥10 regressed to non-elevated measurements without intervention. These findings suggest a role for combining multiple screening methods for early identification of BCRL; furthermore, BCRL diagnosis must incorporate patient symptoms and clinical evaluation with objective measurements obtained from techniques such as perometry and bioimpedance spectroscopy.

Using a quitline plus low-cost nicotine replacement therapy to help disadvantaged smokers to quit.

OBJECTIVES: To trial an intervention in a real-life setting to motivate low-income smokers to try to quit. The intervention under trial was the addition of subsidised nicotine replacement therapy (NRT) to a standard population quitline service. DESIGN: Participants were low-income smokers, recruited "cold" via either a letter in the mail or a flyer inserted in a local newspaper. The intervention group received the usual service of multisession counselling from the quitline plus access to heavily subsidised NRT. A comparison group received the usual quitline service only. Participants were followed up at 3, 6, and 12 months. Trial participants were also compared with a sample of general callers to the quitline. RESULTS: The offer of subsidised NRT recruited more than twice as many low-income smokers than the offer of the cessation service alone (intervention group n = 1000; comparison group n = 377). 63% were first-time callers to the quitline. Intervention respondents showed higher levels of nicotine dependence than comparison group respondents. Comparisons of quitting data were confounded by the differences in the respondents at baseline. 73.5% of smokers in the intervention group tried to quit compared to 61.0% in the comparison group. Unadjusted quit rates were higher in the intervention group than in the comparison group at 3 months and 6 months but not at 12 months. CONCLUSIONS: Disadvantaged smokers were easily engaged to call the quitline, particularly when offered subsidised NRT. Disadvantaged smokers using the quitline, with and without subsidised NRT, achieved cessation outcomes comparable to other studies of "mainstream" smokers.

Impact on the Australian Quitline of new graphic cigarette pack warnings including the Quitline number.

BACKGROUND: In March 2006, Australia introduced graphic pictorial warnings on cigarette packets. For the first time, packs include the Quitline number. OBJECTIVE: To measure the combined effect of graphic cigarette pack warnings and printing the Quitline number on packs on calls to the Australian Quitline service. METHODS: Calls to the Australian Quitline were monitored over 4 years, 2 years before and after the new packets were introduced. RESULTS: There were twice as many calls to the Quitline in 2006 (the year of introduction), as there were in each of the preceding 2 years. The observed increase in calls exceeds that explained by the accompanying television advertising alone. While call volume tapered back in 2007, it remained at a level higher than before the introduction of new packets. No change was observed in the proportion of first time callers. CONCLUSION: Introducing graphic cigarette packet warnings and the Quitline number on cigarette packets boosts demand for Quitline services, with likely flow on effects to cessation.

Changes in lymphocyte activity after thermal injury. The role of suppressor cells.

The high incidence of fatal septicemia associated with severe thermal injury is believed to result from a loss of immunocompetence. To detect burn-mediated immune defects, lymphocyte function in peripheral blood leukocytes from 18 individuals sustaining 20-80% full thickness thermal burns was investigated. We examined the kinetics of the mitogen responses, the development of suppressive activity, and the correlation of mononuclear cell functional abnormalities with the incidence of sepsis. Patients were divided into three groups corresponding to their clinical course. The phytohemagglutinin responses of Ficoll-Hypaque purified leukocytes from eight of these patients (group III) were normal at day 1-2 after injury, but were significantly depressed (mean 16% of normal) at days 5-10 after injury. All of these group III patients experienced multiple, severe, septic episodes, and septic mortality was 75%. The other 10 burned individuals showed either augmented (group II) or unaltered (group I) mitogen responsiveness. Concomitant with evaluation of their mitogen responses, the cells of burn patients were assessed for development of suppressive activity by addition to on-going normal mixed leukocyte reactions (MLR). Only the addition of mononuclear cells with depressed phytohemagglutinin responsiveness (group III) significantly decreased MLR proliferation (mean 80% reduction) by the previously highly responsive, normal MLR combinations. Addition of cells from group III burn patients collected immediately after injury had no suppressive effect. Addition of cells from patients in group I or II or of normal individual's cells had no suppressive effect. These experimental results strongly suggest that a suppressive mononuclear cell is at least partially responsible for the decreased immunocompetence of burn patients.

Quality control for microarray analysis of human brain samples: The impact of postmortem factors, RNA characteristics, and histopathology.

The quality of results from microarray studies depends on RNA quality, which can be significantly influenced by postmortem factors. The aim of this study was to determine which postmortem factors and/or RNA electropherogram characteristics best correspond to microarray output and can be used to prospectively screen RNA prior to microarray analysis. Total RNA was extracted (N=125) from gray and white matter of postmortem frontal and occipital lobe tissue, acquired from normal controls, and patients with schizophrenia, bipolar disorder or major depression. Electropherograms were generated by the Agilent BioAnalyzer 2100, allowing calculation of the 28S/18S ratio, the 18S/baseline peak ratio and the RNA Integrity Number (RIN). These values were compared to post-hybridization image analysis of Affymetrix microarrays. The postmortem variables correlated with some quality measures but could not be used as effective screening tools. Logistic regression demonstrated that all three electropherogram measures were predictive for microarray quality, and that the RIN threshold predictive of "good quality" (>35% present calls) was most consistent with that of prior studies. The optimal RIN must be determined by the investigator's specifications for false inclusion and false exclusion. In contrast to RIN, the quality threshold for the 28S/18S ratio has proven unacceptably variable, due to sensitivity to slight differences in protocol and/or tissue source. In conclusion, the measures we found useful as screening criteria do not replace the need to exclude samples after a microarray analysis is performed, as an acceptable percent call rate and other measures of microarray quality represent the desired endpoint.

An activation-dependent, T-lymphocyte-specific transcriptional activator in the mouse mammary tumor virus env gene.

Transcription of the complete mouse mammary tumor virus (MMTV) proviral genome in mouse cells is controlled by a strong promoter in its long terminal repeat. In the mouse T lymphoma EL4, there is a second, activation-dependent transcriptional initiation site within the envelope (env) gene, from which a short mRNA is generated, encoding the open reading frame of the long terminal repeat. We now report the isolation of a segment of the MMTV env gene (called META, for MMTV env transcriptional activator) which has the expected transcription-activating properties seen in EL4.E1 cells. Namely, it induces activation-dependent, T-lymphocyte-specific transcription of a chloramphenicol acetyltransferase reporter gene. It is active in mouse or human T-helper lymphocyte lines when they are stimulated to transcribe lymphokine genes but is inactive in unstimulated T-helper cells, fibroblasts, a cytotoxic T-lymphocyte line, and a mastocytoma cell line. Its activity is inhibited by cyclosporin A, a specific inhibitor of lymphokine transcription. Several forms of the META have been isolated from EL4.E1 cells, a mouse T-helper cell hybridoma, and from BALB/c spleen cells. Linked to the heterologous thymidine kinase promoter, a 400-bp portion of it is an inducible, orientation-independent, and cyclosporin A-sensitive transcriptional activator in T-helper cells.

Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

Effect of H-7 and Lat-B on retinal physiology.

PURPOSE: To investigate the effects of H-7 and Latrunculin B (Lat-B) on retinal vascular permeability and electrophysiology at concentrations that increase outflow facility in monkeys. METHODS: One eye of 1 rhesus and 22 cynomolgus monkeys received an intravitreal bolus injection of H-7 or Lat-B; the opposite eye received vehicle. Multifocal electroretinograms (mfERGs), and photopic and scotopic full-field electroretinograms (ffERGs, sERGs) were recorded in subsets of monkeys at baseline and at multiple time-points post-H-7 or Lat-B. Vitreous fluorophotometry (VF) and fluorescein angiography (FA) were also performed. RESULTS: No differences between the H-7 or Lat-B treated and control eyes were found in ffERGs, mfERGs, sERGs, or in FAs in any monkey. No significant difference was found in vitreous fluorescein levels between H-7 treated or Lat-B treated vs. control eyes. CONCLUSIONS: No effect on retinal vascular permeability or retinal electrophysiology was apparent after intravitreal administration of H-7 or Lat-B at doses that increase outflow facility and lower IOP when given intracamerally.

In vivo-in vitro correlation of myelotoxicity of 9-methoxypyrazoloacridine (NSC-366140, PD115934) to myeloid and erythroid hematopoietic progenitors from human, murine, and canine marrow.

BACKGROUND: 9-Methoxypyrazoloacridine (PZA) is an anticancer agent that shows selectivity of action for carcinomas over leukemias. It also has nearly equal potency against cycling and quiescent or hypoxic and normoxic target cells. Phase I trials of PZA in humans are nearing completion. PURPOSE: This study was conducted to determine (a) if PZA is directly inhibitory to hematopoietic cells and, if it is, to characterize the inhibition pharmacodynamically, (b) whether species-specific differences in direct toxicity could explain differences in myelosuppression in mice, dogs, and humans, and (c) whether in vitro data correlate with in vivo myelosuppression data. METHODS: In vitro clonogenic assays of hematopoietic progenitors of myeloid and erythroid lineages from human, canine, and murine femoral marrow were used to measure the direct toxicity of PZA. Results from these assays were compared on an area-under-the-curve (AUC) basis to clinical myelosuppression data. RESULTS: On the basis of maximum tolerated concentrations, canine hematopoietic progenitors are most susceptible to PZA, followed by human and then murine progenitors. We found no difference in susceptibility to PZA toxicity between the human progenitors of myeloid and erythroid lineages. Both concentration and duration of exposure contribute to the in vitro toxicity of PZA. In contrast to antimetabolites, the in vitro toxicity of PZA could be minimized at a given AUC by lowering drug concentration and prolonging the period of exposure. On an AUC basis, the in vitro data are consistent with limited in vivo myelosuppression data from preclinical models and correlate with neutropenia data from a phase I trial. CONCLUSIONS: PZA directly inhibits hematopoietic progenitors, an action that is responsible for the myelosuppression observed in humans. Human marrow appears able to compensate for the loss of up to 35% of its myeloid progenitors, in that peripheral neutrophil counts remain unchanged at that level of loss. Although in vivo studies show that prolonged infusion reduces myelosuppression at a given total dose in both rodent and canine models, pharmacokinetic differences make it unlikely that this approach will benefit human patients. IMPLICATIONS: The in vitro data quantitatively predict the AUCs at maximum tolerated dose in preclinical models and human patients. Thus, in vitro clonogenic assays of myelotoxic agents can provide data that make both preclinical toxicology testing and clinical trial planning and interpretation more efficient and accurate.

Effects of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide, 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, and L-phenylalanine mustard on B16, Cloudman S91, and Harding-Passey mouse melanomas.

The effects of 5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU), and L-phenylalanine mustard (L-PAM) have been compared by using three i.p. transplanted mouse melanomas: the B16 melanoma in C57BL/6 mice; the Harding-Passey (HP) malanoma in BALB/c X DBA/2F1 (hereafter called CD2F1) mice; and the Cloudman S91 melanoma in DBA/2 mice. HP melanoma responds well to all three drugs. S91 responds only to L-PAM and MeCCNU. DTIC may accelerate death in mice bearing this tumor. B16 responds well to L-PAM and moderately well to MeCCNU and to multiple injections of DTIC. The best response to DTIC and MeCCNU is given by HP, while the best response to L-PAM is given by S91. Tumor cell-doubling times were found to be 1.5 days for B16, 2 DAYS FOR HP, and 3 days for S91. HP would seem to be the most responsive malanmoma with respect to the 3 agents studied. This may be due to an interaction between the chemotherapeutic agents and the host immune response, since the HP tumor arose in a noninbred mouse and is thus nonsyngeneic with the CD2F1 host. All three tumors appear to be interesting biological models for studying drug combinations and combined therapeutic modalities against melanoma.

Regulation of Epstein-Barr virus latency by latent membrane protein 2.

Like other herpesviruses, Epstein-Barr virus persists in its host through its ability to establish a latent infection that periodically reactivates. Latent membrane protein 2A (LMP2A) regulates reactivation from latency by interfering with normal B cell signal transduction processes, and may define a new class of regulators of herpesvirus latency.

Sodium transport in turtle erythrocytes. Apparent stimulation of exchange diffusion by anaerobiosis.

Studies were performed on Na and K transport by red blood cells of the freshwater turtle under anaerobic and aerobic conditions. Although it had previously been assumed that cation transport in turtle red blood cells was dependent on respiration, the present data show greater Na efflux rates in N(2) than in O(2). However, ouabain inhibited Na transport by the same amount quantitatively in O(2) and N(2) gas phases. Thus there was no difference in ouabain-sensitive or "pump" Na transport rates. Na influx rates were higher in nitrogen than in air and potassium influx rates were not significantly different under aerobic and anaerobic conditions. Moreover in the absence of sodium in the bathing medium no difference between air and nitrogen could be discovered. Finally with ethacrynic acid plus ouabain there was an additional decrease in Na efflux but there was a persisting difference between air and nitrogen. These studies do not rule out the existence of a ouabain-insensitive ethacrynic acid-inhibitable flux; however, they suggest that at least part of the activation of Na efflux observed in N(2) was due to increased exchange diffusion.

HIMeg-1, a cell line derived from a CML patient, is capable of monocytic and megakaryocytic differentiation.

We have characterized HIMeg-1, a subclone of the promegakaryoblastic cell line HIMeg, in terms of its capability of proliferation and differentiation when it is exposed to various agents. We observed that phorbol 12-myristate 13-acetate (PMA) arrested HIMeg-1 growth and induced expression of monocytic surface antigens CD11c and CD14, but not the megakaryocytic surface antigen CD14a. In addition, PMA treatment of HIMeg-1 led to rapid activation of mRNA expression of egr-1, a transcription factor involved in regulating differentiation of hematopoietic progenitor cells. On the other hand, treatment of HIMeg-1 with the activated peripheral blood lymphocyte-conditioned medium (PBL-CM) resulted in greatly enhanced incorporation of 3H-thymidine into newly synthesized DNA. This enhanced 3H-thymidine incorporation appears to be specific to HIMeg-1 since the same concentrations of PBL-CM had little effect on the growth of the megakaryoblastic leukemia cell line SAM-1. The PBL-CM-induced DNA synthesis in HIMeg-1 was associated with activation of CD41a and CD41b surface antigen expression and down-regulation of expression of the erythroid marker glycophorin A and the early myeloid surface antigen CD33. HIMeg-1 capable of responding differentially to PMA and PBL-CM by changing its growth rate as well as its differentiation patterns will provide an ideal model to study the underlying mechanism regulating lineage restriction of hematopoietic progenitor cells.

Antigen presentation by the CD4 positive monocyte subset.

Although CD4 antigen is expressed on monocytes (MO), its functional role is uncharacterized. In this study, isolated human MO were separated into CD4+ and CD4- MO subsets and assessed for presentation of tetanus toxoid. The CD4- MO subset had decreased antigen presenting cell (APC) capacity as well as increased PGE2 production when compared to the CD4+ MO subset. Addition of a cyclo-oxygenase inhibitor (Indomethacin) did not restore the CD4- MO subset's APC capacity to that of the similarly treated CD4+ MO subset, eliminating differential PGE2 production as the primary cause of differential APC capacity. Production of monokines such as IL-1 and plasminogen activator, which affect APC capacity, was similar in the CD4 MO subsets. However, tumor necrosis factor (TNF) production (IFN gamma plus MDP-induced) of the CD4+ MO subset was slightly greater than that of the CD4- MO. CD4- MO's lower APC capacity is not totally explained by their differential IL-1, TNF, or PGE2 production.

Induction of a proteoglycan core protein mRNA in mouse T lymphocytes.

The mouse T lymphocyte cell line EL4.E1 synthesizes a proteoglycan core protein (PGCP) mRNA which is identical to serglycin mRNA found in mouse bone marrow-derived mast cells and a mouse mastocytoma cell line. PGCP mRNA was strongly induced in EL4.E1 cells by phorbol myristate acetate, which also induces mRNAs for several cytokines in these cells. In contrast to the induction of cytokine mRNAs, however, the induction of PGCP mRNA was not inhibited by Cyclosporine. PGCP mRNA was also inducible by allogeneic stimulation of normal mouse spleen cells, and by Con A stimulation of an Interleukin 2-producing T hybridoma cell line. A number of other cell lines expressed an identical or similar, mRNA, including two cytotoxic T cell lines, and three tumor cell lines related to bone marrow-derived cells. The levels of several proteoglycans have previously been reported to increase in cells of bone marrow origin under activating conditions, but this appears to be the first report of an induction of the corresponding PGCP mRNA by immune stimulation of T lymphocytes.

Differential ability of flt3-ligand, interleukin-11, and Steel factor to support the generation of B cell progenitors and myeloid cells from primitive murine fetal liver cells.

A variety of factors produced by stromal fibroblasts, including Flt3-ligand (FL), interleukin-11 (IL-11), Steel factor (SF), and IL-7, have been implicated in stimulating the production of pre-B cells and myeloid cells from primitive hematopoietic precursors. To investigate their relative roles in this process, either as single-acting or synergistic agents, we compared the yield and types of cells produced after 2 weeks from small numbers of Sca-1+ Lin- (i.e., B220-, Ly-1-, Gr-1-, and Ter-119-) day 14.5 murine fetal liver cells placed in stromal cell-free cultures containing all possible combinations of FL, SF, IL-7, and IL-11. None of these factors alone supported the production (or survival) of any cells beyond 1 week: only pairs of factors consisting of either FL or SF plus either IL-11 or IL-7 were effective in this regard, with FL plus IL-11 being the most potent pair (approximately 7 x 10(4) cells obtained per 100 Sca-1+ Lin- input cells). The maximum numbers of cells were produced in the presence of FL, IL-11, and IL-7: these included both B220+ and Mac-1+/Gr-1+ cells (approximately 10(6) and approximately 2 x 10(5), respectively, per 100 Sca-1+ Lin- input cells). Both of these lineages were also obtained with each of the other possible three-factor combinations, albeit with variable effectiveness. Omission of either FL or IL-7 caused the greatest reduction in the yield of B220+ cells (approximately 130-fold and approximately 80-fold, respectively). Omission of IL-11 and, to a lesser extent, FL caused the greatest reduction in the yield of Mac-1+/Gr-1+ cells (approximately 90-fold and approximately 3-fold, respectively). When fetal calf serum was replaced with a defined serum substitute, the out put of B220+ cells remained the same but myelopoiesis was consistently enhanced (approximately 5- to 20-fold). These findings support a model involving factor redundancy in the extracellular signals required to stimulate the production and amplification of both lymphoid and myeloid cells from early Sca-1+ Lin- cells. They also reveal quantitative differences in the abilities of different competent factor combinations to promote this process, which may be further modulated by the presence of undefined serum components.

Purification of GCT cell-derived human colony-stimulating factors.

We have purified human-active colony-stimulating factors from the human cell line, GCT, using sequential ultrafiltration; cation exchange, gel permeation, and reverse-phase high performance liquid chromatography (RHPLC); and ion-exchange HPLC. Activity eluted from sodium dodecyl sulfate-polyacrylamide gels with a peak at 17,500 daltons. Similar results were obtained by processing 35S-methionine-labeled conditioned medium, which showed a labeled band in the same region of activity. This purified HPLC fraction, which had a specific activity of greater than 1 x 10(7) colonies/mg protein, stimulated neutrophil colonies at day 7 and neutrophil, neutrophil-macrophage, and eosinophil colonies at day 14 of culture, suggesting that it contained both granulocyte (G-CSF) and granulocyte-macrophage (GM-CSF) colony-stimulating factors. It also promoted the growth of erythroid progenitors, and the GM-CSF fraction purified by hydrophobic chromatography had erythroid-enhancing activity. Separation of G-CSF from GM-CSF was accomplished by the addition of trifluoroacetic acid to the mobile phase at the reverse-phase HPLC step.