Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo.

BACKGROUND: A variety of anticancer chemotherapeutics induce adverse side effects including myelotoxicity. Dried roots of Phragmites communis Trinius, Phragmitis rhizoma, have been clinically used in traditional folk medicine to relieve various symptoms like fever. In this study, we evaluated the protective effect of the aqueous extract of Phragmitis rhizoma (EPR) against docetaxel-induced myelotoxicity in vitro and in vivo. METHODS: The in vitro myelo-protective effect of EPR was evaluated using the colony forming unit (CFU) assay with hematopoietic progenitor cells. The in vivo efficacy of EPR was evaluated in myelosuppressed C57BL/6 male mice which were induced by repeated intraperitoneal injections of 30 mg/kg docetaxel for 3 times. EPR was orally administered for 4 days to docetaxel-induced myelosuppressed C57BL/6 male mice which were induced by intraperitoneal injection of 30 mg/kg docetaxel for 3 times: Group 1 (vehicle control, n = 10), Group 2 (docetaxel plus vehicle, n = 10), Group 3 (docetaxel plus EPR 30 mg/kg, n = 10), Group 4 (docetaxel plus EPR 100 mg/kg, n = 10) and Group 5 (docetaxel plus EPR 300 mg/kg, n = 10). Whole blood counts were measured automatically, and immune organs were histologically examined. Expression of immunomodulatory cytokines was measured by quantitative real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The toxicity of EPR itself was evaluated in normal human cell lines including IMR-90, foreskin fibroblast and human umbilical vein endothelial cells. The hepatotoxicity of EPR was predicted by multi-parametric assays involving cell viability, caspase 3/7 activity, GSH contents and LDH leakage using the HepaRG hepatic cell line. RESULTS: Co-treatment of EPR or its major component, p-hydroxycinnamic acid, increased the numbers of hematopoietic CFU counts in the docetaxel-induced in vitro myelotoxicity assay system. The in vitro protective effect of EPR against docetaxel toxicity was replicated in a myelosuppressed animal model: white blood cells, neutrophils, lymphocytes and red blood cells rebounded; bone marrow niche and structural integrity of the thymus were preserved; and the expression of immune-stimulating cytokines including IL3, IL6, SCF and GM-CSF was enhanced. Furthermore, EPR and p-hydroxycinnamic acid promoted the proliferation of primary splenocytes and thymocytes. In the toxicity assays, no remarkable signs related with toxicity were observed in all tested normal human cells and HepaRG. CONCLUSIONS: EPR has the potential to ameliorate docetaxel-mediated myelotoxicity in both in vitro and in vivo models. However, the identification of the responsible active components and the precise underlying myelo-protective mechanism of EPR need to be elucidated before novel drug development using EPR can precede.

In-Depth Analysis of the Hidradenitis Suppurativa Serum Proteome Identifies Distinct Inflammatory Subtypes.

Hidradenitis suppurativa is a chronic inflammatory dermatosis with presentations ranging from painful nodules and abscesses to draining tunnels. Using an unbiased proteomics approach, we assessed cardiovascular-, cardiometabolic-, and inflammation-related biomarkers in the serum of patients with moderate-to-severe hidradenitis suppurativa. The serum of patients with hidradenitis suppurativa clustered separately from that of healthy controls and had an upregulation of neutrophil-related markers (Cathepsin D, IL-17A, CXCL1). Patients with histologically diagnosed dermal tunnels had higher serum lipocalin-2 levels compared with those without tunnels. Consistent with this, patients with tunnels had a more neutrophilic-rich serum signature, marked by Cathepsin D, IL-17A, and IL-17D alterations. There was a significant serum‒skin correlation between proteins in the serum and the corresponding mRNA expression in skin biopsies, with healthy-appearing perilesional skin demonstrating a significant correlation with neutrophil-related proteins in the serum. CSF3 mRNA levels in lesional skin significantly correlated with neutrophil-related proteins in the serum, suggesting that CFS3 in the skin may be a driver of neutrophilic inflammation. Clinical significantly correlated with the levels of lipocalin-2 and IL-17A in the serum. Using an unbiased, large-scale proteomic approach, we demonstrate that hidradenitis suppurativa is a systemic neutrophilic dermatosis, with a specific molecular signature associated with the presence of dermal tunnels.

Chlorella vulgaris modulates immunomyelopoietic activity and enhances the resistance of tumor-bearing mice.

We studied the effects of Chlorella vulgaris (CV) on the interaction between stromal and hematopoietic stem cells in normal and Ehrlich ascites tumor (EAT)-bearing mice. Long-term bone marrow culture (LTBMC), cytokine production, spleen mononuclear cells (SMC) proliferation (SCP), colony stimulating activity (CSA), and NK cells activity were evaluated. In tumor bearers, reduced capacity of stromal cell layer to support the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM), concomitantly to decreased numbers of total nonadherent cells in LTBMC and reduced local production of IL-6 and IL-1α, were observed. Presence of the tumor has not altered the number of stromal adherent cells. CV treatment restored the ability of stromal cells from EAT-bearing mice to produce IL-6 and IL-1α, which was consistent with increased number of nonadherent cells and higher ability to display CFU-GM in vitro. EAT growth increased SCP, serum CSA, and IL-10 production and concurrently depressed NK cell activity and the secretion of IL-2, IFN-γ, and TNF-α. Treatment of tumor-bearing mice with CV augmented CSA, SMC proliferation, NK cell activity, and the production of IL-2, IFN-γ, and TNF-α, whereas IL-10 levels where reduced. Our results suggest that CV modulates immunehematopoietic cell activity and disengages tumor-induced suppression of these responses.

Differences in Serum Biomarkers Between Combined Glucosamine and Chondroitin Versus Celecoxib in a Randomized, Double-blind Trial in Osteoarthritis Patients.

BACKGROUND: Non-steroidal anti-inflammatory drugs, e.g., celecoxib, are commonly used for inflammatory conditions, but can be associated with adverse effects. Combined glucosamine hydrochloride plus chondroitin sulfate (GH+CS) are commonly used for joint pain and have no known adverse effects. Evidence from in vitro, animal and human studies suggest that GH+CS have anti-inflammatory activity, among other mechanisms of action. OBJECTIVE: We evaluated the effects of GH+CS versus celecoxib on a panel of 20 serum proteins involved in inflammation and other metabolic pathways. METHODS: Samples were from a randomized, parallel, double-blind trial of pharmaceutical grade 1500 mg GH + 1200 mg CS (n=96) versus 200 mg celecoxib daily (n=93) for 6- months in knee osteoarthritis (OA) patients. Linear mixed models adjusted for age, sex, body mass index, baseline serum protein values, and rescue medicine use assessed the intervention effects of each treatment arm adjusting for multiple testing. RESULTS: All serum proteins except WNT16 were lower after treatment with GH+CS, while about half increased after celecoxib. Serum IL-6 was significantly reduced (by 9%, P=0.001) after GH+CS, and satisfied the FDR<0.05 threshold. CCL20, CSF3, and WNT16 increased after celecoxib (by 7%, 9% and 9%, respectively, P<0.05), but these serum proteins were no longer statistically significant after controlling for multiple testing. CONCLUSION: The results of this study using samples from a previously conducted trial in OA patients, demonstrate that GH+CS reduces circulating IL-6, an inflammatory cytokine, but is otherwise comparable to celecoxib with regard to effects on other circulating protein biomarkers.

Hematopoietic response of rats exposed to the impact of an acute psychophysiological stressor on responsiveness to an in vivo challenge with Listeria monocytogenes: modulation by Chlorella vulgaris prophylactic treatment.

In this study, we investigated the hematopoietic response of rats pretreated with CV and exposed to the impact of acute escapable, inescapable or psychogenical stress on responsiveness to an in vivo challenge with Listeria monocytogenes. No consistent changes were observed after exposure to escapable footshock. Conversely, the impact of uncontrollable stress (inescapable and psychogenical) was manifested by an early onset and increased severity and duration of myelossuppression produced by the infection. Small size CFU-GM colonies and increased numbers of clusters were observed, concurrently to a greater expansion in the more mature population of bone marrow granulocytes. No differences were observed between the responses of both uncontrollable stress regimens. CV prevented the myelossuppression caused by stress/infection due to increased numbers of CFU-GM in the bone marrow. Colonies of cells tightly packed, with a very condensed nucleus; in association with a greater expansion in the more immature population of bone marrow granulocytes were observed. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected/stressed rats treated with the algae. CV treatment restored/enhanced the changes produced by stress/infection in total and differential bone marrow and peripheral cells counts. Further studies demonstrated that INF-gamma is significantly reduced, whereas IL-10 is significantly increased after exposure to uncontrollable stress. Treatment with CV significantly increased INF-gamma levels and diminished the levels of IL-10. Uncontrollable stress reduced the protection afforded by CV to a lethal dose of L. monocytogenes, with survival rates being reduced from (50%) in infected rats to 20% in infected/stressed rats. All together, our results suggest Chlorella treatment as an effective tool for the prophylaxis of post-stress myelossupression, including the detrimental effect of stress on the course and outcome of infections.

Comparative studies of the effects of Tabebuia avellanedae bark extract and beta-lapachone on the hematopoietic response of tumour-bearing mice.

The effects of Tabebuia avellanedae (TACE), traditionally prescribed in the treatment of cancer, and the naphtoquinone beta-lapachone (beta-lap) on the growth and differentiation of granulocyte and macrophage progenitor cells (CFU-GM) were studied in Ehrlich ascites tumour-bearing mice. Myelosuppression concomitant with increases in spleen CFU-GM and in serum colony-stimulating activity (CSA) were observed in these animals. Treatment with TACE (30-500 mg/kg) and beta-lap (1-5mg/kg) reversed these effects in a dose-dependent manner. The optimal biologically active doses of 120 mg/kg TACE and 1mg/kg beta-lap prolonged life span of tumour-bearing mice, both producing the same rate of extension in the duration of survival. Toxic manifestations were produced by the higher doses of beta-lap in normal and tumour-bearing mice. In spite of similarities between treatments, TACE concentrations used to treat the animals presented no traces of beta-lap, as measured by TLC and HPLC analyses. Our findings suggest that the antitumour effect of TACE and beta-lap, acting synergistically with other factors, such as specific cytokines, may result from enhanced macrophage activation against tumour cells. In addition, it is clear from our results that hematopoietic disorders produced by tumours are an important pathological condition that must be considered in drug development.

Use of colony-stimulating factors for chemotherapy-associated neutropenia: review of current guidelines.

Chemotherapy-associated neutropenia is often dose-limiting and may compromise treatment efficacy. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are increasingly used to prevent febrile neutropenia (FN) or to increase dose-density. This review discusses recent changes in treatment guidelines for chemotherapy-associated neutropenia. Primary prophylactic use of CSFs is now recommended as a treatment option at an overall risk of FN of 20%, not taking into account cost-effectiveness. To estimate the risk of FN, patient-, disease-, and treatment-related factors predicting an adverse outcome of FN have been determined. Dose-dense chemotherapy has become feasible with the use of CSFs. However, clinical benefit has been shown only for specific chemotherapy regimens in breast cancer, small cell lung cancer (SCLC), and non-Hodgkin's lymphoma (NHL), for the latter particularly for patients above 60 years of age. Strategies are being developed to tailor the use of CSFs to patients with a high risk of adverse outcome of FN.

Purification and partial characterization of a megakaryocyte colony-stimulating factor from human plasma.

Human plasma obtained from patients with hypomegakaryocytic thrombocytopenia contains a factor that promotes megakaryocyte colony formation by normal human marrow cells. This megakaryocyte colony-stimulating factor was purified from such a plasma specimen. A four-step purification scheme which included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, affinity chromatography on wheat germ lectin-Sepharose 6MB, and reverse-phase high performance liquid chromatography resulted in a recovery of 16.6% of the initial biological activity and an increase in specific activity by 3,489-fold. The purified protein produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified megakaryocyte colony-stimulating factor was capable of promoting megakaryocyte colony formation at a concentration of 7.6 X 10(-8) M. Megakaryocyte colony-stimulating factor was shown to be a glycoprotein and had an apparent 46,000 mol wt. Deglycosylation of megakaryocyte colony-stimulating factor by treatment with trifluoromethane-sulfonate resulted in the loss of its ability to promote megakaryocyte colony formation. Megakaryocyte colony-stimulating factor appears to be an important regulator of in vitro human megakaryocytopoiesis at the level of the colony-forming unit megakaryocyte and may be of importance physiologically.

Long-term exposure to retrovirally expressed granulocyte-colony-stimulating factor induces a nonneoplastic granulocytic and progenitor cell hyperplasia without tissue damage in mice.

Murine marrow cells infected with a retroviral vector (MPZen) bearing a granulocyte-colony-stimulating factor (G-CSF) cDNA insert were transplanted into lethally irradiated recipients to study the effects of autocrine production of G-CSF in normal hemopoietic cells. Most animals remained healthy with no evidence of tissue damage throughout the observation period (4-30 wk) despite high circulating G-CSF levels (range 2,000-26,000,000 U/ml). A dramatic neutrophilic granulocytosis was observed in all hemopoietic tissues with neutrophilic infiltration occurring in the lung and liver. Spleen, peritoneal, and peripheral blood cellularity increased approximately three-, two-, and eightfold, respectively, but total bone marrow cell counts remained unchanged. Progenitor cell numbers granulocyte-macrophage colony-forming cell (GM-CFC), granulocyte colony-forming cell (G-CFC), burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E) and mixed colony-forming cells (Mix-CFC) were elevated between 10-100-fold in the spleen, peritoneal cavity, and peripheral blood, but were unaffected or slightly depressed in the marrow. No tumors developed in syngeneic recipients transplanted with bone marrow or spleen cells from such mice, confirming the nonneoplastic nature of the hyperplasia induced by chronic G-CSF stimulation. These experiments also indicated the stable integration of MPZen vectors in infected cells, as evident from the continuous expression of the inserted gene for at least 6 mo, and from the ability of infected stem cells from the primary recipients to express the gene in lethally irradiated secondary recipients.

Hemopoietic colony growth-promoting activities in the plasma of bone marrow transplant recipients.

Plasma samples were obtained from 34 bone marrow transplant (BMT) recipients before and after administration of the preparative regimen and tested for their ability to promote and/or support growth of hemopoietic colonies. The ability of plasma samples to promote colony formation on their own was tested on normal nonadherent target cells without addition of exogenous growth factors. The growth-supporting activity was examined in the presence of medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) and/or erythropoietin (EPO). A series of kinetic changes was routinely observed. Pretransplant samples rarely gave rise to colonies without addition of exogenous growth factors. Plasma samples obtained after completion of the preparative regimen demonstrated increments of growth-promoting activities for megakaryocyte and granulocyte-macrophage progenitors (CFU-Meg and CFU-GM), respectively, that peaked between 7 and 21 d after transplantation. By day 30, activity levels of some patients had returned to pretransplant values, whereas in other patients, activities remained elevated. Persisting activity levels were associated with delayed engraftment. In contrast, activities for progenitors committed to erythropoiesis (BFU-E) and pluripotent precursors (CFU-GEMM) were only rarely observed. The activities were independent of febrile episodes. Their growth-promoting influence on CFU-GM could be neutralized completely by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies. These data suggest that at least some of the observed activities in post-BMT plasma are related to GM-CSF. The growth-supporting activities of pretransplant plasma samples are lower than normal plasma when tested on CFU-Meg and CFU-GM. The growth-supporting activities improved transiently within the first month after BMT. A decline during the second and third month was followed by a gradual return to activity levels that were comparable to normal plasma. The effects of these plasma samples on BFU-E and CFU-GEMM were assessed with PHA-LCM and EPO. Similar to CFU-Meg- and CFU-GM-supporting capabilities, they improved transiently after BMT with a return of normal support function after 5-6 mo. The observed endogenous production of growth-promoting and growth-supporting activities for hemopoietic progenitors may serve as a background to design clinical trials for the timely administration of recombinant hemopoietic growth factors to BMT recipients.

Early hematopoietic events during tumor growth in mice.

Lewis lung carcinoma (LLC) of C57BL/6 mice, a transplantable tumor widely metastatic by 6-7 days post implant (PI), caused hematopoietic alterations such as progressive anemia (hemoglobin: day 1 PI, 11.0 g/dl; day 19 PI, 7.8 g/dl), neutrophilia (neutrophils: day 1 PI, 2 X 10(3)/microliter; day 19 PI, 22 X 10(3)/microliter), and marrow and splenic myeloid hyperplasia (marrow myeloid-to-erythroid ratio: day 1 PI, 1:1; day 7 PI, 3:1). Accompanying these changes were an increased concentration of marrow granulocyte-macrophage colony-forming units (culture) (GM-CFUC) (day 3 PI, LLC 185 +/- 27% of control; day 19 PI, LLC 265 +/- 10% of control) and accelerated cycling of these myeloid progenitors [day 3 PI, LLC 45.3 +/- 6.5% GM-CFUC in cycle vs. sham (media) injected 17.5 +/- 10.5%; day 7 PI, LLC 52.2 +/- 2.5% vs. sham (media) injected 29.8 +/- 9.8%; day 11 PI, LLC 56.2 +/- 4.4% vs. sham (media) injected 22.2 +/- 14%]. This study questioned whether enhanced hematopoiesis was a result of progressive tumor growth or whether the injection of tumor cells could evoke the response. By use of groups of C57BL/6 mice given an injection of live LLC cells, x-irradiated killed LLC cells, or media, the hematopoietic response to live LLC cells versus dead LLC cells could be dissected. A biphasic colony-stimulating activity (CSA) response in the sera of tumor bearers was found to account for the myelopoietic changes. The first wave of CSA from days 1 to 3 PI stimulated 168 +/- 3.7% more GM-CFUC than control sera and was likely released by dead cells of the tumor inoculum; the second wave from day 7 onward stimulated 220 +/- 7.6% more colonies and was a result of the enlarging tumor mass. Tumor growth was necessary for GM-CFUC proliferation, and the declining growth fraction at day 19 in LLC-bearing mice suggested that hematopoietic exhaustion was a consequence of tumor growth.

Bone marrow colony-forming cells in mice with virus-induced lymphoid leukemia: relation to serum colony-stimulating activity and blood granulocytes.

Mice with advanced lymphoid leukemia have elevated peripheral blood granulocytes and elevated serum colony-stimulating activity, which promotes the in vitro growth of granulocyte and/or macrophage colonies. The number of bone marrow precursor cells of the in vitro granulocyte and/or macrophage colonies varied from normal to 10% of normal. The elevation of colony-stimulating activity correlated best with a combination of increased blood granulocytes and a deficiency of bone marrow precursor cells, which suggested that colony-stimulating activity is a leukopoietin that increases the efficiency and rate of production of granulocytes.

Serum inhibitor activity of granulocyte-macrophage colony formation in patients with cancer.

The serum inhibitor activities of granulocyte-macrophage colony formation were evaluated by the in vitro culture technique in 60 patients with cancer and control subjects including 24 normal adults and 27 patients with a variety of nonneoplastic disorders. The inhibitor activity in cancer patients (mean, 59%) was significantly higher (p less than 0.001) than was that in normal adults (mean, 31%) and patients with nonneoplastic diseases (mean 36%). There was no difference in the inhibitor activity between the latter two groups of subjects. There was no correlation between the serum inhibitor activity in cancer patients and the histological type or primary site of tumor, the estimated duration of extent of disease, and serum albumin levels. Preliminary observations indicated that the inhibitor activity may be associated with serum lipoproteins. There was no significant difference in serum colony-stimulating activity among cancer patients, normal subjects, and patients with nonneoplastic diseases.

Induction of colony-stimulating activity in mice by injection of liposomes containing lipophilic muramyl peptide derivatives.

Colony-stimulating factor (CSF) activity induction by lipophilic derivatives of three muramyl peptides, glyceryl dipalmitate-MDP derivatives, was studied in vivo and in vitro and compared to the activity of the same compounds incorporated within freeze-dried liposomes. Two lipophilic derivatives (MDP-GDP and MDPGBe-GDP) were able to induce CSF activity in vivo and in vitro. The incorporation of these compounds within appropriately designed liposomes composed of distearoylphosphatidylcholine and phosphatidylserine (DSPC/PS) increased their ability to induce CSF activity in vivo but completely abrogated their ability to induce CSF activity in vitro. Furthermore, the phospholipid composition of liposomes influenced the efficacy of glycopeptide liposomal incorporation. Thus, the serum CSF-inducing effect of MDP-GDP was considerably enhanced by incorporation of this compound within liposomes composed of DSPC/PS at a molar ratio 7:0.3 but was not modified if the DSPC/PS molar ratio was 7:3. The lipophilic derivative of MDP (D-D), MDP (D-D)-GDP, was unable to induce CSF activity in vivo or in vitro but surprisingly became active in vivo after entrapment within DSPC/PS liposomes (molar ratio 7:0.3). Our results show that appropriate liposomes may be suitable carriers to deliver CSF activity-inducing agents to macrophages in vivo.

Altered toxicity of 5-fluorouracil following treatment with Corynebacterium parvum.

Recent studies have demonstrated that systemic Corynebacterium parvum increases serum granulocyte-macrophage colony-stimulating factor and stimulates the proliferation of granulocyte-macrophage progenitor cells. It was hypothesized that more rapid cycling of granulocyte-macrophage progenitor cells would render the cells more sensitive to a cell cycle-specific chemotherapeutic agent. The colony-forming ability of bone marrow granulocyte-macrophage progenitor cells was assayed in vitro with soft agar cultures. C. parvum given before 5-fluorouracil in C57BL/6 mice increased the granulocyte-macrophage progenitor cell toxicity, the lymphopenic effect, and the lethality of 5-fluorouracil. When C. parvum was given after 5-fluorouracil, there was more rapid rebound of granulocyte counts to normal or supranormal levels.

Leishmania donovani amastigote component-induced colony-stimulating factor production by macrophages: modulation by morphine.

The neuroimmunomodulatory effects of opiates during microbial infections are now well known; however, not much is known during leishmaniasis. Here, we report the effects of morphine on purified approximately 12-kDa component of Leishmania donovani amastigote antigen (LDAA-12)-induced colony-stimulating factor (CSF) production by mouse peritoneal macrophages (PMs) in vitro. Low concentrations (1 x 10(-9) and 1 x 10(-11) M) of morphine significantly (P < 0.05) augmented the production of CSFs, whereas high concentrations (1 x 10(-3) and 1 x 10(-5) M) inhibited CSF production. Morphine exerted a similar concentration-dependent biphasic effect on the LDAA-12-induced elaboration of granulocyte (G)-macrophage (M)-CSF (GM-CSF) and M-CSF by PMs in their conditioned medium, as quantified by using enzyme-linked immunosorbent assay. Furthermore, selective agonists of mu-(DAGO) and delta-(DPDPE) opioid receptors also, respectively, augmented and inhibited the production of CSFs. Pretreatment of PMs with naloxone (1 x 10(-5) M) significantly (P < 0.05) blocked the augmenting effect of morphine. In contrast, at 1 x 10(-5) M, naloxone lacked any effect on the inhibitory effect of morphine; however, its 100-fold higher concentration partially blocked it. This study, apparently for the first time, demonstrates that morphine, via surface opioid receptors, biphasically modulates the LDAA-12-induced CSF production by PMs, in vitro. These results thus show the implications of opiate abuse on the outcome of therapeutic interventions in areas where both visceral leishmaniasis and drug abuse are rampant.

A myelosclerotic syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor (LIF).

DBA/2 mice engrafted with FDC-P1 cells producing high levels of the leukemia-inhibitory factor (LIF) developed high circulating levels of LIF and a fatal syndrome including the accumulation of excess osteoblasts in the marrow and new bone formation. The mice developed a neutrophil leucocytosis, an enlarged spleen, and excess numbers of hemopoietic cells in the spleen and liver. Marrow cellularity was reduced with selective survival of granulocytic cells, but the frequency of hemopoietic progenitor cells in both the marrow and spleen was higher than in control mice. Megakaryocyte numbers were reduced in marrows with pronounced sclerosis. The disease state may represent a useful model of myelosclerosis, but it remains to be established whether the hemopoietic abnormalities in these mice are direct effects of LIF or secondary changes following occlusion of the marrow by osteosclerotic tissue.

Production of hematopoietic regulatory factors in cultures of adult and fetal mouse organs: measurement by specific bioassays.

Factor-specific cell line bioassays were used to monitor the production in vitro by adult and fetal mouse organs of GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3), IL-6 and leukemia inhibitory factor (LIF). No tissue was observed to produce Multi-CSF. Highest producers of the other regulators were lung, muscle, thymus, heart and bone shaft and all tissues producing detectable growth factors produced all five with the same rank order of activity. Adult tissues produced more GM-CSF than G-CSF and less M-CSF than either, no differences being observed between male, female and pregnant female tissues. In contrast, the pregnant uterus produced high levels of M-CSF as did the fetal membranes and tissues with only low GM-CSF and no G-CSF production. Pre-irradiation did not alter the pattern of regulator production by adult tissues. The intravenous injection of endotoxin elevated serum levels of GM-CSF, G-CSF, M-CSF and IL-6 but the dominant rise was in G-CSF levels. The data indicating that multiple organs produce the regulators monitored in a common rank order suggest some overall linkage in their production with major differences between adult and fetal tissues.

Leishmania donovani amastigote components-induced colony-stimulating factors production.

Increased hematopoiesis, driven by colony-stimulating factors (CSFs), is known to occur in infectious diseases. However, whether Leishmania donovani component(s) can directly induce the synthesis and secretion of CSFs is not known. We report that L. donovani amastigote antigens soluble in culture medium (LDAA; 0.01-10 mg/kg), injected intravenously in BALB/c mice, induced the production of serum CSFs; maximum induction (128>16 colonies) occurred at 1 mg/kg. In vitro also, LDAA (0.01-1 mg/ml) induced mouse peritoneal macrophages (MØs) to elaborate CSFs in the conditioned medium (CM); 0.1 mg/ml LDAA appeared optimal (68+/-9 colonies). Both in vivo and in vitro, the kinetics of CSF production were similar with peak response occurring 24 h after stimulation and return to background levels by 72 h. A predominant approximately 12 kDa LDAA protein (LDAA-12) also induced CSF production, both in serum and CM, in a dose-and time-dependent manner. Rabbit anti-LDAA-12 antibody significantly (p<0.05) reduced both the LDAA-and LDAA-12-induced CSF production, in vitro. Functionally, the LDAA-12-induced CSFs, both in the serum and CM, appeared to be similar as they supported the formation of granulocyte (G), MØ (M) and GM colonies, in vitro, in similar proportion; GM colonies were maximum (>80%). Further, LDAA-12 induced significantly (p<0.05) high GM-CSF levels both in serum and CM (19+/-3 and 15+/-2 ng/ml, respectively), as compared to the controls. Neutralizing (100%) goat anti-mouse tumour necrosis factor-alpha (TNF-alpha) immunoglobulin G did not affect the LDAA-12-induced CSF production by MØs, indicating it to be TNF-alpha-independent. LDAA-12 induced de novo CSF production, as MØs co-treated with LDAA-12 and cycloheximide (50 microg/ml) did not elaborate CSFs. The CSF-inducing capability of LDAA-12 appeared to be heat (70 C; 1 h)-labile, destroyed by proteases (pronase E and trypsin) and was unaffected by sodium periodate treatment. In LDAA-12-treated mice, the splenic and femur colony forming unit-GM counts showed a maximum of 2.2- and 1.9-fold increase, respectively, as compared to the controls. These data are the first to directly demonstrate that L. donovani amastigote components can induce the production of CSFs that may play important role(s) in the pathogenesis of visceral leishmaniasis.

Colony stimulating activity in normal human serum tested against human bone marrow.

Two hundred and thirty normal human sera have been tested for colony stimulating factor (CSF) under carefully controlled conditions using human bone marrow and the semi-solid agar-gel technique. It has been shown that CSF can be detected in most sera when incorporated into underlayers to remove potential inhibitors. The effect of storage temperature on serum CSF has been determined. Storage at 4 degrees, --15 degrees, and --66 degrees for up to 80 days resulted in only a mild decrease in CSF levels. After 240 days of storage, CSF values fell to 50% of that found before storage. Repeated freeze-thawing of serum has not been shown to decrease CSF levels when done in Pyrex glassware. These studies will serve as a baseline for those wishing to study human serum CSA in hematopoietic disorders.