Semisolid Agar Cultures Research Articles

Protective effect of bleomycin on 5-azacitidine induced cytotoxicity and apoptosis in mice hematopoietic stem cells via Bcl-2/Bax and HMGB1 signaling pathway

Antineoplastic drugs cause severe cytotoxicity for normal cells, especially hematopoietic stem cells (HSCs). However, bleomycin (BLM) is glycopeptide antibiotic that is effective on various cancers and has either low or no myelosuppression effects. The aim of the present study was to investigate the effect of BLM on 5-Azacitidine (5-AZA) induced cytotoxicity in bone marrow HSCs. 5-AZA reduced HSC cell viability in a time and dose-dependent manner with an IC50 value of 16 μM. However, pretreatment of the cells with BLM for 4 h induced an antagonistic cytotoxicity with an increased IC50 of 64 μM. 5-AZA decreased the colony formation ability of HSC cells in semi-solid agar culture and this effect was attenuated by BLM. 5-AZA significantly downregulated high mobility group Box1 (HMGB1) and Bcl-2 gene expression but upregulated Bax gene expression, while BLM impeded the action of 5-AZA. Pretreatment with BLM remarkably decreased HMGB1 release into culture media that was induced by 5-AZA. The cells were distribution at the sub/G1 phase. Annexin/PI staining of the cells, poly (ADP-ribose) polymerase (PARP) cleavage, and anion superoxide production indicated that BLM limited 5-AZA induced apoptotic cell death. In conclusion, BLM in combination with 5-AZA effectively reduces the adverse cytotoxic effects of 5-AZA on bone marrow hematopoietic stem cells, providing a new chemotherapeutic strategy.

Mechanisms and molecular typing of carbapenem-resistant Proteus mirabilis strains deficient in swarming motility

Objective To investigate the mechanism and epidemiological characteristics of carbapenem-resistant Proteus mirabilis (PM) strains deficient in swarming motility. Methods PM strains were isolated from Hangzhou General Hospital of CAPF (Chinese People′s Armed Police Forces) during January 2013 to December 2014. Bacterial motility and flagella of the PM strains were observed through semi-solid agar culture and flagella staining. Pulsed-field gel electrophoresis (PFGE) was performed for homology analysis. Antimicrobial susceptibility test and phenotypic confirmatory test were also carried out. PCR analysis and DNA sequencing were performed to confirm the genotype of resistant genes. Plasmid electroporation and S1-PFGE in combination with Southern blot hybridization were used to determine the location of the carbapenem-resistant genes. Genetic structure of the blaKPC-2 gene was obtained by PCR mapping. Results A total of 42 PM isolates deficient in swarming motility were screened out and the resistance rates to imipenem and meropenem were 57.1% and 52.4%, respectively. PCR analysis and DNA sequencing confirmed that 24 carbapenem-resistant PM isolates deficient in swarming motility carried blaKPC-2 gene and belonged to three clones as indicated by the results of PFGE. Southern blot hybridization indicated that the blaKPC-2 gene was located on plasmids varying in size (26 kb, 55 kb and 139 kb). In addition, some of the strains harbored several resistant genes, such as blaTEM-1, blaCTX-M-65 and rmtB. The genetic structures of strains carrying blaKPC-2 gene were ISKpn8, blaKPC-2 and ISKpn6-like from upstream to downstream. Conclusion Compared with the PM strains with swarming motility, the carbapenem-resistance rate was significantly higher in these PM strains deficient in swarming motility. Carbapenemases KPC-2 played an important role in the carbapenem-resistant PM strains deficient in swarming motility. There was a cloning spread trend for carbapenem-resistant PM strains in our hospital. Clinicians should pay more attention to the risk of spreading. Key words: Proteus mirabilis; Swarming motility; Carbapenemase; Flagellum; Nosocomial infection

Do two different stem cell grafts: G-CSF stimulated and unstimulated bone marrow differ according to hematopoietic colony forming capacity?

The study was designed to compare colony forming capacity of granulocyte-colony stimulating factor (G-CSF) stimulated bone marrow (G-BM) with standard unstimulated bone marrow (U-BM) of healthy donors of pediatric patients. CFU-Assay results of 26 healthy donors of pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) were analyzed retrospectively. 13 of donors received 10 μg/kg per day of G-CSF as a single injection for 3 consecutive days and other 13 of donors had unstimulated BM. Colony forming capacity of hematopoietic stem cells evaluated with Colony Forming Unit-Assay (CFU-Assay) with in semi-solid agar culture medium after 14-18 days of culture period. CFU-Assay results of G-BM and U-BM (expressed as means) were; Burst Forming Unit-Erythroid (BFU-E): 15.20 × 10(4)/kg and 8.38 × 10(4)/kg, Colony Forming Unit-Granulocyte Macrophage (CFU-GM): 10.35 × 10(4)/kg and 5.67 × 10(4)/kg, Colony Forming Unit-Erythroid (CFU-E): 0.59 × 10(4)/kg and 0.33 × 10(4)/kg, CFU-Granulocyte Erythroid Macrophage Megakaryocyte (CFU-GEMM): 0.52 × 10(4)/kg and 0.53 × 10(4)/kg respectively. BFU-E and CFU-GM capacity of G-BM was increased and statistically significantly different than standard U-BM (p ⩽ 0.01). In conclusion, increased colony forming capacity of hematopoietic stem cells of G-BM when compared with standard unstimulated BM could be a major advantage for transplantation.

Rubus parvifolius L. inhibited the growth of leukemia K562 cells in vitro and in vivo

To determine the antiproliferative activity of Rubus parvifolius L. (RP) extract, its medicinal serum and RP total saponins (RPTS) against K562 cells in vitro and in vivo. Nude mice models bearing leukemia tumors were treated with different concentrations of RP extract. The size, weight and histopathological change of leukemic tumors were determined. Semi-solid agar culture and methylthiazolyl tetrazolium (MTT) assay were used to determine in vitro the inhibition of colony formation and proliferation of K562 cells respectively by different concentrations of RP medicinal serum and RPTS. RP extract had a tumor inhibition rate of 84.8% when administered to mice at a dose of 1.0 g/day of crude RP root equivalent. Semi-solid agar culture of K562 cells in the presence of 20% (v/v) of RP medicinal serum and 150 mg/L RPTS demonstrated a 50.8% and 100% inhibition of the colony forming unit (CFU)-K562, respectively. The same doses of RP medicinal serum and RPTS showed a proliferation inhibition of 31.4% and 86.3%, respectively against K562 cells in MTT assay. RP extract and RPTS show effective antiproliferative activity against myeloid leukemia cells in vitro and in vivo.

Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures

In vitro shoot regeneration of gladiolus in three different culture systems, viz., semi-solid agar (AS), membrane raft (MR), and duroplast foam liquid (DF) cultures was evaluated following the kinetics of shoot multiplication and hyperhydricity at optimized growth regulator combinations. Compared to the AS system, matrix-supported liquid cultures enhanced shoot multiplication. The peak of shoot multiplication rate was attained at 18 days of incubation in the MR and DF systems, whereas the maximum rate in the AS system was attained at 21 days. An early decline in acceleration trend was observed in liquid cultures than the AS culture. The hyperhydric status of the regenerated shoots in the different culture systems was assessed in terms of stomatal attributes and antioxidative status. Stomatal behavior appeared to be normal in the AS and MR systems. However, structural anomaly of stomata such as large, round shaped guard cells with damage in bordering regions of stomatal pores was pronounced in the DF system along with a relatively higher K+ ion concentration than in the AS and MR systems. Antioxidative status of regenerated shoots was comparable in the AS and MR systems, while a higher incidence of oxidative damages of lipid membrane as evidenced from malondialdehyde and ascorbate content was observed in the DF system. Higher oxidative stress in the DF system was also apparent by elevated activities of superoxide dismutase, ascorbate peroxidase, and catalase. Among the three culture systems, liquid culture with MR resulted in maximum shoot multiplication with little or no symptoms of hyperhydricity. Shoots in the DF system were more prone to hyperhydricity than those in the AS and MR systems. The use of matrix support such as membrane raft as an interface between liquid medium and propagating tissue could be an effective means for rapid and efficient mass propagation with little or no symptoms of hyperhydricity.

Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro.

Low dose of cigarette smoke condensate induces chronic malignant transformation of immortalized human bronchial epithelial cells

Objective:To establish a chronic malignant transformation model of immortalized human bronchial epithelial cell line BEP-2D by low dose cigarette smoke condensate(simulating smoking environment).Methods:The chronic dose of cigarette smoke condensate was determined by MTT assay and the colony formation test of BEP-2D cells.BEP-2D cells were exposed to cigarette smoke condensate once or for multiple times;unexposed cells were taken as control.The malignant tendency of BEP-2D cells was identified by anti-serum experiment and the malignant features of transformed BEP-2D cells were identified by semisolid agar culture.The differentiation ability of BEP-2D cells in anti-serum experiment and the colony forming rates of BEP-2D cells were compared between different groups.Results:The BEP-2D cells were exposed to 0.5,1 and 2 μl/ml of cigarette smoke condensate once or for multiple times and were cultured for 25 generations;the differentiation abilities of BEP-2D cells(the 25th generation)was significantly different between the cigarette smoke condensate exposed groups(at 0.5,1 and 2 μl/ml)and the normal control group(P0.05).The cell malignant transformation model was successfully established in the cells of the 38th generation;the cells had multi-layer growth and had no contacting inhibition,with chromosome abnormality.The colony forming rates in the semisolid agar culture test was significantly higher in all smoke condensate exposed groups than in control group(P0.05).The dose-response relationship showed a good linear correlation(r=0.969,y=42x,P0.05).Conclusion:The malignant transformation of immortalized human bronchial epithelial cells can be successfully induced by cigarette smoke condensate at 0.5-2 μl/ml,which offers an ideal model for simulating smoking environment induced chronic malignant transformation.

CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar

The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4.

In vitro shoot regeneration of gladiolus in semi-solid agar versus liquid cultures with support systems

Multiple shoot clusters of gladiolus were established in semi-solid agar (AS) and liquid media appended with membrane raft (MR) and Duroplast foam (DF) support matrix. A difference in optimum combination of α-napthaleneacetic acid (NAA)/6-benzylaminopurine (BAP) for shoot multiplication was noted with culture systems. Maximum regeneration of 33.15 shoots/cluster was obtained with 1 mg l−1 NAA and 2 mg l−1 BAP in liquid medium with membrane support. There were 33.46 and 25.37% increase in shoot regeneration in MR system than AS and DF systems, respectively. Shoot multiplication was rapid in AS and MR systems (relative differentiation rate of 5.7% per 7 days of incubation) when compared to DF system (relative differentiation rate of 4.6% per 7 days of incubation). Shoot multiplication followed a linear trend with incubation time, whereas shoot elongation fitted well with quadratic functions. Such analysis suggests that shoot number increases steadily with time, whereas the rate of shoot elongation was different at different phases of incubation in different culture systems. Shoots in DF system exhibited faster elongation than those in AS and MR systems.

Bone Marrow-derived Cells Require a Functional Glucose 6-Phosphate Transporter for Normal Myeloid Functions

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the ubiquitously expressed glucose 6-phosphate transporter (Glc-6-PT). Glc-6-PT activity has been shown to be critical in the liver and kidney where a deficiency disrupts glucose homeostasis. GSD-Ib patients also have defects in the neutrophil respiratory burst, chemotaxis, and calcium flux. They also manifest neutropenia, but whether Glc-6-PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib remains controversial. To address this, we transferred bone marrow from Glc-6-PT-deficient (Glc-6-PT(-/-)) mice to wild-type mice to generate chimeric mice (BM-Glc-6-PT(-/-)). As a control, we also transferred bone marrow between wild-type mice (BM-Glc-6-PT(+/+)). While BM-Glc-6-PT(+/+) mice have normal myeloid functions, BM-Glc-6-PT(-/-) mice manifest myeloid abnormalities characteristic of Glc-6-PT(-/-) mice. Both have impairments in their neutrophil respiratory burst, chemotaxis response, and calcium flux activities and exhibit neutropenia. In the bone marrow of BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice, the numbers of myeloid progenitor cells are increased, while in the serum there is an increase in granulocyte colony-stimulating factor and chemokine KC levels. Moreover, in an experimental model of peritoneal inflammation, local production of KC and the related chemokine macrophage inflammatory protein-2 is decreased in both BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice along with depressed peritoneal neutrophil accumulation. The neutrophil recruitment defect was less severe in BM-Glc-6-PT(-/-) mice than in Glc-6-PT(-/-) mice. These findings demonstrate that Glc-6-PT expression in bone marrow and neutrophils is required for normal myeloid functions and that non-marrow Glc-6-PT activity also influences some myeloid functions.

Effects of the growth retardant paclobutrazol on large-scale micropropagation of daylily (Hemerocallis spp.)

Meristematic clusters were induced from daylily scape explants (pedicel-scape junction) in the presence of the growth retardant Paclobutrazol on semisolid agar medium. Liquid shake culture was used to proliferate meristematic clusters. Highly efficient regeneration of adventitious shoots occurred on clusters after subculture on a 0.8% agar strength semisolid medium with the addition of activated charcoal. Paclobutrazol and sucrose levels in the media were found to significantly affect starch accumulation, growth value, and dry weight percentage of liquid-cultured meristematic clusters. The use of liquid shake cultures for mass proliferation of meristematic clusters followed by regeneration of adventitious shoots on semisolid agar culture could be an efficient system for large-scale micropropagation of daylily.

Characterization of the interactions between stromal and haematopoietic progenitor cells in expansion cell culture models

Development of the long-term culture models of haematopoietic stem cells (HSCs) is one of the important tasks in modern biotechnology. It has been suggested that stromal presence is important for haematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive and definitive haematopoietic cells, or direct cell-to-cell contacts of the cultured material with underlying stromal base would be required. During present studies, influence of various feeder layers and feeder layer conditioned media on proliferative, differentiative and clonogenic activity of human AC133+ derived from human umbilical cord blood was investigated. Cell extracts for feeder layers were prepared from 4-6 weeks old human embryos and co-cultured feeder cells. Effects of the conditioned media were also determined. Culture and feeder layer media were additionally supplemented with commonly implemented factors such as GM-CSF, IL-3 and LIF. Estimation of morpho-functional properties of AC133+ cultivated suspension cultures was performed in subculture experiments using semisolid agar culture conditions. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analyzed. Our data suggest that haematopoiesis can be sustained for prolonged cultivation periods in the presence of feeder layer cells or conditioned media supported culture models. Prolonged support of primitive haematopoietic cells and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and suggests that direct cell-to-cell contacts may not be exclusively required for successful long-term in vitro haematopoiesis.

Imatinib inhibits the in vitro development of the monocyte/macrophage lineage from normal human bone marrow progenitors.

The antileukaemic tyrosine kinase inhibitor, imatinib, has been reported to inhibit specifically the growth of bcr-abl expressing CML progenitors at levels of 0.1-5.0 microM, by blocking the ATP-binding site of the kinase domain of bcr-abl. Inhibition of the c-abl, platelet-derived growth factor receptor and stem cell factor receptor (c-kit) tyrosine kinases by imatinib has also been reported. Here, we demonstrate that imatinib significantly inhibits in vitro monocyte/macrophage development from normal bone marrow progenitors, while neutrophil and eosinophil development was less affected. Monocyte/macrophage inhibition was observed in semisolid agar and liquid cultures at concentrations of imatinib as low as 0.3 microM. The maturation of monocytes into macrophages was also found to be impaired following treatment of cultures with 1.0 microM imatinib. Imatinib blocked monocyte/macrophage development in cultures stimulated with and without M-CSF, suggesting that inhibition of the M-CSF receptor, c-fms, by imatinib was unlikely to be responsible. Imatinib may therefore have an inhibitory activity for other kinase(s) that play a role in monocyte/macrophage differentiation. This inhibition of normal monocyte/macrophage development was observed at concentrations of imatinib achievable pharmacologically, suggesting that imatinib or closely related derivatives may have potential for the treatment of diseases where monocytes/macrophages contribute to pathogenesis.

Micropropagation of apple rootstock M.9 EMLA using bioreactor

SummaryA simple protocol for in vitro mass propagation of apple rootstock M.9 EMLA has been developed using an automated, low-cost bioreactor system. Comparative studies between semi-solid agar and bioreactor culture (continuous immersion with net and temporary immersion in liquid media using ebb and flood, with and without air exchanges inside the bioreactor chamber) revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor culture. Although the multiplication rate was highest in continuous immersion culture, a large number of micropropagated apple shoots showed symptoms of hyperhydricity. In case of temporary immersion culture (ebb and flood system) hyperhydricity was reduced as compared with continuous immersion. The percentage of shoots with hyperhydric leaves could be further reduced by air exchanges made over the culture period in a temporary immersion culture (ebb and flood) in air-lift-balloon type bubble bioreactor (BTBB). In vitro nodal cuttings of apple rootstock M.9 EMLA from bioreactor culture were hydroponically cultured for 30 d and more than 90% plants were rooted and acclimatized successfully. To assess the genetic fidelity of plantlets regenerated from nodal explants during bioreactor culture, the RAPD patterns were compared with that of greenhouse grown control plantlet and revealed genetic uniformity of the regenerants.

New cytokines and their clinical application.

The production of mature blood cells is controlled by a network of proteins called hematopoietic growth factors (HGFs). A number of HGFs were identified by their ability to stimulate colony formation in semi solid agar culture of normal bone marrow (1-2), and were called colony stimulating factors (3). It was shown that colonies of mature granulocytes were formed in the presence of granulocyte colony stimulating factor (G-CSF) (4), colonies of mature macrophages were formed in the presence of macrophage colony stimulating factor (M-CSF or CSF-1) (5) and mixed colonies of macrophages and granulocytes were formed in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) (6). Subsequently, some of the CSFs have been taken to clinical trials and are now used routinely in clinical treatments. G-CSF for example has been shown to stimulate recovery of neutrophils after chemotherapy and is used clinically to shorten the period of neutropenia after chemotherapy (7,8). Both G-CSF and GM-CSF are routinely used alone or in combination with chemotherapy to mobilize peripheral blood progenitor cells (PBPC) from the bone marrow to the peripheral circulation (9,10). The use of PBPC has replaced the use of bone marrow for cellular support following high dose chemotherapy, due to more rapid recovery in neutrophils and platelets resulting in a considerable decrease in overall costs of the transplant (11).KeywordsGranulocyte Colony Stimulate FactorStem Cell FactorAllogeneic Hematopoietic Stem Cell TransplantationHematopoietic Growth FactorPeripheral Blood Progenitor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Inhibition and recovery of GM-CSF production in the lung after hyperthermia

The purpose of this work was to study the response that is invoked by hyperthermia in the production of Granulocyte-Macrophage colony-stimulating factor (GM-CSF) by the lung. Rats were heated regionally at chest area by a RF generator operating at 27.397MHz for 1h at various temperatures and then allowed to recover or repair in various periods of time after heat application. Lung tissue from these animals was then removed and cultured for the production of GMCSF. GM-CSF was assayed by the production of colonies in the semi-solid agar cultures of bone marrow cells. Immediately after heat treatment hyperthermia had no significant effect on the production of GM-CSF by the lung. A delayed effect was observed about 3.5h after heat treatment. This effect consisted of a temperature dependent decrease in GM-CSF production. The damage was recoverable and required 1-50 days of post heat treatment time for animals to reach normal level of GM-CSF production. The results suggest that in-vivo application of hyperthermia invokes temporary reduction in GM-CSF production by the lung, which has not been reported previously.

Biological activities and clinical application of M-CSF.

Macrophage colony-stimulating factor (M-CSF) was found to be a glycoprotein with a molecular weight of 85 kDa which stimulated macrophage colony formation of mouse bone marrow cells in a semisolid agar culture system in 1978. M-CSF stimulates differentiation of progenitor cells to mature monocytes, and prolongs the survival of monocytes. It enhances expression of differentiation antigens and stimulates chemotactic, phagocytic and the killing activities of monocytes. Macrophage CSF also stimulates production of several cytokines such as granulocyte-macrophage CSF, granulocyte CSF and interleukin (IL)-6 by priming monocytes, and directly stimulates production and secretion of IL-8 and reactive nitrogen intermediates. In addition to the stimulation of hematopoiesis, M-CSF also stimulates differentiation and proliferation of osteoclast progenitor cells and cytotrophoblasts. Proteoglycan type M-CSF, which contains chondroitin sulfate chains, was found in 1992. In a large-scale double-blind controlled study on acute myeloid leukemia (AML), it has been shown that the administration of M-CSF to patients after consolidation chemotherapies shortens the periods of neutropenia and thrombopenia after chemotherapy and reduces the incidence and shortens the duration of febrile neutropenia, as well as shortening the period required to finish three courses of intensive consolidation therapy.

Indirect suppression of IL-7-responsive B cell precursors by vasoactive intestinal peptide.

Bone marrow is supplied with nerves and neuropeptides that influence a variety of cellular responses. This study represents an initial evaluation of vasoactive intestinal peptide (VIP) as a possible regulator of B lineage lymphocyte formation. As little as 10(-10) M concentrations of VIP inhibited the IL-7-driven clonal proliferation of pre-B cells in semisolid agar cultures. The response was blocked by a VIP antagonist and augmented by the ectoenzyme inhibitor, phosphoramidon. Suspensions of highly enriched B lineage precursors were unaffected by VIP unless they were cocultured with macrophage-like cells and conditioned medium from VIP-treated macrophages contained inhibitory activity. Neutralizing Abs were used to determine that IFN-alpha is at least one substance that is elicited by exposure of macrophages to VIP. These findings suggest that a neuropeptide can potentially modulate lymphopoiesis through a regulatory circuit that involves macrophages and IFN-alpha. They also raise the possibility that VIP can participate in antiviral defense.

Antibody Ligation of CD9 Modifies Production of Myeloid Cells in Long-Term Cultures

The KMC8.8 monoclonal antibody was made by immunizing rats with the BMS2 stromal cell clone, and was selected for further study because its ability to inhibit production of myeloid cells in Dexter cultures but not that of lymphoid cells in Whitlock-Witte cultures. The influence on myeloid progenitors might have been indirect, since the antibody did not prevent responsiveness to colony-stimulating factors in semisolid agar cultures. Furthermore, there was no inhibition, and some augmentation, of cell production when the antibody was added to established Dexter cultures. A cDNA clone that encoded the KMC8.8-recognized molecule was isolated by expression cloning and found to be identical in sequence to a previously published murine CD9 homologue. The antibody and cDNA clone were used to establish that CD9 is expressed by stromal cells, megakaryocytes, platelets, myeloid cells, and subpopulations of mature lymphocytes in mice. Treatment with the KMC8.8/CD9 antibody slightly augmented adhesion between myeloid cells and stromal cells, consistent with previous reports that this member of the tetraspan family of proteins can transmit proadhesive signals to human platelets and lymphoid cells. CD9 might participate in cell-cell interactions critical for correct orientation and movement of maturing myeloid cells in bone marrow.

Inhibition of enriched stem cells in vivo and in vitro by the hemoregulatory peptide SK&F108636.

Replacement of the labile sulfhydryl group (-SH) of the hemoregulatory peptide monomer pyroGluGluAspCysLys (HP5b) with an isosteric methylene group yields a chemically stable compound, SK&F108636. In this study, we describe the effects of SK&F108636 on highly enriched Lin-Sca1+ hematopoietic stem cells. SK&F108636 significantly reduced the fraction of cycling progenitor cells, granulocyte macrophage colony-forming cells (GM-CFC), in vitro and in vivo. There was no effect on GM-CFC or Mix-CFC colony formation. SK&F108636 significantly inhibited proliferation of high proliferative potential (HPP)-CFC in semisolid agar cultures stimulated by stem cell factor + interleukin 3 (IL-3) + IL-1, but had no effect in cultures stimulated with M-CSF + IL-3 + IL-1. SK&F108636 was shown to act directly on the stem cells since SK&F108636 inhibited proliferation of Lin-Sca1+ cells in single cell assays. Administration of SK&F108636 to lethally irradiated mice transplanted with 2000 Lin-Sca1+ cells significantly inhibited proliferation/differentiation of cells developing into colony forming units-spleen (CFU-S) (preCFU-S) and the reconstitution of HPP-CFC and GM-CFC. There was no effect of SK&F108636 on CFU-S colony formation or mature cell regeneration in bone marrow, spleen and blood. Hence, the hemoregulatory peptide monomer SK&F108636 is a potent primitive stem cell inhibitor in vivo and in vitro. Inhibition of stem cell proliferation by small specific inhibitors may protect hematopoiesis from myelotoxic side effects during chemotherapy treatment.