Biomedical subjects

R K Shadduck

Publications and source records attributed to R K Shadduck.

At least 91 records · Page 5Linked to original sources

PubMed1989

Lithium stimulation of diffusion chamber colony growth is mediated by factors other than colony-stimulating factor.

Lithium is a recognized, potent stimulator of granulopoiesis. The present study used the model of clonal growth of granulopoietic precursors in diffusion chambers to investigate the relevance of certain colony-stimulating factors to lithium stimulation in vivo. In this system, lithium stimulation of granulopoiesis could not be attributed to changes in serum or chamber fluid colony-stimulating factor levels. Antibody to colony-stimulating factor-1 administered during culture markedly reduced morphologic expression of colonies in control and lithium-pretreated host mice, yet subculture of chamber contents revealed that lithium stimulation of a granulopoietic progenitor, perhaps of primitive potentiality, had nevertheless occurred. Therefore, we hypothesize that lithium acts in an indirect, hormonal fashion and that these colony-stimulating factors, while necessary for morphologic expression, play no role in the stimulatory effect. This hypothesis raises the possibility that lithium in combination with recombinant colony-stimulating factors may result in clinically effective synergistic stimulation of granulopoiesis.

Animals↗

PubMed1989

A double buffy coat method for red cell removal from ABO-incompatible marrow.

Hemolytic reactions caused by transfusion of ABO-incompatible marrow can be ameliorated by either reduction of isohemagglutinins in the recipient or depletion of incompatible red cells from the harvested marrow. This article describes a rapid and reliable method for removal of incompatible marrow red cells on a blood cell processor using a double buffy coat technique. In five allogeneic bone marrow transplants, the maximum value of transfused incompatible red cells was 8.8 ml. There was no evidence of a hemolytic transfusion reaction in any patient. The median cell recovery for nucleated marrow cells and progenitor cells was 77 and 104 percent, respectively. Engraftment occurred at a median of 13 days (range, 11-21 days) after transplantation. The double buffy coat method of red cell depletion is an acceptable method for processing ABO-incompatible marrow.

ABO Blood-Group System↗

PubMed1989

Development of a radioimmunoassay for human macrophage colony-stimulating factor (CSF-1).

Purified human urinary CSF-1 was used for production of polyclonal CSF antibodies in rabbits. The purified CSF was iodinated by a modified chloramine-T technique with retention of biologic activity. Dilutions of anti-CSF were reacted with 15,000 cpm of 125I-CSF in EDTA-phosphate buffer for 48 hr. Sheep antirabbit serum was added for 3 hr to precipitate the tracer-anti-CSF complex. A 1:1000 dilution of anti-CSF caused 60-90% precipitation of tracer; optimal conditions were observed with a 1:30,000 dilution. Linear displacement curves were obtained with 2-50 U of pure CSF-1. Related hormones did not cross-react in the assay; no displacement was seen with human GM-CSF, IL-1, IL-2, IL-3, EP, LH or FSH. Reactivity was also not observed with murine GM-CSF or IL-3. Ten normal human sera yielded CSF values of 91-138 U/ml in 5 assays. Urine values were 72-105 U/ml. When 32 U of pure CSF-1 was added to normal serum and urine samples, quantitative recovery was observed. Serial assays revealed a rise in serum and urinary CSF during marrow aplasia in a patient undergoing autologous BMT; CSF values returned to normal during the recovery phase. This sensitive and specific radioimmunoassay should prove useful in the further study of CSF-1 responses in vivo.

Animals↗

PubMed1989

Mechanisms of tumor-induced neutrophilia: constitutive production of colony-stimulating factors and their synergistic actions.

Transplantation of a murine mammary carcinoma (CE maca) into mice induces marked granulocytosis and hypercalcemia secondary to excessive bone resorption. Such responses are not induced by another murine mammary carcinoma Bc66. In order to understand the mechanisms of these unique phenomena, we analyzed mRNA of tumor cells for expression of murine granulopoietic growth factors and studied interactions of tumor-derived factors using antiserum to a growth factor in vitro and in vivo. The Northern blot analysis of CE tumor clones revealed the expression of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF), but no other CSF genes, while the Bc66 clone expressed only M-CSF. The G-CSF and M-CSF gene expression in CE tumor clones was accompanied by secretion of these proteins in culture. The granulocyte stimulating activity of CE tumor-derived G-CSF or recombinant human G-CSF was markedly enhanced by purified M-CSF in vitro. Significant but variable neutrophilia was observed in mice inoculated with CE tumor clones. Anti-M-CSF treatment of CE tumor-bearing mice significantly reduced neutrophilia, but did not affect hypercalcemia. These studies document that G-CSF and M-CSF are produced constitutively from the CE maca, and G-CSF is likely responsible for granulocytosis induced by this tumor. G-CSF and M-CSF function synergistically in granulocyte stimulation in vitro and this synergism may also play a role in marked granulocytosis of tumor-bearing animals, providing further evidence of the effect of CSFs in vivo.

Adenocarcinoma↗

PubMed1989

Demonstration of a blood-bone marrow barrier to macrophage colony-stimulating factor.

Several previous studies suggested that murine macrophage colony-stimulating factor (CSF-1) might have impaired access to hematopoietic cells in the marrow. The apparent lack of hematopoietic responses to exogenous CSF and the finding of available or unoccupied CSF receptors despite saturating CSF levels in the serum led to studies of a potential blood-bone marrow barrier for this factor. Groups of mice were injected with pure unlabeled CSF-1 by either intravenous (IV) or intraperitoneal (IP) routes. Marrow and spleen cells were obtained at intervals after injection, held at 0 degree C, and assessed for changes in binding of 125I-CSF. Saturation of all available CSF receptors is achieved in vitro with 100 to 150 U CSF/mL. Despite achieving serum levels of 5,000 to 7,000 U/mL after IV injection of 25,000 units of CSF, less than 50% of the marrow receptors and less than 85% of the splenic receptors were saturated or downregulated. The decline in receptor availability was transient, with return of receptor sites in two to four hours. Increasing the IV dose to 125,000 units increased serum CSF values to approximately 20,000 U/mL and led to a virtual disappearance of available receptors for two to three hours. When administered IP, only approximately 40% of marrow and 80% of splenic receptors were affected for two hours. It was necessary to increase the dose of CSF to 250,000 units IP to saturate or downregulate receptors for three to four hours after injection. These observations indicate a marked blood-bone marrow barrier and lesser blood-spleen barrier for the transfer of serum CSF to responsive hematopoietic cells in vivo.

Animals↗

PubMed1989

Effect of recombinant human granulocyte-macrophage colony-stimulating factor on myelopoiesis in patients with refractory metastatic carcinoma.

The effect of human recombinant GM-colony-stimulating factor (CSF) was evaluated in ten patients with refractory metastatic carcinoma. Initially they received an intravenous (IV) bolus injection of 5 or 25 micrograms/m2 for assessment of acute responses. Six days later, continuous IV infusions of 100 or 500 micrograms/m2 were initiated for a 14-day treatment course. All patients developed profound leukopenia within five to 30 minutes of the bolus injection. This appeared to result from increased expression of an adhesion-promoting glycoprotein (GP) on neutrophils and monocytes as judged by increased reactivity to the Mo1 monoclonal antibody (MoAb). Leukocyte counts returned to normal levels within two hours as cells were released from marrow stores. With the continuous infusion, leukocyte counts increased by 24 hours; peak values of 22,960 and 75.900/microL were achieved after ten to 14 days of treatment with the two dose levels of GM-CSF. This leukocytosis was due to an increase in virtually all cell types. At the high dose level, there was a striking increase in neutrophils (49,400/microL) and eosinophils (20,905/microL) with a sixfold increase in monocytes and two- to threefold increase in lymphocytes. Leukocyte counts declined promptly after cessation of the infusion but remained above baseline for as long as 2 weeks in some patients. These results suggest that GM-CSF may be useful as an adjuvant therapy by stimulating myelopoiesis in cancer patients.

Bone Marrow↗

PubMed1989

Purification of human urine colony-stimulating factor by affinity chromatography.

Antisera from rabbits immunized with murine macrophage colony-stimulating factor (CSF-1) were evaluated for cross-reactivity with human urine CSF-1. One cross-reactive antiserum was used to purify CSF from human urine. The IgG fractions from normal rabbit serum and the anti-CSF serum were bound to cyanogen bromide-activated sepharose. Ten-liter pools of human urine were concentrated by ultrafiltration and applied sequentially to the normal IgG and antiserum columns. Cross-reactive proteins were removed by the IgG column, whereas CSF was bound by the anti-CSF column. After extensive rinsing of the antibody column, the CSF was eluted with 4 M sodium thiocyanate. This fraction contained four to five contaminating proteins as judged by migration in sodium dodecyl sulfate-acrylamide gel. In a further purification step, the CSF was retained selectively by concanavalin A sepharose and eluted with alpha-methylglucoside. This purified CSF had a specific activity of 0.8-2.3 x 10(7) U/mg protein. A single major contaminant was removed by reversed phase high performance liquid chromatography. Final specific activity of the purified CSF ranged from 2.5 to 4.4 x 10(7) U/mg protein. Each 10-liter pool of urine yielded 18-20 micrograms of pure material with a 15%-25% recovery. This technique is more rapid and provides a higher yield of pure human CSF-1 than the more tedious multi-step procedures that have been described previously.

Anemia, Aplastic↗

PubMed1988

Characterization of two clones isolated from the TC-1 murine marrow stromal cell line: growth factor and retrovirus production and physical support of hemopoiesis.

We previously reported the isolation of an adherent murine marrow cell line termed TC-1, and the initial characterization of two subclones derived from this line. In this study we report a further characterization of two subclones from the non-cloned TC-1 cell line. One subclone, TC-1-C-3, consisted of large, slow-growing syncytial polypoid cells that grew to relatively low saturation densities, did not form colonies in soft agar and showed desmosome-like junctions. The other subclone, TC-1-C-11, consisted of smaller, rapidly growing fibroblast-like diploid cells which showed anchorage-independent growth in soft agar. Both these subclones produced growth factors which stimulated giant macrophage colonies in soft agar culture in vitro, but only the TC-1-C-3 subclone produced a retrovirus, whose source was most likely the endogenous ecotropic Emv-2 provirus present in chromosomal DNA in C57BL mice. This retrovirus from the TC-1-C-3 subclone did not appear capable of transforming TC-1-C-11 cells. Together, these data suggest that TC-1-C-3 cells have a special capacity for supporting hemopoiesis. The question of whether the mechanism of this support relates to an intrinsic property of the cell or is possibly related to retrovirus production remains unanswered.

Animals↗

PubMed1988

A functional analysis of hematopoietic growth factor production from class I and class II human alloreactive T cell clones.

Class I and Class II human alloreactive T cell clones or their conditioned media were mixed with progenitor cell-enriched null cells to assess their ability to stimulate human hematopoietic progenitor cell (HPC) growth. Optimal release of erythroid, myeloid or megakaryocyte colony-stimulating factors occurred after 72 hours and required contact of cloned T cells with irradiated stimulator cells expressing the appropriate major histocompatibility complex (MHC) determinants recognized by the T cells. Individual clones were quite heterogeneous in their capacity to release hematopoietic growth factors. Clones that produced optimal levels of factors that stimulated granulocyte-macrophage colony growth did not always produce equivalent amounts of factors that stimulated erythroid colony growth and vice versa when tested against identical target cells. Class II clones released nearly twice as much interleukin 3 activity as Class I clones. Class II clones that lacked cell-mediated lympholytic (CML) activity against B or T lymphoblastoid targets were consistent stimulators of HPC growth. In contrast, Class I or Class II clones that contained CML activity either poorly stimulated or inhibited HPC growth. These CML-positive clones produced greater amounts of gamma interferon. Our findings may have important implications for HPC growth following allogeneic mismatched bone marrow transplantation.

Cell Division↗

PubMed1988

Effect of different modes of dialysis on serum erythropoietin levels in pediatric patients. A report of the Southwest Pediatric Nephrology Study Group.

The relative importance of erythropoietin (Ep) and inhibitors of erythropoiesis in the development of anemia in pediatric patients with end-stage renal disease (ESRD) was assessed in 82 patients: 41 treated with peritoneal dialysis (PD) and 41 with hemodialysis (HD). Serum Ep was determined with a sensitive radioimmunoassay. Potential serum inhibition of erythroid (CFU-E) and granulocytic (CFU-GM) progenitor cell growth was assessed using human bone marrow cell cultures. The mean Ep level for all 82 patients was 33.1 +/- 3.1 mU/ml, which was significantly higher (P less than 0.05) than the values obtained in 29 normal children (26.2 +/- 2.4 mU/ml). Serum Ep in the PD group (41.6 +/- 5.6 mU/ml) was significantly higher (P = 0.007) than that of the HD group (24.6 +/- 2.1 mU/ml). The mean hematocrit in the PD group (25.2 +/- 0.8%) was also significantly higher (P less than 0.002) than that of the HD group (22.2 +/- 0.5%). The mean serum parathyroid hormone (PTH) level as measured by a mid-terminal radioimmunoassay was not significantly different (P = 0.79) in the HD group (17,298 +/- 3,998 pg/ml) from that of the PD group (15,747 +/- 4,227 pg/ml). Neither serum Ep nor PTH concentration correlated with hematocrit or degree of inhibition of erythroid progenitor cell colony (CFU-E) formation in either group of dialysis patients, nor did the hematocrit correlate with the degree of serum inhibition of CFU-E formation. The higher level of Ep in the PD group may indicate more effective removal by PD of some enzymatic substance which reduces the immunologic and biologic activities of Ep.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1988

Characterization of a human monocyte cytotoxicity-inducing factor (MCF).

Recently, we reported a lymphokine, monocyte cytotoxicity-inducing factor (MCF), which is distinct from interferon-gamma (IFN-gamma). In this report, we provide further characterization of MCF. MCF is inactivated by chymotrypsin, but not trypsin, RNase, or DNase. The production of MCF is abolished in a dose-dependent manner by actinomycin D and is diminished by puromycin, and cycloheximide. Native MCF produced under serum-free conditions demonstrated charge heterogeneity with three species having isoelectric points at 2.7, 5.6, and 6.7 respectively, and two molecular weight species of 29 Kd and 14.7 Kd. MCF-activated monocytes were not only able to lyse both NK sensitive and resistant targets, but also secreted IL 1, but not TNF. In summary, MCF is a lymphokine distinct from TNF, IL 1, IL 2, the IFNs, and the CSFs, which is able to activate monocytes to lyse tumor targets.

Chymotrypsin↗

PubMed1988

Effect of pH on binding and dissociation of colony-stimulating factor.

125I-labeled colony-stimulating factor (CSF) binds to granulocytic and monocytic cells in the bone marrow in an irreversible manner. Addition of a 1000-fold excess of unlabeled CSF does not displace the bound material. The present studies showed that brief exposures to pH 2.7-5.0 caused a marked release of the bound material. Such treatments were nontoxic to the marrow cells as judged by trypan blue dye exclusion, assay of colony-forming cells, and by analysis of rebinding of fresh 125I-CSF to the acid-treated cells. The CSF released from marrow cells by low pH revealed two peaks of radioactivity on SDS-acrylamide gel. The first peak (67,500 Da) corresponded to native CSF; a second peak of 53,500 Da was observed. Despite this apparent mild degradation of CSF, the released material showed greater binding to marrow and greater precipitation by anti-CSF than the native 125I-CSF. Further studies showed that acid treatment of marrow cells led to stabilization of the CSF receptors. Pretreatment at pH 4.0 led to retention of binding sites after conversion of marrow cultures to pH 7.5 and incubation at 22-37 degrees C. In contrast, cells that were not exposed to low pH lost receptors rapidly at these temperatures. The extent of preservation of the binding sites was related to the duration of acid exposure. These studies indicate that CSF is retained on the cell surface after binding at 0 degree C and that the CSF can be eluted by acid conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Enhancement of release from MHC class II antigen-positive monocytes of hematopoietic colony stimulating factors CSF-1 and G-CSF by recombinant human tumor necrosis factor-alpha: synergism with recombinant human interferon-gamma.

The influence of purified recombinant human tumor necrosis factor-alpha (rhuTNF-alpha) was assessed alone and in combination with purified recombinant human interferon gamma (rhuIFN-gamma) for its effects on enhancing release from human monocytes of activities that stimulate colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. RhuTNF-alpha or rhuIFN-gamma enhanced release of colony stimulating factors (CSFs), which were determined by a combination of human and mouse colony assays, morphological assessment of colony types and neutralization studies with anti-human macrophage CSF (CSF-1) and anti-human granulocyte (G)-CSF to be CSF-1 and G-CSF. The activity in the uninduced and induced monocyte conditioned media (CM) for CFU-GM-type colonies and clusters was attributed to the presence of both CSF-1 and G-CSF, while the activity in the monocyte CM for BFU-E and CFU-GEMM colonies was attributed to the presence of G-CSF. Monocytes were separated by two-color fluorescence using a dye laser flow cytometry system with cells labeled with anti-leu M3 conjugated with fluorescein isothiocyanate and anti-HLA-DR conjugated with phycoerythrin. While "constitutive" release of CSFs from monocytes was apparent from both the leu M3+, HLA-DR+ and the leu M3+, HLA-DR- (low density or negative DR) fractions, enhanced release of CSFs in response to rhuTNF-alpha or rhuIFN-gamma was confined to the leu M3+, HLA-DR+ population of cells. RhuTNF-alpha and rhuIFN-gamma synergized to enhance release of CSFs such that low concentrations of each molecule, which were inactive when used alone, were active when the two molecules were used together. These studies suggest a role, at least in vitro, for TNF-alpha and IFN-gamma in the release of CSFs from cells of the mononuclear phagocytic lineage.

Animals↗

PubMed1988

In vitro evidence for disappearance of erythroid progenitor T suppressor cells following allogeneic bone marrow transplantation for severe aplastic anemia.

In vitro coculture studies were performed in five patients with severe aplastic anemia (SAA) and their normal HLA-matched donors before and after allogeneic bone marrow transplantation (BMT) to determine whether the erythropoietic function of T cells is abnormal in this disorder. These coculture studies used fresh or cryopreserved marrow T lymphocytes with fresh or cryopreserved marrow T cell-depleted target cells. Four of five aplastic patients had little or no transfusion exposure before studies. The composite results showed that, in comparison to the erythropoietic effects of normal HLA-identical marrow T lymphocytes or engrafted T lymphocytes, T lymphocytes collected from the aplastic patients before BMT consistently suppressed or failed to support CFUE and BFUE growth optimally from autologous marrow, HLA-identical marrow, or engrafted aplastic T cell-depleted marrows. This T cell abnormality was not observed in four multiply transfused leukemics and three patients with myelodysplastic syndrome. Marker analyses of SAA marrow T lymphocytes performed before and after BMT suggested that the erythropoietic functional abnormality was due to abnormal marrow T cell composition reflecting an excess of activated Tac+, T3+, T11+ lymphocytes. Collectively, these in vitro studies provide firmer in vitro evidence implicating T cells in the pathogenesis of SAA. The erythropoietic T cells abnormalities in SAA are fully corrected by allogeneic BMT.

Anemia, Aplastic↗

PubMed1988

Transfusion of bone marrow red cells during bone marrow harvests.

Large volumes of bone marrow may be required for certain types of autologous bone marrow transplants. The present study was done to determine whether red cells obtained during a bone marrow harvest would be useful in reducing homologous transfusion requirements. A group of patients receiving standard transfusion support during the harvest (group 1) was compared to a group that received processed bone marrow red cells (PBMRBC) (group 2). Using the Cobe 2991 cell processor, 90% of the harvested bone marrow red cells were extracted and transfused during the procedure. Group 2 received a median of 1500 ml of blood processed from the bone marrow or 413 ml (volume of marrow processed x hematocrit) of red cells. Infusion of the PBMRBC reduced the homologous transfusion requirement from 6.5 units to 3.0 units (p = 0.02). In addition, group 1 had a 20% decrease in hematocrit following transfusion compared to the pre-harvest hematocrit, as opposed to an 8% decrease in group 2 (p = 0.02). This study indicates that PBMRBC can reduce the homologous transfusion requirements during an autologous bone marrow harvest.

Adolescent↗

PubMed1987

Synergistic myelopoietic actions in vivo after administration to mice of combinations of purified natural murine colony-stimulating factor 1, recombinant murine interleukin 3, and recombinant murine granulocyte/macrophage colony-stimulating factor.

Combinations of low dosages of purified murine hematopoietic colony-stimulating factors (CSFs)--L-cell CSF type 1 (CSF-1), recombinant interleukin 3 (IL-3), and recombinant granulocyte/macrophage CSF (GM-CSF)--were compared with single CSFs for their influence on the cycling rates and numbers of bone marrow granulocyte/macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with human lactoferrin. Lactoferrin was used to enhance detection of the stimulating effects of exogenously administered CSFs. Concentrations of CSFs that were not active in vivo when given alone were active when administered together with other types of CSF. The concentrations of CSF-1, IL-3, and GM-CSF needed to increase progenitor cell cycling rates were reduced by factors of 40-200, 10-50, and 40- greater than 400, respectively; the concentrations needed to increase progenitor cell numbers were reduced by factors of 40-500 (CSF-1), 20-80 (IL-3), and greater than 40- greater than 200 (GM-CSF) when these forms of CSFs were administered in combination with low dosages of one of the other forms of CSFs. The results demonstrate that different CSFs can synergize when administered in vivo to increase the cycling rates and numbers of marrow hematopoietic progenitor cells. These findings may be of relevance physiologically to the regulation of myeloid blood cell production by CSFs.

Animals↗

PubMed1987

Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.

Macrophage colony-stimulating factor (CSF-1) stimulates the production of macrophages from bone marrow progenitor cells. We have identified a cDNA clone for murine CSF-1 by antibody screening of a mouse L-cell cDNA library in the expression vector lambda gt11. A screen of about 150,000 recombinant plaques yielded 6 clones that reacted well with an antibody raised against denatured and reduced mouse L-cell CSF-1. These clones were further screened with synthetic oligonucleotides based on the amino-terminal amino acid sequence of CSF-1. One clone, which hybridized to the oligonucleotides, was sequenced and found to contain a single open reading frame. This encompassed 68 amino acids of the mature protein, including the entire amino-terminal sequence we previously reported. This is preceded by what appears to be a 31 amino acid signal peptide. Blot analysis showed that this cDNA hybridizes to a major mRNA species of about 4.5 kilobases (kb) as well as several smaller, less abundant mRNA species (3.8, 2.3, and 1.4 kb) present in mouse L cells. A similar pattern of hybridization was observed with mRNA from a human pancreatic carcinoma cell line that produces CSF-1. Striking differences in the qualitative and quantitative expression of mRNA species for CSF-1 were observed in various mouse tissues. Liver expressed primarily a 1.4-kb species, heart and lung expressed primarily a 4.5-kb species, brain expressed high levels of both the 4.5-kb and 1.4-kb species, and intestine lacked detectable CSF-1 transcripts. Southern blot analysis suggests that the CSF-1 gene is present as a single copy in the mouse haploid genome and that it is not rearranged or amplified in L cells.

Amino Acid Sequence↗

PubMed1987

Comparative effects in vivo of recombinant murine interleukin 3, natural murine colony-stimulating factor-1, and recombinant murine granulocyte-macrophage colony-stimulating factor on myelopoiesis in mice.

Purified murine colony-stimulating factors (CSF) recombinant interleukin 3 (IL-3), natural CSF-1, and recombinant granulocyte-macrophage (GM) CSF were assessed in vivo for their effects on BDF1 mouse bone marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in untreated mice and in mice pretreated with purified iron-saturated human lactoferrin (LF). The CSF and LF preparations did not contain detectable endotoxin (less than 0.1 ng). Mice pretreated with LF were more sensitive to the effects of CSF. In mice pretreated with LF, 2,000 U IL-3 or 20,000 U CSF-1 significantly enhanced the cycling status and absolute numbers of all progenitors, whereas 20,000 U GM-CSF significantly increased the cycling status of CFU-GM and CFU-GEMM, but had no effect on cycling of BFU-E or on numbers of any of the progenitors. The effects of CSF in mice pretreated with LF were not mimicked by 0.1-100 ng E. coli lipopolysaccharide.

Animals↗