Marrow culture in diffusion chambers in rabbits. I. Effect of mature granulocytes on cell production

Factors influencing granulopoiesis have been evaluated using diffusion chambers implanted in the peritoneal cavity of rabbits. An increase in granulopoiesis in chambers implanted in hosts made neutropenic by nitrogen mustard occurs in mice made neutropenic by x-ray or drug. The intraperitoneal injection of leukocytes inhibited the growth of cells in chambers implanted in rabbits. Removal of mature granulocytes from marrow prior to chamber inoculation produced a marked in-increase in cell growth, especially of granulocytes. Mature granulocytes clearly inhibited cell replication and this inhibition involved both myeloid and erythroid elements, although the data suggest a greater effect on myelopoiesis. In contrast to the mouse, erythropoiesis in chambers in rabbits remained prominent for over 1 wk.     

Marrow culture in diffusion chambers in rabbits. II. Effect of competing demands for red cell and white cell production on cell growth

Studies were done of cell production by marrow in diffusion chambers implanted in the peritoneal cavity of rabbits subjected to various stimuli to hematopoiesis. In chambers in neutropenic hosts and in hosts injected with endotoxin, animals presumed to have an increased stimulus to granulopoiesis, there was increased production of granulocytes but there was also increased production of red cells. Although red cell production was decreased in chambers in polycythemic hosts, granulocyte production was not different from that in controls. Stimulation of erythropoiesis by erythropoietin injections or by exposure to hypoxia increased red cell production by marrow in the implanted diffusion chambers without diminishing granulopoiesis. Only in chambers in hosts made anemic by bleeding was there an increase in red cell production accompanied by a decrease in granulocyte production. In these anemic hosts induction of neutropenia led to an increase in granulopoiesis without any depression of erythropoiesis.     

Human Tissues as Source of Colony-Stimulating Factor in Human Bone Marrow Cultures

Human bone marrow cells were cultured by the agar method using feeder layers containing human tissue fragments of various origin. Colony stimulating factor (CSF) was shown to be released from all tissues tested (adipose tissue, skeletal muscle, peritoneum and vascular wall), and primary cultures of human fibroblasts could also stimulate colony growth. Colony growth of murine bone marrow cells was also stimulated by all feeder layers. Thus, the study demonstrates that monocytes and macrophages might not be the only source of CSF of possible importance for human granulopoiesis in vivo.     

Effect of endotoxin on arachidonic acid release and thromboxane B2 production by human platelets

Plasma thromboxane A₂, which is elevated during endotoxemia, has previously been shown to be a major factor contributing to the mortality and morbidity that occurs in endotoxin shock in the experimental animal. Using a minimal dose of Escherichia coli endotoxin (1 μg/ml), we have demonstrated that the preincubation of human platelets with endotoxin induces changes in platelet arachidonic acid release and the subsequent conversion of the released arachidonic acid to thromboxane B₂, the stable end product of thromboxane A₂. In paired experiments, in the presence of endotoxin, the addition of the aggregating agent thrombin (0.5 U/ml) caused human platelets to release 29.1 ± 3.4% of ¹⁴C-arachidonic acid from prelabeled platelet phospholipids. This value was significantly elevated (P < 0.02) when compared with the release of ¹⁴C-arachidonic acid from platelets in the absence of endotoxin (21.9 ± 3.6%). Similarly, comparison of the results of the conversion of the released arachidonic acid to platelet thromboxane B₂ (TxB₂) revealed that TxB₂ production was significantly increased (P < 0.01) when human platelets were preincubated with endotoxin prior to the addition of thrombin (6.1 ± 0.6%) when compared with TxB₂ formation observed in the absence of endotoxin (3.4 ± 0.5%). The absolute amount of released arachidonic acid that was converted to TxB₂ in the presence of endotoxin (1.8 ± 0.3%) was also significantly higher (P < 0.01) than the value observed in its absence (0.8 ± 0.2%). This study suggests that one of the tissue sources of the proaggregatory vasoconstrictor thromboxane A₂ during endotoxemia is the platelet.     

Alteration of colony-stimulating factor output, endotoxemia, and granulopoiesis in cyclic neutropenia.

Cellular and humoral factors involved in the regulation of granulopoiesis were evaluated in two patients with cyclic neutropenia by utilizing the agar-gel marrow culture technique to serially study marrow granulocytic colony-forming capacity (CFC) and the urinary output of colony-stimulating factor (CSF). CSF output varied inversely with peripheral neutrophil counts and directly with monocyte counts and evidence for infection (endotoxemia and/or staphylococcal abscesses). Following autologous infusion of one patient's plasma obtained during a period of neutropenia, increased urinary excretion of CSF occurred concomitant with increments in both marrow CFC and the proportion of granulocytic progenitor cells in DNA synthesis. Neutrophil periodicity was not altered by the administration of the neutropenic plasma. These findings are consistent with the hypothesis that cyclic neutropenia is caused by a quantitatively decreased entry of stem cells or granulocytic progenitor cells into granulopoiesis.     

Effect of endotoxin pretreatment on hepatic stellate cell response to ethanol and acetaldehyde

BACKGROUND AND AIM: The role of endotoxin in alcohol-induced liver damage is well recognized. How pre-exposure to endotoxin might affect alcohol injury is not known. We herein studied the effect of endotoxin pretreatment on hepatic stellate cell (HSC) response to ethanol and acetaldehyde. METHODS: Rat HSC (CFSC-2G) were exposed to media supplemented with 1 microg/mL lipopolysaccharide (LPS). This was followed by a 24 h exposure to media containing LPS plus 50 mmol/L ethanol or 175 micromol/L acetaldehyde. Lipid peroxidation, collagen, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and transforming growth factor (TGF)-beta1 secretion were determined at the end of both periods of exposure. RESULTS: Lipopolysaccharide pretreatment did not modify lipid peroxidation induced by ethanol or acetaldehyde alone. Glutathione (GSH) content decreased to 4.2 +/- 0.5 and 16.3 +/- 0.8 nmol protein after exposure to ethanol or acetaldehyde alone, and decreased further with LPS pretreatment (2.4 +/- 0.2 and 2.7 +/- 0.3 nmol/mg protein, respectively). Oxidized GSH (GSSG) content increased in ethanol and acetaldehyde LPS-pretreated cells only. Collagen secretion increased to 988 +/- 82 and 1169 +/- 91 microg/10(6) cells after exposure to acetaldehyde or LPS alone. Lipopolysaccharide pretreatment enhanced collagen secretion significantly in both ethanol- and acetaldehyde-treated cells (969 +/- 56 and 1360 +/- 72 microg/10(6) cells, respectively). Interleukin-6 production increased to 288 +/- 48, 1195 +/- 86 and 247 +/- 35 pg/mL per 10(6) cells after ethanol, acetaldehyde and LPS exposure, and increased further with LPS pretreatment in ethanol-exposed cells (680 +/- 23 pg/mL 10(6) cells). CONCLUSION: Lipopolysaccharide pretreatment of HSC adds to the damage produced by ethanol and acetaldehyde by diminishing GSH content and increasing GSSG content, collagen and IL-6 secretion.     

阿糖胞苷对人骨髓细胞长期培养的影响

人骨髓细胞长期培养加入３５μｇ／ｍｌ阿糖胞苷并孵育１小时后中止，然后继续培养，４周龄ＬＴＨＭＣ的造血被抑制；但１８周龄ＬＴＨＭＣ则表现为培养上清液中非粘附细胞数上升，１４天达高峰，基质层出现造血区，原已退缩的基质层有新的基质细胞生长。提示１８周龄ＬＴＨＭＣ仍有造血潜力，其基质层仍存在造血干细胞。     

The in vivo diffusion chamber technique for bone marrow or blood cell culture

Conclusions In vivo diffusion chamber culture technique provides a system that isolates implanted bone marrow or peripheral blood stem cells from host cells while allowing exchange of nutrients, waste produces, and presumed humoral stimulators and inhibitors of cell growth.It includes the advantages of the in vivo model, in which humoral and cellular interactions between host and implanted bone marrow cells can be studied in a system where mature cells can not move away. However, the reproducibility problem, the difficulties in identifying the responsible stimulatory and inhibitory humoral factors, and the costs, do not make this culture technique suitable for routine use. A major application can be the study of the physiological relevance of in vitro observations.This work is supported by a grant from the Koningin Wilhelmina Fonds, the Dutch Cancer Fund, and the Rothrock Foundation     

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture

Summary Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C₅₇Bl mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.     

Pressure measurements and histological investigations after subcutaneous implantation of diffusion chambers in rabbits

Summary In separate experiments conducted in rabbits the pressure was measured in a subcutaneously implanted diffusion chamber system for collecting interstitial fluid, and a histological examination was made of the surrounding tissue. Three days after implantation the pressure was negative in all animals (- 4.23±2.5 mmHg). The diffusion chambers were well tolerated in tissue with minimal cell reaction. The lumen of the chambers remained free of cellular elements for up to 30 days.

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Druckmessungen und histologische Untersuchungen bei subkutan implantierten Diffusionskammern in Kaninchen

Zusammenfassung In getrennten Versuchen wurden bei Kaninchen Druckmessungen an einem subkutan implantierten Diffusionskammersystem zur Gewinnung interstitieller Flüssigkeit sowie histologische Untersuchungen des umgebenden Gewebes durchgeführt. Drei Tage nach Implantation war der Druck mit einem Mittelwert von - 4,23±2,5 mmHg bei allen Tieren negativ. Die Diffusionskammern zeigten eine gute Gewebsverträglichkeit bei minimaler Zellreaktion. Das Kammerlumen war bis zum 30. Tag frei von zellulären Elementen.

     

Effect of endotoxin and immunoglobulin on the course of experimental hepatic encephalopathy

A single intraperitoneal dose of endotoxin (500 g) shortened the time for development of hepatic coma by 27% in 300-g rats that had an end-to-side portacaval shunt followed within 48 hr by hepatic artery ligation. The body temperature of the rats was maintained at 37°C, and the endotoxin was injected just after the hepatic artery was ligated. Controls were injected similarly with saline. The time to death was also shortened by 27%. A single intravenous dose of immunoglobulin (150mg) delayed the time from the massive hepatic ischemia to the onset of hepatic coma by 19%. The immunoglobulin was injected just after the portacaval shunt was completed. Controls were injected similarly with 0.6ml of 25% human serum albumin. While not large, these opposite effects of endotoxin and immunoglobulin were highly significant statistically. These observations complement the findings in human liver failure.     

采用1.068 g/ml分离液对犬骨髓间充质干细胞的分离纯化与体外培养方法的探讨

目的探讨更为可靠、纯净度高的犬骨髓间充质于细胞分离、培养方法。方法将犬骨髓抽取液分别以1．068g／ml及1．073g／ml分离液进行密度梯度离心，采用贴壁培养法获得骨髓间充质干细胞（MSCs）；以LG-DMEM加15％FBS培养；应用于细胞表面标志蛋白，多向诱导分化等方法进行细胞鉴定。结果采用1．068g／ml分离液可获得纯度较高的单核细胞，接种细胞生长良好，孵育24h即可见细胞贴壁生长，平均倍增周期为1d，细胞呈纺锤状，螺旋梳状排列。连续培育8代以上，未见细胞形态、增殖特性改变。MSCs表面标志SH2阳性，CD45阴性，可定向分化为心肌细胞、成骨细胞及脂肪细胞。结论采用1．068g／m1分离液能够较好地进行犬MSCs的体外分离、培养与扩增，分离得到的细胞具备MSCs的特性。     

Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture

Summary. Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive- or negative-acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma-stem cell interactions.
We have used a sensitive bioassay to investigate production of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and stem cell growth factor (SCF), by normal and AA stroma In long-term bone marrow culture (LTBMC). LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension. These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34⁺ cells, in clonogenic assays, in the absence of exogenous cytokines. Cytokines responsible for the colony-stimulating activity (CSA) and burst-promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines. All normal stroma populations produced G-CSF and GM-CSF, 93% produced IL-3, 80% produced IL-6, and 70% produced SCF. Similarly, all AA stroma produced G-CSF and GM-CSF, and 71% produced SCF. In contrast, only 71% of AA stroma produced IL-3 and 36% produced IL-6. Target cell stimulation was not dependent on direct stroma-target cell contact, suggesting production of soluble cytokines. However, although both IL-6 and G-CSF were detected in LTBMC supernatants by enzyme-linked immunoassay (ELISA), IL-3 and GM-CSF were undetectable, perhaps indicating low-level local production of these factors.     

Evaluation of diffusion in gel entrapment cell culture within hollow fibers

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.     

Platelets attenuate production of cytokines and nitric oxide by macrophages in response to bacterial endotoxin

Considerable evidence has been accumulated concerning the roles of platelets in immune responses. In the present study, we examined the functional modulation of macrophages by platelets. When mouse bone marrow-derived macrophages (BMDMs) were co-cultured with platelets, BMDMs produced lower levels of nitric oxide (NO), tumor necrosis factor-α (TNF)-α, and interleukin (IL)-6 in response to a bacterial endotoxin (LPS) and zymosan. The attenuation in the macrophage susceptibility to LPS appeared to be mediated by soluble factors secreted from platelets. The mRNA levels of NOS2 (iNOS), TNF-α, and IL-6 in LPS-stimulated BMDMs that had been cultured with a conditioned medium of platelets were also decreased as analyzed by RT-qPCR. The ability of the platelet-conditioned medium to suppress macrophage NO production was recovered in a high-molecular-weight fraction (>670 kDa) after gel-filtration chromatography on a Superose 6 column. These results suggest that platelets control the susceptibility of macrophages to prevent excessive responses to LPS and provide mechanistic insight into a previous report that experimental thrombocytopenia aggravated organ failure in LPS-induced endotoxemia.     

Composition of fluids from diffusion chambers implanted in the soft tissue and kidneys of rabbits

Summary The fluids from diffusion chambers implanted in soft tissue and kidneys of rabbits were analysed for total protein, albumin, enzymes, ions, glucose, creatinine, urea, uric acid, bilirubin and cholesterol. These data were compared with the corresponding values in plasma. Our data for chamber fluid are in good agreement with data reported for interstitial fluids. The composition of the kidney chamber fluid is nearly constant from three to ten weeks after implantation. The low urea, uric acid and creatinine concentrations indicate that the chamber is not located in the urine collecting area of the kidney. Three days after subcutaneous implantation of chambers, the fluid contains less protein than plasma but has an equal concentration of ions, thus meeting the principal requirements for interstitial fluid. There are indications that the healing process lasts up to ten days after the surgical implantation. In order to examine the permeability of the diffusion chambers, the equilibration half-life times of antibiotics and substances of high and low molecular weights were determined in vitro.

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Zusammensetzung der aus in Weichteilgewebe und Nieren implantierten Diffusionskammern bei Kaninchen gewonnenen Flüssigkeiten

Zusammenfassung Es wurden Flüssigkeiten aus subkutan und in die Niere von Kaninchen implantierten Diffusionskammern gewonnen und folgende Parameter bestimmt: Gesamteiweiß, Albumin, Enzyme und Ionen sowie Glukose, Kreatinin, Harnstoff, Harnsäure, Bilirubin und Cholesterin. Diese Daten wurden mit den entsprechenden Plasmawerten verglichen. Unsere Werte für Kammerflüssigkeiten stimmten gut mit berichteten Daten für interstitielle Flüssigkeit überein. Die Zusammensetzung der Nierenkammerflüssigkeit ist zwischen drei und zehn Wochen nach Implantation annähernd konstant. Die niedrigen Konzentrationen an Harnstoff, Harnsäure und Kreatinin beweisen, daß die Kammer nicht im urinableitenden Teil der Niere liegt. Drei Tage nach subkutaner Implantation der Kammern enthält die Flüssigkeit weniger Eiweiß als das Plasma, hat gleiche Konzentrationen an Ionen und entspricht daher den prinzipiellen Anforderungen für Gewebsflüssigkeit. Es gibt Hinweise, daß der Heilungsprozeß bis zu zehn Tagen nach dem chirurgischen Eingriff der Implantation der Kammern andauert. Um die Permeabilität der Diffusionskammer zu prüfen, wurden Halbwertszeiten für den Konzentrationsausgleich von Antibiotika sowie nieder- und hochmolekularen Substanzen in vitro ermittelt.

     

Effect of Phenytoin on DNA Synthesis by Human Bone Marrow

Diphenylhydantoin (phenytoin) when incubated with human marrow interfered with the synthesis of deoxyribosenucleic acid (DNA) by those cells. This interference was dose related but was present at all concentrations, 25 μg/ml to 100 μg/ml. Cells unable to complete DNA synthesis would die in the marrow, lead to ineffective haemopoiesis and an increased folate requirement.     

The fine structure of monoclonal Hodgkin cells cultured in diffusion chambers

Summary Pleural effusion cells from two patients with stage IV Hodgkin's disease have been cultured continuously in diffusion chambers in mice and studied by electron microscopy after a culture period exceeding 100 days. Cell identity and monoclonal growth in culture has been documented by marker chromosomes (Hossfeld and Schmidt, 1978).These cultured cells grow in close connection, projecting pseudopode-like processes into the intercellular spaces. Most nuclei are lobulated. They always are of low electron density with a narrow rim of condensed chromatin confined to the nuclear membrane. One large prominent nucleolus and up to four smaller nucleoli are found. Nuclear pockets in case 1 and deep cytoplasmic invaginations into the nuclear area in both cases frequently occur.In the cytoplasm, besides microtubuli and fibrils, the Golgi apparatus and mitochondria are the predominant organelles. Most mitochondria appear to be dilated containing fragmented cristae. Free ribosomes and polysomal aggregates are randomly distributed.The ratio nucleoplasm:cytoplasm, on the average, is 0.7 in both cases and the cell diameters lie distinctly above those of lymphocytes.At the electron microscope level these cultured monoclonal cells of Hodgkin's disease are not distinguishable from those described in genuine Hodgkin material. Their probable origin and apparent relation to true histiocytic lymphoma cells will be discussed.We gratefully acknowledge the competent technical assistance of Ms. Beate Menge and Ms. Regina Granitzki     

Comparative Evaluation of Simultaneous Bone Marrow Aspiration and Bone Marrow Biopsy: An Institutional Experience

Bone marrow aspirations and bone marrow biopsies are important diagnostic procedures. A comparative study of both the procedures done simultaneously was retrospectively reviewed in 160 cases where the clinical history is correlated with BMA and BMB results. The advantage of each method is analyzed. Correlation of our findings with that given in the literature is done to give a guideline for both techniques. We have found that 61.25% of the cases showed a positive correlation between bone marrow aspiration and bone marrow biopsy. However, we found that tuberculous granulomas and Hodgkin disease involvement of the marrow were detected better in bone marrow biopsies. The advantage of both the procedures done together provided more material and enabled us to study the cytomorphology of the cells, with the pattern of distribution of the cells depending on the cases. However, when both the procedures are done simultaneously, a proper technique is required so as to yield good diagnostic material.