Effects of gadolinium contrast agents in naïve and nephrectomized rats: relevance to nephrogenic systemic fibrosis.

BACKGROUND: Human nephrogenic systemic fibrosis (NSF) is a rare condition reported in patients with severe renal insufficiency exposed to a gadolinium (Gd)-based contrast agent. An animal model of NSF could help to investigate its mechanisms and lead to prevention and treatment. PURPOSE: To evaluate a possible animal model of NSF using naive and partially nephrectomized rats to induce conditions similar to those in patients at risk of NSF. MATERIAL AND METHODS: Naive rats received intravenous doses of 5 or 10 mmol/kg Omniscan; 5 mmol/kg Magnevist; 1 mmol/kg caldiamide; 1, 2.5, or 5 mmol/kg gadodiamide; 25 micromol/kg GdCl(3); or 25 micromol/kg Gd citrate. Partially nephrectomized rats received 5 mmol/kg Omniscan; 5 mmol/kg Magnevist; 1 mmol/kg caldiamide; 1 mmol/kg gadodiamide; 25 micromol/kg GdCl(3); or 25 micromol/kg Gd citrate. There were three or four animals per group. Clinical signs were recorded during treatment. At termination, clinical biochemistry, histopathology, and tissue Gd and Zn concentrations were investigated. RESULTS: Similar responses to treatment were seen in naive and nephrectomized rats. High doses of gadodiamide were toxic, necessitating early termination of the affected animals. Skin lesions appeared in naive and nephrectomized groups treated with gadodiamide or Omniscan, coinciding with the onset of signs of pruritus, i.e., intensive scratching. The histomorphological features of the skin lesions were also consistent with superficial physical trauma. Dermal fibrosis was not a feature of these skin lesions in any of the groups, i.e., no increased collagen density, CD34+ cells, or increased fibroblasts. This was supported by skinfold measurements that demonstrated no increased skin thickness. Treatment with the gadolinium-based contrast agents and Gd salts resulted in increased Gd content of several tissues. The Gd salts were mainly taken up by the liver and spleen, possibly reflecting formation of insoluble particles and macrophage uptake. Zn tissue concentrations were normal or increased. Other major treatment-related changes included increased serum rat C-reactive protein and histamine; mineralization affecting the dermis, stomach, and blood vessels; and renal proximal tubule vacuolation. CONCLUSION: The visible skin lesions seen in this study appeared to be caused by excessive scratching in response to pruritus. As there was no evidence of dermal fibrosis, the cardinal feature of human NSF, this did not appear to be a model of human NSF.

Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels.

Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed. The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

Bioactive cytidine deaminase, an inhibitor of granulocyte-macrophage colony-forming cells, is massively released in fulminant meningococcal sepsis.

Cytidine deaminase (CDD) catalyzes the hydrolytic deamination of cytidine, which thereby is converted to uridine. CDD is found in serum and different tissues, with particularly high concentrations in polymorphonuclear neutrophils (PMN). We measured the CDD levels in plasma from patients with systemic meningococcal disease. Thirty-seven patients had significantly higher plasma levels of CDD than did 29 healthy control subjects (P=.0001). CDD levels in plasma or serum increased from a median of 96 ng/mL in healthy control subjects to medians of 168 ng/mL in patients without persistent shock (n=23; P=.001) and 422 ng/mL in patients with fulminant meningococcal septicemia (n=14; P=.0001). In most patients with fulminant septicemia, CDD levels in plasma increased during the first 3-53 h after the initiation of therapy (P=.003). CDD alone had no immediate harmful effect when injected into mice during a 4-day period. CDD may modulate the stimulatory effect of colony-stimulating factors on PMN in patients.

Digital image analysis of hematopoietic colonies in vitro.

A system for automatic analysis of in vitro hematopoietic colonies is described and evaluated. With the standard resolution provided by video cameras, the improvement in visualization obtained using features other than size and darkness when classifying potential colonies appears to be limited. We confirmed this by comparing results obtained with the test system with those obtained with a commercial one. However, for some applications it may be useful to supplement the system with specific methods, e.g., to separate merged colonies. Digital image analyses provide new possibilities, for instance of measuring the total cellularity of the dish or analyzing colonies according to the size and cell density of each colony. Examples provided are time course studies of colony development, cellularity feedback effects on colony sizes, and bell-shaped dose-response curves for the growth stimulation obtained by certain conditioned media on a subpopulation of progenitor cells that gives rise to large colonies.

Growth inhibition of granulocyte-macrophage colony-forming cells by human cytidine deaminase requires the catalytic function of the protein.

Previous studies have indicated that cytidine deaminase (CDD) is a potent growth inhibitor of granulocyte-macrophage colony-forming cells (GM-CFC). In this study, we have undertaken molecular cloning and purification of recombinant human CDD to elucidate the growth regulatory potential and mechanism behind the growth suppressive effect. The purified protein had a specific activity of 1.35 x 10(5) U/mg and a Km value of 30 micromol/L. In the GM-CFC assay, the recombinant protein was shown to reduce colony formation to 50% at 16 pmol/L concentration. Similarly, as was observed with CDD derived from granulocyte extract, the effect depended on the presence of thymidine (>/= 4 x 10(-5) mol/L). These results imply that CDD is an extremely potent inhibitor of GM-CFC and that no additional factor from the granulocyte extract is required for the growth inhibitory effect. Modification of CDD by truncation from the C-terminal end, or by amino acid substitution of an active site glutamate residue, eliminated both the enzyme activity and the growth regulatory potential of CDD. Furthermore, CDD from Escherichia coli was found to be even more effective than human CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory activity corresponding to a 10-fold higher enzymatic activity. Taken together, these results show that the catalytic nucleoside deaminating function of the protein is essential for the growth suppressive effect of CDD. Most probably, CDD exerts growth inhibition by depleting the cytidine and deoxycytidine pool required for DNA synthesis, as addition of deoxycytidine monophosphate, which is not a substrate for CDD, neutralizes the inhibiting effect.

Mannuronan enhances survival of lethally irradiated mice and stimulates murine haematopoiesis in vitro.

Mannuronan (poly-beta-(1-->4)-D-mannuronate or poly-M), produced by Pseudomonas aeruginosa as a mucoid exopolysaccharide, has previously been shown to exhibit immunostimulating activity. The authors investigated the in vivo and in vitro effects of mannuronan on murine haematopoiesis. In vivo, prophylactic (-24 h, intraperitoneal) administration of mannuronan enhanced survival of lethally irradiated mice from zero day 40 survivors (NaCl) to 20, 80 and 70% survival at 0.5, 1 and 2 mg/kg bw mannuronan, respectively. In vitro, primary stromal cultures stimulated with mannuronan produced high levels of interleukin(IL)-1, IL-6 and colony stimulating activity. Mannuronan alone did not have any colony stimulating activity on GM-CFC, BFU-E, Mix-CFC or HPP-CFC progenitors in clonogenic assays, but acted synergistically with suboptimal amounts of growth factors on GM-CFC, Mix-CFC and HPP-CFC colony formation. Limiting dilution analysis showed that 1 of 423 bone marrow cells formed colonies in response to suboptimal GM-CSF plus mannuronan compared to 1 of 592 for suboptimal GM-CSF alone. The primitive Lin-Sca-1+ haematopoietic progenitors showed increased day 10 colony size in the presence of mannuronan in single cells assays. These stimulating effects of mannuronan on haematopoiesis may prove to have clinical importance.

Design of low molecular weight hematoregulatory agents from the structure-activity relationship of a dimeric pentapeptide.

We report herein, a new class of simple hematoregulatory semipeptides, formally derived from the cystine-dimerized peptide pGlu-Glu-Asp-Cys-Lys-OH, where the disulfide bond has been replaced by an isosteric dicarba bridge. The structure-activity relationship (SAR) of a series of analogues incorporating replacements at positions 1 and 2 of peptide 1 led to the design of active conformationally constrained cyclic peptides (12, 13). Ring closure was achieved by cyclization of the N-terminal amino groups at position 2 of peptide 2 using pyrazine-2,3-dicarboxylic acid. Subsequent excision of the putative C-terminal scaffold domain from the active cyclic peptides resulted in the discovery of a new class of low molecular weight hematoregulatory agents exemplified by compound 16. This semipeptide analogue, comprising two D-Ser residues connected via amide bonds to the acid groups of pyrazine-2,3-dicarboxylic acid, had comparable biological activity to the lead peptide 1. The stereochemical requirements for the observed biological activity of these novel compounds were examined. Furthermore, the hematopoietic synergistic activity induced by compound 16 in stromal cell cultures was blocked by an antibody known to neutralize the hematoregulatory effect of 1, indicating a common mechanistic end point. Compounds of the class typified by 16 may form the basis for the development of novel therapeutic agents within the area of immunoregulation.

Structure-activity relationships of novel hematoregulatory peptides.

Hematopoiesis is a lifelong cell renewal process regulated by a family of lineage specific hematopoietic growth factors. Several hematopoietic growth factors such as G-CSF, GM-CSF, and M-CSF have been clinically evaluated for enhancement of host defense in normal and immunocompromised patients and for the treatment of infectious diseases. This paper reports the structure-activity relationships of low molecular weight hematoregulatory peptides based on a nonapeptide (1, SK&F 107647). Like the macromolecular growth factors, these peptides modulate host defense. A molecular target for this class of compounds has not yet been identified. However, the structure-activity relationships established by this study implicate a very specific molecular recognition event that is pivotal for the biological activities of 1 and its analogues.

Identification of cytidine deaminase as inhibitor of granulocyte-macrophage colony formation.

Mature human blood granulocytes produce regulatory factors that inhibit colony formation by human and murine granulocyte-macrophage colony-forming cells (GM-CFC). The inhibition of GM-CFC by granulocyte extract (GRE) was strongly enhanced by the addition of thymidine (3 to 6 x 10(-5) M for human cells) and by the presence of fetal calf serum (FCS) in the growth medium. Deoxycytidine and deoxyuridine produced effects similar to those of thymidine, but at higher concentrations (2 to 4 x 10(-4) M). It was further observed that GRE prevented the antiproliferative effects of cytosine arabinoside (Ara-C) and azadeoxycytidine, suggesting that GRE contained cytidine deaminase (CDD) activity, since CDD is known to abolish the effects of these nucleoside analogs. Accordingly, the GRE was tested for and shown to contain an enzymatic activity that converted deoxycytidine to deoxyuridine, confirming the presence of CDD activity in GRE. The GM-CFC inhibition factor was found to copurify with CDD activity during three succeeding chromatographic separations, indicating that CDD was indeed the inhibiting factor itself. This conclusion was further substantiated by gel filtration experiments demonstrating a molecular weight (MW) of approximately 50 kd, which corresponds to the MW previously published for CDD activity. Furthermore, addition of tetrahydrouridine (THU), a known specific inhibitor of CDD, abolished the suppressive effect of GRE on GM-CFC, which independently confirmed the identification of CDD as an inhibitor of GM-CFC. The growth-regulating property of CDD could be explained by depletion of deoxycytidine nucleotides necessary for DNA synthesis or by a direct effect of CDD binding to specific receptors on progenitor cells.

Monoclonal antibodies distinguish between carboxylesterase isoenzymes in different tissues of rat and guinea pig.

The carboxylesterase (CarbE) activity in the main tissues (lung, liver, plasma and small intestine) of both the rat and guinea pig was separated by chromatofocusing. The three CarbE isoenzymes in the small intestine from both species showed nearly identical pI values. Monoclonal antibodies (MAbs) raised against rat lung CarbE (pI 5.8) were used in enzyme-linked immunosorbent assays to distinguish between these closely related CarbE isoenzymes. None of the MAbs did bind to the active site as no inhibition of the enzyme was seen when the MAbs were added. The immunological study showed a strong relationship between lung CarbE (pI 5.8) and the rat liver CarbE (pI 6.0). The MAbs were also strongly bound to the high pI forms of the CarbE isoenzymes in plasma and small intestine from both rat and guinea pig, but not with the low pI forms. These results indicate that two immunochemically distinct categories of CarbE isoenzymes are present in the plasma and small intestine.

Indirect stimulation of hemopoiesis by hemoregulatory peptide (HP5b) dimer in murine long-term bone marrow cultures.

The hemoregulatory dipeptide (pEEDCK)2 was shown to stimulate production of a synergistic activity (SA) in 6- to 8-week-old primary stromal cell cultures. The SA increased colony formation by murine bone marrow cells (approximately 50% above control levels) in cultures stimulated by optimal concentrations of L-cell-derived macrophage colony-stimulating factor (M-CSF). An increased number of granulocyte-macrophage colony-forming cells (GM-CFC) was also observed in long-term bone marrow cell cultures following daily administration of dipeptide for 5 days. The increase in GM-CFC was approximately 90% above control as assessed by colony formation in soft agar and coincided with SA production. It appears that the dipeptide augments the production of myeloid progenitor cells through an indirect mechanism mediated by accessory cells.

Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality.

This paper briefly reviews commonly used procedures for separation of mononuclear cells (MNC) and granulocytes from human blood with X-ray contrast media as gradient material, and also presents new and modified procedures for leucocyte preparation. Standard techniques for human blood do not always yield satisfactory results with blood from other species. In general pure MNC are easily obtained (top fraction), but often the granulocyte fraction has a low purity, due to contamination with MNC that move to the bottom during centrifugation and contaminate the granulocyte suspension. Obviously the density distribution of MNC differs between species. However, the separation can be improved by fine adjustment of gradient medium osmolality. For this purpose we have used Nycodenz, a non-ionic X-ray contrast medium. A favourable property of Nycodenz solutions is that the osmolality and density can easily be varied over a broad range. The cells react promptly to a change of medium osmolality. In hypertonic medium the cells expel water, shrink, their density increases and they sediment faster, in spite of a smaller radius. Further, the cells may pass what was initially a density barrier. A hypotonic environment has the opposite effect. In the present work we were able to show that a slight change of medium osmolality clearly improved different techniques for separation of leucocyte subgroups. For instance, the Isopaque-Ficoll (IF) technique consistently yielded MNC and granulocytes of high purity with human blood. However, with blood from rabbits, rats and mice the granulocyte suspensions were contaminated by 40-60% MNC. By utilizing Nycodenz, and lowering the osmolality by 10-12 per cent (at constant density--1.077 g/ml) we obtained satisfactory separation of MNC as well as granulocytes with blood from these species. A problem in the routine separation of granulocytes (IF) is a high contamination of erythrocytes (2-5 per cell) in the granulocyte suspension. With a two-layer technique with Nycodenz solutions of different densities it was possible to separate granulocytes almost devoid of erythrocytes, after proper adjustment of osmolality. By appropriate combination of density and osmolality, Nycodenz was a suitable gradient material in other separation procedures as well, e.g. the separation of monocytes and mast cells. To facilitate the use of Nycodenz as a versatile gradient material, a computer program providing recipes for various Nycodenz solutions is included as an appendix.

Purification and characterization of carboxylesterases from rat lung.

Carboxylesterase (EC 3.1.1.1) has played an important part in our understanding of the toxicokinetic behaviour of the organophosphorus cholinesterase inhibitors. Carboxylesterases are a heterogeneous group of enzymes that can be separated on the basis of their isoelectric points and by their substrate-specificity. We have purified the isoenzyme (pI 5.8) present in greatest activity in rat lung to near homogeneity. The enzyme was purified by (NH4)2SO4 precipitation, gel filtration, chromatofocusing, separation on anion- and cation-exchangers and hydrophobic-interaction chromatography. The purified enzyme has a molecular mass of approx. 180 kDa with subunits of 60 kDa. The substrate and inhibitor specificities of the enzyme have been characterized. Edman degradation revealed the first 19 amino acid residues of the enzyme. The N-terminus was found to be tyrosine. Inhibition of the enzyme by organophosphorus compounds differed greatly from that of cholinesterases, despite the partial analogy at the N-terminal region.

Effect of VIP on the respiratory burst in human monocytes ex vivo during prolonged strain and energy deficiency.

VIP-stimulated cyclic AMP production and VIP effect on the production of reactive oxygen compounds in human monocytes activated by serum opsonized zymosan (respiratory burst) were studied during a ranger training course lasting for five days with almost continuous physical activity, and deficiency of sleep and energy. Respiratory burst was inhibited and cyclic AMP production was stimulated by VIP on all days. Maximum cyclic AMP production stimulated by VIP (0.1 microM) on the day of control was 148.6% of basal, and 255.3%, 213.8%, 218.9% and 198.7% on Days 1, 2, 3 and 5. Maximum inhibition was observed 20 min after addition of the peptide on the day of control, after 5 min on Days 1, 2 and 3, and after 10 min on Day 5. Inhibition at the 5-min time point was 33.1% on the day of control, and 34.7%, 53.6%, 53.3% and 36.2% on the different days during the training course. The observed increment in VIP effect adds to prior reported data about increased VIP secretion during the training course, and may indicate enhanced physiological significance of VIP during stress.

Leucopoiesis versus concentration of cytokines in diffusion chamber cultures of mouse bone marrow cells: clues to the physiological roles of growth factors.

Physiological mechanisms that regulate formation of neutrophil granulocytes, macrophages and their precursor cells were studied with the diffusion chamber (DC) technique. DC inoculated with mouse bone marrow cells were implanted intraperitoneally into host mice. When these in vivo cultures had been established and their marrow populations were expanding (2-day cultures), the DC were transferred to different environments: new, normal mice, lethally irradiated mice, or incubation flasks with optimal concentrations of growth factors. Culture development during the following final culture period was correlated to the concentration of some select candidate growth regulators in DC. After 3 d the cellularity of DC in irradiated hosts had increased significantly more than in the normal hosts. A difference was detectable already after 1-2 d when preculturing was omitted. The increased growth appeared to take place at several stages of cell maturation and not only at the progenitor cell level. Colony stimulating factor(s) for granulocyte and macrophage progenitors, as well as cytokines stimulating the bone marrow-derived cell line, 14 M.1, were present in DC fluid (DCF) at higher concentrations in irradiated than in normal mice throughout the final culture period. On the other hand, DCF concentrations of tumour necrosis-like factor (that may either induce CSF secretion or directly inhibit myelopoiesis), were not significantly different in irradiated compared with normal DC hosts. The cytokines detected in the DC may at least in part stem from inflammatory cells accumulating around the chambers. This animal model should be useful in further investigation of the highly complex regulatory network governing formation of white blood cells in the intact organism.

Up-regulation of the expression of secretory component and HLA molecules in a human colonic cell line by tumour necrosis factor-alpha and gamma interferon.

Secretory component (SC) acts as a receptor for polymeric IgA (pIgA) and pIgM on the basolateral face of secretory epithelia. This study showed that both the production and surface membrane expression of SC were additively up-regulated by tumour necrosis factor-alpha (TNF) and gamma interferon (IFN-gamma) in a human colonic cell line (HT-29m2). TNF likewise enhanced membrane expression of HLA class I determinants. Moreover, TNF augmented synergistically the IFN-induced de novo synthesis of HLA class II (DR) molecules. These data suggest that there are different regulatory mechanisms for the action of TNF and IFN and for the expression of HLA-DR and SC/HLA class I. Addition of actinomycin D abolished the cytokine-mediated increase of SC synthesis and expression. This observation suggested that transcriptional events are required for the cytokine-mediated up-regulation of SC.

Tumor necrosis factor-alpha up-regulates expression of secretory component, the epithelial receptor for polymeric Ig.

Secretory component (SC) is the receptor that facilitates transcytosis of polymeric IgA and polymeric IgM through secretory epithelial cells and into exocrine fluids. The present study showed that rTNF-alpha enhanced the cellular pool, membrane expression, and secretion of functionally SC in a human colonic carcinoma cell line (HT-29m2) which is known to express and process SC like normal glandular cells. TNF-alpha also up-regulated membrane expression of the constitutive HLA class I molecules, whereas the cells remained HLA class II-negative.

Thymidine-dependent effect of granulocyte-derived inhibitor on granulocyte-macrophage progenitor cells (GM-CFC).

We have compared the effect of granulocyte extract (GRE) on proliferation of hemopoietic cells from various sources, using two different cultures media, CMRL 1066 and McCoy 5A. GRE caused a strong (80-90%) inhibition of granulocyte-macrophage colony forming cells (GM-CFC) in cultures with medium CMRL 1066. GM-CFC in human blood and human and mouse bone marrow were equally sensitive to the inhibitor. The inhibitor had a maximal effect in the concentration range corresponding to GRE from 2 x 10(5) to 2 x 10(6) cells per 1 ml culture dish. At higher GRE concentration the inhibition was reduced. GM-CFC from human blood and mouse marrow were suppressed in cultures with McCoy's medium as well, but to a lesser extent than in CMRL 1066 cultures. On the other hand, in cultures with human bone marrow cells (BMC) and McCoy's medium, GRE had no inhibitory effect. CMRL 1066 medium contains a number of components not present in McCoy's medium. In a systematic study where these substances were added one by one to McCoy's medium we found that inhibition by GRE depended upon the presence of thymidine. At a thymidine concentration of 3 x 10(-5) mol/l GRE strongly suppressed GM-CFC in human blood and bone marrow. This thymidine concentration itself had no effect. Other nucleosides or components of the CMRL 1066 did not potentiate the suppressive effect of GRE.

Colony inhibiting factor in mature granulocytes from normal individuals and patients with chronic myeloid leukemia.

Inhibitory activity in extract from human blood granulocytes was tested on granulocyte-macrophage colony formation in vitro. The inhibition depended on the type of serum used. With mouse BMC and FCS in the cultures, extract corresponding to 2.5 X 10(4) granulocytes/ml reduced the colony number by 35%, and extract from 2 X 10(5) cells caused maximal inhibition (80-90%). With HS and mouse BMC the colony number was reduced by only 11-12%, but stronger inhibition (55%) was observed when the serum concentration was reduced. With both types of sera the total cell number per culture plate was reduced relatively more than the colony number. Human GM-CFC were as sensitive as mouse GM-CFC, and extract from CML granulocytes inhibited less (p less than 0.01) than extract from normal cells. Biochemical studies indicated that the inhibitor is a protein with a molecular weight of 30-60,000. Lactoferrin, a putative inhibitor of CSF production, did not inhibit spontaneous or CSF-induced colony formation in these studies.