Attenuation of CXCR4 responses by CCL18 in acute lymphocytic leukemia B cells.

CCL18 and CXCL12 are homeostatic chemokines with high constitutive concentrations in serum. Elevated levels of CCL18 have been described in various diseases including childhood acute lymphocytic leukemia (ALL) but its functions remain poorly characterized. Its receptor has not been identified, but functional cellular responses like lymphocyte chemotaxis have been described. CXCL12 is a pivotal chemokine for hematopoiesis and B cell homing processes. We demonstrate that CCL18 interferes with CXCL12-mediated pre-B ALL cell activation. CXCL12-induced calcium mobilization, chemotaxis, pseudo-emperipolesis and cellular proliferation could be significantly reduced by CCL18 in pre-B ALL cell lines. The results could be observed in primary cells from patients suffering from pre-B ALL, but not in cells from patients suffering from common ALL. Direct effects of CCL18 on the receptor for CXCL12, CXCR4, could be excluded. Moreover, we found that CCL18 modulations of CXCL12-induced responses are mediated through the chemokine-like receptor GPR30. CCL18 bound to GPR30 expressing cells, and antibodies against GPR30 abolished this binding as well as CCL18-mediated functional effects. We also observed that, CCL18 interferes with the activation of GPR30 by previously identified ligands (17ß-estradiol and chemical agonists). We therefore suggest that CCL18 is an important modulator of CXCR4-dependent responses in pre-B ALL cells via interactions with GPR30.

A protease-free assay for peptidyl prolyl cis/trans isomerases using standard peptide substrates.

Peptidyl prolyl cis/trans isomerases (PPIases) are ubiquitous and abundant enzymes catalyzing peptide bond cis/trans isomerization adjacent to proline in peptides and proteins. An uncoupled protease-free assay of PPIase activity has been developed using the standard tetrapeptide substrates of the proteolytically coupled test system. Differences in the UV/vis absorption spectra of cis and trans conformations of Suc-Ala-Xaa-Pro-Phe-(Y-) anilide (Xaa = Ala, Leu, Phe; Y = 4-nitro, 2,4-difluoro) were exploited to monitor the time course of the cis/trans isomerization subsequent to a solvent jump from 0.47 M LiCl/trifluoroethanol into aqueous solution. The utility of the assay has been demonstrated by the determination of the Michaelis-Menten constants of cytosolic cyclophilin (Cyp18) and of the proteolytically sensitive FK506-binding protein-like PPIase SlyD from Escherichia coli. Furthermore, similar inhibition constants were estimated for the reversible inhibition of human Cyp18 by cyclosporin A (CsA) with both the proteolytically coupled and the novel uncoupled PPIase assay.

Extended binding sites of cyclophilin as revealed by the interaction with HIV-1 Gag polyprotein derived oligopeptides.

Oligopeptides derived from the gag polyprotein (Pr55gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag218-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-1 Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The IC50 value of 184 microM for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1-4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.

Inhibition of peptidyl-prolyl cis/trans isomerase activity by substrate analog structures: thioxo tetrapeptide-4-nitroanilides.

The ubiquitous cyclophilins belong to peptidyl-prolyl cis/trans isomerases (PPIases; EC 5.2.1.8). They are able to catalyze the cis/trans isomerization about peptidyl-prolyl amide bonds. The mode of action of human cytosolic cyclophilin (Cyp18cy) has been studied on substrate analog tetrapeptide-4-nitroanilides containing the thioxo peptidyl-prolyl bond. Five peptides of the general structure Ala-Xaa-psi [CS-N]-Pro-Phe-NH-Np (Xaa = Gly, Ala, (S)-2-aminobutyric acid, Phe, and Leu) containing the thioxo peptidyl-prolyl bond were synthesized. The kcat values for the chymotryptic cleavage of 4-nitroanilide bond of the thioxo tetrapeptide-4-nitroanilides ranged from 1.7 to 9.0 s-1 and were sufficiently high to analyze the conformational equilibria by isomer-specific proteolysis. The rate constants of the cis/trans isomerization of the thioxo peptidyl-prolyl bond were found to be 25-100-fold lower due to the O/S substitution. Cyp18cy binds both thioxo peptides and oxo peptides in similar manner in the active center but cannot utilize the sulfur analogs as substrates. Instead, competitive inhibition occurs, which was further characterized for Ala-Gly-psi[CS-N]-Pro-Phe-NH-Np. The inhibition was nearly independent of the pH value in the range of pH 4.5-9, exhibiting apparent Ki values ranging from 200 to 600 microM. In comparison to Ala-Gly-trans-psi[CS-N]-Pro-Phe-NH-Np, the cis thioxo peptide Ala-Gly-cis-psi[CS-N]-Pro-Phe-NH-Np was found to possess an approximately 30-fold higher affinity for the active site of the enzyme. Thus, in the presence of stoichiometric amounts of Cyp18cy, the total amount of Ala-Leu-cis-psi[CS-N]-Pro-Phe-NH-Np in solution, detectable by isomer-specific proteolysis, was considerably enhanced.

Acting via A2 receptors, adenosine inhibits the upregulation of Mac-1 (Cd11b/CD18) expression on FMLP-stimulated neutrophils.

Modulation of neutrophil responses by adenosine may have an important role in limiting tissue injury during inflammation or ischemia-reperfusion. Mac-1 (CD11/CD18), a member of the leukocyte integrin family, participates in neutrophil adhesion to endothelium, in transendothelial migration, and in phagocytosis. Using monoclonal antibodies and flow cytometry, we investigated the effect of adenosine on the increase in plasma membrane expression of Mac-1 which occurs following stimulation of neutrophils with the chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP). Adenosine and 5'N-ethylcarboxamido-adenosine, a potent A2 agonist, each produced a dose-dependent inhibition of as much as 50% of the increase in Mac-1 expression on neutrophils stimulated with FMLP, with an IC50 of approximately 1 nM. The effect of adenosine was blocked by 8-p-sulfophenyltheophylline, an adenosine-receptor antagonist, N6-cyclopentyl-adenosine, an A1-selective agonist, had no effect on FMLP-stimulated Mac-1 expression in the concentration range expected for its action on neutrophil adenosine receptors of the A1 type. We also found that dibutyryl cyclic adenosine monophosphate inhibited the upregulation of Mac-1, and that the effect of adenosine on Mac-1 expression was not reversed by colchicine or vinblastine. We conclude that adenosine acts via A2 receptors to inhibit the upregulation of Mac-1 expression of FMLP-stimulated neutrophils, and that A1 receptors are not involved. This effect of adenosine may help to limit Mac-1-dependent neutrophil exudation at sites of inflammation or ischemia-reperfusion.

Physical properties and testing methods for PTFE cardiovascular patches.

Patching after endarterectomy, especially carotid artery surgery, is a common procedure to repair and close the surgical site. Both synthetic and natural materials can be used, but saphenous vein is preferred due to its greater long-term patency. In situations where it is not possible to use the saphenous vein, both Dacron and expanded polytetrafluoroethylene (ePTFE) patches have been used successfully. Expanded PTFE patches are readily available, soft and pliable, have excellent biocompatibility and do not require preclotting prior to implantation. Comparison of two types of ePTFE patches versus natural vessel show that they have more than adequate properties for their intended use.

Serum amyloid P-component levels in patients with malignancy.

The P component of amyloid is a normal serum protein designated SAP. SAP has substantial homology with C-reactive protein (CRP). However, unlike CRP, SAP is not an acute-phase reactant in man. Recent studies have established SAP as a major acute-phase protein in mice. Moreover, mice which have received tumour implants have also been found to have raised serum concentrations of SAP. The aim of the present study was to determine possible association between the serum level of SAP and human cancer. We found that patients with carcinoma of the breast have significantly increased serum concentrations of SAP. Moreover, in these patients SAP levels correlated with the severity of the disease. Patients with carcinoma of the colon, however, did not differ from healthy individuals in the serum level of SAP. Possible explanations for this discrepancy are discussed.

Serum IgE levels in patients with inflammatory bowel disease.

The role of allergy in the pathogenesis of inflammatory bowel disease (IBD) is unclear. The present study was performed to evaluate serum levels of IgE and other immunoglobulin classes in patients with IBD. Patients with IBD had significantly elevated serum levels of both IgG and IgM in the presence of normal levels of IgA. Serum concentration of IgE, as well as the prevalence of patients with "high IgE" were significantly increased in IBD. Among patients with IBD, those with Crohn's disease or those in relapse had the highest levels of IgE. The possibility that allergy plays a pathogenic role in a subset of IBD is discussed.

Serum amyloid P-component as a marker of liver involvement in measles infection.

The P-component of amyloid (SAP) is a normal serum protein. Recent studies have pointed to the liver as the site of synthesis of SAP. Moreover, we have recently demonstrated a close correlation between serum level of SAP and the degree of liver impairment in patients with acute hepatitis, chronic active hepatitis, and cirrhosis. In the present study we have investigated liver involvement and serum SAP level during measles infection. Up to 80% of patients with measles had evidence of hepatic dysfunction. Serum SAP level was markedly decreased in patients with measles and correlated with the presence of liver involvement. Taken together, this finding suggests that serum SAP level might be a useful and sensitive indicator of liver disease.

Serum IgE levels in patients with cancer.

Serum levels of IgE and carcinoembryonic antigen (CEA) were determined in 100 patients with carcinoma of the breast and 81 with colon carcinoma in different stages of their disease. CEA levels reflected the stage of the disease and increased progressively from stage 1 to 4. In contrast, all patients regardless of their type of malignancy, stage of the disease, or CEA level had a similar IgE blood level which did not differ from that of the control group.

In vivo clearance and tissue uptake of an anti-DNA monoclonal antibody and its complexes with DNA.

In vivo clearance and tissue localization of a purified mouse anti-DNA monoclonal antibody (MoAb) (A52 IgG2b) and its complexes with DNA were studied in normal BALB/c and autoimmune NZB/NZW mice. The plasma half-life of the autoantibody in both mouse strains was significantly shorter (T 1/2 = 10-15 min), compared with that of purified NZB myeloma proteins (T 1/2 greater than or equal to 180 min). DNA antigen and DNA-A52 IgG complexes in antibody excess were cleared very rapidly (T 1/2 = 4-8 min), while complexes formed in antigen excess persisted in the circulation much longer (T 1/2 = 60 min). Organ studies showed that the anti-DNA MoAb was transiently retained by the liver and the spleen but demonstrated a particular affinity for the kidney tissue. We suggest that tissue damage in SLE glomerulonephritis may be facilitated by direct interaction of anti-DNA antibodies with glomerular components.

Effects of serum amyloid P component on human lymphocytes.

The P component of amyloid is a normal serum protein designated SAP. SAP has substantial homology with C-reactive protein (CRP). Recent studies have established the structure, tissue distribution and binding reactivities of SAP; however, as yet, very minimal insight into its function has been achieved. Recent studies, though somewhat controversial, have suggested a regulatory role for CRP on the immune response. In view of these studies, we wanted to evaluate the in vitro effects of SAP on several immunological properties of human lymphocytes. We found that SAP had a marked inhibitory effect on the proliferative response of lymphocytes to a variety of T-dependent mitogens. In addition, SAP markedly enhanced the formation of active E rosettes, a marker of activated T cells.

Immunoglobulin E response during measles.

Serum IgE levels were determined in 41 patients with measles infection. 23 patients (56%) had an increased level (greater than 20%) during the acute phase of the disease in comparison with the recovery phase. 14 showed no change, and decreased levels were observed in 4 patients only. In contrast, both IgG and IgA levels decreased or remained unchanged in the majority of the patients during disease activity. The changes in the pattern of serum immunoglobulins during measles might be attributed to selective depletion of T-cell subpopulations.

Cryoglobulinemia in acute type-A hepatitis.

Serum cryoglobulins were detected in 18 of 19 patients with type-A, and six of eight with type-B hepatitis. The predominant immunoglobulin in the cryoprecipitates was polyclonal IgM. Patients with type-A hepatitis had a significantly higher mean serum level of cryoglobulins when compared to those with type-B (106.9 vs. 20.5 micrograms/ml). IgM anti-hepatitis-A virus activity was detected in all cryoprecipitates obtained from hepatitis-A patients. Cryoglobulinemia in these patients was transient, associated with disease activity and accompanied by a marked increase in serum level of IgM. These findings are compatible with a recent hypothesis, which predicts the appearance of cryoglobulins under circumstances of enhanced stimulation of B cells in the presence of defective clearance of desialylated glycoproteins by their specific liver receptors.

Immunological evaluation of asymptomatic carriers of hepatitis B virus.

The immune system of 69 asymptomatic HBsAg carriers with normal liver function tests was evaluated. B cell function, as documented by serum immunoglobulin levels, number of mouse rosette-forming lymphocytes and lymphocyte reactivity to staphylococcal protein A, was intact. On the other hand, T cell function was markedly impaired. This was manifested by a significant decrease in E rosette-forming lymphocytes, an increase in stable rosette-forming cells and decreased reactivity to phytohaemagglutinin and concanavalin A. These data rule out the possibility that the immunological aberrations associated with hepatitis B infection are secondary to liver injury. The abnormal immune state either precedes the viral infection, thus predisposing to the acquisition of a carrier state or, alternatively, is a direct result of the infection.

Immunological study of patients with the Papillon--Lefevre syndrome.

Papillon-Lefevre syndrome (PLS) is characterized by palmoplantar hyperkeratosis and by premature periodontal breakdown. In view of recent suggestions that this syndrome is associated with increased susceptibility to infections we decide to evaluate several immunological parameters in PLS patients and their healthy siblings and parents. PLS patients, as well as their siblings and parents, had a significantly impaired reactivity to both T and B cell mitogens. This in vitro abnormality was not associated with increased susceptibility to infections. The relationship between the impaired in vitro response to mitogens and the predilection for dermatologic and dental manifestations of PLS is discussed.

Increased collagen metabolism in granulomata induced in rats deficient in endogenous prostaglandin precursors.

Collagen metabolism was measured (in terms of various hydroxyproline (HP), DNA and protein ratios) in granulomata obtained after s.c. implantation of carrageenan-impregnated and untreated polyether sponges into normal and essential fatty acid deficient (EFAD) rats for 8 and 15 days. Collagen synthesis (HP/protein) in day 8 and 15 untreated granulomata was the same for both normal and EFAD rats, though collagen breakdown (total HP) appeared to be greater in EFAD granulomata on day 15. With carrageenan-impregnated sponges, collagen synthesis in EFAD granulomata was much greater than in normal granulomata on both day 8 and day 15. Ratios of protein and/or HP to DNA (probably indicative of cellular infiltration) were increased in EFAD rats with both sponge types, though this increase was less pronounced with carrageenan-impregnated sponges. It is suggested that endogenous prostaglandin (PG) production (marledly reduced during EFA deficiency) may exert a negative feedback effect on collagen metabolism during proliferative inflammation.