TACI is a TRAF-interacting receptor for TALL-1, a tumor necrosis factor family member involved in B cell regulation.

We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.

Selective association of the p59fyn tyrosine kinase with murine T lymphoma membrane phosphoproteins.

The p59fyn oncogene product, a tyrosine kinase of the src family, is a key enzyme in T-lymphocyte transmembrane signalling that is not known to bind with high stoichiometry to any surface receptor protein. By contrast, the p56lck, another important T-cell tyrosine kinase, is directly linked to CD4 or CD8 surface glycoproteins with high stoichiometry. To document the mode of p59fyn interaction with proteins of the plasma membrane, proteins co-precipitating with the kinase in extracts of purified membranes were phosphorylated in vitro, resolved by two-dimensional electrophoresis and compared with those co-precipitating with the p56lck kinase. The p59fyn, but not the p56lck kinase, associates with three phosphotyrosyl-proteins among which a 95- to 100-kDa component also binds to the src-homology 2 (SH2) domain of the v-crk oncoprotein. This p95-100 phosphoprotein is not the p95Var product of the Vav proto-oncogene; it is recovered much more effectively from 32Pi-metabolically labelled cells after treatment with the tyrosine phosphatase inhibitor phenylarsenoxide, suggesting that its phosphorylation state is controlled by tyrosine phosphatases. The p59fyn may be integrated into the plasma membrane by specific phosphotyrosylproteins and so differ from the p56lck tyrosine kinase, which binds preferentially to the CD4 and CD8 transmembrane adhesion glycoproteins.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice.

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

Platelets play a role in the pathogenesis of the irritant reaction in mice.

The irritant reaction is a model of local inflammation that results from the epicutaneous application of a small molecule with irritating properties such as trinitrochlorobenzene (TNCB). The irritant reaction is mediated by tumor necrosis factor (TNF) and is characterized by skin edema and neutrophil (PMN) infiltration. The aim of this study was to explore the role of platelets in the pathogenesis of the irritant reaction. Mice depleted of platelets by an anti-platelet antibody showed a decrease in edema upon the application of a low dose of TNCB--chosen for causing an irritant reaction that is not complicated by intravascular fibrin deposition and hemorrhage--but no significant change in PMN infiltration. There was platelet trapping in TNCB-treated ears that was maximum between 2 and 6 h after TNCB application. Platelets lined the venular endothelium, which was intact in the absence of hemorrhage, and were not accompanied by fibrin. Mice treated with anti-TNF, anti-CD11a, anti-CD18, or anti-CD54 antibodies showed a decrease in platelet trapping, edema, and PMN infiltration. Platelets contribute to the pathogenesis of the irritant reaction and are necessary for edema to develop but not for PMN infiltration. The role of platelets implicates their early localization in the dermal venules, which depends, at least in part, on TNF and on the adhesion molecules involved in the interaction between CD11a/CD18 and CD54.

Protective effect of N-acetylcysteine in hapten-induced irritant and contact hypersensitivity reactions.

The hapten-induced irritant and contact hypersensitivity reactions are experimental models of cutaneous inflammation in which tumor necrosis factor-alpha is an important mediator. N-acetylcysteine is an anti-oxidant that inhibits the action of the nuclear factor-kB, which promotes the transcription of many genes, including the gene for tumor necrosis factor-alpha. We tested the ability of N-acetylcysteine to antagonize the development of the irritant and contact hypersensitivity reactions induced by the epicutaneous application of trinitrochlorobenzene in mice. Systemic and topical treatment with N-acetylcysteine reduced skin swelling in both the irritant and contact hypersensitivity reactions; in the latter it also reduced the dermal leukocyte infiltrate. It also reduced the cutaneous expression of the mRNA for tumor necrosis factor-alpha in both conditions. These results show that N-acetylcysteine antagonizes the development of irritant and contact hypersensitivity reactions and that its action includes a reduction in the expression of tumor necrosis factor-alpha mRNA. N-acetylcysteine may be useful in the treatment of cutaneous inflammation mediated by tumor necrosis factor-alpha.

Detection of HTLV-III-specific IgG bands in the CSF from a patient with AIDS and encephalitis.

Recent evidence would suggest that HTLV-III may be neurotropic. We have found oligoclonal IgG bands by isoelectric focusing in the CSF of a homosexual man with AIDS and encephalitis. Subsequent analysis revealed that such bands contained anti-HTLV-III activity, suggesting that neurologic symptoms in AIDS patients may be caused by replication of HTLV-III inside the CNS.

Methods for assessing complement activation in the clinical immunology laboratory.

Complement activation is a key component of the pathogenesis of immune-mediated tissue damage in many diseases. Assessment of complement activation in current practice is largely based on the measurement of intact C3 and C4 or the determination of complement haemolytic function. These parameters reflect activation only indirectly, are insensitive and open to influence by factors other than complement conversion. New approaches to evaluate complement activation directly using sensitive techniques have been developed, and several could be adopted easily in most laboratories. These concentrate on the detection of activation fragments, neoantigens or complexes that only arise as a direct result of complement activation. The wide application of these techniques in research and clinical practice would enhance our understanding of the pathogenesis of a range of inflammatory and infectious diseases.

Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen.

Field medicine in tropical areas needs laboratory assays which are inexpensive and easy to perform. To meet this need a semi-quantitative dot-enzyme immunoassay (EIA) was developed for the detection of an L. donovani-related circulating antigen and tested for clinical relevance in the diagnosis of visceral leishmaniasis (VL). The dot-EIA probes serum spotted on nitrocellulose for the presence of the antigen using a monoclonal antibody raised against L. donovani promastigotes, a peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum and chloronaphthol as peroxidase substrate. The intensity of dot staining by chloronaphthol is read by eye and scored. The dot-EIA was used to test the following groups: 69 patients with VL from Brazil, Kenya, China and France, nine patients with cutaneous leishmaniasis, 38 patients with tropical diseases other than VL, five patients with rheumatoid arthritis and 40 health blood donors. The specificity of the assay was 96.7% (3/92 false positive) and the sensitivity 98.5% (1/69 false negative). A quantitative EIA for the detection of serum antibodies which makes use of a crude, soluble L. infantum promastigote extract as capture antigen and which was used as the reference method, proved to be more specific (98.9%) but similarly sensitive (98.5%). It should be possible to produce a kit, suitable for large scale application at low cost in order to facilitate routine use of the dot-EIA in the diagnosis of VL.

Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay.

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.

Increased total serum IgE concentration in patients who have undergone splenectomy after trauma.

Serum immunoglobulin and complement concentrations were evaluated in 20 patients who had undergone splenectomy after trauma. The concentrations of IgE and IgA in the patients were significantly increased compared with those in controls. The IgE values were not correlated with the time after splenectomy or IgA, IgG, IgM, IgD, C3, and C4 values.

Class I and class II major histocompatibility complex antigens on hepatocytes: importance of the method of detection and expression in histologically normal and diseased livers.

Methodological differences in major histocompatibility complex (MHC) antigen detection were investigated on isolated, viable hepatocytes and cryostat hepatic sections from 27 children with liver disorders, six of whom had normal histology. Class I antigens were constantly found on sections using a three step immunoperoxidase technique after acetone/chloroform fixation, other techniques being less sensitive, or on isolated hepatocytes by indirect immunofluorescence alone. With mechanical isolation the percentage of positivity ranged from 85 to 100%, while with collagenase isolation it ranged from 22 to 49% on immediate testing, and from 53 to 80% after 24 hour incubation. Class II antigens were only detected in one patient with autoimmune chronic active hepatitis and two with primary sclerosing cholangitis. Flow cytofluorimetric analysis in 11 cases confirmed class II or class I positivity, or both, on isolated hepatocytes, allowing MHC antigen expression on hepatocytes to be measured. Class I and II antigen detection on hepatocytes is influenced by the technique used. Although class I antigens are invariably expressed on hepatocytes, class II antigens are only found on hepatocytes from patients with immune mediated liver disorders.

Activation of the alternative complement pathway: clinical application of a new technique to measure fragment Ba.

A new laser nephelometric technique for the measurement of the alternative complement pathway fragment Ba has been developed. Activation of the alternative complement pathway was assessed in 16 patients with Gram negative bacteraemia, six with Gram positive bacteraemia, 20 with rheumatoid arthritis, and 18 healthy subjects. Patients with Gram negative bacteraemia had significantly higher values of Ba (median 14.8%) than controls (9.3%) (p less than 0.01), while patients with Gram positive bacteraemia and rheumatoid arthritis had values similar to those of controls (10.1% and 9.5%). The technique proved sensitive and precise, and is suitable for the routine laboratory evaluation of complement activation through the alternative pathway.

Low serum haemolytic function of the fourth complement component (C4) in insulin dependent diabetes.

Low serum concentrations of the fourth component of complement (C4) are found in insulin dependent diabetes, and may be important in the aetiology of the disease. To ascertain whether function of C4 is also impaired both its haemolytic activity and its concentration were measured in 34 insulin dependent diabetics, 15 non-insulin dependent diabetics, 20 healthy subjects, and 12 pairs of monozygotic twins discordant for insulin dependent diabetes. C4 function was measured by a radial immune haemolytic assay, and C4 concentration by laser nephelometry. Both measurements were significantly lower in insulin dependent diabetics (C4 function: median 47%, range 4-100%; C4 concentration: 0.22 g/l, 0.10-0.38 g/l) than in non-insulin dependent diabetics (67%, 33-138%, p less than 0.01; 0.27 g/l, 0.16-0.50 g/l, p less than 0.02) and controls (74%, 33-138%, p less than 0.01; 0.27 g/l, 0.18-0.40 g/l, p less than 0.03). C4 function and concentration were lower in both diabetic (48%, 12-100%; 0.17 g/l, 0.08-0.31 g/l) and non-diabetic twins (47%, 12-100%; 0.17 g/l, 0.07-0.36 g/l) than controls (p less than 0.01; p less than 0.01). Thirteen (38%) of the insulin dependent diabetics had a reduction in either C4 function or concentration, but in only five were both features reduced. Values of function and concentration were strongly correlated in both diabetic and non-diabetic twins (r = 0.95, p less than 0.001; r = 0.92, p less than 0.001). These results show defects in C4 function and concentration in insulin dependent diabetes, which--being present in the non-diabetic co-twin of diabetics--may represent a genetic predisposition to the disease.

Clinical application of new technique that measures C4d for assessment of activation of classical complement pathway.

A new laser nephelometric technique that measures C4d for the assessment of the activation of the classical complement pathway was developed. C4d was isolated from other larger C4 related molecules at a final concentration of polyethylene glycol of 12% and then quantitated by laser nephelometry using a commercially available antiserum, which reacts with C4d determinants. C4d standard (100%) was produced by exhaustive activation of the classical pathway in pooled normal human serum using heat aggregated human immunoglobulin. Serial dilutions of the standard provided a reference curve against which clinical samples were read. Patients with rheumatoid arthritis showed significantly higher C4d values (mean 53.8%) than controls (21.7%; p less than 0.001). The technique proved accurate, rapid, and suitable for the routine laboratory evaluation of complement activation through the classical pathway, and it may be useful in the management of those conditions in which complement activation has a pathogenic role.

Circulating levels of mannose binding protein in human immunodeficiency virus infection.

Mannose binding protein (MBP) is a serum lectin which, upon binding to a carbohydrate extremity, acquires the ability to activate the classical complement pathway. MBP binds human immunodeficiency virus (HIV) in vitro via glycans on gp120 and thus, it may play a defensive role in HIV infection and contribute to virus clearance through the activation of complement associated with this condition. We measured serum MBP and activation indices of the classical complement pathway (plasma C4d and C3d) in HIV-seropositive patients at different stages of disease severity, and in normal subjects. MBP was higher in HIV patients as a whole and in each Centers for Disease Control (CDC) group than controls (P<0.01). MBP was not significantly different between CDC groups and and did not significantly correlate either with CD4-positive lymphocytes, neopterin or beta2-microglobulin or with C4d and C3d. The possibility that MBP plays a defensive role in HIV infection cannot be excluded, but, it it is, it does not appear to act by recruiting complement for vital elimination.

Relationship between the tumour-associated antigen 90K and cytokines in the circulation of persons infected with human immunodeficiency virus.

A tumour-associated antigen known as 90K has been found in high concentrations in the serum of patients infected with human immunodeficiency virus (HIV) even in the absence of neoplastic complications. In order to investigate the relationship between the production of 90K and soluble inflammatory mediators, we studied serum concentrations of the antigen, tumour necrosis factor-alpha (TNF-alpha), interleukin-I-alpha (IL-I-alpha), interferon-gamma (IFN-gamma), IFN-alpha, neopterin and beta 2-microglobulin (beta 2-m) in patients with non-neoplastic HIV infection at various stages of disease and in control persons. The antigen was detected in all those studied but its concentration was higher in HIV-infected patients compared with controls (P < 0.001), increasing progressively with advancing stages of disease. There was a negative correlation between concentrations of 90K and IL-I-alpha in patients in U.S.A. Centers for Disease Control groups II and III (P < 0.02) and also between that of 90K and both TNF-alpha (P < 0.01) and IL-I-alpha (P < 0.05) in control persons. The results indicate that 90K is not merely a tumour-associated antigen and that its production may be part of immune and inflammatory responses in the absence of neoplasia. The correlation between the concentrations of 90K and of some cytokines in asymptomatic patients and healthy persons suggests that 90K may be part of a network of immune and inflammatory reactants.