TSG-6 - a double-edged sword for osteoarthritis (OA).

PURPOSE: To explore mechanisms underlying the association of TSG-6 with osteoarthritis (OA) progression. METHODS: TSG-6-mediated heavy chain (HC) transfer (TSG-6 activity) and its association with inflammatory mediators were quantified in knee OA (n=25) synovial fluids (SFs). Paired intact and damaged cartilages from the same individuals (20 tibial and 12 meniscal) were analyzed by qRT-PCR and immunohistochemistry (IHC) for gene and protein expression of TSG-6 and components of Inter-alpha-Inhibitor (IαI) and TSG-6 activity ± spiked in IαI. Primary chondrocyte cultures (n=5) ± IL1ß or TNFα were evaluated for gene expression. The effects of TSG-6 activity on cartilage extracellular matrix (ECM) assembly were explored using quantitative hyaluronan (HA)-aggrecan binding assays. RESULTS: TSG-6 activity was significantly associated (R > 0.683, P < 0.0002) with inflammatory mediators including TIMP-1, A2M, MMP3, VEGF, VCAM-1, ICAM-1 and IL-6. Although TSG-6 protein and mRNA were highly expressed in damaged articular and meniscal cartilage and cytokine-treated chondrocytes, there was little or no cartilage expression of components of the IαI complex (containing HC1). By IHC, TSG-6 was present throughout lesioned cartilage but HC1 only at lesioned surfaces. TSG-6 impaired HA-aggrecan assembly, but TSG-6 mediated HA-HC formation reduced this negative effect. CONCLUSIONS: TSG-6 activity is a global inflammatory biomarker in knee OA SF. IαI, supplied from outside cartilage, only penetrates the cartilage surface, restricting TSG-6 activity (HC transfer) to this region. Therefore, unopposed TSG-6 in intermediate and deep regions of OA cartilage could possibly block matrix assembly, leading to futile synthesis and account for increased risk of OA progression.

Tumor necrosis factor-inducible gene 6 protein: A novel neuroprotective factor against inflammation-induced developmental brain injury.

Inflammation is an important factor contributing to developmental brain injury in preterm infants. Although tumor necrosis factor-inducible gene 6 protein (TSG-6) has immunomodulatory effects in several inflammatory conditions of adult animals, nothing is currently known about the role of TSG-6 in the developing brain, its impact on perinatal inflammation and its therapeutic potential. The aim of the current work was 1) to characterize the developmental expression of TSG-6 in the newborn rat brain, 2) to evaluate the impact of LPS exposure on TSG-6 expression and 3) to assess the therapeutic potential of exogenous TSG-6 administration. Brain hemispheres of healthy Wistar rats (postnatal day 1-postnatal day 15 (P1-P15)) were evaluated with regard to the physiological expression of TSG-6. LPS-treated rats (0.25mg/kg LPS i.p. on P3) were analyzed for inflammation-induced changes in TSG-6 and cytokine expression. To evaluate whether exogenous recombinant human (rh)TSG-6 affects inflammation-induced brain injury, newborn Wistar rats, exposed to LPS on P3, were treated with rhTSG-6 i.p. (four repetitive doses of 2.25mg/kg every 12h, first dose 3h before LPS injection). PCR, Western blotting and multiplex ELISA were performed according to standard protocols. TSG-6 is physiologically expressed in the developing brain with a linear increase in expression from P1 to P15 at the mRNA level. At P6, regional differences in TSG-6 expression in the cortex, thalamus and striatum were detected at mRNA and protein level. Furthermore, TSG-6 gene expression was significantly increased by inflammation (induced by LPS treatment). Combined treatment with LPS and TSG-6 vs. LPS exposure alone, resulted in significant down-regulation of cleaved caspase-3, a marker of apoptosis and neuronal plasticity. In addition, several inflammatory serum markers were decreased after TSG-6 treatment. Finally, TSG-6 is physiologically expressed in the developing brain. Changes of TSG-6 expression associated with inflammation suggest a role of TSG-6 in neuroinflammation. Reduction of cleaved caspase-3 by TSG-6 treatment demonstrates the putative neuroprotective potential of exogenous TSG-6 administration in inflammation-induced developmental brain injury.

Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression.

OBJECTIVE: We investigated the relationship between the molecular weight (MW) distribution of hyaluronan (HA) in synovial fluid (SF) and risk of knee osteoarthritis (OA) progression. METHODS: HA MW was analyzed for 65 baseline knee SFs. At 3-year follow-up, knees were scored for change in joint space narrowing (JSN), osteophyte (OST) progression, or occurrence of total knee arthroplasty (TKA). HA MW distribution was analyzed using agarose gel electrophoresis (AGE), and its relationship to OA progression was evaluated using logistic regression. The association between HA MW and self-reported baseline knee pain was analyzed using Pearson's correlation coefficients. RESULTS: Knee OA was categorized as non-progressing (OST-/JSN-, 26 knees, 40%), or progressing based on OST (OST+/JSN-, 24 knees, 37%), OST and JSN (OST+/JSN+, 7 knees, 11%) or total knee arthroplasty (TKA, 8 knees, 12%). The MW distribution of HA in baseline SFs was significantly associated with the odds of OA progression, particularly for index knees. After adjusting for age, gender, BMI, baseline X-ray grade and pain, each increase of one percentage point in %HA below 1 million significantly increased the odds of JSN (odds ratios (OR) = 1.45, 95% CI 1.02-2.07), TKA or JSN (OR = 1.24, 95%CI 1.01-1.53) and the odds of any progression (OR = 1.16, 95% CI 1.01-1.32). HA MW distribution significantly correlated with pain. CONCLUSION: These data suggest that the odds of knee OA progression increases as HA MW distribution shifts lower and highlight the value of reporting MW distribution rather than just average MW values for HA.

TSG-6 activity as a novel biomarker of progression in knee osteoarthritis.

OBJECTIVE: To establish whether there is an association between TSG-6 activity and osteoarthritis progression. DESIGN: TSG-6 activity was determined in 132 synovial fluids from patients with OA of the knee, using a novel quantitative TSG-6 activity assay. The association between TSG-6 activities at baseline and four distinct disease progression states, determined at 3-year follow-up, was analyzed using logistic regression. RESULTS: There was a statistically significant relationship between TSG-6 activity at baseline and all OA progression states over a 3-year period. Patient knees with TSG-6 activities in the top tenth percentile, compared to the median activity, had an odds ratio (OR) of at least 7.86 (confidence interval (CI) [3.2, 20.5]) for total knee arthroplasty (TKA) within 3 years, and of at least 5.20 (CI [1.8, 13.9]) after adjustment for confounding factors. Receiver operating characteristic (ROC) analysis for knee arthroplasty yielded a cut-off point of 13.3 TSG-6 activity units/ml with the following parameters: area under the curve 0.90 (CI [0.804, 0.996]), sensitivity 0.91 (CI [0.59, 0.99]), specificity 0.82 (CI [0.74, 0.88]) and a negative predictive value (NPV) of 0.99 (CI [0.934, 0.994]). CONCLUSION: The TSG-6 activity is a promising independent biomarker for OA progression. Given the high NPV, this assay may be particularly suitable for identifying patients at low risk of rapid disease progression and to assist in the timing of arthroplasty.

Reduced susceptibility to collagen-induced arthritis in DBA/1J mice expressing the TSG-6 transgene.

OBJECTIVE: Expression of TSG-6 (tumor necrosis factor-stimulated gene 6) is induced by proinflammatory cytokines. This study was undertaken to examine the effects of local expression of TSG-6 in arthritic joints of TSG-6 transgenic mice, in the collagen-induced arthritis (CIA) model. METHODS: We generated transgenic mice that harbored the TSG-6 gene under the control of the T cell-specific lck promoter. Arthritis was induced by immunization with bovine type II collagen (CII), and its progression was monitored based on the incidence of arthritis, the arthritis index, and footpad swelling. Anti-CII antibodies and cytokine production were determined by enzyme-linked immunosorbent assay. Gene expression arrays were used to compare gene expression profiles of transgenic and control mice at various stages of CIA. RESULTS: TSG-6 was expressed in limbs of transgenic mice after immunization with CII, while its expression in nontransgenic animals was insignificant. The incidence of CIA was reduced in TSG-6 transgenic animals, its onset delayed, and all parameters of clinical arthritis significantly reduced. However, the immune response against CII was not significantly inhibited in TSG-6 transgenic mice. CONCLUSION: TSG-6 expression has been demonstrated in patients with rheumatoid and other forms of arthritis. Our data show that local expression of TSG-6 at sites of inflammation results in potent inhibition of inflammation and joint destruction in a model of autoimmune arthritis in mice. Therefore, it is likely that TSG-6 plays a similar modulatory role in human rheumatoid arthritis and related diseases and may have potential for the treatment of autoimmune arthritis in humans.

Amelioration of collagen-induced arthritis in DBA/1J mice by recombinant TSG-6, a tumor necrosis factor/interleukin-1-inducible protein.

OBJECTIVE: To examine the effect of recombinant TSG-6 on collagen-induced arthritis (CIA) in DBA/1J mice. TSG-6 is a tumor necrosis factor (TNF)/ interleukin-1 (IL-1)-inducible hyaluronan-binding protein produced by synovial cells and chondrocytes that is present in synovial fluids of patients with rheumatoid arthritis. METHODS: To determine the effect of TSG-6 on chronic inflammatory joint disease, we induced CIA in DBA/1J mice by immunization with bovine type II collagen. Animals were treated with 12 intraperitoneal doses of 200 microg of recombinant TSG-6, beginning 3 days before the expected onset of disease symptoms. Progression of arthritis was monitored by determining the disease incidence, arthritis index, and footpad swelling. Levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen and serum concentrations of IL-6 were determined at various time points. Histologic examination of affected joints was performed approximately 20 days after the onset of arthritis. RESULTS: Treatment with recombinant TSG-6 protein had a potent ameliorative effect, manifested by decreases in the disease incidence, arthritis index, and footpad swelling. Histologic examination of affected joints in TSG-6-treated animals revealed little pannus formation and cartilage erosion, features which were conspicuous in control mice. Animals treated with recombinant TSG-6 developed significantly reduced levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen. CONCLUSION: The antiinflammatory effect of the TNF/IL-1-inducible TSG-6 protein in murine CIA suggests a role for this protein as an endogenous regulator of the inflammatory process.

Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines.

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal-induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.

TSG-6: an IL-1/TNF-inducible protein with anti-inflammatory activity.

The pro-inflammatory cytokines IL-1 and TNF-alpha are primary mediators of the acute phase response, the complex reaction of the mammalian organism to infection and injury. Among the genes activated by TNF-alpha and IL-1 in a variety of cells is TNF-stimulated gene 6 (TSG-6). The TSG-6 cDNA encodes a secreted 35 kDa glycoprotein which is abundant in synovial fluids of patients with various forms of arthritis and detectable in serum of patients with different inflammatory or autoimmune disorders. TSG-6 protein consists of two structural domains: a hyaluronan-binding link module, the characteristic domain of the hyaladherin family of proteins, and a C-terminal CUB domain, present in a variety of diverse proteins. TSG-6 forms a stable complex with components of the plasma protein inter-alpha-inhibitor (I[alpha]I), a Kunitz-type serine protease inhibitor. TSG-6 and I(alpha)I synergize to inhibit plasmin, a serine protease involved in the activation of matrix metalloproteinases which are part of the proteolytic cascade associated with inflammation. Recombinant human TSG-6 protein exerts a potent anti-inflammatory effect in a murine model of acute inflammation. Modulation of the proteolytic network associated with inflammatory processes may be a mechanism whereby TSG-6, in cooperation with I(alpha)I, inhibits inflammation. Activation of the TSG-6 gene by pro-inflammatory cytokines, presence of TSG-6 protein in inflammatory lesions and its anti-inflammatory effect suggest a role for TSG-6 in a negative feed-back control of the inflammatory response.

Long pentraxins: an emerging group of proteins with diverse functions.

The earliest described pentraxins, C reactive protein (CRP) and serum amyloid P component (SAP), are cytokine-inducible acute phase proteins implicated in innate immunity whose concentrations in the blood increase dramatically upon infection or trauma. The highly conserved family of pentraxins was thought to consist solely of approximately 25 kDa proteins. Recently, several distinct larger proteins have been identified in which only the C-terminal halves show characteristic features of the pentraxin family. One of the recently described "long" pentraxins (TSG-14/PTX3) is inducible by TNF or IL-1 and is produced during the acute phase response. Other newly identified long pentraxins are constitutively expressed proteins associated with sperm-egg fusion (apexin/p50), may function at the neuronal synapse (neuronal pentraxin I, NPI), or may serve yet other, unknown functions (NPII and XL-PXN1). Evidence obtained by molecular modeling and by direct physicochemical analysis suggests that TSG-14 protein retains some characteristic structural features of the pentraxins, including the formation of pentameric complexes.

TSG-6 expression in human articular chondrocytes. Possible implications in joint inflammation and cartilage degradation.

OBJECTIVE: The hyaluronan-binding protein TSG-6 (tumor necrosis factor-stimulated gene 6) forms a stable complex with the serine protease inhibitor, inter-alpha-inhibitor, potentiates the inhibition of plasmin activity, and has antiinflammatory effects in vivo. This study examines the expression of TSG-6 in human articular chondrocytes and cartilage. METHODS: Human articular chondrocytes and cartilage explants were stimulated with cytokines, growth factors, and other agents. TSG-6 expression was analyzed by imaging-assisted Northern and Western blotting. RESULT: TSG-6 messenger RNA (mRNA) expression was upregulated by cytokines and growth factors, predominantly interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), platelet-derived growth factor AA (PDGF-AA), and transforming growth factor beta 1 (TGF beta 1). TSG-6 mRNA induction by TGF beta 1 was delayed as compared with IL-1beta. Treatment of the cells with the glucocorticoid dexamethasone neither induced TSG-6 mRNA nor did it affect IL-1 beta-induced transcript levels. TSG-6 mRNA induction may involve several signal transduction pathways. The strong transcriptional stimulation by phorbol myristate acetate suggests protein kinase C (PKC)-mediated signaling. In contrast, PKA- and Ca- dependent signals are only marginally involved as messengers leading to increased TSG-6 levels after IL-1beta and TNF alpha treatment. In chondrocyte and cartilage organ cultures, both free TSG-6 (35 kd) and the complex with inter-alpha-inhibitor (120 kd) were present and upregulated by IL-1 beta, TNF alpha, or TGF beta 1. CONCLUSION: Chondrocytes are a source of TSG-6 which may play a role in cartilage remodeling and joint inflammation.

TNF/IL-1-inducible protein TSG-6 potentiates plasmin inhibition by inter-alpha-inhibitor and exerts a strong anti-inflammatory effect in vivo.

TNF-stimulated gene 6 (tsg6), encoding a 35-kDa secretory glycoprotein (TSG-6), is induced in fibroblasts, chondrocytes, synovial cells, and mononuclear cells by the proinflammatory cytokines TNF-alpha and IL-1, or by LPS. Large amounts of TSG-6 protein were found in synovial fluids of patients with rheumatoid arthritis. TSG-6 protein forms a stable complex with components of the serine protease inhibitor, inter-alpha-inhibitor (I alpha I). In this work, we show that TSG-6 potentiates the inhibitory effect of l alpha l on the protease activity of plasmin. The plasmin/plasminogen activator system is important in the protease network associated with inflammation. To test the hypothesis that through their cooperative inhibitory effect on plasmin TSG-6 and l alpha l can modulate the protease network and thus inhibit inflammation, we examined the effect of TSG-6 on experimentally induced inflammation. Human recombinant TSG-6 protein showed a potent anti-inflammatory activity in the murine air pouch model of carrageenan- or IL-1-induced acute inflammation. The inhibitory effect of locally administered TSG-6 on the IL-1-induced cellular infiltration was comparable with that of systemic dexamethasone treatment. Two mutant TSG-6 proteins with single amino acid substitutions close to the N terminus showed a complete or partial loss of anti-inflammatory activity. The anti-inflammatory effect of the TNF/IL-1-inducible TSG-6 protein, along with its ability to inhibit protease action through interaction with l alpha l, suggests that TSG-6 production during inflammation is part of a negative feedback loop operating through the protease network.

TSG-6, a glycoprotein associated with arthritis, and its ligand hyaluronan exert opposite effects in a murine model of inflammation.

TSG-6 is an arthritis-associated hyaluronan binding protein whose production in synovial cells, chondrocytes, fibroblasts and mononuclear cells is stimulated by TNF-alpha and IL-1. The purpose of this study was to gain insights into the role of TSG-6 and its functional interactions with hyaluronan in inflammation. In the murine air pouch model of carrageenan/IL-1-induced inflammation TSG-6 showed a dramatic inhibitory effect on the cellular infiltration of the inflammatory site by neutrophilic PMN, while hyaluronan enhanced cellular infiltration. There was no indication of a neutralizing or cooperative effect when TSG-6 and hyaluronan were injected together. The potent antiinflammatory effect of TSG-6 along with its induction by proinflammatory cytokines suggests that TSG-6 is part of a negative feedback loop in the control of the inflammatory response.

TSG-6, an arthritis-associated hyaluronan binding protein, forms a stable complex with the serum protein inter-alpha-inhibitor.

TSG-6 is a secreted 35-kDa glycoprotein, inducible by TNF and IL-1. The N-terminal portion of TSG-6 shows sequence homology to members of the cartilage link protein family of hyaluronan binding proteins. The C-terminal half of TSG-6 contains a so-called CUB domain, characteristic for developmentally regulated proteins. High levels of TSG-6 protein are found in the synovial fluid of patients with rheumatoid arthritis and some other arthritic diseases. Here we show that TSG-6 readily formed a complex with a protein present in human, bovine, rabbit, and mouse serum. This complex was stable during SDS-PAGE under reducing conditions, and in the presence of 8 M urea. The protein that binds TSG-6 was purified from human serum and identified as inter-alpha-inhibitor (I alpha I) by N-terminal microsequencing. Microsequencing of the complex itself revealed the presence of TSG-6 and two of the three polypeptide chains of I alpha I (bikunin and HC2). Experiments with recombinant TSG-6 and I alpha I purified from human serum showed that the TSG-6/I alpha I complex is rapidly formed even in the apparent absence of other proteins at 37 degrees C, but not at 4 degrees C. The TSG-6/I alpha I complex was cleaved by chondroitin sulfate ABC lyase, suggesting that cross-linking by chondroitin sulfate is required for the stability of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

TSG-6: a TNF-, IL-1-, and LPS-inducible secreted glycoprotein associated with arthritis.

TSG-6 (TNF-stimulated gene 6) was originally discovered by differential screening of a cDNA library prepared from TNF-stimulated human diploid FS-4 fibroblasts. We show that the 35-kDa protein encoded by TSG-6 was undetectable in the medium of untreated FS-4 cultures, whereas its production reached approximately 1400 and 700 ng/10(6) cells after 24-h treatment with IL-1 or TNF, respectively. Stimulation of TSG-6 protein and mRNA levels was also demonstrated in normal human mononuclear cells by treatment with TNF and, especially, by LPS. In view of the inducibility of TSG-6 by inflammatory cytokines and its earlier demonstrated affinity for hyaluronan, we examined the presence of TSG-6 protein in the synovial fluids from patients with various forms of arthritis. TSG-6 protein was undetectable in the joint fluids of persons with no known history of arthritis, but high levels of TSG-6 oere demonstrated in the synovial fluids of a majority of arthritis patients. TSG-6 protein was also detected in the sera of some of the arthritis patients, albeit at concentrations that were less than in the joint fluids. To investigate the source of TSG-6 in the synovial fluids, we examined the production of TSG-6 protein in cultures of synovial cells. Synoviocytes from rheumatoid arthritis patients produced TSG-6 protein constitutively, and this production was increased by treatment with TNF or IL-1, but not with TGF-beta. Steady-state levels of TSG-6 mRNA were also increased in synoviocytes after treatment with TNF or IL-1. The presence of high levels of TSG-6 protein in the synovial fluids of arthritis patients and its inducibility by inflammatory cytokines in fibroblasts, mononuclear cells, synoviocytes, and chondrocytes suggest a role for TSG-6 in arthritis and inflammation.

A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44.

TSG-6 cDNA was isolated by differential screening of a lambda cDNA library prepared from tumor necrosis factor (TNF)-treated human diploid FS-4 fibroblasts. We show that TSG-6 mRNA was not detectable in untreated cells, but became readily induced by TNF in normal human fibroblast lines and in peripheral blood mononuclear cells. In contrast, TSG-6 mRNA was undetectable in either control or TNF-treated human vascular endothelial cells and a variety of tumor-derived or virus-transformed cell lines. The sequence of full-length TSG-6 cDNA revealed one major open reading frame predicting a polypeptide of 277 amino acids, including a typical cleavable signal peptide. The NH2-terminal half of the predicted TSG-6 protein sequence shows a significant homology with a region implicated in hyaluronate binding, present in cartilage link protein, proteoglycan core proteins, and the adhesion receptor CD44. The most extensive sequence homology exists between the predicted TSG-6 protein and CD44. Western blot analysis with an antiserum raised against a TSG-6 fusion protein detected a 39-kD glycoprotein in the supernatants of TNF-treated FS-4 cells and of cells transfected with TSG-6 cDNA. Binding of the TSG-6 protein to hyaluronate was demonstrated by coprecipitation. Our data indicate that the inflammatory cytokine (TNF or IL-1)-inducible, secretory TSG-6 protein is a novel member of the family of hyaluronate binding proteins, possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis.

Critical role of the C-terminus in the biological activities of human tumour necrosis factor-alpha.

Alterations of the C-terminal amino acid sequence of human tumour necrosis factor-alpha (hTNF-alpha) caused significant changes in its biological activity. Thus shortening of the C-terminus by removal of two or three amino acids led to a very marked loss of cytotoxic activity. Other, more subtle changes introduced by site-directed mutagenesis resulted in a less drastic reduction in cytotoxicity. The mitogenic activity towards human fibroblasts of the hTNF-alpha was reduced in parallel with the loss of cytotoxicity. These results suggest that the C-terminal amino acids of hTNF-alpha are critical for its biological actions and that they may be part of the receptor-binding site.

Age-dependent antileukemic action and suppression of immune response by 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone) in mice.

The antineoplastic activity of 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone) against murine leukemia P388 was found to be markedly reduced in 12- and 18-month-old mice as compared to young animals. The immune response against sheep red blood cells (SRBC), a T cell-dependent antigen, was also strongly diminished in tumor-free old mice and was further suppressed after ambazone treatment. Since the antileukemic effect of ambazone disappeared more or less in congenitally athymic nude mice, in neonatally thymectomized or silica-pretreated animals, it has been concluded that the action of the compound seems to be limited to young adult immunocompetent tumor-bearing hosts. Therefore immunosenescence, primarily of T cell functions of old tumor-bearers, may represent a decisive factor influencing the antileukemic, especially curative effect of ambazone in aged animals. A combined treatment with ambazone and immunomodulators (thymalin or a splenopentin derivative) failed to improve the antileukemic effect in young and old leukemia P388-bearing mice.

Antigen spot test for the detection of specific antibodies in the nanogram range by means of the PAP-technique.

A modification of a method is described for detection of antigens and antibodies by means of nitrocellulose paper discs as high-capacity solid phase antigen carrier and mouse PAP. Mouse IgG spotted on nitrocellulose paper may be easily detected at a level of 1 ng. Detection of specific antibodies is possible down to a concentration of 80 ng per ml. This method is of practical importance for the screening of hybridoma supernatants.