Repair of extensive articular cartilage defects in horses by autologous chondrocyte transplantation.

Damaged adult articular cartilage has very limited capacity to heal. Autologous chondrocyte transplantation (ACT) has been used clinically and studied in experimental animals in an attempt to provide biologically based cartilage regeneration. This study evaluated cartilage repair following ACT in a large animal model over a period of 2 years. Articular cartilage defects (10 mm in diameter, full-thickness) were created in the minor load-bearing area on the lateral talus of tibiotarsal joints of eight adult horses. In each animal, the right joint was repaired using autologous chondrocytes injected beneath the periosteum, as in the original ACT procedure (Brittberg, M., A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson N. Engl. J. Med. 331:889-895, 1994): the left joint remained untreated to serve as a control. Clinical and pathological evaluation was within the range of normal for all horses at both time points. Compared to untreated defects, ACT resulted in significantly improved defect filling with a well-integrated neocartilage and comparable expression of cartilage-specific markers. The histological score (Peterson, L., T. Minas, M. Brittberg, A. Nilsson, E. Sjogren-Jansson, and A. Lindahl Clin. Orthop. 374:212-234, 2000) (10.4 +/- 0.9 for ACT and 5.6 +/- 3.9 for controls, all animals, p = 0.016) indicated that ACT contributed to the reparative process. For the first time, the efficacy of ACT was demonstrated in a large animal model supporting the potential of ACT for cartilage regeneration in patients.

Biocompatible alginate from freshly collected Laminaria pallida for implantation.

A simple procedure is described for the extraction and purification of alginate from the inner stipes of the kelp Laminaria pallida. Alginate yield was about 10-15% of the dry mass, with a 70:30 mannuronic/guluronic acid ratio. Analysis of the purified alginate revealed a low polyphenol content while proteins were below detection level. The purified alginate was highly viscous, with 10-15 mPa s and 281 mPa s for a 0.1% and 0.5% solution, respectively, indicating a very high molecular mass (larger than 250 kDa). Bead formation occurred in the presence of divalent cations, but also in the presence of artificial serum (FCSIII) without added divalent cations. The biocompatibility of the alginate was tested with the in vitro mice lymphocyte test as well as by implantation of Ba2+ cross-linked beads beneath the kidney capsule of BB/OK rats. There was no evidence for significant mitogenic activity or fibrotic reaction. Biocompatibility of the alginate was also demonstrated by the encapsulation of human chondrocytes into Ca2+ cross-linked alginate beads. Immobilized chondrocytes grew and remained functional (i.e. they produced collagen).

Internalization of phospholipids from the plasma membrane of human osteoblasts depends on the lipid head group.

The redistribution of spin- or fluorescence-labeled phospholipid analogs across the plasma membrane of human osteoblast cells, either in suspension or grown as monolayers, was investigated. After incorporation into the outer membrane leaflet, analogs of the aminophospholipids phosphatidylserine and phosphatidylethanolamine moved rapidly to the inner monolayer, whereas the choline-containing analogs of phosphatidylcholine and sphingomyelin disappeared more slowly from the outer leaflet. The fast inward movement of the aminophospholipids became reduced after lowering the intracellular ATP, suggesting the presence of an aminophospholipid translocase activity in the plasma membrane of these cells. From these data, a transverse phospholipid asymmetry in osteoblasts can be inferred with the aminophospholipids mainly concentrated in the inner monolayer and the choline-containing phospholipids in the outer leaflet. A similar pattern of phospholipid internalization was inferred for osteoblasts from human osteoporotic bones and for a human osteosarcoma cell line. The relevance of the enrichment of phosphatidylserine in the cytoplasmic membrane leaflet for calcification in skeletal tissues is emphasized.

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells.

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.

Increased serum levels of soluble IL-2 receptor, CD30 and CD8 molecules, and gamma-interferon in angioimmunoblastic lymphadenopathy: possible pathogenetic role of immunoactivation mechanisms.

Abnormal immunoreaction associated with increased cell activation phenomena might play a role in the pathogenetic events leading to the development of angioimmunoblastic lymphadenopathy (AILD). In the present study we investigated the serum levels of some soluble molecules related to cell activation in 24 patients with AILD at presentation. In particular, we measured by immunoenzymatic or immunoradiometric techniques the levels of the Tac peptide (sIL-2R), soluble CD30 (sCD30) and CD8 (sCD8) antigens, and gamma-IFN (gIFN). The results show that all the above molecules are increased as compared to normal controls, with a different pattern of increase for the different molecules. The sIL-2R levels were very high in all cases with no overlap between AILD and control samples (mean 6315 +/- 3374 U/ml, controls 271 +/- 112 U/ml, P less than 0.001). Very high values of the sCD30 antigen (722 +/- 895 U/ml) were detected in all cases but five, as opposed to the lack of detectable levels in controls. A significant increase of sCD8 (978 +/- 646 U/ml, controls 334 +/- 95 U/ml, P less than 0.01) and gIFN (329 +/- 236 U/ml, controls 97 +/- 43 U/ml, P less than 0.01) was also observed with some overlap between AILD samples and controls. The above findings further support the view that a condition of abnormally enhanced cell activation is likely to play a central role in the pathogenetic events leading to the composite clinicopathological picture of AILD.

Soluble interleukin 2 receptors in patients after bone marrow transplantation.

An enzyme-linked immunosorbent assay was used to quantify soluble interleukin 2 receptor (IL2R) in the serum of 25 patients prior to allogeneic or autologous bone marrow transplantation and in the early post-transplantation period. A significant correlation between IL2R and the occurrence of fever and/or graft-versus-host disease (GVHD) could be shown. Patients with moderate to severe GVHD (grade II-IV) had significantly higher IL2R levels (median 480 U/ml) than patients without or with acute GVHD grade I (median 139 U/ml). In patients without or with acute GVHD grade I, significant differences in the maximum IL2R levels depended on the duration of fever greater than or equal to 38 degrees C. Evaluation of the peak IL2R levels in patients with fever lasting longer than 6 days led to a median of 260 U/ml and in patients with fever lasting less than 6 days to a median of 118 U/ml. In patients without or with acute GVHD grade I, who developed fever lasting longer than 6 days, IL2R levels started to rise with the onset of fever, reached peak values during temperature maximum and declined parallel with temperature normalization. In patients without or with acute GVHD grade I who developed fever lasting for only 6 days or less IL2R levels remained within the normal range. In patients with acute GVHD grade II-IV, IL2R levels began to rise with the onset of fever, and then continued to rise despite temperature normalization. The peak levels were reached in the early period of acute GVHD. Our observations in BMT patients show that severe infections and acute GVHD are associated with a stimulation of the immune system leading to elevated IL2R serum levels.

Serum levels of soluble CD30 molecule (Ki-1 antigen) in Hodgkin's disease: relationship with disease activity and clinical stage.

In all cases of Hodgkin's disease (HD) Reed-Sternberg (RS) cells express the CD30 antigen. It has been recently demonstrated that this molecule can be released from the cell membrane of CD30+ neoplastic cells in a soluble form (sCD30), detectable in culture supernatants and body fluids. In this paper we investigated by an immunoassay the serum levels of sCD30 in 58 patients with HD, in order to define the possible relationship of this molecule to the clinical and pathological findings. sCD30 molecule was found at detectable levels in 24 out of 50 patients (48%) with active disease, whereas it was always absent in control sera and in cases in complete remission. Among the patients with active HD, the incidence and mean values of detectable levels (+/- SEM) of sCD30 were higher in cases with progressive or relapsing disease (61.5%, mean 458 +/- 190 U/ml) as compared to those at presentation (43.2%, mean 116 +/- 33 U/ml). Among the cases at presentation, detectable levels were observed more often in patients with advanced stages (III + IV: 61%) and constitutional symptoms ('B': 61.5%) than in early stages (I + II: 26%) and without symptoms ('A': 33.3%). In addition, higher mean values were found in stages III + IV (182 +/- 60 U/ml) and in 'B' cases (208 +/- 73 U/ml) than in stages I + II (65 +/- 30 U/ml) or 'A' patients (64 +/- 26 U/ml). The above findings suggest a possible role for the sCD30 as a tumour marker in HD.

Soluble interleukin 2 receptor and tissue polypeptide antigen serum concentrations in end-stage renal failure.

Serum concentrations of soluble interleukin 2 receptor (IL-2R) were measured in 65 hemodialysis patients and compared with serum levels of beta 2-microglobulin and tissue polypeptide antigen (TPA). Elevated IL-2R levels, found in 85% of examined patients, correlated with elevated TPA serum concentrations (p less than 0.05). Patients with high IL-2R levels were significantly younger (p less than 0.05) than patients with low levels. Primary renal disease and residual renal function had no significant influence on TPA or IL-2R serum concentrations. In 16 patients with carpal tunnel syndrome, increased serum concentrations of IL-2R (p less than 0.005) and TPA (p less than 0.001) were found. We conclude that a non-specific dialysis-induced activation of epithelial and lymphoid cells rather than a specific immune response could explain the concomitant elevation of IL-2R and TPA serum concentrations in hemodialyzed patients. Patients with pronounced cell turnover, reflected by elevated IL-2R and TPA levels, may show an increased susceptibility to dialysis-associated amyloidosis.

The therapeutic efficacy of an anti-IL-2 receptor monoclonal antibody correlates with an increase in serum soluble IL-2 receptor levels.

We tested the capacity of three rat monoclonal antibodies (MoAb), recognizing different epitopes on the L chain of mouse interleukin-2 receptor (IL-2R), to block delayed-type hypersensitivity (DTH) and local graft-versus-host reaction (GVHR). Furthermore, we investigated the effect of IL-2R-targeted immunotherapy on serum soluble IL-2R levels by ELISA technique. Following administration of AMT-13 MoAb, in sufficient amounts to suppress both DTH (60-90% inhibition) and local GVHR (55-70% inhibition), an increase in soluble IL-2R levels up to 12-fold was observed. In contrast, the administration of 7D4 MoAb did not show any immunosuppressive effect in vivo and even decreased the soluble IL-2R levels. The third anti-IL-2R MoAb AMT45-20 had an intermediate effect. AMT45-20 MoAb marginally suppressed the DTH as well as local GVHR (15-35% inhibition) and induced only a slight increase in soluble IL-2R levels (up to 4-fold). Both cyclosporin A, a conventional immunosuppressive drug, and the anti-L3T4 MoAb, which defines the entire T helper cell subset, suppressed GVH and DTH response but did not increase the soluble IL-2R serum levels. The increased concentration of soluble IL-2R in the serum of successfully treated mice may be due to destruction of IL-2R-positive cells by anti-IL-2R-targeted immunotherapy and seems to be a sensitive indicator for the success of such a therapy.

Demonstration of two distinct forms of released low affinity-type rat IL-2 receptors.

The release of IL-2 binding proteins, derived from the 55,000 MW low affinity IL-2R (L chain), has been observed for virtually all L chain-bearing cells in either humans, the mouse or the rat. Based on the characterization of the released human L chain as a molecule 10,000 MW smaller than the cell surface receptor, either proteolytic cleavage or differential splicing of the L chain encoding mRNA have been suggested as mechanisms underlying the receptor release. Combining affinity labelling of the L chain with [125I]IL-2 and immunoprecipitation with L chain-specific monoclonal antibody (mAb) applied for the detection of soluble rat IL-2R revealed the existence of two classes of soluble receptors, one being of the same size as cell surface-expressed L chain, the other of 40,000 apparent molecular mass. These findings raise the possibility of other mechanisms of receptor release than those discussed for human L chain.

Factors influencing the response to hepatitis B vaccination of hemodialysis patients.

The response rate and HBsAG antibody concentrations were examined after hepatitis B vaccination in 78 hemodialysis patients aged between 29 and 79 years. The values were related to age, duration of hemodialysis, body weight, creatinine, urea nitrogen, serum concentrations of beta 2-microglobulin and soluble interleukin-2 receptor (IL-2R). Patients with low anti-HBsAG antibody concentrations (10-100 mU/ml) had significantly higher IL-2R serum concentrations than those with high anti-HBsAG antibody concentrations (greater than 3,000 mU/ml; p less than 0.05). Discriminant multivariate analysis (p = 0.032) revealed the influence (62%) of IL-2R on the response rate while other factors were similar in all patient groups. It is concluded that preactivation of T cells with an increased release of IL-2R may contribute to impaired immune response after hepatitis B vaccination.

Ki-1 (CD30) antigen is released by Ki-1-positive tumor cells in vitro and in vivo. I. Partial characterization of soluble Ki-1 antigen and detection of the antigen in cell culture supernatants and in serum by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) has been developed that allows the quantitative determination of the Ki-1 (CD30) antigen in soluble form. Similar levels of sensitivity of this new Ki-1 ELISA and the ELISA previously described for measuring the soluble 55-kDa chain of the interleukin 2 receptor were seen. As assessed with this ELISA, the investigated Ki-1+ permanent cell lines released the Ki-1 antigen into the culture supernatant. In culture supernatants of concanavalin A-activated human peripheral blood lymphocytes, however, this antigen could not be detected. The released Ki-1 antigen has an apparent molecular weight (Mr) of 85,000, whereas the cell-associated Ki-1 antigen has an Mr of 105,000. We investigated sera from 30 normal donors, 15 patients with systemic infections, and 63 patients suffering from lymphomas for soluble Ki-1 antigen. In all sera from normal donors and patients with systemic infectious diseases, soluble Ki-1 antigen was below the detection limit (i.e., less than 70 pg). In contrast, high amounts of the soluble Ki-1 antigen were found in sera from 18 malignant lymphomas containing Ki-1+ tumor cells. This finding demonstrates that the release of the Ki-1 antigen takes place not only in vitro, but in vivo as well. Moreover, these results imply that the Ki-1 antigen may be used as a serum tumor marker.

Demonstration of two distinct forms of released low-affinity type interleukin 2 receptors.

The release of interleukin 2 (IL2)-binding proteins, derived from the 55-kDa low-affinity IL2 receptor (IL2R; L chain), has been observed for virtually all L chain-bearing cells in either humans, the mouse or the rat. Based on the characterization of the released human L chain as a molecule 10 kDa smaller than the cell surface receptor, either proteolytic cleavage or differential splicing of the L chain-encoding mRNA have been suggested as mechanisms underlying the receptor release. Combining affinity labeling of the L chain with 125I-labeled IL2 and immunoprecipitation with L chain-specific monoclonal antibodies applied for the detection of soluble mouse IL2R revealed the existence of two classes of soluble receptors, one being of the same size as cell surface expressed L chain, the other of 45-kDa apparent molecular mass. These findings raise the possibility of mechanisms of receptor release other than those discussed for human L chain.

Interleukin 2 receptor in patients with localized and systemic parasitic diseases.

An enzyme-linked immunosorbent assay was used to quantify soluble interleukin 2 receptor (IL-2R) in the serum of patients with helminthic and protozoal infections. The results demonstrated that levels of IL-2R were normal in patients with helminthic infections limited to the intestinal tract (ascariasis, trichuriasis), but significantly elevated in patients with systemic or long-lasting infections (strongyloidiasis, schistosomiasis, fascioliasis, opisthorchiasis). In patients infected with Schistosoma mansoni levels of IL-2R were higher in those with the hepatosplenic than in those with the intestinal form of the disease. Patients with malaria also showed increased serum levels of IL-2R, irrespective whether the infection was caused by Plasmodium falciparum or P. vivax. No difference was observed between patients with acute or history of malaria. The highest levels of IL-2R were observed in patients with visceral leishmaniasis. Interestingly, in these patients the concentration of IL-2R correlated to specific antibody titre. The results are discussed in the context of preferential activation of T lymphocytes, B lymphocytes and/or macrophages during the course of the different parasitic infections investigated.

Elevated titers of cell-free interleukin 2 receptor in serum of lupus mice.

Activation/proliferation of mouse and human T and B cells is associated with expression and subsequent release of interleukin 2 receptors (IL 2R) into the milieu. The soluble form of IL 2R, at least in part, retains its ability to bind to IL 2 and to anti-receptor antibodies, but its exact structure remains unknown. Because systemic lupus erythematosus (SLE) is associated with T and B cell activation, we have used monoclonal anti-IL 2R antibodies in an ELISA to measure levels of IL 2R in sera of various lupus strains. High levels of the released receptor were found at an active clinical stage in sera of four autoimmune strains of mice homozygous for the lpr (lymphoproliferation) gene that causes T cell expansion, massive lymphoid organ enlargement, and promotes the autoimmune process. High levels were also found in lupus mice characterized primarily by B cell proliferation (BXSB males) and in (NZB X W)F1 mice characterized by T and B cell activation. Similarly high IL 2R serum levels could be induced experimentally in normal mice injected with immunostimulants such as bacterial lipopolysaccharide or Freund's complete adjuvant. The results indicate that IL 2R serum levels may provide a good marker of ongoing lymphoid cell activation/proliferation, and thus might be useful in the follow-up of patients with systemic autoimmune or other lymphoproliferative disorders. The biologic roles, if any, of the soluble form of IL 2R and its effects in normal and abnormal conditions remain to be determined.

Determination of cell-free interleukin 2 receptor level in the serum of normal animals and of animals bearing IL-2 receptor positive tumours with high or low metastatic capacity.

Serum levels of cell-free interleukin-2 receptors were elevated above normal in mice bearing the IL-2R positive T-cell lymphoma Eb or its highly metastatic variant ESb. Although ESb cells expressed less IL-2R molecules than Eb cells on their cell surface, serum receptor levels were raised more quickly in ESb than in Eb tumour bearing animals. Elevated IL-2R serum levels were a sensitive tumour marker in animals bearing the aggressive variant ESb but not in animals bearing the low metastatic line Eb. Peritoneal ascites tumour-bearing animals had higher serum IL-2R levels than corresponding animals with subcutaneously growing tumours. Thus, serum IL-2R levels in tumour-bearing animals were dependent on the tumour line and influenced by the site and mode of tumour growth.

Soluble interleukin 2 receptors are released by long term-cultured insulin-specific T cells transiently after contact with antigen.

An enzyme-linked immunosorbent assay was used to quantitate soluble interleukin 2 receptors (IL 2R) released by antigen-dependent, insulin-specific murine T cells into the culture supernatant, as well as cell-associated IL 2R present in cell lysates. IL 2R were released solely after T cell activation by antigen. The release of IL 2R was transient, reaching optimal levels within 72 hr after antigen challenge and gradually declining to background levels thereafter, when the cells were subcultured in IL 2-enriched medium. The decrease in the amount of IL 2R released during culture in IL 2-containing medium paralleled the decrement in cellular IL 2R detected in cell lysates, in cell surface-expressed IL 2R as determined by cytofluorometry, as well as in high-affinity IL 2R. In contrast, IL 2R were constitutively released by an IL 2-dependent T cell clone. Soluble IL 2R might exert an immunoregulatory function by competing with cellular IL 2R for IL 2 binding.

Regulation of interleukin-2 receptor expression and receptor release.

The generation and cell surface expression of IL-2 receptors was monitored by: (i) an ELISA that permits quantitative determination of detergent-solubilized or soluble IL-2 receptors; and (ii) detection of the binding of 125I-labelled recombinant IL-2 and of anti-IL-2 receptor antibodies to receptor bearing cells. Upon lectin stimulation both high and low affinity IL-2 receptors became expressed in parallel at the cell surface. Both high and low affinity receptors were upregulated by IL-2. Upon lectin activation the amount of cell-associated receptors increased and on day 2 of the culture period IL-2 receptors were detectable in the culture supernatant. IL-2 upregulated both high and low affinity IL-2R expression on T-lymphoblasts. IL-2R bearing leukemic cells and T lymphoblasts released IL-2R when cultured in vitro. IL-2R release by T lymphoblasts was enhanced dramatically by IL-2. On the other hand, IL-2-receptor positive leukemic cells released receptors in an IL-2 independent manner. Release of receptors could also be detected in serum-free medium. At least a part of the released receptors could be specifically bound to immobilized pure recombinant IL-2 and to monoclonal anti-IL-2-receptor antibodies. Small but significant amounts of soluble IL-2 receptors were detectable in the sera of normal mice. In sera of mice inoculated with IL-2-receptor positive syngeneic leukemic cells, elevated levels of IL-2 receptors were detectable. Release of IL-2 receptors seems to represent one of the major routes by which the receptors are cleared from the cell surface.