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Objectives Previously, we have shown the involvement of cellular communication network factor 4/Wnt-activated protein Wnt-1-induced signaling protein 1 (CCN4/WISP1) in osteoarthritic (OA) cartilage and its detrimental effects on cartilage. Here, we investigated characteristics of CCN4 in chondrocyte biology by exploring correlations of CCN4 with genes expressed in human OA cartilage with functional follow-up.Design Spearman correlation analysis was performed for genes correlating with CCN4using our previously established RNA sequencing dataset of human preserved OA cartilage of the RAAK study, followed by a pathway enrichment analysis for genes with ρ ≥|0.6.| Chondrocyte migration in the absence or presence of CCN4 was determined in a scratch assay, measuring scratch size using a live cell imager for up to 36 h. Changes in expression levels of 12 genes, correlating with CCN4and involved in migratory processes, were determined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR).Results Correlation of CCN4with ρ ≥|0.6| was found for 58 genes in preserved human OA cartilage. Pathway analysis revealed “neural crest cell migration” as most significant enriched pathway, containing among others CORO1C, SEMA3C, and SMO. Addition of CCN4 to primary chondrocytes significantly enhance chondrocyte migration as demonstrated by reduced scratch size over the course of 36 h, but at the timepoints measured no effect was observed on mRNA expression of the 12 genes.Conclusion CCN4 increases cell migration of human primary OA chondrocytes. Since WISP1 expression is known to be increased in OA cartilage, this may serve to direct chondrocytes toward cartilage defects and orchestrate repair.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients.Impact statementWe describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.

Articular cartilage makes smooth movement possible and destruction of this tissue leads to loss of joint function. An important biomolecule that determines this function is the large aggregating proteoglycan of cartilage, aggrecan. Aggrecan has a relatively short half-life in cartilage and therefore continuous production of this molecule is essential.

Background: Rheumatoid arthritis (RA) is one of the most prevalent and debilitating joint diseases worldwide. RA is characterized by synovial inflammation (synovitis), which is linked to the development of joint destruction. Magnetic resonance imaging and ultrasonography are widely being used to detect the presence and extent of synovitis. However, these techniques do not reveal the activation status of inflammatory cells such as macrophages that play a crucial role in synovitis and express CD64 (Fc gamma receptor (FcγR)I) which is considered as macrophage activation marker. Objectives: We aimed to investigate CD64 expression and its correlation with pro-inflammatory cytokines and pro-damaging factors in human-derived RA synovium. Furthermore, we aimed to set up a molecular imaging modality using a radiolabeled CD64-specific antibody as a novel imaging tracer that could be used to determine the extent and phenotype of synovitis using optical and nuclear imaging. Methods: First, we investigated CD64 expression in synovium of early- and late-stage RA patients and studied its correlation with the expression of pro-inflammatory and tissue-damaging factors. Next, we conjugated an anti-CD64 antibody with IRDye 800CW and diethylenetriamine penta-acetic acid (DTPA; used for 111In labeling) and tested its binding on cultured THP1 cells, ex vivo RA synovium explants and its imaging potential in SCID mice implanted with human RA synovium explants obtained from RA patients who underwent total joint replacement. Results: We showed that CD64 is expressed in synovium of early and late-stage RA patients and that FCGR1A/CD64 expression is strongly correlated with factors known to be involved in RA progression. Combined, this makes CD64 a useful marker for imaging the extent and phenotype of synovitis. We reported higher binding of the [111In]In-DTPA-IRDye 800CW anti-CD64 antibody to in vitro cultured THP1 monocytes and ex vivo RA synovium compared to isotype control. In human RA synovial explants implanted in SCID mice, the ratio of uptake of the antibody in synovium over blood was significantly higher when injected with anti-CD64 compared to isotype and injecting an excess of unlabeled antibody significantly reduced the antibody-binding associated signal, both indicating specific receptor binding. Conclusion: Taken together, we successfully developed an optical and nuclear imaging modality to detect CD64 in human RA synovium in vivo.

Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that is important in the regulation of joint homeostasis and disease. TGFβ signalling is induced by loading and has an important function in maintaining the differentiated phenotype of articular chondrocytes. Concentrations of active TGFβ differ greatly between healthy and osteoarthritic joints, being low in healthy joints and high in osteoarthritic joints, leading to the activation of different signalling pathways in joint cells. The characteristic pathology of osteoarthritic joints, such as cartilage damage, osteophyte formation and synovial fibrosis, seems to be stimulated or even caused by the high levels of active TGFβ, in combination with altered chondrocyte signalling pathways (which are also observed in ageing joints). In this Review, the changing role of TGFβ in normal joint homeostasis, ageing and osteoarthritis is discussed: TGFβ counteracts pathological changes in a young healthy joint, alters its signalling during ageing and is a driving force of pathology in osteoarthritic joints.

Objective RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-β (TGF-β)–mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1β (IL)-1β and TGF-β superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis.Methods Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase–quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry.Results RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-β inhibited RBPMS expression while BMP2 and IL-1β increased its expression. TGF-β-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis.Conclusions Expression of RBPMS in chondrocytes is regulated by TGF-β superfamily members and IL-1β, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage.

Proteins from the Wnt signaling pathway are very important for joint development. Curiously, osteoarthritis (OA) is thought to be a recapitulation of developmental processes. Various members of the Wnt signaling pathway are overexpressed in the synovium during experimental OA. Here, we investigated the potency of specific Wnt proteins, when expressed in the synovium, to induce OA pathology. We overexpressed Wnt5a, Wnt8a, Wnt16, and WISP1 in the synovium using adenoviral vectors. We determined whether overexpression resulted in OA pathology by histology, and we measured whether Wnt signaling led to increased protease activity in the joint. Synovial overexpression of Wnt8a and Wnt16 led to canonical Wnt signaling in the cartilage, whereas overexpression of Wnt5a did not. Canonical Wnt signaling increased protease activity and induced cartilage damage shortly after overexpression. Specific blocking of the canonical Wnt signaling pathway with Dickkopf-1 reduced the Wnt-signaling–induced cartilage damage. By contrast, the noncanonical signaling Wnt5a did not cause cartilage lesions. Overexpression of WISP1, a downstream protein of canonical Wnt signaling, resulted in increased cartilage damage. In conclusion, our data show that canonical Wnts and WISP1, which we found overexpressed in the synovium during experimental OA, may conduce to OA pathology.

Objective:To rescue chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in osteoarthritic conditions by inhibition of protein kinases.Methods:hMSCs were cultured in pellets. During early chondrogenic differentiation, these were exposed to osteoarthritic synovium-conditioned medium (OAS-CM), combined with the Janus kinase (JAK)-inhibitor tofacitinib and/or the transforming growth factor β-activated kinase 1 (TAK1)-inhibitor oxozeaenol. To evaluate effects on chondrogenesis, the glycosaminoglycan (GAG) content of the pellets was measured at the time that chondrogenesis was manifest in control cultures. Moreover, mRNA levels of matrix molecules and enzymes were measured during this process, using real-time polymerase chain reaction (RT-PCR). Initial experiments were performed with hMSCs from a fetal donor, and results of these studies were confirmed with hMSCs from adult donors.Results:Exposure to OAS-CM resulted in pellets with a much lower GAG content, reflecting inhibited chondrogenic differentiation. This was accompanied by decreased mRNA levels of aggrecan, type II collagen, and Sox9, and increased levels of matrix metalloproteinase (MMP)1, MMP3, MMP13, ADAMTS4, and ADAMTS5. Both tofacitinib (JAK-inhibitor) and oxozeaenol (TAK1 inhibitor) significantly increased the GAG content of the pellets in osteoarthritis (OA)-like conditions. The combination of both protein kinase inhibitors showed an additive effect on GAG content. In agreement with this, in the presence of OAS-CM, both tofacitinib and oxozeaenol increased mRNA expression of sox9. The expression of aggrecan and type II collagen was also up-regulated, but this only reached significance for aggrecan after TAK1 inhibition. Both inhibitors decreased the mRNA levels of MMP1, 3, and 13 in the presence of OAS-CM. Moreover, oxozeaenol also significantly down-regulated the mRNA levels of aggrecanases ADAMTS4 and ADAMTS5. When combined, the inhibitors caused additive reduction of OA-induced MMP1 mRNA expression. Counteraction of OAS-CM-induced inhibition of chondrogenesis by these protein kinase inhibitors was confirmed with hMSCs of two different adult donors. Both tofacitinib and oxozeaenol significantly improved GAG content in cell pellets from these adult donors.Conclusions:Tofacitinib and oxozeaenol partially prevent the inhibition of chondrogenesis by factors secreted by OA synovium. Their effects are additive. This indicates that these protein kinase inhibitors can potentially be used to improve cartilage formation under the conditions occurring in osteoathritic, or otherwise inflamed, joints.

In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitrochondrogenesis, it was found that supplementing transforming growth factor beta (TGFβ) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFβ; however, the observed results could not be attributed to TGFβ, as either capturing TGFβ with an antibody or blocking the canonical TGFβ signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFβ family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.

Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that is central in the regulation of joint health and disease. Inhibition of TGFβ activity/signaling in experimental osteoarthritis (OA) has been performed to modulate OA severity and progression. In this narrative review we discuss the potential reasons for the variable results of TGFβ inhibition in these models.

Due to the complexity and heterogeneity of osteoarthritis (OA) pathophysiology, studying the interaction between intrinsic molecular changes in chondrocytes after hyper-physiological mechanical stress (MS) and aberrant signalling of OA risk genes remains a challenge. In this study we set out to set up an in vitro3D neo cartilage pellet model that enables us to explore the responses of OA risk genes to hyper-physiological MS.

Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.

The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitroby chondrogenic differentiation of BMSCs or studied ex vivoduring murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitroby articular chondrocytes or studied in ex vivoarticular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.

Cytokines are involved in osteoarthritis (OA) at several levels. They are involved in primary cartilage damage, but also in synovial activation that is observed in osteoarthritic joints. From in vitro studies and animal models for OA, several cytokines have been identified that are potential targets for OA therapy. Two promising targets are the destructive cytokine Interleukin-1 (IL-1) and the anabolic growth factor transforming growth factor (TGF)β and these will be discussed in more detail. Inhibition of IL-1 has been proven to result in amelioration of osteoarthritis-like pathology in animal models and the role of IL-1 is substantiated in studies in IL-1 deficient mice. In contrast, application of the anabolic growth factor TGFβ may provide an alternative approach to promote cartilage integrity and repair. TGFβ is a potent stimulator of chondrocyte matrix production, and therefore has a potency to repair already damaged cartilage. However, TGFβ induces tissue fibrosis and osteophytes at the joint margins and can only be applied to promote cartilage repair when these side effects can be blocked. This appears possible with concomitant, compartmentalized application of selective inhibitors of TGFβ in soft tissues, using local gene therapy with inhibitory Smad 6 and 7. Since OA is often limited to a few joints, local gene therapy may provide a suitable way to treat OA patients. Depending on the phenotype of a particular OA patient, e.g. with or without marked synovial activation, treatment may be focused mainly on suppression of catabolism or stimulation of anabolism, but combination therapy seems most warranted.

Degradation of type II collagen is thought to be a key step in the destruction of articular cartilage in patients with rheumatoid arthritis or osteoarthritis. The aim of this study was to investigate whether type II collagen degradation is associated with cartilage destruction. Type II collagen degradation was studied in two murine arthritis models, zymosan‐induced arthritis (ZIA), which develops reversible articular cartilage damage based on proteoglycan analysis, and antigen‐induced arthritis (AIA), in which there is irreversible damage to the cartilage. Type II collagen degradation was assayed immunohistochemically using the COL2‐3/4m antibody which recognizes denatured type II collagen, such as is produced by collagenase cleavage. In both models, degradation of type II collagen was observed in the non‐calcified articular cartilage of arthritic but not of control knees. In the patella‐femoral compartment, collagen denaturation started to increase on day 3 (ZIA) and day 7 (AIA) and remained high on day 14. In contrast, in the tibia‐femoral compartment, type II collagen breakdown was not increased before 14 days in either model. By 28 days, collagen denaturation was strongly reduced in the patella‐femoral compartment in the ZIA model, but persisted in the tibia‐femoral compartment in both models. In conclusion, increased type II collagen degradation was found in articular cartilage of both ZIA and AIA animals. Since ZIA does not develop irreversible cartilage destruction, this indicates that cartilage may have the ability to withstand a limited degree of type II collagen degradation without developing irreversible damage. Copyright © 1999 John Wiley & Sons, Ltd.

Objective: Articular cartilage has only limited capacities for repair and it is not known what is the exact mechanism of matrix restoration. It was investigated whether the reparative process in murine articular cartilage after moderate proteoglycan depletion is accompanied by a change in the chondrocyte phenotype either to hypertrophy or to a less differentiated phenotype as assayed by the expression of specific collagen subtypes.

Transforming growth factor-β (TGF-β) is a potent regulator of cell metabolism, proliferation, and differentiation. To study the role of endogenous TGF-β in processes such as tissue repair and inflammation, potent and specific inhibitors are required. Because the type II TGF-β receptor (TGFβRII) has a high affinity for TGF-β, the extracellular domain of TGFβRII (TGF-βsRII) was expressed inPichia pastorisandEscherichia coli.Expression of the soluble TGFβsRII usingP. pastorisresulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium. Although expression of TGFβsRII inE. coliresulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein. Because in both systems a C-terminal 6×His coding sequence was inserted behind the coding sequence for the extracellular domain of TGFβRII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni–NTA agarose. The purified proteins appeared to be potent inhibitors of TGF-β1 and TGF-β3. In contrast, TGFβsRII was less effective in neutralization of TGF-β2. In conclusion, biologically active TGFβsRII can be produced usingP. pastorisandE. coliexpression systems. The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGFβsRII in large amounts to further elucidate the role of TGF-β1 and TGF-β3 in physiological processes like tissue repair and inflammation.

Objective: The related molecules bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1) have both been shown to stimulate chondrocyte proteoglycan (PG) synthesisin vitro. We investigated thein-vivoeffects of these factors on articular cartilage PG metabolism.

Osteoarthritis is characterized by focal cartilage destruction and marked formation of osteophytes. We have investigated the possible relationship between site specific occurrence of cartilage damage and osteophytes in the collagenase induced murine osteoarthritis model. The degree of instability of the joint correlated with the amount of cartilage loss. Moreover, cartilage damage in the medial tibial plateau correlated only strongly with the osteophyte at the medial plateau, whereas a similar, site directed trend was noted for lateral damage and lateral osteophytes. A separate study with intraarticular injection of TGFβ1 in normal murine knee joints revealed that this factor can induce osteophytes at characteristic sites, suggesting a role of endogenous TGFβ in this phenomenon.

Background: Synovitis-associated pain is mediated by inflammatory factors that may include S100A8/9, which is able to stimulate nociceptive neurons via Toll-like receptor 4. In this study, we investigated the role of S100A9 in pain response during acute synovitis. Methods: Acute synovitis was induced by streptococcal cell wall (SCW) injection in the knee joint of C57Bl/6 (WT) and S100A9−/−mice. The expression of S100A8/A9 was determined in serum and synovium by ELISA and immunohistochemistry. Inflammation was investigated by 99mTc accumulation, synovial cytokine release, and histology at days 1, 2, and 7. To assess pain, weight distribution, gait analysis, and mechanical allodynia were monitored. Activation markers in afferent neurons were determined by qPCR and immunohistochemistry in the dorsal root ganglia (DRG). Differences between groups were tested using a one-way or two-way analysis of variance (ANOVA). Differences in histology were tested with a non-parametric Mann–Whitney Utest. pvalues lower than 0.05 were considered significant. Results: Intra-articular SCW injection resulted in increased synovial expression and serum levels of S100A8/A9 at day 1. These increased levels, however, did not contribute to the development of inflammation, since this was equal in S100A9−/−mice. WT mice showed a significantly decreased percentage of weight bearing on the SCW hind paw on day 1, while S100A9−/−mice showed no reduction. Gait analysis showed increased “limping” behavior in WT, but not S100A9−/−mice. Mechanical allodynia was observed but not different between WT and S100A9−/−when measuring paw withdrawal threshold. The gene expression of neuron activation markers NAV1.7, ATF3, and GAP43 in DRG was significantly increased in arthritic WT mice at day 1 but not in S100A9−/−mice. Conclusions: S100A8/9, released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.

Children with ADA deficiency have an impairment of both cellular and humoral immunity. We performed a detailed study of the relative toxicities of the ADA substrates adenosine and deoxyadenosine with regard to 3H-thymidine incorporation in mitogen-stimulated human peripheral blood lymphocytes and malignant lymphoid cell lines. Lymphocytes, isolated by Percoll gradient centrifugation and counterflow elutriation, were stimulated with various mitogens (PHA, ConA, PWM, SpA, StA) known to activate different lymphocyte subpopulations. Both lymphocytes and lymphoid cell lines were incubated with various adenosine and deoxyadenosine concentrations in presence of the ADA inhibitor deoxycoformycin. Combinations of deoxycoformycin and adenosine or deoxyadenosine gave inhibition of 3H-thymidine incorporation in both lymphocytes and cell lines. Adenosine affected 3H-thymidine incorporation of mitogen-stimulated lymphocytes to a similar extent as deoxyadenosine. However, to the malignant T cell line Molt 4 deoxyadenosine was more toxic than adenosine. This was not observed with the cell lines REH (nonBnonT), KM-3 (nonBnonT) and RAJI (B).Lymphocytes stimulated with B- or T cell mitogens were affected in their thymidine incorporation to a comparable extent by adenosine and deoxyadenosine. The cell line Molt was more sensitive to deoxyadenosine than both stimulated lymphocytes and the cell lines REH, KM-3 and RAJI.

Accumulation of adenosine is detected in plasma of ADA-deficient children(1). We incubated human lymphoid cell lines with adenosine in presence of the ADA inhibitor deoxycoformycin. The malignant T cell line Moltland the non-malignant B cell line RPMI 1788 were both more affected by adenosine than the malignant B cell line RAJI was. However, adenosine had a remarkable biphasic effect on cell growth of all three cell lines. After 48 hours incubation with 40µM adenosine growth of all three cell lines was severely inhibited (Molt 493%, RPMI 1788 87%, RAJI 63%). When 100µM adenosine was used the inhibitory effect was less severe (Moltµ58%, RPMI 1788 72%, RAJI 49%). This biphasic effect was also reflected in the cell cycle. After 24 hours incubation addition of 20µM adenosine to the cell cultures resulted in accumulation of cells in the G1phase while addition of 50µM adenosine resulted in a decrease of cells in the G1phase. Drawing conclusions about the inhibitory effects of adenosine should be done with caution.

Guanosine and deoxyguanosine are detected in urine of PNP-deficient patients (1). We have studied the effects of these compounds on 3H-thymidine incorporation in human peripheral blood lymphocytes and malignant lymphoid cell lines. Peripheral blood lymphocytes, isolated by Percoll gradient centrifugation and counterflow elutriation were stimulated with various mitogens (PHA, Con A, PWM, SpA, StA) known to activate different lymphocyte subpopulations.In all cell cultures tested deoxyguanosine inhibited 3H-thymidine more strongly than guanosine. Stimulated lymphocytes and cell lines were cultured in presence of different concentrations of guanosine and deoxyguanosine. Lymphocytes stimulated with B cell mitogens were as sensitive to guanosine and deoxyguanosine as lymphocytes stimulated with T cell mitogens were. Incorporation of 3H-thymidine in the cultured cell line Molt 4 by guanosine and deoxyguanosine was inhibited more severe than in the cultured RAJI (B) and REH (nonBnonT) cell lines and in stimulated peripheral blood lymphocytes. When possibilities for antileukemic chemotherapy using deoxyguanosine or guanosine are considered, deoxyguanosine seems to be a better candidate than guanosine.