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T Ternynck

Publications and source records attributed to T Ternynck.

At least 19 recordsLinked to original sources
PubMed2000

Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms.

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.

Animals↗
PubMed1999

Natural antibody status in patients with Hashimoto's thyroiditis.

Raised levels of circulating natural antibodies (NABS) have been found in association with systemic lupus erythematosus (SLE) and chronic active hepatitis (CAH), indicative of polyclonal B-cell activation associated with these relatively non-organ specific autoimmune diseases. This study examined the natural antibody response in the organ-specific autoimmune disease Hashimoto's thyroiditis. Serum samples obtained from 69 women with newly diagnosed Hashimoto's thyroiditis together with 64 controls were analysed for IgG and IgM NABS directed at DNA, actin, myoglobin, myosin, trinitrophenyl hapten (TNP) and tubulin as the NAB antigen panel using an established ELISA. The same technique was also used to estimate thyroglobulin and thyroid microsomal autoantibody activities. Compared to a reference panel of normal serum samples, 31 of the Hashimoto's samples showed a greater than 2SD elevation of IgG and/or IgM NABS against one or more of the panel antigens together with elevated IgG thyroglobulin and thyroid microsomal antibody levels. The cases positive for one or more of the NAB panel also showed a greater incidence of active Hashimoto's thyroiditis as indicated by the presence of antibodies directed against the thyroid specific antigens. The above findings suggest that raised levels of NABS are also a feature of this organ-specific autoimmune disease. The wide ranging NAB specificities involved are consistent with an underlying or epiphenomenal state of polyclonal B-cell activation.

Antibodies, Antinuclear↗
PubMed1998

Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules.

Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB x NZW)F1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab')2 and Fab' fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes.

Amino Acid Sequence↗
PubMed1998

Interleukin-1 receptor defect in autoimmune NZB mouse brain.

Interleukin-1 receptors (IL-1R type I and II) have been characterized in murine nervous structures (hippocampus and frontal cortex), in vascular structures (vessels, choroid plexus), and in the anterior pituitary. Because interleukin-1 (IL-1), injected or induced in the brain, is a powerful regulator of the stress axis and immune functions, it was of interest to investigate IL-1Rs and IL-1 in autoimmune mice. In control mice, bacterial lipopolysaccharide (LPS), administered i.p. or i.c.v., induces a sharp decrease in available brain IL-1 receptors, in spite of a moderate increase in mRNAs for both receptor types. This is concomitant with an increase in IL-1 alpha, beta, and ra mRNA. Ligand production clearly overcomes receptor turnover. In autoimmune mice (NZB and NZB/NZW F1), a strong defect in IL-1R (type I) is demonstrated in the dentate gyrus. This tissue-specific defect cannot be explained by increased occupancy by endogeneous ligands as for LPS-treated mice. The transmission of the defect is Mendelian and suggests the involvement of a single gene. However patterns of IL-1R mRNAs (evaluated by RT-PCR) are similar in NZB and in controls, suggesting a translational or post-translational abnormality. The contribution of this genetic disorder in the development of autoimmunity remains to be clarified. Because the brain IL-1 system sends inhibitory signals towards immune functions, this lack of functional IL-1 binding sites might participate in the disregulations observed in NZB autoimmune mice.

Animals↗
PubMed1998

Interleukin-1 receptor deficiency in the hippocampal formation of (NZB x NZW)F2 mice: genetic and molecular studies relating to autoimmunity.

Interleukin-1 receptor (IL-1R) deficiency has been previously described in the dentate gyrus of autoimmune NZB and (NZB x NZW) F1 (or BWF1) mice. In this study, the genetic and molecular characterization of this defect were investigated in BWF2 mice in relation to anti-DNA antibody production and microsatellite D1Nds4 (near the IL1r1 gene) polymorphism. IL-1R density was quantified in the brain, spleen and pancreas, using in vitro quantitative autoradiography with recombinant human [125I]-IL-1alpha as the ligand. This study of the dentate gyrus of F2 mice revealed three phenotypes: NZW-like, NZB-like and F1-like, which occurred in a ratio of 1:1:2, with IL-1R densities of 100%, 17% and 59%, respectively as compared to control NZW mice (100%). In contrast, IL-1R densities observed in the choroid plexus and peripheral organs were similar. Moreover a high production of IgG2a anti-DNA antibodies was observed in F2 mice, as in their parents, particularly those with the NZB-like phenotype. Microsatellite mapping of D1Nds4 revealed polymorphism in both parents and BWF2 mice in relation to the level of IL-1R density in the dentate gyrus. In spite of the acute defect in IL-1 binding in the dentate gyrus of NZB mice, molecular analysis of IL-1R mRNA (type I, II and accessory protein) showed similar amounts of mRNA, measured following RT-PCR amplification, in the hippocampal formation of both NZB and control C3H/He mice. In conclusion, the transmission of the IL-1R defect in the dentate gyrus of NZB mice is monofactorial and the defect appears to be at the post-transcriptional level of IL-1R synthesis. The lack of IL-1R in the dentate gyrus seems to correlate with some autoimmune characteristics. Correlation of D1Nds4 polymorphism with the level of IL-1R density suggests that it could be a predisposing gene to disease or a marker for other closely linked predisposing genes.

Animals↗
PubMed1998

Penetrating anti-DNA monoclonal antibodies induce activation of human peripheral blood mononuclear cells.

Different studies have shown that some autoantibodies are able to penetrate into living cells and that this phenomenon has functional consequences, including apoptosis. We have explored the effect of anti-DNA antibodies (Ab) on the in vitro activation of peripheral blood mononuclear cells (PBMNC) and found that a human polyclonal anti-DNA, IgG, which efficiently penetrated living cells, was able to induce the expression of different cell activation antigens in vitro such as CD69, CD71 or CD98 by PBMNC from normal individuals. However, the cell activation phenotype induced by anti-DNA Ab was considered anomalous since the expression of some activation antigens was not up-regulated, and others showed aberrant behaviour (such as down-regulation of ICAM-1 expression). Similar results were obtained using different murine anti-DNA monoclonal antibodies (mAb). In addition, mAb that showed an efficient ability to penetrate living cells tended to have a greater effect on PBMNC activation. Anti-DNA Ab were also able to induce a noticeable expression of CD95/Fas. These data indicate that penetrating anti-DNA Ab are able to induce an anomalous activation state in vitro in a significant fraction of PBMNC. We believe this effect may occur in vivoand could have an important function in the pathogenesis of the immune dysregulation seen in SLE.

Animals↗
PubMed1998

Immunochemical, structural and translocating properties of anti-DNA antibodies from (NZBxNZW)F1 mice.

Many monoclonal antibodies (mAb) derived from the spleens of (NZBxNZW)F1 mice react strongly with dsDNA and also other self antigens, although more weakly. When added to cell cultures, these polyreactive anti-DNA mAb penetrate into various cell types and accumulate in the nucleus within a few hours. Almost all anti-DNA mAb bind to cell membrane antigens but the extent of their binding does not directly correspond to their penetration capabilities. Sequence analysis of anti-DNA mAb indicated the use of various germ-line VH families. The complementary-determining regions (CDR) 3 differ but they all contain a relatively high number of tyrosines and positively charged amino acids (lysine and arginine). Haptens (biotin, fluor-escein, oligonucleotides) and macromolecules (peroxidase, IgG) covalently coupled to the mAb or their F(ab')2 and Fab fragments were translocated through the cytoplasm and into the cell nucleus. Furthermore, 61% of peripheral blood lymphocytes were labelled when mice were injected with fluorescein-labelled mAb. Peptides corresponding to CDR2, CDR3 and CDR2 linked to CDR3 (CDR2-3) of several penetrating mAb were prepared and their intracellular translocating capacity was assessed. The CDR2-3 peptide, but not the individual peptides, was able to penetrate cells and could be used as a vector to transport macromolecules. Although all CDR2-3 reacted with dsDNA and other self antigens, each one exhibited a distinct polyreactivity profile.

Amino Acid Sequence↗
PubMed1997

Mimotopes of polyreactive anti-DNA antibodies identified using phage-display peptide libraries.

Three monoclonal IgG2a anti-DNA polyreactive autoantibodies, derived from lupus-prone mice (NZB x NZW)F1, were studied by surface plasmon resonance (BIAcore) analysis using three different synthetic double-stranded (ds) oligonucleotides of 25, 30, and 25 base pairs (bp). These monoclonal antibodies (mAb) exhibited dissociation rate constants (k(off)), ranging from 0.0001 (mAb F14.6 and F4.1) to 0.01/s (mAb J20.8) and k(on) ranging from 2 x 10(5) to 2 x 10(6) /M/s. The screening of a constrained random peptide library displayed on M13 bacteriophages on these mAb allowed the determination of the specific consensus motifs (mimotopes) for mAb F14.6 and J20.8, but not for mAb F4.1. No cross-reaction was observed between F14.6- and J20.8-specific peptides (and vice versa). Binding of all phages selected on F14.6 was inhibited with 700 ng/ml soluble DNA. The binding of one group of peptides selected on J20.8 was inhibited by 400 ng/ml soluble DNA, of a second group by 2500 ng/ml, while binding of a third group could not be inhibited. The determined consensus sequences do not match with known sequences. Peptides specific for F14.6 share negative charges and aromatic rings that may mimic a DNA backbone, while peptides selected with J20.8 do not bear any negative charge, implying a different kind of molecular recognition, for example hydrogen or salt bonds. The peptides selected on J20.8 also bind serum antibodies from human patients with systemic lupus erythematosus. In addition, BALB/c mice immunized with some of the selected phages exhibit high serum titers of IgG3 anti-dsDNA antibodies, further supporting the hypothesis that peptide epitopes may mimic an oligonucleotide structure.

Amino Acid Sequence↗
PubMed1996

Recycling of ELISA plates for the serological diagnosis of tuberculosis using a Mycobacterium tuberculosis-specific glycolipid antigen.

A Mycobacterium tuberculosis-specific glycolipid antigen (diacyl-trehalose-DAT) and a battery of defined sera from tuberculous patients were used to evaluate the reliability of recycling ELISA microplates for serodiagnosis of tuberculosis. The reuse of plates was evaluated by the same ELISA technique before and after recycling with an acid buffer to dissociate and release antibodies from antigen-antibody complexes fixed on used plates. The correlation coefficients for comparisons between several new plates used on different days were always higher than r = 0.90, and the variation between tests was always lower than 12%. For comparisons performed systematically between new plates and recycled plates, the values were variable. Up to the fifth recycling, the correlation coefficient was higher than r = 0.60. From the sixth recycling, correlation coefficient values fell to r = 0.381. The results obtained with recycled plates washed manually were much poorer than those obtained after automatic washing. The results described in this report demonstrate that an ELISA test can be performed on recycled plates under the conditions described.

Enzyme-Linked Immunosorbent Assay↗
PubMed1996

Characterization of autoantibody activities in sera anti-DNA antibody and circulating immune complexes from 12 systemic lupus erythematosus patients.

To examine autoantibodies present in patients with active systemic lupus erythematosus (SLE), sera, circulating immune complexes (CIC), and antibodies purified on DNA-immunoadsorbent were tested by enzyme immunoassay. A panel of self-antigens, including DNA, histones (HIS), glomerular basal membrane (GBM), thymus cell extract (TCE), actin (ACT), myosin (MS), and tubulin (TUB), was used to define their specificities. IgM antibodies against all antigens of the panel were detected in sera, CIC, and in antibodies eluted from the DNA-immunoadsorbent and demonstrated a large polyreactivity. IgG antibodies showed restricted activities against DNA, HIS, GBM, and TCE in sera and a large polyreactivity in CIC. Inhibition experiments were performed to assess their mono- or polyreactivities. Among the IgG autoantibody population recognizing DNA, two populations of IgG antibodies were detected in the sera and in the affinity purified anti-DNA: one recognizes DNA, HIS, and GBM, and the other binds to DNA and to cytoskeletal proteins. These autoantibody populations were found in CIC, which also often contained high amounts of IgG antibodies recognizing ACT and MS. A third population of IgG antibody that recognizes only TCE and could not be inhibited by DNA or other antigens was found in serum and CIC. Our data demonstrate the existence of several populations of autoantibody in serum and CIC of SLE patients: (1) IgM polyreactive autoantibodies, (2) IgG polyreactive autoantibodies recognizing DNA and cytoskeletal proteins, (3) IgG specific to DNA, which cross react with HIS and GBM, and (4) IgG specific to TCE antigens.

Adolescent↗
PubMed1995

Defects in the regulation of anti-DNA antibody production in aged lupus-prone (NZB x NZW)F1 mice: analysis of T-cell lymphokine synthesis.

(NZB x NZW)F1 (B/W) mice spontaneously develop a lupus-like syndrome characterized by an increased level of autoantibodies in old mice. We analysed the role of T cells in the regulation of anti-DNA antibody production by B cells in vitro as a function of age. In cultures of old mouse T and B cells, IgG and IgM anti-DNA antibodies were synthesized at high levels, in contrast to consistently lower amounts, particularly of IgG, measured in cultures of young mouse cells. Addition of young mouse T cells to old B cells inhibited IgG, but not IgM, anti-DNA production, whereas T cells from old mice stimulated IgG synthesis by young mouse B cells. Addition of supernatants harvested from concanavalin A (Con A)-stimulated T cells to B-cell cultures induced similar effects. Therefore, we evaluated possible modifications of lymphokine synthesis compared to that of the healthy NZW parent. T cells from old mice were able to secrete normal levels of interferon-gamma (IFN-gamma) and interleukin (IL)-10; however, secretion of IL-2 and IL-4 was dramatically decreased. Semi-quantitative polymerase chain reaction analysis of constitutive RNA messengers showed increased IFN-gamma levels in young and old B/W mice, and normal IL-10 mRNA levels in young and higher levels in old mice. Constitutive IL-2 and IL-4 mRNA were detected only after Con A stimulation and their levels decreased in old compared to young B/W mice; in particular IL-2 mRNA was considerably lower in old B/W than in control NZW mice. Taken together, these results suggest that, despite constitutive T-cell abnormalities, young B/W mice are able partially to control their lymphokine production, whereas aged mice exhibit a deficient synthesis, associated with an increased capacity to produce IFN-gamma.

Aging↗
PubMed1994

[Physiological autoimmunity].

The serum of normal individuals contains antibodies directed against their own antigens (autoantigens). These autoantibodies are the main and the best known component of normal autoimmunity. They mostly involve polyreactive (one antibody recognising several antigens) IgM, but also IgG, usually having low affinity for antigens. The role of natural autoimmunity is becoming better understood. It participates in the equilibrium of the immune system, contributing to controlled production of antibodies, accelerated elimination of external antigens or aged autoantigens, and to triggering of a specific immune response. Some data suggest that the action of autoantibodies in homeostasis can extend beyond the immune system. Many pathological states, of immune nature or not, are associated with an increase or diminution of the titre of natural autoantibodies. The mechanisms and consequences of these changes remain to be elucidated.

Animals↗
PubMed1994

Beneficial effect of polyclonal immunoglobulins from malaria-infected BALB/c mice on the lupus-like syndrome of (NZB x NZW)F1 mice.

We previously reported that infection of BALB/c mice with the parasite Plasmodium chabaudi induces high production of natural autoantibodies. Here we demonstrate that such an infection of lupus-prone (NZB x NZW)F1 (B/W) mice retards the development of their autoimmune disease. Survival and disease hallmarks (high-grade proteinuria and IgG anti-DNA antibodies) were delayed for 6 months when parasite inoculation was given at either 3 or 7 months of age, i.e. before or after the onset of the clinical symptoms. Similar beneficial effects, although less pronounced, were obtained when mice were treated with a total of 800 micrograms of IgG (P-IgG) or IgM (P-IgM) or 300 micrograms of cryoglobulin preparations isolated from P. chabaudi-infected BALB/c mice while similarly prepared fractions from uninfected mice had little effect. Compared to these fractions, P-IgG and P-IgM contained higher levels of natural antibodies bearing the D23 idiotype characteristic of polyreactive natural autoantibodies with enhanced activity against Fab and Fc fragments of IgG. In surviving mice, the level of anti-DNA antibodies, particularly those of IgG1 isotype, were significantly decreased. Flow cytometric analysis of various T cell subsets showed that the number of cells expressing gamma delta T cell receptor (TcR) antigens which did not vary with age was not modified after P-IgG or P-IgM treatment. In contrast, the number of T cells expressing V beta 8.1,2,V beta 10 and V beta 14 TcR antigens, which increased with age, were significantly reduced. Taken together, these results indicate that parasite infection of mice induces the synthesis of populations of IgM and IgG natural autoantibodies with immunoregulatory properties and that these antibodies attempt, at least transitorily, to rescue a natural autoantibody network that is deficient in B/W mice.

Animals↗
PubMed1994

Natural autoantibodies are involved in the haemolytic anaemia of NZB mice.

NZB is a mouse strain that spontaneously develops autoimmune haemolytic anaemia at 10-12 months of age. We analysed the autoantibodies present throughout their life and compared them to natural autoantibodies found in the normal mouse. Sera and Coombs' antibodies eluted from red blood cells (RBC) were tested for their activities against RBC and a panel of antigens: actin, myoglobin, myosin, tubulin, spectrin, DNA and trinitrophenyl bovine serum albumin (TNP-BSA), F(ab')2 and Fc fragments of IgG by using enzyme immunoassays (EIA) and Western blotting analysis of RBC membrane extracts. In NZB mouse sera, activities of IgM and IgG against the whole panel, compared to those of sera from age-matched BALB/c mice, increased progressively throughout life with oscillating values in parallel with the anti-RBC activity. Two periods of autoantibody production seem to exist: the first is characterized by a fluctuating high level of IgM and stable level of IgG natural autoantibodies, and the second by a rise of IgG natural autoantibodies in parallel with IgG anti-RBC antibodies. The presence of idiotype D23 (IdD23), which is characteristic of natural polyspecific autoantibodies, was high on serum IgM and low on IgG autoantibodies throughout life. To further analyse autoantibody level oscillations, we tested IgM and IgG fractions after their separation from whole serum and observed highly enhanced autoantibody activities of both IgM and IgG. These autoreactivities markedly diminished when the separated IgM and IgG fractions were recombined, suggesting humoral control of the autoreactivity as we had already noted for IgG in normal animals. During the first period of autoantibody production, IgM and IgG antibodies eluted from RBC (Combs' antibodies) and those eluted from serum using an RBC-immunoadsorbent (circulating antibodies) reacted with all RBC membrane components, with all antigens of the panel and with F(ab')2 and Fc. Some of these reactivities were comparable to those exhibited by a monoclonal antibody recognizing bromelain-treated RBC. In the second period, both IgM and IgG Coombs' antibodies reacted more strongly with spectrin, and exhibited new specificities, for example against the band 3 polypeptide. IdD23 was abundant on Combs' IgG antibodies in the second period. Taken together, these data suggest that IgM and IgG natural autoantibodies, able to recognize not only RBC antigens but also other antigens, particularly F(ab')2 and Fc fragment of IgG, predominate in Coomb's antibody population.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1993

Autoantibodies in the sera of Trypanosoma cruzi-infected individuals with or without clinical Chagas disease.

Variations in the levels and the specificities of autoantibodies directed against a panel of antigens (cytoskeleton proteins, DNA, laminin) were analyzed in the sera from two groups of humans infected with Trypanosoma cruzi. One group was constituted of apparently healthy blood donors (BD) and the other of patients with clinically confirmed Chagas disease (CCH). In both infected groups, a high proportion but not all sera exhibited dramatic enhancement of IgM and IgG autoantibodies directed against all antigens tested. Sera positive for IgG autoantibodies were generally found more frequently in the CCH than in the BD group, except for anti-actin antibodies more often present in BD sera. Anti-laminin IgG antibodies were present in a similar number of individuals in both groups. Although the titers of anti-laminin IgG antibodies were in general higher in CCH, their dissociation constants were in the same range (7 x 10(-8) - 10(-7) M) in both groups. IgG autoantibodies were demonstrated to be polyreactive with laminin and other self antigens as well. Circulating immune complexes were present in sera from both groups and the activity of the antibodies dissociated from these complexes was directed against all the antigens of the panel. Although the IgE concentration was significantly enhanced in several subjects from both groups, the incidence of positive sera was higher in the CCH (60%) than in the BD (39%) group. Our results demonstrate that autoantibodies with the characteristics of natural autoantibodies are found in both T. cruzi-infected apparently healthy individuals and patients.

Antibody Affinity↗
PubMed1993

Monoclonal IgG and IgM autoantibodies obtained after polyclonal activation, show reactivities similar to those of polyclonal natural autoantibodies.

In this study, we described the properties of two groups of polyreactive IgG monoclonal antibodies (mAb) derived from splenocytes of a Plasmodium chabaudi-infected BALB/c mouse. These IgG mAb reacted with self antigens, but not with the parasite. Depending upon the antigen under study, they showed low (10(-5) M) to high (10(-8) M) affinities. When examined by immunoblotting using mouse organ extracts as the antigen source, each IgG mAb had a different reactivity profile. The IgG2b, but not the IgG2a mAb group, reacted with F(ab')2 fragments of IgG from normal mouse sera, as well as with F(ab')2 fragments of other mAb from the same fusion. Their anti-F(ab')2 activity was inhibited by various antigens, suggesting that the binding sites for these antigens and F(ab')2 fragments were close or overlapping. We also characterized two monoclonal IgM that were strongly and almost exclusively reactive with polyclonal F(ab')2 fragments of normal IgG, and that inhibited the binding of normal polyclonal IgG to self antigens. We previously described such properties for polyclonal IgM from normal mouse sera. The binding of the F(ab')2-reactive IgG mAb to self antigens was also inhibited by normal polyclonal IgM. These results indicate that the IgG and IgM obtained after polyclonal stimulation, exhibit characteristics similar to those of autoantibodies present in normal mouse sera. Furthermore, they confirm our previous studies showing the polyreactivity of polyclonal IgG. Finally, they also show that such IgG mAb exhibit properties similar to those of natural IgM, namely polyreactivity, affinity and connectivity.

Animals↗