Correction of T cell deficiency in ZAP-70 knock-out mice by simple intraperitoneal adoptive transfer of thymocytes.

The tyrosine kinase zeta chain-associated protein of 70 kDa (ZAP-70) plays a key role in T cell development and signalling. In the absence of ZAP-70, T cell development is arrested in the CD4+ CD8+ double-positive stage, thus ZAP-70 homozygous knockout (ZAP-70-/- ) mice have no mature T cells in their peripheral lymphoid organs and blood, causing severe immunodeficiency. We investigated the early kinetics and long-term effects of wild-type thymocyte transfer on T cell repopulation in ZAP-70-/- mice. We used a single intraperitoneal (i.p.) injection to deliver donor thymocytes to the recipients. Here, we show that after i.p. injection donor thymocytes leave the peritoneum through milky spots in the omentum and home to the thymus, where donor-originated CD4- CD8- double-negative thymocytes most probably restore T cell development and the disrupted thymic architecture. Subsequently, newly developed, donor-originated, single-positive αß T cells appear in peripheral lymphoid organs, where they form organized T cell zones. The established chimerism was found to be stable, as donor-originated cells were present in transferred ZAP-70-/- mice as late as 8 months after i.p. injection. We demonstrate that a simple i.p. injection of ZAP-70+/+ thymocytes is a feasible method for the long-term reconstitution of T cell development in ZAP-70-deficient mice.

Proteoglycan aggrecan conducting T cell activation and apoptosis in a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease and its targeting of the joints indicates the presence of a candidate autoantigen(s) in synovial joints. Patients with RA show immune responses in their peripheral blood to proteoglycan (PG) aggrecan. One of the most relevant animal models of RA appears to be proteoglycan-induced arthritis (PGIA), and CD4(+) T cells seem to play a crucial role in the initiation of the disease. In this review, the role of various T cell epitopes of aggrecan in the induction of autoreactive T cell activation and arthritis is discussed. We pay special attention to two critically important arthritogenic epitopes, 5/4E8 and P135H, found in the G1 and G3 domains of PG aggrecan, respectively, in the induction of autoimmune arthritis. Finally, results obtained with the recently developed PG-specific TCR transgenic mice system showed that altered T cell apoptosis, the balance of activation, and apoptosis of autoreactive T cells are critical factors in the development of autoimmunity.

ZAP-70 tyrosines 315 and 492 transmit non-genomic glucocorticoid (GC) effects in T cells.

ZAP-70 kinase is a key regulator of early T-cell signaling; moreover, it also participates in non-genomic glucocorticoid (GC) signaling. Short-term high-dose GC-analogue treatment induces the phosphorylation of the kinase, and its association with the GC receptor (GR). In the present work, first, we identified those tyrosine (Y) residues of the ZAP-70 kinase which were involved in non-genomic GC signaling using an array of P116 cells (ZAP-70-deficient Jurkat subclone) lentivirally-transfected with wild type or point-mutated ZAP-70 constructs where Y-residues were replaced with phenylalanine (F) at positions 069, 126, 178, 238, 292, 315, 492 or 493. Then, we characterized the GC-analogue-induced Y-phosphorylation of 3 key substrates of the ZAP-70 kinase: SLP-76, LAT and Cbl. Finally, we studied the cross talk between the non-genomic GC- and TcR/CD3 signaling pathways. Y-F mutations at positions 315 or 492 abolished the short high-dose Dexamethasone (DX) treatment-induced ZAP-70 phosphorylation suggesting that these Y-residues were involved in ZAP-70-mediated non-genomic GC actions. DX treatment alone induced Y-phosphorylation of LAT, SLP-76 and Cbl; moreover, in F315- and F492-ZAP-70 mutated cells decreased DX-induced Y-phosphorylation of SLP-76 and Cbl was observed indicating that these molecules might transmit downstream non-genomic GC signals in a ZAP-70 dependent manner. Short, high dose DX treatment influenced significantly the anti-CD3-induced signaling events: we observed alterations in LAT, SLP-76 and Cbl Y-phosphorylation and a decreased Ca(2+)-signal. These results confirm that ZAP-70 represents an important link between the non-genomic GC and TcR/CD3 signaling pathways. Importantly, the DX-induced effects on resting and activated T-cells are differentially mediated. These fine molecular details help to better understand the complex mechanism of non-genomic GC effects in T-cells.

Disease-promoting and -protective genomic loci on mouse chromosomes 3 and 19 control the incidence and severity of autoimmune arthritis.

Proteoglycan (PG)-induced arthritis (PGIA) is a murine model of rheumatoid arthritis. Arthritis-prone BALB/c mice are 100% susceptible, whereas the major histocompatibility complex-matched DBA/2 strain is completely resistant to PGIA. To reduce the size of the disease-suppressive loci for sequencing and to find causative genes of arthritis, we created a set of BALB/c.DBA/2-congenic/subcongenic strains carrying DBA/2 genomic intervals overlapping the entire Pgia26 locus on chromosome 3 (chr3) and Pgia23/Pgia12 loci on chr19 in the arthritis-susceptible BALB/c background. Upon immunization of these subcongenic strains and their wild-type (BALB/c) littermates, we identified a major Pgia26a sublocus on chr3 that suppressed disease onset, incidence and severity via controlling the complex trait of T-cell responses. The region was reduced to 3 Mbp (11.8 Mbp with flanking regions) in size and contained gene(s) influencing the production of a number of proinflammatory cytokines. Additionally, two independent loci (Pgia26b and Pgia26c) suppressed the clinical scores of arthritis. The Pgia23 locus (∼3 Mbp in size) on chr19 reduced arthritis susceptibility and onset, and the Pgia12 locus (6 Mbp) associated with low arthritis severity. Thus, we have reached the critical sizes of arthritis-associated genomic loci on mouse chr3 and chr19, which are ready for high-throughput sequencing of genomic DNA.

T cell receptor (TCR) signal strength controls arthritis severity in proteoglycan-specific TCR transgenic mice.

T cell receptor transgenic (TCR-Tg) mice specific for the arthritogenic 5/4E8 epitope in the G1 domain of cartilage proteoglycan were generated and back-crossed into arthritis-prone BALB/c background. Although more than 90% of CD4(+) T cells of all TCR-Tg lines were 5/4E8-specific, one (TCR-TgA) was highly sensitive to G1-induced or spontaneous arthritis, while another (TCR-TgB) was less susceptible. Here we studied whether fine differences in TCR signalling controlled the onset and severity of arthritis. Mice from the two TCR-Tg lines were immunized side by side with purified recombinant human G1 (rhG1) domain for G1 domain of cartilage proteoglycan (PG)-induced arthritis (GIA). TCR-TgA mice developed severe and early-onset arthritis, whereas TCR-TgB mice developed weaker arthritis with delayed onset, although TCR-TgB CD4(+) T cells expressed approximately twice more TCR-Vß4 chain protein. The more severe arthritis in TCR-TgA mice was associated with higher amounts of anti-G1 domain-specific antibodies, larger numbers of B cells and activated T helper cells. Importantly, TCR-TgB CD4(+) T cells were more sensitive to in vitro activation-induced apoptosis, correlating with their higher TCR and CD3 expression and with the increased TCR signal strength. These findings indicate that TCR signal strength determines the clinical outcome of arthritis induction: 'optimal' TCR signal strength leads to strong T cell activation and severe arthritis in TCR-TgA mice, whereas 'supra-optimal' TCR signal leads to enhanced elimination of self-reactive T cells, resulting in attenuated disease.

Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells.

Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling.

Galactoside-specific misletoe lectin modulates dexamethasone-induced apoptosis and glucocorticoid receptor level in Balb/c thymocytes.

The galactoside-specific plant lectin, Viscum album agglutinin-(VAA)-I has been shown to activate the natural immune system and modulate the maturation of thymocytes in vivo. However the mechanism of this immunobiological action is not yet understood. In our previous study we demonstrated the VAA-I-induced enhancement of proliferation and selection of thymocytes which inhibited the dexamethasone (DX)-induced thymocyte depletion. In this present work we investigated the effect of 1, 4 and 21 days of VAA-I treatment on DX-induced apoptosis of thymocytes in Balb/c mice. The number of early apoptotic cells was detected with Annexin V staining while the late apoptotic cells were identified according to their propidium iodide incorporation into DNA using flow cytometry. The expression of glucocorticoid receptor (GCR) in double-negative (DN), double-positive (DP) and CD4 or CD8 single-positive (SP) cell populations was assessed. The additive effect of lectin on DX-induced apoptosis of thymocytes consisted of two different actions of VAA-I and DX. One-day VAA-I treatment caused enhanced apoptosis in SP mature cells in contrast to the apoptotic effect of DX, which was mainly directed towards immature DN and DP cells. Treatment with 30 ng/kg VAA-I for four days elevated the GCR level (mean fluorescence intensity) in DP thymocytes. Lectin treatment for 21 days caused more than 20% elevation of GCR expression in all thymocyte subpopulations (DN, DP, CD4+ and CD8+). These results suggest that VAA-I may alter the sensitivity of thymocytes to glucocorticoids and this effect may play a role in the bell-shaped dose-response curve of lectin-induced immunological effects.

[EEG-restitution and brain metabolism during electroconvulsive treatment in relaxation (author's transl)]. / EEG-Restitution und Hirnmetabolismus während modifizierter Elektroschockbehandlung.

Blood-gas (pO2, pCO2) and pH-changes of venous (v.jugularis interna) and arterial (A.femoralis) blood samples, furthermore glucose utilization and lactate-, pyruvate-production of brain were investigated during electroconvulsive treatment in relaxation of 45 psychotic patients. The blood-gas values and substrate concentrations were statistically evaluated and represented in a function of the characteristic phases of the postconvulsive EEG-activity. A correlation was found between the glucose metabolism of the brain and the postconvulsive recovery of EEG. The restitution of postconvulsive brain metabolism runs discontinuously in the first 12 minutes of postconvulsive state. In the phase of electric silence and periodic delta-waves the brain metabolism was shifted to anaerobic direction. During the treatment no anoxic anoxia or acidosis takes place during the seizure activity and restitution, the measurable metabolic changes are moderate and supposedly play no important role in the "effect" of treatment.

Correlation between blood gases, glycolytic enzymes and EEG during electroconvulsive treatment in relaxation.

Twenty-three psychiatric patients were investigated during electroconvulsive treatment in relaxation. The blood gases, pH and serum bicarbonate levels in blood samples from the internal jugular vein and the femoral artery were measured radiometrically. The LDH fractions were separated electrophoretically and their activity, along with the activity of aldolase, was then determined on test materials. EEG recordings were made during the seizure and also during postconvulsive restitution. The following conclusions were drawn: (1) There was no evidence of anoxic anoxia in the brain during and after seizures. (2) A close relationship was found between the corresponding phases of electrical activity and brain metabolism as indicated by the blood gas changes during postconvulsive restitution. (3) On the basis of the increased glycolytic activity in the sera it is probable that glucose metabolism was shifted in the anaerobic direction during postconvulsive restitution of the brain tissues.

[Electron microscopic study of striatodental calcification (Fahr) (author's transl]. / Elektronenoptische Untersuchungen bei "striato-dentaler Calcifikation" (Fahr)

In a woman, aged 52, who had impaired phosphate excretion and low serum calcium levels, abundant calcium deposits (Pseudokalk) were found between the basement membranes of blood vessels in the regions of corpus striatum and nucleus dentatus as well as in the subcortical white matter and centrum semiovale. Calcium deposits were found also outside blood vessels but always in the vicinity of the basement membrane. These can be phagocytozed by makrophages or astrocytes. Calcium deposits have a characteristic ultrastructure. They are built up of 140-400 A electron lucent filaments (acid mucopolysaccharid?) within which electron dark segments built up of 40-80 A units (calcium deposits?) are found. The concentric rings of calcified deposits are of reflection of differing density of aggregation of dark filaments. The growth of the deposits takes place by additional precipitation. In the development of calcification of the cerebral blood vessels in Fahr's disease, the role of high serum phosphate levels, the increased permeability and dysfunction of mesenchymal cells of the vessel walls are discussed.