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PubMed2004

Pathological theta oscillations in idiopathic generalised epilepsy.

OBJECTIVE: To investigate spectral power, inter- and intra-hemispheric coherence in the interictal scalp electroencephalography (EEG) of 41 patients with idiopathic generalised epilepsy. METHODS: Two minutes of eyes-closed waking interictal EEG activity was analysed. Fast Fourier transformation was performed. Raw and age-regressed, Z-transformed values were computed for 19 electrodes and 4 frequency bands: absolute power (AP, ZAP), percent power (RP, ZRP), band mean frequency (MF, ZMF), inter-hemispheric (CO, ZCO) and intra-hemispheric (IC, ZIC) coherence. Compressed values (scalp averages) were computed for each parameter and 4 frequency bands. Compressed data of the patients (GE group) and the healthy controls (C group) were compared. RESULTS: ZAP across the entire 1.5-25.0 Hz range was greater in the GE than in the C group. Delta and theta ZRP was greater, alpha ZRP was less in GE than in C. ZMF and ZIC was about the same in the GE and C groups. The crucial, band-specific finding was significantly greater theta-ZCO in the GE group, in contrast to normal or decreased ZCO in the other bands. In addition, within-group correlation between ZAP of the frequency bands, correlation of ZAP and ZCO, correlation of ZIC and ZCO were different for the two groups. CONCLUSIONS: The pattern of enhanced ZAP, ZRP, ZCO, together with normal ZIC and ZMF in the theta range was a peculiar, novel finding in GE. It was incompatible with any of the known patterns of altered power and coherence due to lesional or metabolic aetiology. This pattern presumably indicates the presence of a powerful, diffuse, hypersynchronous, hypercoherent theta oscillation at the thalamo-cortical level of the brain. The role of inter-hemispheric connections in maintaining this oscillation was suggested. The other findings suggest disturbed central regulation of EEG power and coherence in the interictal state.

Adolescent↗
PubMed2004

Differential role of neutrophils and monocytes during subcutaneous plasma extravasation.

We examined the behavior of polymorphonuclear leukocytes (PMNs) and monocytes during subcutaneous plasma extravasation in guinea-pigs. Plasma extravasation was induced by intradermal injection of zymosan-activated plasma (ZAP). The degree of extravasation correlated logarithmically with the concentration of injected ZAP, and was composed of PMN-dependent and -independent components. The latter was mediated primarily by histamine. The former accounted for 40-50% of the total plasma extravasation, peaked within 15 min, and then rectilinearly decreased with a half-life between 30 and 40 min. Histological examination of skin at 15 min after ZAP injection demonstrated PMN attachment to the luminal surface of venule endothelial cells, without evidence of PMN extravasation. We next examined whether monocyte infiltration of subcutaneous tissue played a causal role in plasma extravasation. Monocyte-predominant infiltration was initially caused by an intradermal injection of a monocyte-specific chemotactic factor, the S19 ribosomal protein (RP S19) dimer. Monocyte infiltration did not induce plasma extravasation even in guinea-pigs with elevated peripheral blood monocyte levels following administration of a macrophage-colony stimulating factor. A simultaneous injection of prostaglandin E2, a vasodilating agent, with RP S19 dimer also did not induce plasma extravasation. In contrast, a simultaneous injection of RP S19 dimer with ZAP changed the leukocyte infiltration pattern from PMN-predominant to monocyte-predominant, and almost completely suppressed the PMN-dependent component of the ZAP-induced plasma extravasation. The lack of plasma extravasation in the monocyte-predominant pattern was reproduced when a strong monocyte infiltration was induced by an intradermal injection of apoptotic cells. We conclude that leukocyte-induced plasma extravasation is specific for PMN, and is not due to a physical leakage of plasma during leukocyte emigration. Rather, plasma extravasation is probably caused by a cognate interaction between PMNs and postcapillary venule endothelial cells.

Animals↗
PubMed1995

Interaction between lck and syk family tyrosine kinases in Fc gamma receptor-initiated activation of natural killer cells.

Ligation of the Fc gamma R on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that Fc gamma R ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early Fc gamma R-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the zeta chain of the Fc gamma R complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-gamma 2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-gamma 2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling Fc gamma R stimulation to critical downstream signaling events. Taken together, our results suggest not only that Fc gamma R stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade.

Cell Line↗
PubMed2001

Immunohistochemical markers for Barrett's esophagus and associations to esophageal Z-line appearance.

BACKGROUND: Data from previous studies on intestinal metaplasia at the gastroesophageal junction have been conflicting, which makes the diagnosis of Barrett's esophagus less obvious. This may partly be due to the lack of a reliable classification of the Z-line appearance. We previously proposed such a classification (the ZAP classification) that was shown to correlate with the prevalence of intestinal metaplasia. The use of different immunohistochemical techniques has increased in the study of intestinal metaplasia. In the present study our aim was to 1) evaluate the impact of different antibodies, namely cytokeratin (CK) 7, 13, and 20, CaCO3/73, and FBB2/29, in order to differentiate between Barrett's esophagus and cardia intestinal metaplasia, and 2) explore the staining patterns in different ZAP grades. METHODS: Thirty-nine specimens with intestinal metaplasia were compared--9 from Barrett's esophagus, 6 from cardia, and 24 from the Z-line. The Z-line specimens were evaluated with respect to ZAP grade. RESULTS: No differences were encountered regarding staining patterns for CK13 and CaCO3/73 in Barrett's esophagus and cardia. The staining pattern of CK7/20 was significantly different between Barrett's esophagus and cardia. CK7/20 showed a rising frequency of Barrett's esophagus staining pattern with rising ZAP grade. CONCLUSION: CK7/20 is a feasible marker for Barrett's esophagus. Intestinal metaplasia in different ZAP grades differs regarding expression of immunohistochemical markers.

Aged↗
PubMed2001

Requirement for tyrosine residues 315 and 319 within zeta chain-associated protein 70 for T cell development.

Engagement of the T cell antigen receptor (TCR) induces the transphosphorylation of the zeta chain-associated protein of 70,000 Mr (ZAP-70) protein tyrosine kinase (PTK) by the CD4/8 coreceptor associated Lck PTK. Phosphorylation of Tyr 493 within ZAP-70's activation loop results in the enzymatic activation of ZAP-70. Additional tyrosines (Tyrs) within ZAP-70 are phosphorylated that play both positive and negative regulatory roles in TCR function. Phosphorylation of Tyr residues (Tyrs 315 and 319) within the Interdomain B region of the ZAP-70 PTK plays important roles in the generation of second messengers after TCR engagement. Here, we demonstrate that phosphorylation of these two Tyr residues also play important roles in mediating the positive and negative selection of T cells in the thymus.

Animals↗
PubMed1988

Decreased neutrophil thromboxane A2 and endothelial PGI2 production in the postoperative period. An in vitro assay for detection of neutrophil and plasma dysfunction.

Severe trauma results in reversible abnormalities in neutrophil function, but the specific role in the pathogenesis of postoperative sepsis is undetermined. Twenty adult patients undergoing elective surgical procedures were studied. Blood samples were obtained prior to and 24 hours after operation. Blood neutrophils were isolated and incubated (10(7) cells/mL) on bovine vascular endothelial cell monolayers. Untreated plasma or zymosan-activated plasma (ZAP) or 65 C inactivated plasma was added, and TxB2 and 6-keto PGF1 alpha production measured after 2 hours. Endothelial damage was detected by light and scanning electron microscopy beginning 2 and 4 hours after treatment. Preoperatively, neutrophil TxB2 release was less than 200 pg/mL; following ZAP it was 2153 pg/mL (p less than 0.001), with untreated plasma 1055 pg/mL (p less than 0.005) and inactivated plasma 764 pg/mL (p less than 0.01). Neutrophil TxB2 release on a plastic dish was not different from incubation on endothelium. Endothelial 6-keto PGF1 alpha release following addition of untreated plasma preoperatively was 1308 pg/mL (p less than 0.01), and with ZAP 1305 pg/mL (p less than 0.01). Activated neutrophils did not alter 6-keto PGF1 alpha production. Postoperatively, neutrophil TxB2 production in response to ZAP was 1092 pg/mL, which was significantly reduced compared to the preoperative response (p less than 0.01). Endothelial damage by activated neutrophils in the postoperative period demonstrated on scanning electron microscopy was also reduced; 6-keto PGF1 alpha release in the postoperative period inducted by ZAP was 569 pg/mL and by untreated plasma 549 pg/mL, which was significantly lower than in the preoperative period (p less than 0.05 and p less than 0.05, respectively). No difference in chemotaxis was demonstrated. It is concluded that operative trauma is followed by lowered neutrophil TxB2 release, appearance of a plasmatic factor that depresses endothelial 6-keto PGF1 alpha production, as well as decreased neutrophil-induced endothelial damage. The neutrophil-endothelial monolayer system is a sensitive method for detection of neutrophil and plasmatic dysfunction.

Adult↗
PubMed2005

Studies of patients' thymi aid in the discovery and characterization of immunodeficiency in humans.

Studying the molecular and genetic bases of primary immunodeficiency is valuable at several levels. First, such information directly benefits patients in both short- and long-term management. Sophisticated diagnostic tools based on these studies can be used early and lead to appropriate treatment before potentially fatal infections and complications arise. Genotyping is also critical for future development and implementation of gene therapy. Secondly, investigating primary immunodeficiency helps understand the normal immune system in humans. As described in this report, the roles of zeta-associated protein of 70 kDa (Zap-70), CD25, and CD3delta are substantially different in humans when compared with the roles of homologous molecules in other species. Last, information obtained from these studies can be applied to other fields of investigation. Prominent examples for such applications include the intensive effort to design and produce specific inhibitors of Zap-70 and Janus kinase 3 as specific immunosuppressive agents. Most types of primary immunodeficiency in general and severe combined immunodeficiency in particular are rare and therefore cannot be easily studied by using traditional genetic methodology. Instead, biochemical methods were used to explore for candidate genes as was the case in the discovery of Zap-70 deficiency. Critical to the success of these discoveries was the careful analysis of patients' thymus glands. Detection of abnormalities in the thymus in these patients, which preceded identification of the genetic defect, aided in the assessment of the severity and nature of the immune disorder (primary versus secondary). Such assessment is critical before high-risk bone marrow transplantation. Equally important was the contribution of studies of the thymus to the description of novel phenotype of immunodeficiency as clearly demonstrated in defining CD8 lymphocytopenia, Zap-70 deficiency, and CD25 deficiency. Indeed, analysis of the thymus directly pointed to CD25 as candidate gene. Recently, the study of thymocyte-derived transcripts using DNA microarrays was key to discovering CD3delta deficiency. Finally, immunohistochemical analysis of the thymus was critical in pinpointing the roles of Zap-70, CD25, and CD3delta in the development of human T cells.

CD3 Complex↗
PubMed1997

Reconstitution of T cell antigen receptor-induced Erk2 kinase activation in Lck-negative JCaM1 cells by Syk.

The two related protein-tyrosine kinases Syk and Zap are rapidly phosphorylated on tyrosine residues and enzymatically activated upon crosslinking of the T cell antigen receptor. We have previously reported that the activation of Syk is less dependent on the Src family kinase Lck than the activation of Zap. Here we report that overexpression of Syk in the Lck-negative JCaM1 cells enabled the T cell antigen receptor/CD3 complex to induce a normal activation of the mitogen-activated protein kinase (MAPK) pathway and expression of a nuclear factor of activated T cells reporter construct. In contrast, Zap and other protein-tyrosine kinases were unable to reconstitute these signaling pathways when expressed at the same levels. In parallel, Syk was phosphorylated on tyrosine, while Zap was not. The Syk-mediated T cell antigen receptor-induced MAPK activation was detectable within 1 min of receptor stimulation and peaked at 3-5 min. The capacity of Syk to reconstitute the MAPK response required the catalytic activity of Syk, an intact autophosphorylation site (Y518 and Y519), both Src homology 2 domains and it was blocked by the inhibitory N17-mutated dominant-negative Ras construct. A Y341-->F mutant of Syk, which is deficient in its interaction with phospholipase Cy1 and Vav, was less efficient than wild-type Syk. Our results suggest that Syk, in contrast to Zap, can transduce signals from the T cell antigen receptor independently of Lck.

Animals↗
PubMed1994

Role of CD18 in the accumulation of eosinophils and neutrophils and local oedema formation in inflammatory reactions in guinea-pig skin.

1. Intradermal injection of the complement fragment C5a des Arg induces local oedema formation that, in rabbits and man, is dependent on circulating neutrophils. Monoclonal antibodies to the leukocyte adhesion molecule CD11/CD18 block neutrophil accumulation and prevent neutrophil-dependent oedema formation. The role of CD11/CD18 in mediating eosinophil accumulation in vivo is less established. In this study we have used an anti-human CD18 monoclonal antibody, 6.5E, to investigate the neutrophil-dependency of oedema formation induced by C5a des Arg in guinea-pig skin. We also studied the role of CD18 in mediating eosinophil accumulation in the same model. 2. Stimulated adhesion of 111In-labelled guinea-pig neutrophils and eosinophils to serum-coated plastic was inhibited in a dose-dependent manner by 6.5E suggesting that the monoclonal antibody recognizes and blocks the guinea-pig CD18 adhesion molecule. 3. The accumulation of 111In-labelled neutrophils induced by zymosan-activated plasma (ZAP, as a source of C5a des Arg) in skin sites was reduced by up to 89% in animals treated intravenously with F(ab')2 fragments of 6.5E. ZAP-induced accumulation of 111In-labelled eosinophils was also greatly reduced (by up to 78%) by treatment with 6.5E. 4. Despite the inhibition of ZAP-induced neutrophil accumulation by 6.5E, local oedema formation in the same skin sites was unaffected, except at the top dose of ZAP, by treatment with the anti-CD18 monoclonal antibody, suggesting that the oedema response was largely neutrophil-independent. Indeed, ZAP-induced oedema formation was reduced by up to 81% by the H1 receptor antagonist, mepyramine. 5. Accumulation of 111 In-labelled eosinophils in a passive cutaneous anaphylactic (PCA) reaction was also blocked by treatment with 6.5E, while oedema formation in the same skin sites was unaffected.Intradermal injection of cationic protein-containing extracts of Schistosoma mansoni larvae also induced the accumulation of 111 In-labelled neutrophils and eosinophils which was abrogated by intravenous 6.5E.In contrast, extract-induced local oedema formation was similar in control and 6.5E-treated guinea-pigs.6. In summary, the local accumulation of radiolabelled neutrophils at sites of inflammation in guinea pigskin was dependent on the adhesion molecule CD18 while, in contrast, there was no evidence for neutrophil-dependent oedema formation in this species. Accumulation of radiolabelled eosinophils was also dependent on CD18.

Animals↗
PubMed1996

Effects of dexamethasone and cyclosporin A on the accumulation of eosinophils in acute cutaneous inflammation in the guinea-pig.

1. Eosinophils are thought to play an important role in the pathophysiology of allergic diseases and pharmacological suppression of their recruitment is considered to be of therapeutic benefit. In the present study we have assessed and compared the effects of treatment with dexamethasone and cyclosporin A on the accumulation of 111In-labelled eosinophils and local oedema formation in sites of acute inflammation in guinea-pig skin. 2. When injected locally 150 min prior to i.d. administration of antigen in a passive cutaneous anaphylactic (PCA) reaction, dexamethasone (10(-9) to 3 x 10(-7) mol per site) dose-dependently inhibited oedema formation by up to 50%. Similarly, oedema formation induced by PAF and lipopolysaccharide (LPS), but not by zymosan-activated plasma (ZAP), was significantly inhibited by dexamethasone. In contrast, 111In-eosinophil accumulation measured in response to i.d. injection of PAF, LPS and ZAP or in the PCA reaction was not altered. 3. Systemic treatment with dexamethasone (4 mg kg-1, i.v., 150 min pretreatment period) inhibited both oedema formation and 111In-eosinophil accumulation induced by PAF, ZAP, LPS and in the PCA reaction. 4. The effects of i.d. injection of cyclohexamide (2 x 10(-7) mol per site) on 111In-eosinophil accumulation and oedema formation induced by PAF, ZAP or in a PCA reaction were evaluated in order to assess the dependency of these responses on protein synthesis. Cycloheximide had no effect on the responses measured. In contrast, 111In-eosinophil accumulation, but oedema formation, induced by LPS was inhibited by 30%. 5. Acute (10 mg kg-1, i.v., 15 min pretreatment) or prolonged (10 mg kg-1, s.c. daily for 3 days) systemic treatment with cyclosporin A had no effect on 111In-eosinophil accumulation or oedema formation induced by PAF, ZAP, LPS or in the PCA reaction. 6. In conclusion, we demonstrate preferential inhibitory effects of dexamethasone on 111In-eosinophil accumulation according to its site of administration. In addition we show that dexamethasone inhibits protein synthesis-independent acute inflammation in guinea-pig skin. Finally, our results do not support the concept that eosinophils are an important cellular site of action for the inhibitory effects of cyclosporin A in a guinea-pig model of allergic inflammation.

Animals↗
PubMed1991

Oxidant-mediated, CD18-dependent microvascular dysfunction induced by complement-activated granulocytes.

To determine the mechanisms whereby complement-activated granulocytes induce microvascular dysfunction in skeletal muscle, we examined the effect of antineutrophil serum (ANS), IB4 (a monoclonal antibody that inhibits CD18-dependent neutrophil adherence), xanthine oxidase inhibition or inactivation, deferoxamine, and catalase on the increase in canine gracilis muscle microvascular permeability induced by intravascular administration of zymosan-activated plasma (ZAP). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient (sigma) for total plasma proteins, and the extent of neutrophil infiltration was estimated by assessing muscle myeloperoxidase activity. ZAP infusion was associated with a marked increase in vascular permeability compared with control muscles that received no treatment or to muscles treated with zymosan heat-inactivated plasma (ZIP) (sigma = 0.51 +/- 0.04, 0.89 +/- 0.02, and 0.90 +/- 0.01, respectively). Estimates of sigma in animals rendered neutropenic with ANS, or treated with IB4, deferoxamine, or catalase before ZAP infusion were not significantly different from values obtained in control or ZIP-treated muscles (sigma = 0.96 +/- 0.02, 0.88 +/- 0.03, 0.85 +/- 0.02, and 0.79 +/- 0.01, respectively). However, xanthine oxidase inactivation or inhibition provided no protection from this ZAP-induced microvascular dysfunction (sigma = 0.58 +/- 0.02 and 0.58 +/- 0.01, respectively). In addition, neutropenia and inhibition of neutrophil adherence also prevented ZAP-induced increases in vascular resistance and tissue neutrophil infiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effects of L-652,731, a platelet-activating factor (PAF) receptor antagonist, on PAF- and complement-induced pulmonary hypertension in sheep.

Thromboxane-mediated pulmonary hypertension, pulmonary edema, arterial hypoxia and pulmonary leukostasis occur in response to the infusion of plasma containing zymosan-activated complement (ZAP) in sheep. Platelet-activating factor (PAF) is a potential mediator of some of these effects. We investigated the effects of PAF infusions in unanesthetized sheep and the effects of the PAF receptor antagonist L-652,731 [trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran] on the hematologic, hemodynamic and biochemical alterations produced by infusions of both ZAP and PAF. Infusions of 2 to 20 micrograms of PAF in 0.25% ovine serum albumin-saline produced pulmonary hypertension, hypoxia and dose-dependent thrombocytopenia, neutropenia and thromboxane synthesis. The effects of a 2 micrograms of PAF infusion were both qualitatively and quantitatively similar to those produced by a ZAP infusion. Pretreatment with aspirin (10 mg/kg) protected the sheep against the pulmonary vascular response to 20 micrograms of PAF and blocked completely the thromboxane synthesis. L-652,731 at a dose of 8 mg/kg blocked completely the neutropenia, thrombocytopenia, thromboxane synthesis, pulmonary hypertension and hypoxia induced by 5 micrograms of PAF, but this protective effect was not observed in animals infused with ZAP. These results indicate that PAF is probably not a mediator of the neutropenia, thromboxane-mediated pulmonary hypertension and hypoxia which result from the infusion of ZAP into sheep.

Animals↗
PubMed1985

Contribution of circulating formed elements to prostanoid production in complement-mediated lung injury in sheep.

Incubation of plasma with zymosan results in complement activation. Infusion of this "zymosan-activated plasma" (ZAP) into the superior vena cava in sheep results in pulmonary leukostasis, pulmonary hypertension with generation of thromboxane (TXB), and hypoxemia. To examine the contribution of circulating formed elements to TXB generated when ZAP is infused, we studied four pairs of sheep in a cross-circulation model. Acetylsalicylic acid (ASA) irreversibly acetylates cyclooxygenase and blocks TXB production. Four hours after an intravenous dose of ASA (10 mg/kg), no circulating ASA was detectable by high-pressure liquid chromatography. Infusion of ZAP in ASA-treated animals resulted in no pulmonary hypertension and no rise in serum TXB levels. Treated animals were then cross-circulated with untreated sheep through cannulas that attached a carotid artery of each animal to a jugular vein of the other, creating simultaneous arteriovenous fistulas between the animals. A roller pump maintained cross-circulation flow at 450 ml/min for 15 minutes. This resulted in greater than 80% mixing of the circulating blood volumes of each pair of animals, verified by measurement of an intravascular marker. After cross-circulation the ASA-treated animals were still unable to generate TXB in response to ZAP infusion. Untreated animals had a typical response when infused with ZAP after cross-circulation, with elaboration of large amounts of TXB. Since the treated animals with circulating, nonaspirinated formed elements showed no response and the untreated animals with similar blood composition had a normal response, we conclude that circulating formed elements do not contribute significantly to the TXB recovered from sheep infused with activated complement components.

Animals↗
PubMed1984

The in vivo effect of leukotriene B4 on polymorphonuclear leukocytes and the microcirculation. Comparison with activated complement (C5a des Arg) and enhancement by prostaglandin E2.

The effect of synthetic leukotriene B4 (LTB4) on chemotaxis in vivo (51Cr-polymorphonuclear leukocyte [PMN] accumulation) was examined and its potency compared with that of C5a des Arg-containing zymosan-activated plasma (ZAP). On a molar basis the amount of C5a des Arg calculated to be in our preparation of ZAP was found to be up to approximately 80 times more potent than LTB4, although in vitro the two chemotaxins have been reported to be about equipotent. ZAP is more representative of what may happen in vivo than its principal constituent C5a des Arg, but for a more precise comparison the purified and isolated peptide will have to be compared with synthetic LTB4. Whereas ZAP induced severe PMN-dependent microvascular injury (increase in vessel permeability [125I-albumin] and hemorrhage [59Fe-erythrocytes]), LTB4 only induced an increase in vascular permeability, and this occurred only in the presence of simultaneously injected prostaglandin E2 (PGE2). PGE2 also enhanced substantially the number of PMNs and the amount of exuded plasma at injection sites of the chemotaxins. However, unlike in two other reports, LTB4 did not cause an immediate transient increase in vessel permeability, nor did it enhance the permeability-increasing effect of bradykinin. Furthermore, unlike PGE2 LTB4 did not induce an increase in blood flow, but a decrease (57Co-microspheres). It is concluded that LTB4 may act as a host-derived chemoattractant in vivo, but, compared with that of ZAP (primarily activated complement), its role in acute inflammation is probably less significant than that of the complement-derived chemotaxin(s).

Animals↗
PubMed1984

The effect of an infusion of zymosan-activated plasma on hemodynamic and pulmonary function in sheep.

To assess the integrated cardiopulmonary response to acute microvascular lung injury, we infused zymosan-activated plasma (ZAP) into mature, awake sheep. ZAP infusion resulted in an immediate increase in the mean pulmonary artery pressure (PAP) from 17.5 +/- 4.5 mm Hg to a maximum of 33.0 +/- 5.4 mm Hg by 5 min (p less than 0.01) of a 90-min infusion. The pulmonary artery hypertension (PAH) was sustained throughout ZAP infusion, and for up to 30 min after infusion was completed (p less than 0.05). Lung lymph flow increased immediately, but lymph to plasma total protein ([L/P]) ratios remained unchanged, suggesting increased pulmonary microvascular permeability. The PaO2 fell to a minimum by 5 min (106.9 +/- 17 to 88.3 +/- 14 mm Hg; p less than 0.01), but recovered toward preinfusion values in the final 60 minutes of ZAP infusion. The cardiac index (CI) and stroke volume index (SVI) also fell significantly for 15 minutes (p less than 0.01), and then recovered to levels not different from baseline by 30 min. In parallel with all of these changes, the polymorphonuclear leukocyte count (PMN) in arterial blood fell immediately [baseline to 5 min, delta 4.9 +/- 2.5 (X 10(3))/mm3; (p less than 0.01)] due to sequestration within the pulmonary microvasculature, since a baseline PMNs were being released from the lung, -6.0 +/- 19%, while by 15 min the PMNs were being sequestered, +58.4 +/- 31% (p less than 0.01). Therefore, ZAP infusion reproduced the clinical picture of acute microvascular lung injury in humans, specifically acute PAH and increased pulmonary microvascular permeability. These two changes were due to a cascade of events associated with the pulmonary microvascular trapping of PMNs.

Animals↗
PubMed1995

The glycosylphosphatidylinositol-anchored CD59 protein stimulates both T cell receptor zeta/ZAP-70-dependent and -independent signaling pathways in T cells.

The glycosylphosphatidylinositol (GPI)-anchored CD59 protein (human protectin) protects cells against complement-induced lysis, binds to CD2 and also transduces activation signals within T cells. We have further examined the biochemical signals transduced by CD59 and addressed its role in regard to the CD3-mediated signaling cascade. We show here that CD59 cross-linking induces a time-dependent activation of p56lck and of p70zap (ZAP-70) in CD3-positive Jurkat cells, leading to the stimulation of the T cell receptor zeta/ZAP-70 signaling cascade and interleukin-2 (IL-2) synthesis. Cross-linking of CD59 on peripheral T cells and thymocytes induces tyrosine phosphorylations identical to those seen in Jurkat cells and this is followed by lymphokine production and proliferation. In contrast, only activation of CD59-associated p56lck occurs in CD3-negative Jurkat cells, while IL-2 production is impaired, consistent with the lack of ZAP-70 tyrosine phosphorylation observed in these cells. CD59 triggers activation events even in the absence of CD3/T cell receptor expression in Jurkat cells. CD59 cross-linking synergizes with sub-optimal doses of phorbol ester for activation of the protein kinase C and of the p42mapk, as shown by in vitro phosphorylation of histone HIIIS and myelin basic protein, respectively, and leads to CD25 but not CD69 expression. In conclusion, at least two signaling pathways are triggered through CD59, the first one involving ZAP-70 activation and leading to IL-2 secretion and a second pathway observed in the absence of ZAP-70 activation leading to CD25 expression. These two pathways are likely to be involved in the modulation of T cell activation by CD59 protein.

Antigens, CD↗
PubMed1995

The role of tyrosine phosphorylation in the interaction of cellular tyrosine kinases with the T cell receptor zeta chain tyrosine-based activation motif.

Immunoglobulin receptor family tyrosine-based activation motifs (ITAM) define a conserved signaling sequence, EX2YX2L/IX7YX2L/I, that mediates coupling of the T cell antigen receptor (TCR) to protein tyrosine kinases (PTK). In the present study, we explored the role of phosphorylation of the two ITAM tyrosine residues in the interactions of the motif with the PTK ZAP-70 and p59fyn. The data show that the phosphorylation of a single tyrosine within the motif enables binding of p59fyn, whereas phosphorylation of both tyrosines within the motif is required for maximal binding of the PTK ZAP-70. Quantitative binding experiments show that nanomolar concentrations of the doubly phosphorylated zeta 1-ITAM are sufficient for ZAP-70 recruitment, whereas micromolar levels of singly phosphorylated ITAM are necessary for p59fyn binding. ZAP-70 binds with low efficiency to a singly phosphorylated ITAM, but shows preferential binding to the C-terminal phosphotyrosine in the ITAM, whereas p59fyn binds selectively to the N-terminal phosphotyrosine. The present data thus show that there is the potential for a singly phosphorylated ITAM to couple to cellular PTK. Moreover, the data suggest a mechanism for heterogeneity in signal transduction responses by the TCR, since ITAM could differentially couple the TCR to downstream signaling events depending on their phosphorylation state.

Amino Acid Sequence↗
PubMed1998

Nocodazole inhibits signal transduction by the T cell antigen receptor.

The potential role of the cytoskeleton in signaling via the T cell antigen receptor (TCR) was investigated using pharmacological agents. In Jurkat T cells, disruption of the actin-based cytoskeleton with cytochalasin D or disruption of the microtubules with colchicine did not affect TCR induction of proximal signaling events triggered by CD3 mAb. Polymerized actin and tubulin, therefore, were not required for TCR-mediated signal transduction. Nocodazole, however, was found to inhibit dramatically TCR signaling, independently of its ability to depolymerize microtubules. This effect was TCR-specific, because signaling via the human muscarinic acetylcholine receptor 1 in the same cells was unaffected. A mechanism for the inhibition of TCR signaling by nocodazole was suggested by in vitro assays, which revealed that the drug inhibited the kinase activity of LCK and, to a lesser extent, FYN. The kinase activity of ZAP-70 in vitro, however, was unaffected. These results, therefore, suggested that nocodazole prevented initial phosphorylation of the TCR by LCK after stimulation, and as a result, it blocked activation of downstream signaling pathways. Immunofluorescence analyses also revealed that nocodazole and the specific SRC-family kinase inhibitor PP1 delocalized ZAP-70 from its constitutive site at the cell cortex. These effects did not require the SH2 domains of ZAP-70. The localization of ZAP-70 to the cell cortex is, therefore, regulated by the activity of SRC-family kinases, independently of their ability to phosphorylate immunoreceptor tyrosine-based activation motifs of the TCR.

Actins↗