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Background and Purpose: The expression of voltage-dependent K(+) channels (K(v) ) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF-1) and its inducer geranylgeranylacetone (GGA) could affect the expression of K(v) 1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97).Experimental Approach: Transfected mouse atrial cardiomyocytes (HL-1 cells) and COS7 cells were subjected to luciferase reporter gene assay and whole-cell patch clamp. Protein and mRNA extracts were subjected to Western blot and quantitative real-time polymerase chain reaction.Key Results: Heat shock of HL-1 cells induced expression of Hsp70, HSF-1, SAP97 and K(v) 1.5 proteins. These effects were reproduced by wild-type HSF-1. Both heat shock and expression of HSF-1, but not the R71G mutant, increased the SAP97 mRNA level. Small interfering RNA (siRNA) against SAP97 abolished HSF-1-induced increase of K(v) 1.5 and SAP97 proteins. A luciferase reporter gene assay revealed that the SAP97 promoter region (from -919 to -740) that contains heat shock elements (HSEs) was required for this induction. Suppression of SIRT1 function either by nicotinamide or siRNA decreased the level of SAP97 mRNA. SIRT1 activation by resveratrol had opposing effects. A treatment of the cells with GGA increased the level of SAP97 mRNA, K(v) 1.5 proteins and I(Kur) current, which could be modified with either resveratrol or nicotinamide.Conclusions and Implications: HSF-1 induced transcription of SAP97 through SIRT1-dependent interaction with HSEs; the increase in SAP97 resulted in stabilization of K(v)1.5 channels. These effects were mimicked by GGA. [ABSTRACT FROM AUTHOR]

The impact of pretransplant T-cell sensitivity testing using carboxylfluorescein diacetate succinimidyl ester (CFSE)–based flow cytometry was studied in 32 patients with chronic renal failure. There was considerable interindividual variation in the inhibitory effects of cyclosporine (CSA), tacrolimus (TAC), and prednisolone (PRD) but only a small amount of interindividual variation for mycophenolic acid (MPA). Patients with high sensitivity to CSA tended to experience viral reactivation. In addition to post-transplant blood-level monitoring, pretransplant pharmacodynamics could provide useful information on optimal and safe immunosuppressive therapy. [ABSTRACT FROM AUTHOR]

Aim: The purpose of this study was to determine whether anaphylactic hypotension in rabbits is accompanied by hepatic venoconstriction, and the effects of anaphylaxis on hepatic segmental vascular resistances and liver weight in isolated perfused rabbit livers. Methods: The rabbits were sensitized by subcutaneous injection of antigen of 2.5 mg ovalbumin with complete Freund's adjuvant three times at 1 week interval. One week after sensitization, anaphylaxis was induced by an injection of 2.5 mg ovalbumin into the jugular vein of pentobarbital anaesthetized rabbits or the perfusate of rabbit livers perfused via the portal vein at a constant flow. Using the double occlusion technique to estimate the hepatic sinusoidal pressure, pre- ( Rpre) and post-sinusoidal ( Rpost) resistances were calculated for the isolated perfused livers. Results: An antigen injection into the sensitized rabbits caused not only a decrease in systemic arterial pressure from 79 ± 2 to 40 ± 4 mmHg, but also an increase in portal venous pressure ( Ppv) from 9.5 ± 2.2 to 24.1 ± 3.9 cmH2O. Portal hypertension persisted for 8 min after the antigen injection. An injection of antigen into the perfusate caused a marked increase in Ppv from 5.4 ± 0.1 to 28.6 ± 2.4 cmH2O at 6 min, but only a slight increase in double occlusion pressure from 2.2 ± 0.2 to 3.8 ± 0.2 cmH2O, resulting in a selective increase in Rpret rather than Rpost. Concomitant with the hepatic pre-sinusoidal constriction, liver weight loss occurred. Conclusion: Anaphylactic hypotension in rabbits is accompanied by hepatic venoconstriction which is characterized by pre-sinusoidal contraction. [ABSTRACT FROM AUTHOR]

We investigated whether thrombocytopenia in patients with chronic hepatitis C is due to anti-platelet autoantibodies. Platelet-associated IgG (PAIgG) and platelet-associated IgM (PAIgM) were measured by direct immunofluorescent flow cytometric analysis. Elevation of PAIgM level was detected in 70% of chronic hepatitis C patients, while only a mild elevation of PAIgG level was detected in 32% of the cases. The elevation of PAIgM values in these patients was comparable to that in patients with chronic immune thrombocytopenic purpura (ITP). However, elevated PAIgM was also found in both patients with and without thrombocytopenia, and no correlation was found between PAIgM and platelet count. Eluted PAIgM did not react with normal platelets in all cases with a positive PAIgM value, indicating that eluted PAIgM contained no detectable anti-platelet antibodies. During alpha-interferon therapy, the level of PAIgM increased in association with the decrease in platelet counts in 75% of the cases; however, eluted PAIgM at any day point never reacted with platelets from normal donors. PAIgM was elevated in patients with chronic hepatitis C, but contained no detectable anti-platelet autoantibodies. Thrombocytopenia in these patients is not due to anti-platelet autoantibodies. [ABSTRACT FROM AUTHOR]

Castanospermine (CSP) inhibits α-glucusidase, which is involved in the initial step N-linked oligosaccharide processing of secretory and membrane glycoproteins. In Staphylococcus aureus Cowan I (SAC)-stimulated human lymphocyte culture, CSP at a dose of 20 μg/ml caused a twofold increase in immunoglobulin G (IgG) release after 7 days. An initial 48-h exposure to CSP sufficed for this enhancing effect. Plaque-forming cell assays on the seventh day disclosed that CSP caused an increase in the number of IgG-, IgA- and IgM-secreting cells. In cross-culture experiments, only a mixture of B cells pretreated with CSP and untreated T cells showed an increase in IgG production. Tritiated thymidine incorporation studies revealed that CSP enhanced B-cell responses to T cell-derived soluble factor (TSF). When incubated with CSP for 18 h, B cells showed an increased surface binding on [3H]concanavalin A (Con A). These results indicate that the alteration in B-cell membrane oligosaccharides enhances the response to TSF at an early stage of SAC culture, leading loan increase in Ig-secreting cell number at later stages. The present study provides evidence that cell-surface oligosaccharides of B cells play an important role in the responses of B cells to lymphokines. [ABSTRACT FROM AUTHOR]

Electrophysiological effects of ethanol on rabbit sino-atrial node were studied by means of the double-microelectrode voltage clamp method. In spontaneously beating preparations, ethanol (above 1%) decreased the maximum rate of depolarization of the action potential. Furthermore, 3% ethanol produced a significant decrease in the action potential amplitude, the action potential duration at 50% repolarization and the rate of the diastolic depolarization. Of the current systems, voltage clamp studies showed that ethanol reduced the slow inward current, the time-dependent potassium outward current and the hyperpolarization activated inward current. These observations indicate that ethanol does not have an effect on a single current system, but directly modifies the spontaneous discharge of sino-atrial node pacemaker cells due to a reduction of the time-dependent current systems. [ABSTRACT FROM AUTHOR]

The aim of this study was to find ultrasound markers for detecting rare chromosomal aberrations in the first trimester. Measuring nuchal translucency thickness and assessment of the combined test in the first trimester contribute to the detection of rare chromosomal aberrations. [Extracted from the article]

Hepatic xenotransplantation from guinea-pig to rat has not been established. This failure is partly ascribed to differences in hepatic vascular characteristics between two species. However, the differences in hepatic vascular resistance distribution and responses to vasoconstrictors are not known. The present study was designed to determine basal levels of segmental vascular resistances and the responses to histamine and noradrenaline in isolated guinea-pig and rat livers. The livers were haemoperfused (Hct 8.3%) via the portal vein at a constant flow. The sinusoidal pressure was measured by the double occlusion pressure, and was used to determine the pre- ( Rpre) and post-sinusoidal ( Rpost) resistances. There was no significant difference in basal total hepatic vascular resistance ( Rt) between two species, whereas Rpre in rat (69% of Rt) was significantly greater than that in guinea-pig (61% of Rt). The responses to noradrenaline were similar; Rpre increased in a greater magnitude than Rpost, and liver weight was reduced. However, the noradrenaline-induced increase in Rt was greater in rats than in guinea-pigs. In contrast, histamine increased predominantly Rpost over Rpre, and increased liver weight in guinea-pig, while it affected no haemodynamic variables in rat. There exist species differences in the hepatic vasculature between rat and guinea-pig. Basal pre-sinusoidal resistance in rat is greater than that in guinea-pig. Although noradrenaline predominantly contracts pre-sinusoidal vessels in both species, histamine causes predominant post-sinusoidal vasoconstriction in guinea-pig liver, while it has no vasoactive effects on rat liver. [ABSTRACT FROM AUTHOR]

Aims: Theophylline clearance is known to be reduced in patients with chronic liver diseases (CLDs) such as chronic hepatitis (CH) and liver cirrhosis (LC). The Child-Pugh (CP) score is generally used for pharmacokinetic evaluation, whilst a model for end-stage liver disease (MELD) has not yet been fully evaluated. This study aimed to predict theophylline clearance in patients with LC classified based on CP and MELD scores by population pharmacokinetic (PPK) analysis., Methods: PPK analysis included 433 steady-state trough concentrations from 192 Japanese bronchial asthma patients with and without CLDs and was performed using NONMEM. The severity of LC was assessed by CP and MELD scores., Results: The final CP and MELD models which described apparent theophylline clearance (CL/F) were obtained. The CP model showed that the mean CL/F in patients without CLDs, CH patients and LC patients with CP class A, B and C was 0.0473, 0.0413, 0.0330, 0.0280 and 0.0209 L/h kg -1 , respectively. The MELD model predicted that CL/F in patients without CLDs, CH patients and LC patients with MELD scores of <10, 10-14, 15-19, 20-24 and ≥25 was 0.0472, 0.0413, 0.0324, 0.0268, 0.0230, 0.0197 and 0.0155 L/h kg -1 , respectively., Conclusions: CL/F in various stages of LC was evaluated, and a change in CL/F was highly dependent on the severity of CLDs in both models. The MELD model provided a more accurate and precise description of theophylline clearance in LC than the CP model, which may be due to the wider dynamic range of the MELD score., (© 2022 British Pharmacological Society.)

New Findings: What is the central question of this study? Whether anaphylaxis affects sympathetic outflows to the brown adipose tissue (BAT) and adrenal gland and whether anaphylaxis affects some brain areas in association with sympathetic regulation. What is the main finding and its importance? Sympathoexcitatory responses to anaphylaxis occurred regionally in the kidney and adrenal gland, but not in the thermogenesis-related BAT. Further, anaphylactic hypotension also caused increase in c-fos immunoreactivity in the hypothalamic and medullary areas. Moreover, catecholaminergic neurons of the brainstem cause adrenal sympathoexcitation in a baroreceptor-independent manner., Abstract: We previously reported that sympathetic nerve activity (SNA) to the kidney and the hindlimb increases during anaphylactic hypotension in anaesthetized rats. Based on this evidence, we examined effects of anaphylactic hypotension on SNA to the brown adipose tissue (BAT), and the adrenal gland and kidney in anaesthetized rats. We demonstrated that adrenal and renal SNA, but not BAT-SNA, were stimulated. In addition, the effects of anaphylaxis on neural activities of the hypothalamic and medullary nuclei, which are candidates for relaying efferent SNA to the peripheral organs, were investigated via immunohistochemical staining of c-fos. Anaphylaxis increased c-fos expression in the neurons of the paraventricular nucleus (PVN) of the hypothalamus and in those of the nucleus tractus solitarii (NTS) and rostral ventrolateral medulla (RVLM) of the medulla oblongata; c-fos was expressed in γ-aminobutyric acid (GABA)-ergic neurons of the NTS and in the catecholaminergic neurons of the RVLM. In addition, c-fos expression in the rostral NTS and mid NTS during anaphylaxis was reduced by sinoaortic baroreceptor denervation; however, increased c-fos expression in the caudal NTS and RVLM or adrenal sympathoexcitation were not affected by sinoaortic baroreceptor denervation. These results indicated that anaphylactic hypotension activates the hypothalamic PVN and the medullary NTS and RVLM independently of the baroreflex pathway. Further, it stimulated efferent SNA to the adrenal gland and kidney to restore blood pressure., (© 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.)

1. Exercise training attenuates circulatory shock due to haemorrhage, endotoxin or heatstroke. However, it remains unknown whether exercise training attenuates anaphylactic shock. Hepatic venoconstriction is involved in rat anaphylactic hypotension. In the present study, we determined the effects of exercise training on both anaphylaxis-induced segmental venoconstriction in rat perfused livers and systemic anaphylaxis in conscious rats. The role of nitric oxide (NO) in the effect of exercise on the venoconstriction of perfused livers was also examined. 2. Rats were subjected to running training on a motorized treadmill for 4 weeks. Two weeks prior to the anaphylaxis experiment, Sprague-Dawley rats were actively sensitized with the antigen ovalbumin. In isolated livers perfused portally with blood, the portal venous pressure (P(pv)) and sinusoidal pressure were measured to determine the pre- and post-sinusoidal resistances (R(pre) and R(post), respectively). In conscious rats, systemic arterial pressure (SAP) and P(pv) were determined. 3. In the perfused livers of sedentary rats, antigen administration led to a predominant presinusoidal constriction, as evidenced by 4.6- and 1.7-fold increases in R(pre) and R(post), respectively. The anaphylaxis-induced increase in R(pre) was significantly attenuated by 24% by exercise training. Inhibition of NO synthase with N(G)-nitro-L-arginine methyl ester (100 micromol/L) 10 min prior to the injection of antigen enhanced anaphylactic venoconstriction, but did not alter the effect of exercise training on the increase in R(pre). In contrast, exercise training did not attenuate either anaphylactic hypotension or portal hypertension in conscious rats. 4. In conclusion, exercise training attenuates the anaphylaxis-induced presinusoidal constriction in rat isolated perfused livers, independent of NO production. However, this action is not evident in conscious rats and exercise training does not affect anaphylactic hypotension in conscious rats.

1. The effects of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on anaphylaxis-induced venoconstriction were examined in rat isolated livers perfused with blood-free solutions in order to clarify the role of NO in anaphylactic venoconstriction. 2. Rats were sensitized with ovalbumin (1 mg) and, 2 weeks later, livers were excised and perfused portally in a recirculating manner at a constant flow with Krebs'-Henseleit solution. The antigen (ovalbumin; 0.1 mg) was injected into the reservoir 10 min after pretreatment with L-NAME (100 micromol/L) or D-NAME (100 micromol/L) and changes in portal vein pressure (Ppv), hepatic vein pressure (Phv) and perfusate flow were monitored. In addition, concentrations of the stable metabolites of NO ( and ) were determined in the perfusate using an HPLC-Griess system. 3. The antigen caused hepatic venoconstriction, as evidenced by an increase in Ppv from a mean (SEM) baseline value of 7.7 +/- 0.1 cmH2O to a peak of 21.4 +/- 1.1 cmH2O at 3 min in D-NAME-pretreated livers. Pretreatment with L-NAME augmented anaphylactic venoconstriction, as reflected by a higher Ppv (27.4 +/- 0.8 cmH2O) after antigen than observed following D-NAME pretreatment. The addition of L-arginine, a precursor for the synthesis of NO, reversed the augmentation of anaphylactic venoconstricion by L-NAME. This suggests that hepatic anaphylaxis increased the production of NO, which consequently attenuated anaphylactic venoconstriction. However, perfusate NOx levels did not increase significantly after antigen in livers pretreated with either L-NAME or D-NAME. 4. In conclusion, L-NAME potentiates rat anaphylactic hepatic venoconstriction, suggesting that NO contributes to the attenuation of the venoconstriction. However, this functional evidence was not accompanied by corresponding changes in perfusate NOx concentrations.

1. The role of shear stress in nitric oxide (NO)-mediated attenuation of anaphylactic venoconstriction was studied using an isolated ovalbumin-sensitized guinea pig liver. 2. Guinea pigs were actively sensitized by a subcutaneous injection of 1 mg ovalbumin. Two weeks after sensitization, the livers were perfused with diluted blood under constant flow or constant perfusion pressure. The constant flow could result in increased shear stress during constriction, while the constant perfusion pressure could prevent changes in shear stress. Using the double occlusion technique to estimate the hepatic sinusoidal pressure, pre- and postsinusoidal constriction was evaluated. Hepatic anaphylaxis was induced by an injection of ovalbumin (4 microg) into the perfusate, the volume of which was 40 mL. 3. Under either constant flow or pressure, anaphylaxis caused venoconstriction of predominantly presinusoids over postsinusoids, although anaphylactic venoconstriction under constant pressure was significantly greater than that under constant flow. When shear stress was held constant by maintaining constant perfusion pressure, a NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L), potentiated similarly both pre- and postsinusoidal constriction induced by anaphylaxis. This suggests that hepatic anaphylaxis shear stress-independently generates NO, resulting in dilatation of both pre- and postsinusoidal vessels in a similar magnitude. In contrast, when shear stress was allowed to rise under constant flow, anaphylactic presinusoidal constriction was preferentially potentiated by L-NAME. 4. Hepatic anaphylaxis can increase NO production in a shear stress-independent manner and dilates similarly both pre- and postsinusoids, while NO produced in a shear stress-dependent manner attenuates predominantly venoconstriction of the presinusoids where shear stress is preferentially increased.

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patient's platelets showed a greatly reduced GPIbalpha and completely absent GPIX surface expression. Immunoblot analysis disclosed greatly reduced GPIbalpha and residual amounts of GPIbbeta and GPIX in the platelets. DNA sequencing analysis revealed the patient to be homozygous for a novel missense mutation in the GPIX gene that converts Cys (TGT) to Tyr (TAT) at residue 97. Transient transfection studies confirmed that mutant GPIX was not expressed on the transfected cells, showing that the mutation was responsible for the BSS phenotype observed in the patient.

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system. The TPO level (mean +/- SD) in ITP patients was mildly elevated (1.86 +/- 1.17 fmol/ml) compared to that in normal subjects (0.76 +/- 0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35 +/- 6.42 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.

Objective: Autoimmunity-prone (New Zealand white x BXSB)F1 ([NZW x BXSB]F1) mice have been shown to be useful as a model of antiphospholipid syndrome with myocardial infarction. The aim of this study was to examine the cross-reactivity of anticardiolipin antibody (aCL) derived from (NZW x BXSB)F1 mice with oxidized low-density lipoprotein (ox-LDL), which is closely associated with atherosclerosis., Methods: Six monoclonal antibodies (MAb) against CL were established from (NZW x BXSB)F1 mice, and reactivity of aCL with ox-LDL was examined by micro-enzyme-linked immunosorbent assay., Results: Higher titers of anti-ox-LDL autoantibodies were found in adult (NZW x BXSB)F1 mice compared with other autoimmunity-prone mouse strains (P < 0.01) or a control strain (P < 0.005). There was a significant positive correlation between titers of aCL and those of anti-ox-LDL in (NZW x BXSB)F1 mice (r = 0.79, P < 0.001). Of the 6 MAb against CL, 2 clones that showed beta 2-glycoprotein 1-dependent reactivity also cross-reacted with ox-LDL. Binding of monoclonal aCL to solid-phase cardiolipin was inhibited by ox-LDL, but not by native LDL., Conclusion: We confirmed that aCL derived from (NZW x BXSB)F1 mice can cross-react with ox-LDL. This result suggests that aCL, which is closely associated with lupus-associated thrombosis, may also play an important role in atherosclerotic complications in patients with systemic lupus erythematosus.

We studied a female patient with cyclic fluctuation in platelet count following splenectomy for autoimmune thrombocytopenia. The cyclical fluctuation appeared to be in phase with her menstrual cycle and her platelet count was low during menses. Bone marrow examinations performed at the peak as well as the bottom of the platelet count showed normal or increased numbers of megakaryocytes. The patient's platelet count increased rapidly after intravenous gamma-globulin (IVIgG) therapy, suggesting that a failure of platelet production is unlikely to account for the cycle. Platelet-associated IgM (PAIgM) was markedly elevated, whereas PAIgG was normal at any stage of the cycle. MACE assay demonstrated that PAIgM contained IgM anti-glycoprotein (GP) IIb-IIIa autoantibodies. Comparison between MACE assay using untreated and EDTA-treated platelets at 37 degrees C demonstrated that the platelet-associated IgM autoantibodies mainly recognized divalent cation-dependent conformation(s) of GPIIb-IIIa. No antibodies were, however, detected in her serum. The levels of IgM anti-GPIIb-IIIa showed an inverse relationship with the platelet count. In spite of the marked increase in platelet count after IVIgG, however, the levels of IgM anti-GPIIb-IIIa remained elevated. These findings suggest that platelet-associated IgM anti-GPIIb-IIIa autoantibodies are of pathogenic significance in this patient.

In this study employing a sensitive immunoblot assay, we have characterized GPIIb and GPIIIa in thrombasthenic platelets from seven type II and four type I patients from 10 unrelated families. The amounts of GPIIb and GPIIIa were both markedly reduced in all these patients, and abnormal molecular weight GPIIb or GPIIIa was not detected. In all of four type I patients the amount of GPIIb was much lower than that of GPIIIa. In this study, however, we found that the amount of GPIIb was also lower even in six out of seven type II patients. Immunodepletion of patients' platelets with AP2 (a monoclonal antibody specific for the GPIIb-IIIa complex), AP3 (specific for GPIIIa) or AMF7 (specific for alpha v) further confirmed that GPIIIa existed in excess, and demonstrated that excess GPIIIa were mostly in free form and not associated with GPIIb or alpha v. The reduction of GPIIb may represent an abnormality in GPIIb processing in these type II and type I thrombasthenic platelets. It remains unclear whether these two subgroups represent distinct categories.

The level of platelet-associated IgG (PAIgG) is reported to be elevated in patients with systemic lupus erythematosus (SLE). However, the nature of PAIgG is unclear. We have investigated whether the PAIgG of SLE consists of anti-platelet autoantibodies or immune complexes (IC). The PAIgG values measured by flow cytometry were elevated in 11/25 patients with SLE. 3/6 SLE patients with thrombocytopenia had a high level of PAIgG (the mean fluorescence intensity > 10). We used an ether elution technique to determine whether elevated PAIgG consists of anti-platelet antibodies or IC. Preliminary experiments showed that the eluates prepared from platelets sensitized with anti-HPA-4a antibody reacted with normal platelets, while the eluates prepared from platelets sensitized with heat-aggregated IgG or model IC failed to react with normal platelets. These results indicate that the reactivity of eluates can distinguish between platelet-bound antibody and IC. We applied this technique to analysis of the PAIgG of SLE platelets. The eluates from SLE platelets (the mean fluorescence intensity > 10) reacted with normal platelets, indicating that the PAIgG of SLE platelets has the nature of antiplatelet autoantibodies. Furthermore, we investigated the target antigens which bind PAIgGs of SLE, using the direct immunoprecipitation procedure and modified antigen capture ELISA (MACE). Both methods identified GPIIb/IIIa as the target antigens. We conclude that the ether elution technique can distinguish between anti-platelet antibodies and IC, and that the PAIgGs of SLE with a high PAIgG value and thrombocytopenia have the nature of anti-platelet autoantibodies.

By two-colour flow cytometric analysis, we examined the proportion of B lymphocytes bearing CD5 cell surface antigen (CD 5+ B cells), which are capable of producing autoantibodies, both in peripheral blood and spleen from patients with chronic immune thrombocytopenic purpura (ITP). The percentage of CD5+ B cells in peripheral blood lymphocytes (PBLs) was significantly increased (P less than 0.005) in patients with ITP (3.7 +/- 2.2%, n = 30) as compared with normal controls (1.7 +/- 0.7%, n = 28). However, there was no correlation between the percentages of circulating CD5+ B cells and platelet counts. The percentage of splenic CD5+ B cells in ITP patients was much more increased (9.0 +/- 4.5%, n = 9), P less than 0.005) compared with that of other disorders (3.2 +/- 0.5%, n = 5). Furthermore, isolated splenic CD5+ B cells from two out of five ITP patients produced high levels of IgM-type, platelet-bindable antibodies (PBIgM) after stimulation with Staphylococcus aureus Cowan I (SAC), while CD5- B cells isolated from the same spleen or splenic CD5+ B cells from other non-autoimmune disorders failed to produce significant amount of PBIgM. In three ITP patients, no increase in PBIgM was detected despite SAC stimulation. The increased proportion of CD5+ B cells in peripheral blood and spleen, and their ability to produce anti-platelet antibodies indicate that they are directly involved in the autoimmune pathogenesis in ITP.

It is well known that the platelet-specific alloantigen, Baka is carried on glycoprotein (GP) IIb, but little is known about the biochemical characteristics of its epitopes. To clarify the characteristics of the epitopes, we examined the interaction of four anti-Baka sera (Yam, Lin, Kl and MO) with their epitopes, either with or without modifications by sodium dodecyl sulphate (SDS) and/or neuraminidase. By immunoprecipitation, all four antisera bound to the intact GP IIb/IIIa complex from a Baka-positive subject. In contrast, immunoblotting demonstrated that Yam, Lin and Kl bound to SDS-denatured GP IIb, while MO did not. When blotted GP IIb was treated with neuraminidase, Yam and Lin did not bind to desialylated GP IIb, while Kl still did. When the purified GP IIb/IIIa complex or washed platelets were treated first with neuraminidase followed by immunoblotting, the molecular weight of GP IIb decreased from 145 kD to 138 kD; Yam did not bind to desialylated GP IIb, but Kl did. Furthermore, to eliminate the effect of SDS, we examined the interaction of Yam and Lin with neuraminidase-treated platelets using flow cytometry. The results were the same as those obtained using immunoblotting. Our results thus demonstrate that the expression of the Baka epitopes is not uniform and that sialic acid contributes to the expression of some actual allogenic epitopes.

Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.

Employing an immunoblotting procedure, we have identified and characterized an autoantigen carried on glycoprotein (GP) IIb in a patient with chronic idiopathic thrombocytopenic purpura (ITP), and have compared the location of the autoantigen with that of the platelet-specific alloantigen Baka. Immunoblots, using the partially purified GP IIb/IIIa complex as the target antigen, indicated that GP IIb alpha carried both the ITP autoantigen and the Baka alloantigen. The ITP plasma contained another antibody against a 100 kD protein (P100), a trace contaminant in the GP IIb/IIIa sample, which is probably a proteolytic fragment of an internal 124 kD protein. After chymotrypsin treatment, the auto- and alloantigen were found to be located on 65 kD fragments detectable under reducing conditions. In addition, immunoblots made after two-dimensional nonreduced-reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE) directly demonstrated that both 65 kD fragments had a molecular weight of 80 kD under nonreducing conditions; this provides evidence that these fragments were one and the same, and were derived from GP IIb alpha. Immunoblots of platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP IIb alpha was retained by the platelet membrane. We conclude, therefore, that a 65 kD fragment, which represents the membrane side of the chymotrypsin cleavage site on GP IIb alpha, carries a clinically important determinant(s) recognized not only by the anti-Baka alloantibody, but also by the ITP autoantibody.

Cutaneous delayed hypersensitivity to antigens prepared from Sporothrix schenckii, S. schenckii var. luriei (atypical S. schenckii), S. curziconia, S. inflata and S. ghanensis was tested in eleven cutaneous sporotrichotic patients. Cross-reactions were observed in various degrees. Five normal controls were not reactive.