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Background: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β’s in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. Methods: Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. Results: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β’s z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. Conclusions: We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, which inhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.

The ability to sense and move within an environment are complex functions necessary for the survival of nearly all species. The spinal cord is both the initial entry site for peripheral information and the final output site for motor response, placing spinal circuits as paramount in mediating sensory responses and coordinating movement. This is partly accomplished through the activation of complex spinal microcircuits that gate afferent signals to filter extraneous stimuli from various sensory modalities and determine which signals are transmitted to higher order structures in the CNS and to spinal motor pathways. A mechanistic understanding of how inhibitory interneurons are organized and employed within the spinal cord will provide potential access points for therapeutics targeting inhibitory deficits underlying various pathologies including sensory and movement disorders. Recent studies using transgenic manipulations, neurochemical profiling, and single-cell transcriptomics have identified distinct populations of inhibitory interneurons which express an array of genetic and/or neurochemical markers that constitute functional microcircuits. In this review, we provide an overview of identified neural components that make up inhibitory microcircuits within the dorsal and ventral spinal cord and highlight the importance of inhibitory control of sensorimotor pathways at the spinal level.

Localization of kinases to sub-cellular compartments allows them dynamic control over specific subsets of their targets. We previously found GSK-3β could modulate myofilament Ca 2+ sensitivity. However, whether GSK-3β modulates Ca 2+ sensitivity in vivo , if it localizes to the myofilament, and the consequences, are unknown. In myofilament enriched LV tissue from human non-failing and heart failure (HF) patients (n = 66) we found GSK-3β does localize to the myofilament and is altered by sex, age, and HF. To determine its in vivo functional role, we used myocyte specific inducible GSK-3β knock-out mice for skinned myocyte force-calcium experiments and found that GSK-3β reduction reduced calcium sensitivity. Further, we measured function in human samples and found myofilament GSK-3β levels directly correlated to Ca 2+ sensitivity. To establish how GSK-3β binds to the myofilament, we performed co-IP and IHC with phosphorylated forms of GSK-3β. GSK-3β phosphorylated at Y216 had a high affinity for the myofilament and localized to the z-disc. Mutating Y216 to a phospho-mimetic increased binding to the myofilament while mutating it to a phospho-null ablated binding.To identify GSK-3β’s myofilament targets, we performed mass spectrometry on myofilament phospho-enriched samples from GSK-3β KO and WT mice. As GSK-3β modulated Ca 2+ sensitivity, we expected to detect thin filament proteins. However, in agreement with its localization to the z-disc, GSK-3β primarily phosphorylated z-disc proteins.In the GSK-3β KO mice, strain analysis revealed the posterior wall contracted significantly earlier than the anterior wall, indicating baseline mechanical dyssynchrony. The WT mice had synchronous contraction, and interestingly there was significantly higher myofilament GSK-3β in the anterior wall compared to the posterior wall, a difference we hypothesize maintains synchrony. Thus, losing this fine control over Ca 2+ sensitivity as in the GSK-3β KO mice would induce mechanical dyssynchrony. Overall, these findings reveal that GSK-3β dynamically localizes to the myofilament to modulate Ca 2+ sensitivity through Y216 phosphorylation. The consequence of this “fine tuning” maintains chamber level mechanical synchrony.

Contractile remodeling in sustained atrial fibrillation (AF) has been analyzed by limited studies whose results were confounded by either coexisting systolic heart failure or valve disease (valvular AF) or origin of tissue (right chamber instead of left, atrial appendage instead of main wall). We sought to assess the structural changes in contractile apparatus and its physiological implications on single cardiomyocyte mechanics in patients with non-failing non-valvular AF. We utilized left atrial wall tissue from rejected donor hearts from 5 subjects in sinus rhythm (SR) and 3 with AF (age 50, 2♀ vs 60, 1♀), all with no signs of cardiovascular or valvular disease. Isolated single skinned myocytes were mounted to a force transducer and length controller and set to an initial sarcomere length of 2.1 μm. Isometric active and passive forces were recorded using custom software during [Ca2+] solution switching (0.79 - 46.8 μM). Surprisingly, we found that isometric maximal calcium-activated force (Fmax) was almost two times higher in AF compared to SR patients (n: SR = 15 cells, AF = 9 cells, p < 0.0001). This was unexpected, as previous studies found that AF patients had depressed contractile function, although these were confounded by heart failure and valve disease. There were no differences in calcium sensitivity, hill coefficient, or cell cross-sectional area (CSA) between SR and AF. We next performed 1D SDS-PAGE electrophoresis to compare myosin heavy chain (MHC) isoforms. In SR patients, atrial expression of β-MHC was very low (14% of total MHC expression), but this was significantly elevated in AF patients (37%, p = 0.05). The observed rise in contractile force might be a compensatory adaptation to sustain ventricular filling in initial stages of non-valvular non-failing AF patients. Or it may be a maladaptive response to atrial unloading resulting in wasted energy utilization. The contribution of this cellular increase in contractility to whole organ function is unclear. There is strong evidence that fibrotic remodeling and inflammation play an important role in AF, but the clinical challenge is still significant. Conversely, there has been very little work done on the contractile apparatus in AF, and whether it may represent a possible therapeutic target.

Neural circuitry generating locomotor rhythm and pattern is located in the spinal cord. Most spinal cord injuries (SCIs) occur above the level of spinal locomotor neurons; therefore, these circuits are a target for improving motor function after SCI. Despite being relatively intact below the injury, locomotor circuitry undergoes substantial plasticity with the loss of descending control. Information regarding cell type-specific plasticity within locomotor circuits is limited. Shox2 interneurons (INs) have been linked to locomotor rhythm generation and patterning, making them a potential therapeutic target for the restoration of locomotion after SCI. The goal of the present study was to identify SCI-induced plasticity at the level of Shox2 INs in a complete thoracic transection model in adult male and female mice. Whole-cell patch-clamp recordings of Shox2 INs revealed minimal changes in intrinsic excitability properties after SCI. However, afferent stimulation resulted in mixed excitatory and inhibitory input to Shox2 INs in uninjured mice which became predominantly excitatory after SCI. Shox2 INs were differentially modulated by serotonin (5-HT) in a concentration-dependent manner in uninjured conditions but following SCI, 5-HT predominantly depolarized Shox2 INs. 5-HT(7) receptors mediated excitatory effects on Shox2 INs from both uninjured and SCI mice, but activation of 5-HT(2B/2C) receptors enhanced excitability of Shox2 INs only after SCI. Overall, SCI alters sensory afferent input pathways to Shox2 INs and 5-HT modulation of Shox2 INs to enhance excitatory responses. Our findings provide relevant information regarding the locomotor circuitry response to SCI that could benefit strategies to improve locomotion after SCI. SIGNIFICANCE STATEMENT Current therapies to gain locomotor control after spinal cord injury (SCI) target spinal locomotor circuitry. Improvements in therapeutic strategies will require a better understanding of the SCI-induced plasticity within specific locomotor elements and their controllers, including sensory afferents and serotonergic modulation. Here, we demonstrate that excitability and intrinsic properties of Shox2 interneurons, which contribute to the generation of the locomotor rhythm and pattering, remain intact after SCI. However, SCI induces plasticity in both sensory afferent pathways and serotonergic modulation, enhancing the activation and excitation of Shox2 interneurons. Our findings will impact future strategies looking to harness these changes with the ultimate goal of restoring functional locomotion after SCI.

Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.

The ongoing contractile and metabolic demands of the heart require a tight control over protein quality control, including the maintenance of protein folding, turnover and synthesis. In heart disease, increases in mechanical and oxidative stresses, post-translational modifications (e.g., phosphorylation), for example, decrease protein stability to favour misfolding in myocardial infarction, heart failure or ageing. These misfolded proteins are toxic to cardiomyocytes, directly contributing to the common accumulation found in human heart failure. One of the critical class of proteins involved in protecting the heart against these threats are molecular chaperones, including the heat shock protein70 (HSP70), HSP90 and co-chaperones CHIP (carboxy terminus of Hsp70-interacting protein, encoded by theStub1gene) and BAG-3 (BCL2-associated athanogene 3). Here, we review their emerging roles in the maintenance of cardiomyocytes in human and experimental models of heart failure, including their roles in facilitating the removal of misfolded and degraded proteins, inhibiting apoptosis and maintaining the structural integrity of the sarcomere and regulation of nuclear receptors. Furthermore, we discuss emerging evidence of increased expression of extracellular HSP70, HSP90 and BAG-3 in heart failure, with complementary independent roles from intracellular functions with important therapeutic and diagnostic considerations. While our understanding of these major HSPs in heart failure is incomplete, there is a clear potential role for therapeutic modulation of HSPs in heart failure with important contextual considerations to counteract the imbalance of protein damage and endogenous protein quality control systems.This article is part of the theme issue ‘Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective’.

In ischemic cardiomyopathy (ICM), regions with ischemic damage are weaker than surrounding tissue, leading to contractile heterogeneity (ischemia-induced dyssynchrony, IID) that worsens function and mortality. IID is distinct from conduction abnormality-induced dyssynchrony that is treated with Cardiac Resynchronization Therapy (CRT). Our previous work found CRT reactivates glycogen synthase kinase 3β (GSK-3β) and restores myofilament function. Pacing an infarct cannot strengthen it, so CRT is ineffective in IID. However, we hypothesized GSK-3β may modulate myofilament function in ICM and could be leveraged to treat IID. We measured GSK-3β in whole tissue and myofilament-enriched samples from human rejected donor (Control), ICM, and dilated cardiomyopathy (no IID, DCM) LV. GSK-3β was detected in all the myofilament samples, but there was a 71±12% reduction in ICM. In whole tissue, there was very little phospho-Y216 GSK-3β, however it was highly enriched in the myofilament. Immunofluorescence on adult human myocytes showed weak co-localization of total GSK-3β and α-actinin at the z-disc compared to a strong correlation with p-Y216 GSK-3β. Furthermore, co-IP of GSK-3β and the myofilament show total GSK-3β had low-affinity to myofilament proteins, while p-Y216 GSK-3β binds with a high affinity. This led us to hypothesize Y216 phosphorylation modulates GSK-3β binding to the myofilament. To remove the confounding effect of antibodies, we created adenoviral constructs of myc-tagged wild-type, Y216F (unphosporylatable) and Y216E (constitutively phosphorylated) GSK-3β and transfected them into rat neonatal ventricular myocytes (NRVMs). The Y216E construct alone associated with the myofilament in co-IP experiments. We then performed skinned myocyte functional studies on human Control and ICM LV. The ICM myocytes were desensitized to calcium compared to Control and this was restored with exogenous GSK-3β treatment, but had no effect on Control myocytes. While GSK-3β is a promiscuous kinase in the myocyte, we have identified a specific regulatory mechanism involving Y216 phosphorylation, a site with a largely unknown role, that could allow precise therapeutic intervention to improve contractile function in the ICM heart.

Cardiac dyssynchrony arises from conduction abnormalities during heart failure and worsens morbidity and mortality. Cardiac Resynchronization Therapy (CRT) re-coordinates contraction using bi-ventricular pacing, but the cellular and molecular mechanisms involved remain largely unknown. We aimed to determine how dyssynchronous heart failure (HF(dys)) alters the phospho-proteome and how CRT interacts with this unique phospho-proteome by analyzing Ser/Thr and Tyr phosphorylation. Phospho-enriched myocardium from dog models of Control, HF(dys), and CRT was analyzed via mass spectrometry. There were 209 regulated phospho-sites among 1,761 identified sites. Compared to Con and CRT, HF(dys) was hyper-phosphorylated and tyrosine phosphorylation was more likely to be involved in signaling that increased with HF(dys) and was exacerbated by CRT. For each regulated site, the most-likely targeting-kinase was predicted, and CK2 was highly specific for sites that were “fixed” by CRT, suggesting activation of CK2 signaling occurs in HF(dys) that is reversed by CRT, which was supported by western blot analysis. These data elucidated signaling networks and kinases that may be involved and deserve further study. Importantly, we have identified a possible role for CK2 modulation in CRT. This may be harnessed in the future therapeutically to compliment CRT, improving its clinical effects.

Corn (Zea mays L.) planting and the first harvest of perennial forages overlap in the northeastern USA in some years. The use of glyphosate [N-(phosphonomethyl)glycine]–resistant corn may help dairy producers lessen their workload during this time by allowing for a timely glyphosate application after completion of the first harvest of perennial forages. We evaluated 92 and 103-d hybrids to determine the impact of season-long weed interference and the optimum timing for glyphosate application on corn forage yield and quality. Season-long weed interference vs. weed-free corn in two competitive growing seasons reduced dry matter (DM) accumulation at silking (R1 stage) by 50 to 65%, DM yield by 70 to 75%, and calculated milk yield by 75 to 80%. Season-long weed interference reduced milk per megagram, a forage quality index, by 10% in a dry year by preventing grain formation. Glyphosate application at the three to four leaf stage (V3–V4) vs. weed-free corn resulted in similar DM accumulation at the R1 stage, DM content at harvest, DM yield, forage quality, and calculated milk yield in both years. Glyphosate application at the V5–V6 stage vs. weed-free corn increased milk per megagram by 7% in the dry year, but resulted in 20 to 25% less DM yield in both years. Dairy producers in the northeastern USA should apply glyphosate by the V3–V4 stage in competitive growing conditions, regardless of hybrid maturity, which may overlap with the first harvest of perennial forages in some years.

The compatibilization by transesterification of aromatic thermotropic liquid crystal polyesters (Ar-TLCP's) and TLCP's containing aliphatic ethylene-terephthalate (ET) linkages has been studied. Three Ar-TLCP's were mixed with two p(ET/HBA)'s, each having different extents of blockiness, to form a series of binary blends (Bin-TLCP's). The morphology and physical properties of the resultant Bin-TLCP's depend on the block structures, compositions, viscosities, and susceptibility to transesterification of the components. In this work, the influence of TLCP block structure and transesterification on the compatibility of the components of the Bin-TLCP's is described.

A thermotropic liquid crystalline polymer (TLCP) containing aliphatic linkages exhibits properties of both a random-coil and a rigid-rod. By varying its block structure, one can change its ability to form compatible blends with other polymers. In this work, the ability of the TLCP p(ET/HBA60) with a high degree of block structure, X-7G TM , to form compatible blends with polycarbonate and aromatic TLCPs is compared to that of a p(ET/HBA60) with a more random molecular structure, Rodrun TM LC-3000. It was found that both materials formed compatible blends through transesterification of the block structure and that a higher degree of blockiness enhanced the rate of transesterification. NMR spectra and differential thermal analysis were used to quantify the degree of transesterification and scanning electron microscopy was utilized to examine the effects of the blending process on the degree of compatibilization and the morphology of the resulting blends.

The TCR/CD3 receptor complex plays a key role in antigen recognition and T-cell activation. Therefore, the present study investigates TCR alpha/beta (TCR1) and CD3 receptor density (RD, number of receptors per cell) on uremic helper-inducer (CD4) T lymphocytes in relation to T-cell proliferative response induced by anti-CD3 monoclonal antibodies (mAb). We found, that: (1) the number of TCR/CD3 receptors on uremic helper-inducer (CD4) T lymphocytes is decreased and correlated well with the blunted lymphocyte proliferation induced by anti-CD3 mAb; (2) these findings were associated with diminished binding capacity of IL-1 beta and IL-6 to their receptors (IL-1R, IL-6R) on helper-inducer T cells, whereas (3) the IL-2 receptor (IL-2R) and molecule expression of CD4 and lymphocyte function antigen-1 (LFA-1) were increased, and (4) uremic monocytes displayed a decreased density of intercellular adhesion molecule-1 (ICAM-1) expression, which interacts as receptor-ligand pair with LFA-1. The incubation of uremic and control peripheral blood mononuclear cells with uremic serum enhanced these above-mentioned changes in the expression of examined receptors and molecules. These data might also support the hypothesis that the blunted T-cell response to antigen in uremia is due to downregulation of the TCR/CD3 receptor complex by uremic milieu.

Item does not contain fulltext Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by spontaneous chromosomal instability with predisposition to immunodeficiency and cancer. In order to assess the cellular basis of the compromised immune response of NBS patients, the distribution of functionally distinct lymphocyte subsets in peripheral blood was evaluated by means of double-colour flow cytometry. The study involved the 36 lymphopenic patients with a total lymphocyte count < or =1500 microl (group A) and seven patients (group B) having the absolute lymphocyte count comparable with the age-matched controls (> or =3000 microl). Regardless of the total lymphocyte count the NBS patients showed: (1) profound deficiency of CD4+ and CD3/CD8+ T cell subsets and up to fourfold increase in natural killer (NK) cells, almost lack of naive CD4+ T cells expressing CD45RA isoform, unchanged percentage of naive CD8+ cell subset (CD8/CD45RA+) but bearing the CD8 receptor of low density (CD8low); (2) normal expression of CD45RA isoform in the CD56+ lymphocyte subset, profound decrease in alpha beta but up to threefold increase in gamma delta-T cell-receptor (TCR)-positive T cells; (3) shift towards the memory phenotype in both CD4+ and CD8+ lymphocyte subpopulations expressing CD45RO isoform (over-expression of CD45RO in terms of both the fluorescence intensity for CD45RO isoform and the number of positive cells); and (4) an increase in fluorescence intensity for the CD45RA isoform in NK cells population. These results indicate either a failure in T cell regeneration in the thymic pathway (deficiency of naive CD4+ cells) and/or more dominant contribution of non-thymic pathways in lymphocyte renewal reflected by an increase in the population of CD4+ and CD8+ memory cells, gamma delta-TCR positive T as well as NK cell subsets.

The aim of the study was to estimate the results of recombinant human growth hormone (rhGH) treatment in children with end-stage renal disease (ESRD). 60 growth retarded children with ESRD (mean age 11.2 +/- 7.2 years) were treated with rhGH at a dose of 1-1.1 IU/kg/week. The time of observation was 24 months. Thirty children completed first year, 18--second year of treatment. The mean growth velocity prior to the treatment was 3.03 +/- 1.9, during first year of the study--7.52 +/- 2.42, during second year 6.68 +/- 2.87 cm/year. The negative correlation between growth velocity and patient's age (r = -0.39; p0.05) suggest the better growth results in younger children during rhGH treatment. The rhGH therapy is effective method of treatment in growth retarded children with ESRD. Side effects are rare.

Chronic infection with hepatitis C virus (HCV) is estimated to affect almost 170 million individuals worldwide. 20-30% of these individuals develop cirrhosis and its sequelae. Only 15-20% of patients with chronic hepatitis C achieve a sustained virological response to interferon monotherapy. The prevalence of anti-HCV antibodies in dialysis patients varies between 1% and 29% in Western Europe. In patients with ESRD on maintenance HD therapy, in whom a blunted immune response per se is observed, the usefulness of IFN-alpha therapy is usually discussed in the context of subsequent transplantation associated with intensive immunosuppressive treatment regimens. A recent study has shown that in this patient group renal transplantation is associated with a fivefold increase in posttransplantation liver disease as well as a relative risk of death of 3.3 compared to HCV-negative patients. Thus, eradication of HCV infection in patients with ESRD may substantially reduce morbidity and mortality in renal allograft recipients. The imbalance of T-helper (Th) lymphocyte cytokine production may play an important role in the immunopathogenesis of chronic HCV infection. Little is known about the effects of IFN-alpha therapy on Th1/Th2 activity in HD patients. The type of immune response against infectious agents is determined in part by the pattern of cytokines secreted by T lymphocytes. Th1 cells promote cellular immunity against infectious agents, while Th2 cells induce humoral immune response and immune tolerance activity. The measurement of Th1/Th2 profile should increase our understanding of the immune status of patients with HCV infection. Therefore, the recently presented studies were undertaken to evaluate the influence of IFN-a therapy on Th1/Th2 balance in HD patients with chronic HCV infection.

MDR1 gene encodes for a transmembranous glycoprotein, gp-170, which acts as a drug export pump and is also a cyclosporine(CsA)-binding protein. This study aimed at evaluating MDR1 expression in NS sensitive(S) and resistant(R) to therapy (steroids/S/, cyclophosphamide/C/, CsA) patients. Twenty six boys, 13 girls aged 3-8 years were included to the study. MDR1 was analysed using: 1) evaluation of gp-170 activity according to DiC2/3/ [3,3-Diethyloxa-carbocyanine Iodide] by means of flow cytometry and as 2) mRNA expression of MDR1 determined by RT-PCR. The analysis was performed in the lymphocyte subset CD4/CD45RA presenting suppressor-inducer activity. Negative control, Jurkat-T-cell line, not expressing the MDR1 phenotype, was transfected with viral expression vector containing a full-length cDNA for the human MDR1 gene. We found that: in SR-NS the high expression of MDR1 was associated mainly with the suppressor-inducer T-cells (CD45RA+CD4+) and was subsequently enhanced during an ineffective treatment with C and/or CsA. C-R-NS and CsA-R-NS were partially reversible by S- and R-Verapamil; this was in vitro confirmed by inhibition of export pump activity, gp-170. SS-NS, C-S-NS and CsA-S-NS presented the low expression and activity of MDR1 comparing to R-children (p0.001) and healthy controls (p0.00001). Resistance to therapy in NS patients seems to be resulted from the enhanced expression of MDR1 gene and subsequent high activity of export pump P-gp-170. Calcium channel blockers may reverse the MRD1-related resistance in the therapy of NS. Analysis of MDR1 may help to detect of suspected therapy resistance in NS.

T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity(SS) or resistance(SR). Activity of Th1 and Th2 cells was defined by the production of IL-2, IFN-gamma and IL-4, IL-10 (ELISA), respectively, in the supernatants of the culture of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double or triple colour flow cytometry. In SS children with NS we found the cytokine synthesis indicating the predominance of Th2 activity. We conclude that prior to treatment the Th1 and Th2 cell activity provides a useful tool to evaluate the probability of steroid sensitivity in patients with primary NS.

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.

The pathogenesis of chronic renal allograft rejection is still speculative. Amongst other factors immune-mediated graft injury is proposed. Since the allo-antigen is specifically recognized by the variable (V) alpha and beta chains of the T-cell receptor, a restricted T-cell repertoire might support the notion of allo-antigen involvement in chronic rejection.By the means of semiquantitative polymerase chain reaction the V beta families 1-20 were assessed in allograft biopsies with histologically confirmed chronic and acute rejection. At the same time the V beta repertoire was analyzed in PBMC.The intragraft V beta repertoire was limited to 1 to 3 dominant V beta families in chronic and acute rejection. The response was highly individual and did not correlate to the type or degree of HLA mismatches. The T-cell repertoire in PBMC was polyclonal and did not reflect the immune response in the graft.The finding of a restricted V beta repertoire in both forms of rejection might indicate an immunological basis not only for acute, but also for ongoing chronic rejection. Tailor-made antibodies against the dominant V beta clones might provide a tool for selective immunosuppression in both entities of rejection targeting only those T cells which were activated by allo-antigens.

In a patient with metastatic melanoma transmitted by the renal allograft, HLA serves as an alloantigen per se and is associated with tumor antigens at the same time. The influence of this antigeneic pattern on the Vbeta T-cell repertoire in an allogeneic melanoma, allograft, and peripheral blood mononuclear cells (PBMC) was assessed by polymerase chain reaction. Vbeta13.1 and 19 were found in both the melanoma and the graft. Vbeta14 was detected only in the melanoma and Vbeta6 was detected only in the kidney. PBMC revealed an unrestricted Vbeta pattern. Markers for cytotoxic activity of T cells--granzyme B and perforin--were not expressed during immunosuppressive therapy as clinically reflected in a nonrejecting allograft and in a progressing melanoma. In vitro PBMC proliferated to recombinant interleukin-2, whereas recombinant interferon-gamma did not augment this response. Initiation of immune therapy, in addition to discontinuation of immunosuppression, might support the rejection of the allogeneic tumor by dominant Vbeta T cells.

Eight children with thrombo-embolic disease in the course of nephrotic syndrome were treated at II Clinic of Children's Diseases (Institute of Pediatrics, Poznań) between 1991 and 1993. The diagnosis was established on the basis of clinical examination and noninvasive imaging techniques. Two patients had an atypical localisation of the thrombus in the left ventricle and right atrium. In laboratory tests of the coagulation system, all of the children had decreased levels of antithrombin III (AT III). All children were treated with heparin and 4 with fibrinolytic agents. AT III concentrate was administered to 3 children. Total resolution of thrombo-embolic disease was obtained in 5 patients, 3 died during treatment. Thrombo-embolic disease should be taken into account in the differential diagnosis of complications of nephrotic syndrome.

Fifteen children treated with fibrinolytic agents are presented. The most frequent indication was thromboembolic disease (TED). Eleven patients received streptokinase, 5-urokinase and 3-tissue plasminogen activator. Concomitant heparin was administered to 9 patients with TED. Total resolution was achieved in 9 children, partial improvement in 5; 1 child died during treatment without any improvement. Bleeding complications were observed in 6 patients, 1 of them died due to haemorrhagic stroke. According to the literature and our own experience, we recommend fibrinolytic agents as the treatment of choice for severe TED also in children.

Some of CD4+ helper T cells-mediated mechanisms of IgE synthesis were discussed. The present division of helper T cells population into Th1 and Th2 subsets reflects their role in the induction as well as in the control of immune response. The Th1 cells are involved mainly in the cellular immune responses, and Th2 cells in the humoral immune responses, including IgE synthesis. Development of investigations on cytokines and evaluation of their influence on regulation of IgE synthesis may have a role in the treatment of allergic diseases in the future.

We report on 11 patients from 8 independent families (3 pairs of sibs) with a complex clinical pattern including microcephaly, peculiar "bird-like" face, growth retardation, and, in some cases, mild-to-moderate mental deficiency. Most of the patients have recurring respiratory tract infections. One girl has developed B-cell lymphoma. A detailed anthropometric study of 15 physical parameters, including 3 cephalic traits, was performed. It was possible to study the chromosomes of PHA-stimulated lymphocytes in all of the patients. We found structural aberrations with multiple rearrangements, preferentially involving chromosomes 7 and 14 in a proportion of metaphases in all individuals. Profound humoral and cellular immune defects were observed. Serum AFP levels were within normal range. Radioresistant DNA synthesis was strongly increased in all 8 patients who were hitherto studied in this respect. Our patients fulfill the criteria of the Nijmegen breakage syndrome, which belongs to the growing category of ataxia telangiectasia-related genetic disorders. In light of the increased predisposition to malignancy in this syndrome, an accurate diagnosis is important for the patient.

The study was undertaken to evaluate the relationship between non-responsiveness to hepatitis B (HBV) vaccination in haemodialysed patients and HBs antigen (Ag) presentation and recognition depending on TCR/CD3 receptors expression. We have found that the cause of the blunted response to HBV vaccination is multifactorial and seems to be associated with the following: (1) A reduced number of TCR/CD3 antigen receptor complexes on freshly isolated uraemic CD4 T cells, especially in non-responders. (2) The blunted proliferative response of uraemic CD4 T cells isolated from non-responders and stimulated for 6 days by autologous monocytes presenting HBsAg was associated with the decreased density of the TCR/CD3 receptors. (3) Moreover, in uraemic non-responders the expression of adhesion and accessory molecules on monocytes (intercellular adhesion molecule-1/ICAM-1, HLA-DR/Ia/) was significantly decreased following the culture with autologous monocytes serving as HBsAg-presenting cells. CD4 molecules and lymphocyte function antigen-1 beta/LFA-1 beta/ on helper-inducer T cells were increased before and after the culture. (4) These findings were also associated with a diminished binding capacity of IL-1 beta and IL-6 to their receptors on helper-inducer T cells. (5) IL-2, IFN-gamma and IL-4 production was decreased in uraemic non-responders, especially after 72 h of the culture. (6) Inhibited proliferation of helper-inducer T cells in uraemic non-responders was only partially reversible in the presence of exogenous IL-1 beta, IL-6, IL-2 and IFN-gamma. (7) HLA typing of uraemic non-responders was associated with extended haplotype: HLA A1,B8,DR3,DR7,DQ2.

TCR/CD3 receptor complex plays a central role in antigen recognition and T cell activation. Therefore, the present study investigates TCR alpha/beta (TCR-1) and CD3 receptor density (RD, number of receptors per cell) on uremic CD4 T lymphocytes in relation to T cell proliferative response induced by anti-CD3 monoclonal antibodies (mAb). The influence of uremic serum on TCR/CD3 receptor expression of normal and uremic CD4 T lymphocytes was evaluated as well. We found, that: a) percentage of TCR-1 and CD3 positive cells of freshly isolated CD4 T lymphocytes is the same in controls and ESRD-patients, but the TCR/CD3 RD is lower on uremic CD4 T lymphocytes, b) Incubation for 24 h with uremic serum lowers TCR/CD3 RD on normal and uremic CD4 T cells, c) There is positive correlation between TCR/CD3 RD and anti-CD3 induced lymphocyte proliferation. These data might also support the hypothesis that blunted T-cell response to antigen in uremia is due to down-regulation of the TCR/CD3 receptor complex by uremic milieu.

The interdependence of blunted T cell proliferation induced by anti-CD3 monoclonal antibodies (mAb) and the preactivation of T lymphocytes (CD25+) in ESRD patients was investigated in this study. We focused on the density of IL-2 (CD25) receptors [IL-2R] on the lymphocyte surface rather than enumeration of IL-2R positive cells. The effect of exogenous IL-2 on these parameters was also tested. Blunted T lymphocyte proliferation induced by anti-CD3 mAb is only partially corrected by addition of exogenous IL-2 after 24 hrs. Freshly isolated uremic CD4 T cells show higher percentage of IL-2 positive cells and a higher IL-2R density on the cell surface compared to controls. However, after anti-CD3 mAb stimulation the number of IL-2R positive cells and IL-2R density in CD4 T subset was significantly lower than in samples from normal donors. Exogenous IL-2 had no influence on IL-2R expression on CD4 cells in uremic patients. On the other hand, following anti-CD3 mAb stimulation uremic CD8 cells reveal more IL-2R positive cells with higher IL-2R density than in controls. Moreover, exogenous IL-2 enhance IL-2R expression and density on uremic CD8 cells more than in controls. Our results suggest that the blunted T cell proliferation in ESRD patients might result from (a) preactivation of CD4 T cells, (b) diminished response of uremic CD4 T cells to IL-2, and (c) higher suppressor cells activity.

The study was undertaken to establish the clinical and immunological aspects of HBV-mediated membranous glomerulonephritis in children. Out of 54 children with membranous GN treated in the Children's Memorial Hospital 51 children (94.4%) had the disease related to HBV infection. The inhibitory effect of immune complexes: HBsAg-IgG and HBeAg-IgG, isolated from sera of these children on lymphocyte proliferation of blood donors was observed. This may partly explain the status of immune tolerance towards HBV in the infected children.

To evaluate the B cell lineage system in newborns we estimated IL-4 (BCGF/BSF-1) production by lymphocytes isolated from the cord blood and its influence on antibody synthesis. Undertaken experiments were performed in two groups of newborns: stressed newborns mainly with perinatal infection and full-term healthy neonates, comparing to peripheral blood of adults as control. Results revealed 1) the significantly higher percentage of mature B cells (B1) in cord blood of stressed newborns, 2) the significantly higher IL-4 production comparing to full-term neonates, 3) diminished IgG and IgA synthesis in vitro by allogenic activated B cell blasts in the presence of supernatants from cultures of PHA-stimulated lymphocytes isolated from cord blood of stressed newborns. Induction of IgM synthesis by these active supernatants was significantly higher in stressed newborns than in the other examined groups. We suggest that immunoregulatory mechanisms, which control the production of IL-6 (BCDF/BSF-2) are still not completely mature at birth.

Studies were undertaken to evaluate the effect of hepatitis B virus (HBV) immune complexes (HBV-IC) on IL-2 dependent human lymphocyte proliferation. The following parameters were studied: 1) Effect of HBV-IC (HBsAg-IgG or HBeAg-IgG) on PHA-mediated lymphocyte proliferation; 2) Influence of HBV-IC on the ability of PHA-stimulated peripheral blood lymphocytes (PBL) for IL-2 production and IL-2 receptor expression. HBV-IC induced a dose dependent and antigenic dependent suppression of PHA stimulated lymphocytes. The suppressor effect exerted by HBsAg-IgG was irreversible. In contrast, the suppression mediated by HBeAg-IgG was reversible: lymphocytes preincubated with this preparation washed and activated with PHA responded well to mitogen. The presence of HBV-IC in the cultures of PHA-activated PBL decreased their ability to produce IL-2: HBeAg-IgG exerted a stronger suppressor effect. This effect was partially reversible: removal of HBV-IC from the culture by washing and subsequent stimulation of PBL with PHA increased the capacity of lymphocytes to produce IL-2. This was particularly evident with HBeAg-IgG. Decreased activity of IL-2 observed in the cultures, was also partially dependent on the ability of HBV-IC to bind IL-2 present in the culture medium. Experiments performed using ultracentrifugation indicated that HBV-IC, especially HBsAg-IgG, may bind to IL-2 and inactivate it. HBV-IC had also an effect on IL-2 receptor expression: 1) their presence in the cultures of PHA-stimulated PBL decreased the number of Tac positive cells; 2) the response of HTCL to exogenous IL-2 was decreased by HBV-IC present in the culture medium. This was especially observed in the case of HBsAg-IgG. We suggest that the observed inhibition of PHA-induced lymphocyte proliferation exerted by immune complexes containing HBsAg-IgG or HBeAg-IgG may be caused mainly by their influence on IL-2 dependent mechanism of lymphoproliferation.