SURVIVAL AND SUBSET CLASSIFICATION ANALYSIS OF 82 PATIENTS WITH INFLAMMATORY MYOPATHY

Background: Idiopathic inflammatory myopathies (IIM) are characterised by chronic muscle inflammation, various organ involvements and the presence of certain specific autoantibodies. Objectives: We assessed survival and characterized subsets based on muscle biopsy and myositis specific autoantibodies (MSAs). Methods: Eighty-two patients with muscle biopsy proven IIM were included in the study. All cases had MSA and myositis associated antibody (MAA) tests (Jo-1, PL-7, PL-12, Mi-2, SRP, Pm-Scl, Ku, ribosomal, AMA-M2) using Western-blot kits. Survival analysis was performed by Kaplan Meier test. Results: Fift y-nine women and 23 men with a mean age of 49.3 ± 14.6 years and with 7.5 ± 4.5 years of mean follow-up time were included. Interstitial lung disease (ILD) (51.2%), arthritis (51.2%), Raynaud’s phenomenon (42.7%), skin symptoms (45.1%), dysphagia (24.4%) and significant cardiac involvement (15.9%) were the most prevalent disease manifestations. 15 cases were associated with malignancies. Myositis subsets were as follow: 26.8% (n=22) polymyositis /PM/, 30.5% (n=25) dermatomyositis/DM/, 1.2% (n=1) juvenile PM/DM, 8.5% (n=7) inclusion body myositis /IBM/, 22% (n=18) overlap myositis /OM/, and 11% (n=9) immune mediated necrotizing myopathy /IMNM/. Malignancy was most frequently associated with IMNM (7 out of 9 patients). Altogether 18 patients died from which 15 deaths can be connected to myositis related events. Eight patients died of malignancies, 5 patients due to cardiac events (heart failure, arrythmia), 2 due to lung fibrosis and 3 by unknown causes. The worst prognosis with a 10-year survival of 31 % was in the IMNM subgroup (p<0.01), followed by patients with PM (68%), IBM (84%) OM (85.1%) and DM (85.3%). Mi-2 positive patients had a favourable prognosis with a 10-year survival of 100%. Patients with IMNM had the worst prognosis (10-year survival of 31.1%), followed by PM (76%), DM and IBM (85.7% each). Patients with antisynthetase antibody-positivity had worse prognosis compared to patients with other antibodies or no identifiable antibodies (10-year survival of 55%, n=16) (p<0.05). Conclusions: Th e worst survivals were seen in the IMNM and PM groups, due to the high frequency of the underlying malignancies and cardiac manifestations. Although ILD was the most frequent involvement, it was not the main cause of death

Zinc-induced structural changes of the disordered TPPP/p25 inhibits its degradation by the proteasome

AbstractTubulin Polymerization Promoting Protein/p25 (TPPP/p25), a neomorphic moonlighting protein displaying both physiological and pathological functions, plays a crucial role in the differentiation of the zinc-rich oligodendrocytes, the major constituent of myelin sheath; and it is enriched and co-localizes with α-synuclein in brain inclusions hallmarking Parkinson's disease and other synucleinopathies. In this work we showed that the binding of Zn2+ to TPPP/p25 promotes its dimerization resulting in increased tubulin polymerization promoting activity. We also demonstrated that the Zn2+ increases the intracellular TPPP/p25 level resulting in a more decorated microtubule network in CHO10 and CG-4 cells expressing TPPP/p25 ectopically and endogenously, respectively. This stabilization effect is crucial for the differentiation and aggresome formation under physiological and pathological conditions, respectively. The Zn2+-mediated effect was similar to that produced by treatment of the cells with MG132, a proteasome inhibitor or Zn2+ plus MG132 as quantified by cellular ELISA. The enhancing effect of zinc ion on the level of TPPP/p25 was independent of the expression level of the protein produced by doxycycline induction at different levels or inhibition of the protein synthesis by cycloheximide. Thus, we suggest that the zinc as a specific divalent cation could be involved in the fine-tuning of the physiological TPPP/p25 level counteracting both the enrichment and the lack of this protein leading to distinct central nervous system diseases

Osteopontin Levels Are associated with Late‑Time Lower Regional Brain Volumes in Multiple Sclerosis

Osteopontin (OPN) is a proinflammatory marker produced by systemic immune and central nervous system (CNS) resident cells. We examined, if the level of OPN in the cerebrospinal fluid (CSF) and blood is associated with late-time regional brain volumes and white matter (WM) lesion load in MS. Concentrations of OPN in blood and CSF were related to MRI findings 10.1 ± 2.0 years later in 46 patients with MS. OPN concentration was measured by ELISA, while regional brain volumes and lesion load was assessed by magnetic resonance imaging (MRI) using 3D MPRAGE sequence and automated MR volumetry. OPN measured in the CSF was associated with several regional brain volumes and WM lesion load measured 10.1 ± 2.0 years later. CSF OPN concentration correlated with long-term enlargement of lateral- and inferior lateral ventricles and the elevation of gross CSF volume, in conjunction with the reduction of several cortical/subcortical gray matter and WM volumes. Serum OPN showed no long-term association with regional brain volumes. OPN measured from the CSF but not from the serum was associated with lower regional brain volumes measured a decade later, indicating the primary role of inflammation within the CNS in developing long-term brain related alterations

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1β, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3-/- mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation

Complexity of the Immune Response Elicited by Different COVID-19 Vaccines, in the Light of Natural Autoantibodies and Immunomodulatory Therapies

Despite the abundance of data on the COVID-19 vaccine-induced immune activation, the impact of natural autoantibodies (nAAbs) on these processes is less well defined. Therefore, we investigated potential connections between vaccine efficacy and nAAb levels. We were also interested in the impact of immunomodulatory therapies on vaccine efficacy. Clinical residual samples were used for the assessment of the COVID-19 vaccine-elicited immune response (IR) (n=255), as well as for the investigation of the immunization-associated expansion of the nAAb pool (n=185). In order to study the potential interaction between immunomodulatory therapies and the vaccine-induced IR, untreated, healthy individuals and patients receiving anti-TNFα or anti-IL-17 therapies were compared (n total =45). In-house ELISAs (anticitrate synthase, anti-HSP60 and-70) and commercial ELISAs (anti-SARS-CoV-2 ELISAs IgG, IgA, NeutraLISA and IFN-γ release assay 'IGRA') were applied. We found significant differences in the IR given to different vaccines. Moreover, nAAb levels showed plasticity in response to anti-COVID-19 immunization. We conclude that our findings may support the theorem about the non-specific beneficial 'side effects' of vaccination, including the broadening of the nAAb repertoire. Considering immunomodulation, we suggest that anti-TNFα and anti-IL17 treatments may interfere negatively with MALT-associated IR, manifested as decreased IgA titers; however, the modest sample numbers of the herein presented model might be a limiting factor of reaching a more comprehensive conclusion

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

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

Ligation of TLR Homologue CD180 of B Cells Activates the PI3K/Akt/mTOR Pathway in Systemic Sclerosis and Induces a Pathological Shift in the Expression of BAFF Receptors

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) pathways are known to play a key role in B-cell activation and fibrosis in systemic sclerosis (SSc). Receptors of B-cell activator factor (BAFF) utilize these pathways, which can be influenced by Toll-like receptors (TLRs), as TLRs can alter the expression of BAFF-binding receptors. Our results show that B-cell stimulation via TLR homologue CD180 phosphorylates Akt in diffuse cutaneous SSc (dcSSc) to a lower extent than in healthy controls (HCs). We found basal downregulated BAFF receptor (BAFF-R) and enhanced transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) expression in dcSSc B cells, which might enhance the formation of autoantibody-secreting plasma cells. Moreover, this pathological shift was observed in naive B cells, emphasizing the importance of their increase in SSc. Additionally, we measured higher serum levels of autoantibodies to BAFF in dcSSc patients, suggesting that an imbalance in the complex system of BAFF/anti-BAFF autoantibodies/BAFF-binding receptors may contribute to the development of SSc. Anti-CD180 antibody treatment had opposite effects on the expression of BAFF-R and TACI in HC B cells, resulting in similar levels as observed in SSc B cells without stimulation, which argues against the usefulness of such therapy in SSc

Interactions between two regulatory proteins of microtubule dynamics, HDAC6, TPPP/p25, and the hub protein, DYNLL/LC8

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6.Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network

Az autoimmun encephalitisek laboratóriumi vizsgálati lehetőségei = Autoimmune encephalitis: possibilities in the laboratory investigation

Absztrakt: Bevezetés: Az elmúlt 10 évben a nem klasszifikálható neurológiai vagy pszichiátriai tünetegyüttes képében megjelenő encephalitisek esetén egyre gyakrabban igazolódik be, hogy a háttérben a központi idegrendszer valamely fehérjéje ellen induló autoimmun folyamat áll. A paraneoplasiás limbicus encephalitisek esetében intracelluláris antigének (anti-Hu/ANNA1, anti-Ri/ANNA2, anti-CV2/CRMP5 és anti-Ma2/Ta) ellen indul immunreakció, mely mögött tüdő-, ovarium- vagy heredaganat áll, és jellemző a rossz prognózis. Ezzel szemben az utóbbi években felfedezett, színes klinikai képpel megjelenő autoimmun encephalitisek mögött gyakran bizonyítható a neuronalis sejtfelszíni receptor (NMDAR, GABABR, AMPAR) vagy szinaptikus fehérje (LGI1, CASPR2) ellen képződő autoantitestek jelenléte, ami immunszuppressziós kezelésre jól reagál. Célkitűzés: Célunk felhívni a figyelmet a neurológiai, pszichiátriai és intenzív terápiás ellátást igénylő autoimmun encephalitises esetek emelkedő számára, valamint az autoantitestek kimutatásának jelentőségére. Módszer: Laboratóriumunkba az elmúlt 6 évben 836 autoimmun encephalitis irányú, 717 beteghez tartozó vizsgálatkérés érkezett. A betegek szérum- és liquormintáit 6 különböző receptorfehérjével transzfektált sejtvonalból álló BIOCHIP-en vizsgáltuk indirekt immunfluoreszcens technikával. Eredmények: A vizsgált betegek 7,5%-ában tudtunk valamelyik receptorfehérje ellen IgG autoantitestet kimutatni. Gyakorisági sorrendben NMDAR > LGI1 > GABABR > CASPR2 ellen találtunk pozitív eseteket. Következtetés: Az autoantitest kimutatása segít a betegség korai stádiumban való felismerésében és a diagnózis felállításában. Mindez fontos, mert az időben felismert betegek eredményesen kezelhetőek plazmaferézissel vagy immunszuppresszív szerekkel, melyek hatékonyságát ismételt autoantitestmeghatározással lehet követni. Ezért a laboratóriumnak nagy szerepe lehet a gyorsan progrediáló kóros idegrendszeri folyamatok megállításában. Orv Hetil. 2018; 159(3): 107–112. | Abstract: Introduction: The role of autoimmune responses against central nervous system (CNS) antigens in encephalitis presenting with non-classified neurologic or psychiatric symptoms has been appreciated in the past decade. Paraneoplastic limbic encephalitis has a poor prognosis and is most commonly associated with lung, ovarium, and testicular neoplasms, leading to immune reactions against intracellular antigens (anti-Hu/ANNA1, anti-Ri/ANNA2, anti-CV2/CRMP5 and anti-Ma2/Ta). In contrast, the recently described autoimmune encephalitis subtypes present with a broad spectrum of symptoms, respond to autoimmune therapies well and usually associate with autoantibodies against neuronal cell surface receptors (NMDAR, GABABR, AMPAR) or synaptic proteins (LGI1, CASPR2). Aim: Our aim is to bring to awareness the increasing number of autoimmune encephalitis patients requiring neurologic, psychiatric and intensive care and to emphasize the significance of detecting various autoantibodies in diagnosing patients. Method: In the past 6 years, our laboratory received 836 autoimmune encephalitis diagnostic test requests from a total of 717 patients. Serum and cerebrospinal fluid (CSF) samples were analysed with indirect immunofluorescence using a BIOCHIP consisting of cell lines transfected with 6 different receptor proteins. Results: IgG autoantibodies against receptor proteins were present in 7.5% of patients. The frequency of positive samples was the following: NMDAR > LGI1 > GABABR > CASPR2. Conclusion: Detecting autoantibodies facilitates the diagnosis of autoimmune encephalitis in an early stage. Patients diagnosed early can be effectively treated with plasmapheresis and immunosuppressive drugs. The efficiency of therapies can be monitored by autoantibody detection. Therefore, the diagnostic immune laboratory plays an important role in proper diagnosis and in the prevention of rapidly progressing symptoms. Orv Hetil. 2018; 159(3): 107–112

Anti-neuronal autoantibodies (cell surface and onconeuronal) and their association with natural autoantibodies in synthetic cannabinoid induced psychosis

Patients suffering from encephalitis may present psychiatric symptoms; however, the clinical relevance of anti-neuronal antibodies in patients experiencing a psychotic episode without encephalitis is still unclear. In this study, we examined the presence of anti-neuronal cell surface autoantibodies and onconeural autoantibodies in serum samples of 22 synthetic cannabinoid users presenting with psychosis. We found only two positive cases; however, seven patients had borderline results. Nonetheless, we found no significant correlation between anti-neuronal autoantibodies and the intensity of psychosis indicated by the Positive and Negative Syndrome Scale (PANSS) scores. The length of drug use and the combination of other drugs with synthetic cannabinoids have no significant effect on anti-neuronal autoantibody positivity. Nonetheless, the ratio of anti-citrate synthase (anti-CS) IgM and IgG natural autoantibodies was significantly lower (p = 0.036) in the anti-neuronal autoantibody-positive/borderline samples, than in the negative group. Interestingly, anti-CS IgM/IgG showed a significant negative correlation with PANSS-positive score (p = 0.04, r = −0.464). Our results demonstrated that anti-neuronal autoantibody positivity occurs in synthetic cannabinoid users, and the alteration of anti-CS IgM/IgG natural autoantibody levels points to immunological dysfunctions in these cases