Ig Synthesis Research Articles

The kappa free light chains index is an accurate diagnostic biomarker for paediatric multiple sclerosis.

Multiple sclerosis (MS) may occur before the age of 18. Differentiation between paediatric MS (PedMS) and other demyelinating syndromes (ODSs) is challenging. In adult with MS, the kappa free light chain (KFLC) index has proven to be a reliable marker of intrathecal Ig synthesis. To assess the diagnostic value of the KFLC index in a cohort of patients with paediatric-onset, inflammatory disorders of the CNS. We included 73 patients and divided them into four groups: PedMS (n = 16), ODS (n = 17), encephalitis and/or inflammatory epilepsy (EE, n = 15), and controls without inflammatory CNS diseases (n = 25). The KFLC index was calculated and compared with the results of the oligoclonal bands determination. The KFLC index was higher in the PedMS group (median (interquartile range (IQR)): 150.9 (41.02-310.6)) than in the ODS (3.37 (2.22-8.11)), the EE (5.53 (2.31-25.81)) and the control group (3.41 (2.27-5.08)), respectively. The best KFLC index cut-off for differentiating between patients with PedMS and controls was 6.83 (sensitivity: 100%; specificity: 92%). A KFLC index over 93.77 indicated that the patient is very likely to have PedMS (sensitivity: 68%; specificity: 100%). The KFLC index is a reliable tool for the diagnosis of MS in a paediatric population.

Implications of monoclonal gammopathy and isoelectric focusing pattern 5 on the free light chain kappa diagnostics in cerebrospinal fluid.

Oligoclonal bands (OCB) analysis is the reference standard for detecting an intrathecal IgG synthesis. Alongside OCB, free light chains kappa (FLCκ) are considered an additional sensitive biomarker for determining patterns 2 or 3, indicating intrathecal Ig synthesis. However, kFLC IF is not suitable for detecting a monoclonal pattern 5. The primary aim of this study was to evaluate the impact of incorporating FLCκ analysis into routine cerebrospinal fluid (CSF) diagnostics instead of OCB testing on the rate of missed monoclonal IgG detection. A two-center retrospective biomarker study was conducted. OCB were identified using isoelectric focusing in polyacrylamide gels followed by silver staining or in agarose gels followed by immunofixation. FLCκ were quantified using nephelometry and FLCκ assay (Siemens). Out of a combined total of 17,755 OCB analyses conducted between 2011 and 2021, a subset of 269 cases (1.5 %) exhibited pattern 5. 98 samples (36 %), which included 18 samples with intrathecal inflammation as determined by additional OCB pattern 2 were included in the FLCκ analysis. Of those, 16 (89 %) had intrathecal FLCκ synthesis. While FLCκ offers a promising avenue for detecting an intrathecal inflammation, the pattern 5, though rare, remains a valuable additional finding of OCB analysis. A combined approach of FLCκ and OCB analysis is recommended for a comprehensive assessment of the humoral intrathecal immune response.

Significance of immunoglobulins synthesis with central nervous system involvement in Neuro-Behçet’s disease

ObjectivesDemyelination and immunocyte-infiltrated lesions have been found in neuro-Behçet’s disease (NBD) pathology. Lacking satisfying laboratory biomarkers in NBD impedes standard clinical diagnostics. We aim to explore the ancillary indicators for NBD diagnosis unveiling its potential etiology. Methods28 NBD with defined diagnosis, 29 patients with neuropsychiatric lupus erythematosus, 30 central nervous system idiopathic inflammatory demyelination diseases (CNS-IIDD), 30 CNS infections, 30 cerebrovascular diseases, and 30 noninflammatory neurological diseases (NIND) were retrospectively enrolled. Immunoglobulins (Ig) in serum and cerebral spinal fluid (CSF) were detected by immunonephelometry and myelin basic protein (MBP) by quantitative enzyme-linked immunosorbent assay. ResultsIgA index is almost twice enhanced in NBD than NIND with an accuracy of 0.8488 in differential diagnosis, the sensitivity and specificity of which were 75.00 % and 90.00 % when the cutoff was > 0.6814. The accuracy of CSF Ig and quotient of Ig all exceed 0.90 in discerning NBD with damaged and intact blood–brain barrier (BBB). Clustering analyses divided NBD into two different phenotypes: one with BBB damage has lower Ig synthesis, the other with extra-synthesis in parenchymal sites but with intact BBB. MBP index is significantly correlated with kappa (KAP) index and lambda (LAM) index (r = 0.358, 0.575, P < 0.001), hinting the NBD pathogenesis of CNS demyelination in triggering excessive intrathecal Ig productions and humoral responses. ConclusionsIgA index acts as a potential diagnostic indicator in differentiating NBD from NIND and CNS-IIDD. Excessive immunoglobulin production induced by CNS inflammation and demyelination might be latent immunopathogenesis of NBD.

Complement Activation Is Associated With Disease Severity in Multiple Sclerosis.

Histopathologic studies have identified immunoglobulin (Ig) deposition and complement activation as contributors of CNS tissue damage in multiple sclerosis (MS). Intrathecal IgM synthesis is associated with higher MS disease activity and severity, and IgM is the strongest complement-activating immunoglobulin. In this study, we investigated whether complement components (CCs) and complement activation products (CAPs) are increased in persons with MS, especially in those with an intrathecal IgM synthesis, and whether they are associated with disease severity and progression. CC and CAP levels were quantified in plasma and CSF of 112 patients with clinically isolated syndrome (CIS), 127 patients with MS (90 relapsing-remitting, 14 primary progressive, and 23 secondary progressive), 31 inflammatory neurologic disease, and 44 symptomatic controls from the Basel CSF databank study. Patients with CIS/MS were followed in the Swiss MS cohort study (median 6.3 years). Levels of CC/CAP between diagnosis groups were compared; in CIS/MS, associations of CC/CAP levels with intrathecal Ig synthesis, baseline Expanded Disability Status Scale (EDSS) scores, MS Severity Score (MSSS), and neurofilament light chain (NfL) levels were investigated by linear regression, adjusted for age, sex, and albumin quotient. CSF (but not plasma) levels of C3a, C4a, Ba, and Bb were increased in patients with CIS/MS, being most pronounced in those with an additional intrathecal IgM production. In CIS, doubling of C3a and C4a in CSF was associated with 0.31 (CI 0.06-0.56; p = 0.016) and 0.32 (0.02-0.62; p = 0.041) increased EDSS scores at lumbar puncture. Similarly, doubling of C3a and Ba in CIS/MS was associated with 0.61 (0.19-1.03; p < 0.01) and 0.74 (0.18-1.31; p = 0.016) increased future MSSS. In CIS/MS, CSF levels of C3a, C4a, Ba, and Bb were associated with increased CSF NfL levels, e.g., doubling of C3a was associated with an increase of 58% (Est. 1.58; CI 1.37-1.81; p < 0.0001). CNS-compartmentalized activation of the classical and alternative pathways of complement is increased in CIS/MS and associated with the presence of an intrathecal IgM production. Increased complement activation within the CSF correlates with EDSS, future MSSS, and NfL levels, supporting the concept that complement activation contributes to MS pathology and disease progression. Complement inhibition should be explored as therapeutic target to attenuate disease severity and progression in MS.

Effects of tofersen treatment in patients with SOD1-ALS in a “real-world” setting – a 12-month multicenter cohort study from the German early access program

In April 2023, the antisense oligonucleotide tofersen was approved by the U.S. Food and Drug Administration (FDA) for treatment of SOD1-amyotrophic lateral sclerosis (ALS), after a decrease of neurofilament light chain (NfL) levels had been demonstrated. Between 03/2022 and 04/2023, 24 patients with SOD1-ALS from ten German ALS reference centers were followed-up until the cut-off date for ALS functional rating scale revised (ALSFRS-R), progression rate (loss of ALSFRS-R/month), NfL, phosphorylated neurofilament heavy chain (pNfH) in cerebrospinal fluid (CSF), and adverse events. During the observation period, median ALSFRS-R decreased from 38.0 (IQR 32.0-42.0) to 35.0 (IQR 29.0-42.0), corresponding to a median progression rate of 0.11 (IQR-0.09 to 0.32) points of ALSFRS-R lost per month. Median serum NfL declined from 78.0pg/ml (IQR 37.0-147.0pg/ml; n=23) to 36.0pg/ml (IQR 22.0-65.0pg/ml; n=23; p=0.02), median pNfH in CSF from 2226pg/ml (IQR 1061-6138pg/ml; n=18) to 1151pg/ml (IQR 521-2360pg/ml; n=18; p=0.02). In the CSF, we detected a pleocytosis in 73% of patients (11of 15) and an intrathecal immunoglobulin synthesis (IgG, IgM, or IgA) in 9 out of 10 patients. Two drug-related serious adverse events were reported. Consistent with the VALOR study and its Open Label Extension (OLE), our results confirm a reduction of NfL serum levels, and moreover show a reduction of pNfH in CSF. The therapy was safe, as no persistent symptoms were observed. Pleocytosis and Ig synthesis in CSF with clinical symptoms related to myeloradiculitis in two patients, indicate the potential of an autoimmune reaction. No funding was received towards this study.

Intrathecal nonspecific immunoglobulin synthesis in syphilitic infection

Intrathecal Ig synthesis is one of the characteristic laboratory features of neurosyphilis (caused by Treponema pallidum subspecies pallidum able to affect the central nervous system (CNS), but a persisting pathogen-driven immune response is problem of scar effect. This long-term Ig production within the CNS compartment even after timely and proper treatment remains unexplored. We hypothesized that a long-term current chronic inflammation in the CNS caused by syphilitic infection can be a trigger of nonspecific humoral immune response and the scar effect may result from non-specific B cell activation within the CNS in the absence of antigens. The MRZ reaction is a laboratory tool for detection of intrathecal nonspecific humoral immune reaction, which is poorly investigated in syphilis patient. The MRZ reaction (MRZR) is used as a marker of intrathecal nonspecific humoral immune response and is composed of the three antibody indices (AI) against neurotropic viruses: measles, rubella and varicella zoster virus. A positive MRZR, defined as an elevated AIs ( 1.5) against at least one or more viral agents, confirms diagnosis of a chronic or autoimmune disease involving CNS recently declared as a highly specific marker of multiple sclerosis. Insufficient data on neuroinfections motivated us to examine a hypothesis that a long-term chronic inflammation within the CNS compartment caused by syphilitic infection may be a trigger of intrathecal nonspecific humoral immune response. We assessed prevalence of positive MRZR in 147 patients with later-stage syphilis seropositive for MRZ viruses. Study group included 43 patients with first-time diagnosed neurosyphilis, 16 patients with a follow-up visit after neurosyphilis treatment, 88 patients with excluded neurosyphilis. There were no significant differences between categories of patients examined, and presence of positive MRZR was not associated with any demographic, clinical and laboratory characteristics. To sum up, our study (n = 147) showed that 27 patients (18%, 95% CI: 1225% ) were positive for intrathecal Ig production against one, two or three of the M, R, Z viruses, and bi- and tri-specific MRZR was present in 7 of 147 patients (5%, 95% CI: 210%). The data obtained evidence that frequency of the MRZR for syphilitic infection matches that of found in general population and a long-term current syphilitic infection affecting CNS seems to not be a trigger of a nonspecific intrathecal immune response. Among our patients, we also selected 43 patients with previously treated syphilitic infection without any neurological symptoms and excluded neurosyphilis diagnosis, so these patients can be considered as an apparently healthy group.

No evidence of oligoclonal bands, intrathecal immunoglobulin synthesis and B cell recruitment in acute ischemic stroke.

Within the past 10 years, immune mechanisms associated with acute ischemic stroke (AIS) have been brought into focus, but data on B cell activation and intrathecal Ig production is still scarce. In this study, we determined the prevalence of an elevated IgG index, positive oligoclonal bands (OCBs) and chemokine C-X-C motif ligand 13 (CXCL13) levels in the cerebrospinal fluid (CSF) as markers of intrathecal IgG synthesis and B cell activation in patients with AIS. In a retrospective study we analyzed the cerebrospinal fluid (CSF) from 212 patients with AIS from December 2013 to May 2018 assessing intrathecal Ig synthesis, OCBs and CXCL13 concentrations. Overall, 5.7% (12/212) of AIS patients showed an intrathecal IgG synthesis, 0.5% (1/212) with isolated elevated IgG index, 5.2% (7/136) isolated positive OCBs and 2.9% (4/136) both elevated IgG index and positive OCBs. CXCL13 levels were elevated in 3.6% (3/83) of the patients. Approximately one third of these patients had simultaneously chronic inflammatory CNS disease (multiple sclerosis, neuromyelitis optica spectrum disorder, neurosarcoidosis). There was no significant association between CSF findings and stroke characteristics including vascular territory, localization, volume, etiology, acute treatment, or blood-brain barrier dysfunction. Intrathecal IgG synthesis was more common in patients with prior stroke. Longitudinal CSF analysis did not reveal any newly-occurring, but instead mostly persistent or even disappearing intrathecal IgG synthesis after AIS. We found no evidence of a relevant B cell recruitment and intrathecal IgG synthesis in patients with AIS. In fact, the occurrence of intrathecal IgG synthesis was associated with concurrent chronic inflammatory CNS disease or previous stroke. Consequently, in patients with first-ever AIS and intrathecal IgG synthesis, physicians should search for concomitant inflammatory CNS disease.

A FAM46C-Dependent Molecular Circuit Modulates Antibody Synthesis and Proliferation in Plasma Cell Dyscrasias

Plasma cells (PCs) are the terminal effectors of B lymphocyte differentiation in charge of antibody production. Such intensive task requires rapid endoplasmic reticulum (ER) expansion and the concerted induction of the machinery affording ER import, folding, and trafficking along the secretory route. Moreover, the by-products of massive immunoglobulin (Ig) production cause a peculiar dependence of PCs on protein degradative pathways with a crucial clinical consequence: theextraordinary success of proteasome inhibitors against multiple myeloma (MM) and systemic light chain amyloidosis (AL). Notably, an essential player in the proteomic reshaping occurring during PC differentiation is the non-canonical poly(A)polymerase FAM46C/TENT5C. Upon B cell activation, FAM46C is induced to polyadenylate and stabilize mRNAs encoding Igs and ER-targeted proteins, thus promoting humoral immune responses (Mroczek et al., 2017; Bilska et al., 2020). Interestingly, FAM46C locus on chromosome 1p12 is mutated or deleted in up to 20% MM patients (Chapman et al., 2011; Boyd et al., 2011, Barbieri et al., 2016), suggesting that MM cells are subject to a selective pressure to restrict FAM46C expression and activity, to ultimately curb Ig synthesis and secretion in favor of cell survival and tumor growth. Accordingly, exogenous re-expression of FAM46C in mutated MM cells raised the secretory capacity beyond sustainability, resulting in intracellular ATP reduction, ROS accumulation and increased apoptotic susceptibility (Fucci et al., 2020). Our research aims to discover the precise molecular circuits, centered on FAM46C, regulating PC secretory capacity in efficient coordination with degradative pathways, to discover unprecedented vulnerabilities of PCs and new therapeutic opportunities against PC dyscrasias. To clarify the molecular mechanisms underlying the effects of FAM46C on Ig secretion we combined cutting-edge protein biochemistry, imaging and unbiased proteomic assays and adopted in vitro and in vivo models of MM and AL. In these models, we disclosed that FAM46C concertedly promotes the expression of ER and Golgi proteins, potently expanding the secretory apparatus and light chain (LC) secretion. To assess the impact of FAM46C on PC proliferation and disease progression in vivo, we silenced or overexpressed FAM46C in AMLC-2 cells that bear a hemizygous loss in 1p12 region (Arendt et al., 2008). Following intravenous injection into Rag2-/- IL2rg-/- mice, we found remarkably reduced tumor growth and longer survival in recipients of FAM46C-overexpressing cells, despite higher initial circulating LC levels, as compared to other groups. Moreover, we observed intermediate disease progression in mice receiving mock-transduced ALMC-2 cells, attesting to dose-dependent impact of FAM46C. The in vivo association of higher secretory activity with drastically decreased PC proliferation reveals that FAM46C uncouples monoclonal component levels from disease burden. Finally, among FAM46C-modulated proteins, we found SRP components and RNA methyltransferases involved in ribosome assembly and tRNA processing. The data unveil a new role played by FAM46C in PCs, in addition to its mRNA stabilizing activity, indicating the existence of a previously unrecognized integrated network coordinating ribosome biogenesis, protein translation, and ER expansion, potentially crucial to harmonize intensive protein secretion with cellular homeostasis. Altogether, our studies disclose an unexpected molecular circuit orchestrating Ig synthesis and secretion, and regulating PC proliferation and survival that may be exploited to design new therapeutic strategies aimed to reduce pathogenic LC production, to eliminate MM and AL cells, and to improve the efficacy of current therapeutic regimens.

The deubiquitinase OTUD1 regulates immunoglobulin production and proteasome inhibitor sensitivity in multiple myeloma

Serum monoclonal immunoglobulin (Ig) is the main diagnostic factor for patients with multiple myeloma (MM), however its prognostic potential remains unclear. On a large MM patient cohort (n = 4146), we observe no correlation between serum Ig levels and patient survival, while amount of intracellular Ig has a strong predictive effect. Focused CRISPR screen, transcriptional and proteomic analysis identify deubiquitinase OTUD1 as a critical mediator of Ig synthesis, proteasome inhibitor sensitivity and tumor burden in MM. Mechanistically, OTUD1 deubiquitinates peroxiredoxin 4 (PRDX4), protecting it from endoplasmic reticulum (ER)-associated degradation. In turn, PRDX4 facilitates Ig production which coincides with the accumulation of unfolded proteins and higher ER stress. The elevated load on proteasome ultimately potentiates myeloma response to proteasome inhibitors providing a window for a rational therapy. Collectively, our findings support the significance of the Ig production machinery as a biomarker and target in the combinatory treatment of MM patients.

Intrathecal immunoglobin synthesis and its role in patients with neurosyphilis.

Intrathecal protein synthesis (ITS) occurs in various central nervous system disorders, but few quantitative studies have focused on ITS for neurosyphilis (NS) in southwestern China. We made a study to quantitatively assess the ITS in patients with NS and to investigate the association between ITS and the stages of NS. CSF-serum specimen pairs from 142 patients (66 NS and 76 non-NS/syphilis) were collected for routine CSF and serum tests. The NS group was divided into slight and severe subgroups according to the NS stages. Three formulas for the quantitative determination of the intrathecal synthesis were calculated to characterize the specimens, including the Ig index (QIg/Qalb), Ig extended index (Ig_EI), and intrathecally synthesized fraction (IgIF) using the hyperbolic function. The role of QTPPA/QIgG as an antibody index (AI = Q specific Ig/QIgG) was also explored. Sero_TRUST titres (1:16, 1:1-1:256), sero_TPPA titres (1:163840, 1:1280-1:1310720), total protein (MTP), and CSF_Igs (p < 0.05) were found to be significantly elevated in the NS group. Intrathecal Ig synthesis can be identified using all three formulas in the NS group. The pattern of Ig intrathecal synthesis was IgIF-G (48.62%) > IgIF-A = IgIF-M (p < 0.05), with the dominant intrathecal fraction being IgG (median, 48.62%), which was also verified by QIgG> Qalb> QIgM = QIgA. In the slight NS group, the intrathecal fractions of IgM (>0 in 4 out of 20 cases) and IgG (>0 in 16 out of 20) were lower than the intrathecal fractions of IgM (>0 in 19 out of 35 cases) and IgG (>0 in 33 out of 38) in the severe group (p < 0.05). The area under the curve (AUC) of the CSF_TPPA antibody index was 0.867 (0.792, 0.922), with an optimal cutoff point of 0.81, providing a sensitivity of 88.91% and specificity of 84.62%. Although the intrathecal synthesis pattern is IgG dominant in patients with NS, brain-derived IgM and IgA can also be found. Moreover, intrathecal IgM and IgG were associated with a parenchymatous type of neurosyphilis. Syphilis-specific antibodies are a new potential tool for NS diagnosis.

Peripheral Nerve Involvement at First Diagnosis of Multiple Sclerosis: A Prospective MR Neurography Study.

The aim of this study was to assess peripheral nerve involvement in patients with multiple sclerosis (MS) at first clinical presentation using quantitative magnetic resonance (MR) neurography in correlation with clinical, laboratory, electrophysiological, and central nervous MR imaging data. In this prospective monocentric study, 30 patients first diagnosed with MS according to the McDonald criteria (19 women; mean age, 32.4 ± 8.8 years) and 30 age- and sex-matched healthy volunteers were examined with high-resolution 3 T MR neurography using a dual-echo T2-relaxometry sequence covering the tibial and peroneal nerves from proximal thigh to distal calf. Magnetic resonance biomarkers of T2 relaxation time (T2 app ), proton spin density (PSD), and nerve cross-sectional area (CSA) were correlated with clinical symptoms, intrathecal immunoglobulin (Ig) synthesis, nerve conduction study, and lesion load on brain and spine MR imaging. The diagnostic accuracy of MR biomarkers was assessed using receiver-operating characteristic curves. Diffuse nerve changes were detected along the tibial and peroneal nerves in MS patients, who showed decreased PSD ( P < 0.001), increased T2 app ( P < 0.001), and smaller tibial nerve CSA ( P < 0.001) compared with healthy subjects. Tibial PSD was identified as best parameter separating patients from controls (area under the curve = 0.876). Intrathecal IgG and IgM synthesis correlated with PSD values ( r = -0.44, P = 0.016, and r = -0.42, P = 0.022). Contrast-enhancement of brain or spine lesions was related to larger tibial and peroneal CSA ( P < 0.001, P = 0.033). Abnormal electrophysiology correlated with higher tibial and peroneal T2 app ( P < 0.001 and P = 0.033), lower tibial and peroneal PSD ( P = 0.018 and P = 0.002), and smaller peroneal CSA ( P < 0.001). Quantitative MR neurography reveals peripheral nerve changes in patients with initial diagnosis of MS. Correlation of imaging findings with intrathecal immunoglobulin synthesis may indicate a primary coaffection of the peripheral nervous system in MS.

Kappa Free Light Chains in Cerebrospinal Fluid in Inflammatory and Non-Inflammatory Neurological Diseases.

Background: Oligoclonal bands represent intrathecal immunoglobulin G (IgG) synthesis and play an important role in the diagnosis of multiple sclerosis (MS). Kappa free light chains (KFLC) are increasingly recognized as an additional biomarker for intrathecal Ig synthesis. However, there are limited data on KFLC in neurological diseases other than MS. Methods: This study, conducted at two centers, retrospectively enrolled 346 non-MS patients. A total of 182 patients were diagnosed with non-inflammatory and 84 with inflammatory neurological diseases other than MS. A further 80 patients were classified as symptomatic controls. Intrathecal KFLC production was determined using different approaches: KFLC index, Reiber’s diagram, Presslauer’s exponential curve, and Senel’s linear curve. Results: Matching results of oligoclonal bands and KFLC (Reiber’s diagram) were frequently observed (93%). The Reiber’s diagram for KFLC detected intrathecal KFLC synthesis in an additional 7% of the patient samples investigated (4% non-inflammatory; 3% inflammatory), which was not found by oligoclonal band detection. Conclusions: The determination of both biomarkers (KFLC and oligoclonal bands) is recommended for routine diagnosis and differentiation of non-inflammatory and inflammatory neurological diseases. Due to the high sensitivity and physiological considerations, the assessment of KFLC in the Reiber’s diagram should be preferred to other evaluation methods.

Disrupting Mitohormesis As a Novel Therapeutic Strategy for Multiple Myeloma (MM) Including Those with High Risk Disease and Proteosome Inhibitor Resistance

Background: Moderate mitochondrial stress induced by multiple mediators but most notably ROS can lead to activation of persistent mito-protective mechanisms termed “Mitohormesis”. As a result of massive protein synthesis, malignant plasma cells (PCs) from MM patients (pts) undergo substantial ER stress but in addition high rates of Ig synthesis contributes to overproduction of ROS. We hypothesized that MM cells exploit mitohormesis to maintain ROS in the hometic zone, thereby increasing mitochondrial fitness to avoid apoptosis. We therefore set out to determine if the processes of mitohormesis are activated in MM and whether unmitigated mitochondrial stress can be exploited as a therapeutic strategy in MM.Results: Protective stress mechanisms of mitohormesis include the activation of the mitochondrial UPR (UPR MT),a mitochondrial-to-nuclear signaling pathway mediated by CHOP and ATF5 that upregulates mitochondrial import proteins, chaperones and proteases to maintain mitochondrial proteastasis. We first demonstrated that UPR MT activation occurs with progression from precursor to overt MM. Using a UPR MTgene signature derived from published gene-sets we observed upregulation of UPR MT genes in single-cell RNA sequencing (scRNA-seq) data generated from PCs derived from Vκ*MYC mice (a transgenic mouse model of MM) spanning the spectrum of the disease. UPR MT gene signature scores in PCs from mice increased with disease progression with the highest levels found in late-MM> int-MM> early MM>wild type mice. Similarly, analysis of publicly available gene expression datasets (GSE6477) that includes normal donors, MGUS and newly diagnosed MM (NDMM) revealed higher expression of UPR MT genes in the majority of NDMM, weak expression in MGUS and absence in normal PCs. To assess the impact of UPR MT expression on pt outcomes we calculated a UPR MT index score derived from the median expression of 12 mtUPR classifier genes across the MMRF CoMMpass dataset of NDMM pts. Stratifying pts by UPR MT expression score we found that pts in the top quartile had a significantly shorter PFS and OS compared to pts with the lowest quartile weighted score.Next, we postulated that perturbation of the mitochondrial import protein, Translocase of the Inner Membrane 23 (TIM23) would exaggerate mitochondrial stress as mitochondrial import efficiency is a key regulator of the UPR MT. First, we demonstrated that TIM23 complex genes are enriched in pts from the CoMMpass dataset with poor risk (1q gain and PR gene signature) and that shorter PFS and OS is associated with a higher weighted score of TIM23 complex genes. We then demonstrated that genetic (shRNA) knockdown or pharmacologic inhibition of TIM23 with MB-10, a small molecule inhibitor of TIM23 induced apoptosis of MM cell lines and primary pt PCs. Further non-transformed cell lines, CD138 - non-MM cells and normal donor hematopoietic progenitor cells were less susceptible to the effects of MB-10. Consistent with activation of the UPR MT, treatment of MM cells resulted in increased cytosolic ATF4, CHOP and a shift of ATF5 to the nuclear fraction. Activation of the CHOP-dependent branch of the UPR MT resulted in in upregulation of mitochondrial-targeted proteins, cpn10 and ClpP. Interestingly, MB-10 also induced XBP1 splicing demonstrating that inhibition of TIM23 complex can simultaneously activate the IRE1/XBP1 branch of integrated stress response (ISR), This led us to hypothesize that targeting TIM23 as an alternative means of activating the ISR could overcome acquired resistance to proteosome inhibitors (PIs). Indeed, PI-resistant and parental isogenic cell lines were equally susceptible to MB-10 as measured by IC50 values of cell growth. Finally, we demonstrated that doxycycline inducible knockdown of TIM23 in a mouse xenograft model induced tumor regression with significantly small tumor volumes at the end of 17 days of doxycycline treatment compared to tumors expressing an inducible control vector.Conclusions: These data demonstrate that mitohormesis and UPR MT activation is associated with MM progression and worse clinical outcomes. Further we show that disrupting mitochondrial protein import results in unmitigated mitochondrial stress that switches the UPR MT from an adaptive cytoprotective to cytotoxic proapoptotic response. Thus, targeting mitochondrial import proteins such as TIM23 may represent novel therapeutic targets for MM. DisclosuresSchimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Trudel: Janssen: Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Sanofi: Honoraria; Pfizer: Honoraria, Research Funding; Genentech: Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding.

Effects of Natalizumab Therapy on Intrathecal Immunoglobulin G Production Indicate Targeting of Plasmablasts.

ObjectivesTo evaluate the long-term effects of natalizumab (NTZ) on different features of intrathecal immunoglobulin (Ig) synthesis in patients with multiple sclerosis (MS) and to quantify the expression of α4-integrin in stages of B-cell maturation.MethodsWe combined a cross-sectional (49 NTZ-treated MS patients, mean treatment duration 5.1 years, and 47 untreated MS patients) and a longitudinal study (33 patients with MS before and during NTZ, mean treatment duration: 4.8 years), analyzing paired serum and CSF samples for IgG, IgA, and IgM levels, reactivity against selected viruses (measles virus, rubella virus, and varicella zoster virus [MRZ] reaction), and oligoclonal bands (OCBs). Banding patterns before and after therapy were directly compared by isoelectric focusing in 1 patient. In addition, we determined the expression of α4-integrin by FACS analysis on blood-derived B-cell subsets (plasmablasts, memory B cells, and naive B cells) of healthy controls.ResultsIn serum, NTZ decreased IgM and IgG, but not IgA, levels. IgM hypogammaglobulinemia occurred in 28% of NTZ-treated patients. In CSF, NTZ treatment resulted in a strong reduction of intrathecally produced IgG and, to a lesser extent, IgA, whereas IgM indices [(Ig CSF/Serum)/(Albumin CSF/Serum)] remained largely unchanged. Reduction of the IgG index correlated with NTZ treatment duration, as did serum IgM and IgA levels. MRZ reaction was unchanged and OCB persisted. Direct comparison of OCB pattern before and after NTZ revealed the persistence of individual bands. α4-Integrin expression was highest on plasmablasts (CD19+CD38+CD27+).ConclusionOur data indicate that NTZ reduces short-lived plasmablasts in the CNS compartment but has little effect on locally persisting long-lived plasma cells.

Applications of ozone in dentistry and oral health: A systemic review

The concept of dental ozone therapy is significantly becoming a trend nowadays, O3 is pure ozone gas that has a soft sky-blue color with a pungent smell extremely reactive, powerful oxidizing agent. It has many biomedical properties like antibacterial, immune-stimulating, synthesis of Igs, etc. There are various uses of ozone in the field of dentistry, and the objective of this review is to shed light on clinical dental applications of ozone and summarize the research findings regarding the use of ozone in dentistry. This study shows that Oral lichen planus-Lichen planus is an inflammatory condition that affects the oral mucous membranes with clinical features of white, lacy patches or red swollen lesions and open sores in the gum. In dentistry, ozone has proved to be successful in the treatment of root caries, non cavitated fissure carious lesions, early carious lesions in teeth, dental surgery or following tooth extraction processes and reduction of pathogenic micro- organisms of carious dentine.

Restriction d’hétérogénéité des gammaglobulines sur l’électrophorèse des protéines sériques

AbstractElectrophoresis allows the separation of serum proteins into different fractions in order to study their variations and to interpret them. The gamma globulin zone, in which a large proportion of immunoglobulins (Ig) migrate, gives a picture of the immune response. The normal pattern of this zone is a Gaussian distribution of immunoglobulins, corresponding to a polyclonal synthesis of Ig. Other aspects can be observed, restriction of heterogeneity, oligoclonality and monoclonality. Heterogeneity restriction is the most difficult aspect to interpret, should Ig be identified? Is it necessary to check this aspect regularly? It appears that in some cases it is essential to discuss the clinical context with the physician. This article addresses the problem of defining and perceiving the aspects of heterogeneity restriction and their interpretation, illustrated by examples of concrete clinical contexts.

Kappa Free Light Chains in the Context of Blood Contamination, and Other IgA- and IgM-Related Cerebrospinal Fluid Disease Pattern.

In this retrospective, monocentric cohort study, we tested if an intrathecal free light chain kappa (FLC-k) synthesis reflects not only an IgG but also IgA and IgM synthesis. We also analysed if FLC-k can help to distinguish between an inflammatory process and a blood contamination of cerebrospinal fluid (CSF). A total of 296 patient samples were identified and acquired from patients of the department of Neurology, University Medicine Greifswald (Germany). FLC-k were analysed in paired CSF and serum samples using the Siemens FLC-k kit. To determine an intrathecal FLC-k and immunoglobulin (Ig) A/-M-synthesis we analysed CSF/serum quotients in quotient diagrams, according to Reiber et al. Patient samples were grouped into three cohorts: cohort I (n = 41), intrathecal IgA and/or IgM synthesis; cohort II (n = 16), artificial blood contamination; and the control group (n = 239), no intrathecal immunoglobulin synthesis. None of the samples had intrathecal IgG synthesis, as evaluated with quotient diagrams or oligoclonal band analysis. In cohort I, 98% of patient samples presented an intrathecal synthesis of FLC-k. In cohort II, all patients lacked intrathecal FLC-k synthesis. In the control group, 6.5% presented an intrathecal synthesis of FLC-k. The data support the concept that an intrathecal FLC-k synthesis is independent of the antibody class produced. In patients with an artificial intrathecal Ig synthesis due to blood contamination, FLC-k synthesis is lacking. Thus, additional determination of FLC-k in quotient diagrams helps to discriminate an inflammatory process from a blood contamination of CSF.

The dental applications of ozone: A review

Concept of dental ozone therapy is significantly becoming a trend nowadays, O3 is pure ozone gas has a soft sky-blue colour with a pungent smell extremely reactive, powerful oxidizing agent. It has a many biomedical properties like antibacterial, immune-stimulating, synthesis of Igs, etc. There are various uses of ozone in the field of dentistry, and the objective of this review is to shed light on clinical dental applications of ozone and summarize the research findings regarding the use of ozone in dentistry. Keywords: Ozone (O3).

Identification of Novel Regulatory Pathway for Immunoglobulin Production Provides Rational Treatment for Bortezomib-Resistant Multiple Myeloma Patients

Introduction Monoclonal immunoglobulin (Ig) is a valuable diagnostic marker in patients with multiple myeloma (MM). An inevitable consequence of extensive Ig synthesis is overload of misfolded proteins that saturate proteasome capacity making the myeloma cells highly sensitive to proteasome inhibitors (PI). Even though PI are regularly used in the clinic, resistance often emerges leaving clinicians with limited treatment options. Therefore, there is a need for a robust marker selecting MM patients for precise PI-based combination therapy. Methods We performed a multiple database search for genes associated with Ig production and MM patients' survival. Additionally, we compared gene expression profiles (RNAseq) of primary MM cells with low and high Ig levels. Next, we validated the identified hits by shRNA knockdown and overexpression studies using myeloma cell lines, primary MM samples, and mouse models. We also applied mass spectrometry-based proteomic analysis, advanced biochemical approaches, and genetic models to reveal the Ig production pathway components and function. Finally, we performed a limited rational drug screening to select suitable compounds for combination treatment. Results RNAseq and database mining revealed a strong association between the expression of plasma cell-specific deubiquitinase OTUD1, Ig production, and MM patient survival. Suppression of OTUD1 with shRNAs in RPMI8226 and MM1.S cell lines reduced Ig levels, increased proliferation, and induced bortezomib resistance. Conversely, inducible OTUD1 overexpression enhanced Ig production, slowed down proliferation, and increased bortezomib sensitivity. In the xenografts mouse models cells with high OTUD1 levels synthesized more Ig and developed smaller tumors. Intriguingly, the transcription of Ig genes was not influenced by OTUD1 expression suggesting that OTUD1 functions as a posttranslational regulator of Ig assembly. To gain mechanistic insight into the Ig pathway regulation by OTUD1, we utilized the biotin proximity labeling method (Turbo-ID) combined with mass spectrometry analysis. We found several novel OTUD1 interaction partners including the E3 ubiquitin ligase KEAP1 and endoplasmic reticulum (ER) redox protein PRDX4. We demonstrated that KEAP1 acts upstream of OTUD1 by regulating OTUD1 ubiquitination and stability. Consistently, survival analysis revealed that MM patients with high KEAP1 expression (low OTUD1) had a worse prognosis than patients with low levels of KEAP1 (high OTUD1). PRDX4 regulates disulfite bonds formation during protein folding and is uniquely expressed in fully differentiated plasma cells. Here, we revealed that OTUD1 specifically deubiquitinates and thus stabilizes PRDX4 inside the ER. Additionally, we performed rescue genetic experiments and found a direct link between the OTUD1-PRDX4 axis and Ig production. The increase in OTUD1 expression (high Ig) led to a dramatic increase in the total pool of ubiquitinated proteins formed mainly by misfolded Ig, while OTUD1 knockdown (low Ig) had an opposite effect. We showed that changes in the level of ubiquitinated proteins correlated with PI sensitivity. Of note, OTUD1 did not affect the expression of proteasome subunits, either their enzymatic activity. Our mechanistic findings prompted us to propose a novel therapeutic opportunity in PI resistant MM patients. We hypothesize that the resensitization of Ig low MM cells to PI could be achieved by enhancing ER stress leading to an increase in misfolded proteins that would ultimately saturate proteasomes. Indeed, from clinically relevant drugs tested so far, the HSP-90 inhibitor (17-AAG) reverted the PI resistance in OTUD1 low (Ig low) myeloma cells. An in vivo validation of the combination treatment and testing of Ig involvement in PI sensitivity and proliferation of MM cells is ongoing. Conclusion Here we present the discovery of a novel regulatory mechanism for Ig production in plasma cells. Based on our results and previously published studies, we conclude that Ig synthesis is a clinically significant factor related to PI response and MM patient survival. Our findings suggest that the intracellular Ig level is an important biomarker to identify patients benefiting the most from PI-based therapies. Finally, we provide a rational solution for selective, combination therapy to overcome PI resistance in MM patients with a decreased capacity to synthesize Ig. Figure Disclosures Hajek: Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy.

NF-κB signaling induces inductive expression of the downstream molecules and IgD gene in the freshwater carp, Catla catla.

Toll-like receptors (TLRs) in innate immune system act as primary sensors in detecting the microbial components and activate their signaling cascades to induce NF-κB (nuclear factor NF-κB) towards the augmentation of immunoglobulin (Ig) synthesis. To gain insights into the efficacy of NF-κB pathway in immunoglobulin D (IgD) synthesis in the Indian Major Carp Catla catla, cloning and sequencing of TLR-signaling downstream molecules [TRAF3 (TNF receptor-associated factor 3), NEMO (nuclear factor-kappa B essential modulator), NF-κB and BAFF (B cell activating factor)] were performed by infecting the fish with pathogens. mRNA expression analysis of the downstream molecules and IgD showed significant up-regulation of these genes in kidney (P ≤ 0.001) as compared to spleen (P ≤ 0.05). To ascertain the role of NF-κB pathway in IgD synthesis, the primary cell culture of kidney and spleen in monolayer cell suspension was treated with NF-κB inhibitor (BAY 11-7082) and down-regulation of BAFF, NEMO, NF-κB, and IgD gene was observed. These results highlight the importance of NF-κB signaling pathway in augmenting the IgD gene expression in the freshwater carp, Catla catla.