Cerebrospinal fluid biomarkers of superficial siderosis in patients with spontaneous intracranial hypotension.

BACKGROUND AND PURPOSE: Spontaneous intracranial hypotension (SIH) is an important etiology of infratentorial superficial siderosis (iSS) of the central nervous system. However, the prevalence of iSS amongst patients with SIH is unknown and the imaging findings of iSS might represent a late stage of disease. The aim was to identify cerebrospinal fluid (CSF) biomarkers of iSS in patients with SIH. METHODS: Consecutive patients evaluated for SIH at our institution between May 2017 and January 2019 were included. Lumbar CSF samples were analyzed for the presence of ferritin and bilirubin. Magnetic resonance imaging was assessed for the presence of iSS. RESULTS: Twenty-four patients with SIH were included. CSF samples were positive for bilirubin in 2/19 (10.5%). CSF ferritin was elevated in 7/23 (30.4%). Signs of iSS on imaging were present in four patients (16.7%). All patients with imaging signs of iSS demonstrated elevated CSF ferritin. Ferritin level was significantly higher amongst patients demonstrating iSS compared to those without (median 45.0 vs. 11.0 µg/l; p = 0.003). Symptom duration was longer in patients with iSS than in patients without iSS (median 40 months vs. 9 months, p = 0.018). CONCLUSION: Cerebrospinal fluid alterations indicative of iSS are prevalent amongst patients with SIH. It is speculated that a preclinical phase without symptoms or imaging signs but during which elevated biomarkers of the disease are apparent from CSF analysis might exist. It is suggested that measurement of CSF ferritin is incorporated in the work-up of patients with SIH to identify those at risk of developing iSS.

Analysis of IL-6 serum levels and CAR T cell-specific digital PCR in the context of cytokine release syndrome.

INTRODUCTION: Chimeric antigen receptor T-cell (CAR-T) therapies are increasingly used to treat relapsed B-cell lymphomas and acute lymphoblastic leukemia. Considering the frequency of cytokine release syndrome and CAR-T-related encephalopathy syndrome (CRS/CRES) after CAR-T administration, strategies enabling timely prediction of impending CRS/CRES are a clinical need. METHODS: We evaluated the dynamics of serum interleukin (IL)-6 levels and CAR-T transgene copy numbers by digital droplet polymerase chain reaction in the peripheral blood of 11 consecutive patients with aggressive B-cell malignancies. RESULTS: Four of 11 patients developed CRS, and 3 patients had CRES (33%), 2 of them had previous CRS. IL-6 levels increased on the day of clinical manifestation of CRS. All CRS patients had increased IL-6 peak levels (median IL-6 peak 606 pg/mL in CRS patients vs. 22 pg/mL in non-CRS patients, p = 0.0061). Different patterns emerged from the dynamics of CAR-T/µg genomic DNA: "rapid increase and rapid decrease with complete disappearance," "rapid increase and slow decrease with higher persistence," "rapid increase and rapid decrease with lower persistence," and "slow increase and rapid decrease with almost disappearance." Patients with the pattern "rapid increase and slow decrease with higher persistence" of CAR-T/µg genomic DNA concentration seemed to be at higher risk of developing CRS/CRES. CONCLUSION: Thus, the dynamics of CAR-T transgene copy numbers merits further evaluation for a possible association with manifestation of CRS. Increased IL-6 serum levels at CRS manifestation may contribute to the interpretation of symptoms.

Reduced survival after autologous stem cell transplantation in myeloma and lymphoma patients with low vitamin D serum levels.

High-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) is applied for consolidation in myeloma and relapsing lymphoma patients. Vitamin D (VitD) exerts effects during hematopoietic stem cell proliferation, differentiation and interactions with the immune system. VitD deficiency is frequent in patients with hematological malignancies, but its prognostic relevance after ASCT remains unclear. We investigated the effect of VitD serum levels in patients with lymphomas and myeloma at ASCT on progression-free (PFS) and overall survival (OS). The cohort (n = 183) was divided into two groups: 81 (44%) had VitD levels >52 nmol/L and 102 (56%) ≤52 nmol/L at ASCT. VitD levels >52 nmol/L were associated with better PFS (P = 0.0194) and OS (P = 0.011). Similarly, when evaluating myeloma patients alone, patients with lower VitD levels (≤52 nmol/L) had inferior PFS (P = 0.0412) and OS (P = 0.049). Finally, the multivariate analysis was consistent that varying VitD levels were significantly associated with OS (P = 0.0167), also when stratifying patients in groups with VitD levels > versus ≤52 nmol/L (P = 0.0421). Our data suggest that reduced serum VitD levels in myeloma and lymphoma patients undergoing HDCT/ASCT are associated with inferior outcome. Optimizing VitD levels before ASCT may be warranted.

Serum Free Light Chain Assay: Shift Toward a Higher κ/λ Ratio.

BACKGROUND: The analysis of serum free light chains (FLCs) is clinically relevant for the diagnosis and therapeutic management of clonal plasma cell disorders. This study compares the performance of monoclonal and polyclonal FLC κ and λ assays in clinical samples determined in a single academic center. METHODS: Serum FLCs were analyzed from 102 patients using the Freelite (Binding Site) and N Latex (Siemens) assays on the BN ProSpec System (Siemens). When available, data for protein electrophoresis, immunofixation, C-reactive protein, and estimated glomerular filtration rate (eGFR) were combined with FLC results to evaluate performance. RESULTS: Method evaluation showed acceptable imprecision and inaccuracy measures of <4.4% and 12.9%, respectively. Poor agreement between the methods was observed, including constant and proportional bias and poor correlation (Kendall τ, 0.671-0.901). The N Latex assay was not affected by the renal impairment estimated by eGFR, unlike the FLC κ/λ ratio results by the Freelite assay. With the Freelite assay, 98% of putative controls without monoclonal gammopathy (n = 42) showed a κ/λ ratio that was above the median of the standard diagnostic range or renal diagnostic range. A shift toward higher κ/λ ratios was also observed when retrospective data between 2011 and 2017 were compared. CONCLUSIONS: Unlike the Freelite assay, κ/λ ratios analyzed with the N Latex assay were not affected by renal failure. Both methods showed acceptable performances using nephelometry, but they were poorly correlated. A shift toward κ/λ ratios might impair the specificity of borderline increased κ/λ results. This should be considered when interpreting FLC κ and λ results.

High-resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids.

High-resolution capillary zone electrophoresis is used to distinguish transferrin glycoforms present in human serum, cerebrospinal fluid, and serum treated with neuraminidase and N-glycosidase F. The obtained data are compared to mass spectrometry data from the literature. The main focus is on the analysis of the various asialo-transferrin, monosialo-transferrin, and disialo-transferrin molecules found in these samples. The features of capillary zone electrophoresis and mass spectrometry are reviewed and highlighted in the context of the analysis of undersialylated and hypoglycosylated transferrin molecules. High-resolution capillary zone electrophoresis represents an effective tool to assess the diversity of transferrin patterns whereas mass spectrometry is the method of choice to elucidate structural identification about the glycoforms. Hypoglycosylated transferrin glycoforms present in sera of alcohol abusers and normal subjects are structurally identical to those in sera of patients with a congenital disorder of glycosylation type I. Asialo-transferrin, monosialo-transferrin and disialo-transferrin observed in sera of patients with a type II congenital disorder of glycosylation or a hemolytic uremic syndrome, in cerebrospinal fluid and after treatment of serum with neuraminidase are undersialylated transferrin glycoforms with two N-glycans of varying structure. Undersialylated disialo-transferrin is also observed in sera with high levels of trisialo-transferrin.

Analysis of blood degradation products and ferritin in the cerebrospinal fluid of dogs with acute thoracolumbar intervertebral disk extrusion, a prospective pilot study.

BACKGROUND: Hemorrhage in the spinal canal leads to further damage of the spinal cord influencing outcome in dogs with intervertebral disk (IVD) extrusion. The aim of the study was to evaluate blood degradation products and ferritin in the cerebrospinal fluid (CSF) of dogs with thoracolumbar IVD extrusion, and their association to clinical parameters and MRI findings. RESULTS: In the CSF of dogs with IVD extrusion, both net oxyhemoglobin absorption (NOA) and net bilirubin absorption (NBA) were significantly higher compared to the control groups of dogs with steroid responsive meningitis arteritis (SRMA) and idiopathic epilepsy (IE) (P < 0.001), but NOA compared to the idiopathic epilepsy group contaminated artificially with blood (IEc) was not (P = 0.890). Ferritin concentration was significantly higher in dogs with IVD extrusion compared to dogs with IE (P = 0.034), but not to dogs with SRMA (P = 0.526). There was no association between NOA, NBA or ferritin concentration and severity or duration of clinical signs. In dogs with a higher ferritin concentration the outcome was better (P = 0.018). In dogs with evidence of hemorrhage on MRI, NOA and NBA were significantly higher (P = 0.016, P = 0.009), but not ferritin (P = 0.0628). CONCLUSION AND CLINICAL IMPORTANCE: Quantification of blood degradation products and ferritin in the CSF of dogs to assess subarachnoidal hemorrhage is feasible; however, larger case numbers are needed to evaluate the relevance of NBA and ferritin as prognostic indicators.

False low holotranscobalamin levels in a patient with a novel TCN2 mutation.

BACKGROUND: Measurement of holotranscobalamin (holoTC) is increasingly used as a screening test for cobalamin (Cbl) deficiency. A level well below the reference interval strongly supports a deficient state. We examined a 21-year-old woman diagnosed as Cbl deficient because of an extremely low holoTC level as measured by the Abbott Architect Assay. METHODS: The patient was evaluated for Cbl deficiency employing an in-house holoTC method as well as other routine markers of Cbl status. Further analyses included exploration of the Cbl binding proteins employing gel filtration of a serum sample saturated with 57 Co-labeled Cbl and Sanger sequencing of exons 1-9 and the intron-exon boundaries of the TCN2 gene, the gene coding for transcobalamin (TC). RESULTS: The patient had normal hematological variables throughout. Despite initial treatment with Cbl, holoTC as measured by the Abbott assay remained low, while holoTC measured with the in-house assay was normal, and behaved as TC upon gel-filtration. By Sanger sequencing, we detected a homozygous single point mutation c.855T>A in exon 6 of TCN2, corresponding to a asparagine (Asn) to lysine (Lys) substitution in position 267 of the mature protein. CONCLUSIONS: We describe a novel point mutation of the TCN2 gene. The mutation does not seem to interfere with the function of TC, but the mutation may well explain the low level of holoTC detected by the Abbott assay. Our results underscores that mutations of TCN2 have to be considered when implausible holoTC results are obtained.

[Phosphate disorders: hyperphosphatemia or pseudohyperphosphatemia?]. / Phosphat(verw)irrungen: tatsächlich «Hyper¼ oder nur «Pseudo¼?

We report the case of a 79 year old woman presenting with progressive confusion and drowsiness. Renal insufficiency with hyperkalemia as well as hypercalcemia and severe hyperphosphatemia were diagnosed. Renal insufficiency improved with treatment. However, hyperphosphatemia persisted without apparent explanation. We discuss possible causes of hyper- and pseudohyperphosphatemia. Specifically, phosphate analysis may be disturbed by the paraproteins in patients with multiple myeloma, resulting in pseudohyperphosphatemia. We review the standard laboratory phosphate measurement and the mechanisms of interference with paraproteins.

Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation.

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA(Leu(CUN))) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA(Leu(CUN)) and tRNA(Leu(UUR)) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA(Leu(CUN)) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.

Monoclonal gammopathy missed by capillary zone electrophoresis.

BACKGROUND: Serum protein electrophoresis is used as a screening test for monoclonal gammopathies. Here, we present a case of a high-concentration monoclonal immunoglobulin (M-protein) that was missed by serum protein electrophoresis on a Capillarys 2 capillary zone electrophoresis system. The aim of our study was to identify the reason for the failure of the system to detect the M-protein. METHODS: M-protein solubility was examined in response to temperature, pH, ionic strength, the chaotropic agent urea and the reducing agent 2-mercaptoethanol. RESULTS: Precipitation of the M-protein was not cold-induced, but solubility decreased at pH 8.5 or higher, when the pH approached the apparent isoelectric point. The M-protein also precipitated in alkaline Capillarys 2 electrophoresis buffer (pH 10), which was the reason for the false-negative electrophoresis result. Precipitation of the M-protein was not related to the ionic strength of the buffer. Solubility improved in presence of urea. Pre-treatment of serum with 2-mercaptoethanol revealed the missing M-protein peak of 36 g/L on the electropherogram. CONCLUSIONS: This case shows that insolubility of M-proteins in alkaline buffer is one possible cause of false-negative results on capillary zone electrophoresis systems. False-negative results should be considered, especially when accompanying laboratory results are inconsistent with the electropherogram.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin.

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP.

Expression of phosphoproteins and amelotin in teeth.

The organic material of our teeth consists of collagens and a number of calcium-binding phosphoproteins. Six of these phosphoproteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), namely osteopontin, bone sialoprotein, dentin matrix protein (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and enamelin. We prepared a cDNA library from rat incisors in order to identify the genes involved in tooth formation. The library was screened by subtractive hybridization with two probes; one specific for teeth, the other for bone. We found that the vast majority of the clones from our library were expressed at similar levels in bone and teeth, demonstrating the close relationship of the two tissues. Only 7% of all the clones were expressed in a tooth-specific fashion. These included clones for the enamel proteins; amelotin, amelogenin, ameloblastin and enamelin; for the dentin proteins DSPP and DMP1; and for the intermediate filament protein cytokeratin 13. Several typical bone proteins, including collagen I, osteocalcin, alkaline phosphatase and FATSO, were also expressed at significantly higher levels in teeth than in bone, probably due to the extreme growth rate of rat incisors. The amino acid sequence of rat amelotin showed 62% identity with the sequence from humans. It was expressed considerably later than the other enamel proteins, suggesting that amelotin may serve a function different from those of amelogenin, ameloblastin and enamelin.

Aberrant expression of FGFRL1, a novel FGF receptor, in ovarian tumors.

FGFRL1 is a novel member of the FGF receptor family. It is expressed at very low levels in a great variety of cell lines and at relatively high levels in SW1353 chondrosarcoma cells, MG63 osteosarcoma cells and A204 rhabdomyosarcoma cells. Screening of 241 different human tumors with the help of a cancer profiling array suggested major alterations in the relative expression of FGFRL1 in ovarian tumors. Five distinct ovary tumors were therefore analyzed by quantitative and competitive PCR. Several tumors were found to exhibit a significant decrease in the expression of FGFRL1 in the tumor tissue relative to the matched control tissue. One ovarian tumor showed a 25-fold increase in the relative expression. Since FGFRL1 appears to be involved in the control of cell proliferation and differentiation, its aberrant expression might contribute to the development and progression of ovarian tumors.

Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor.

FGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man.

Three members of the connective tissue growth factor family CCN are differentially regulated by mechanical stress.

Expression of connective tissue growth factor (CTGF), a member of the CCN gene family, is known to be significantly induced by mechanical stress. We have therefore investigated whether other members of the CCN gene family, including Cyr61 and Nov, might reveal a similar stress-dependent regulation. Fibroblasts growing under stressed conditions within a three-dimensional collagen gel showed at least a 15 times higher level of Cyr61 mRNA than cells growing under relaxed conditions. Upon relaxation, the decline of the Cyr61 mRNA to a lower level occurred within 2 h, and was thus quicker than the response of CTGF. The regulation was fully reversible when stress was reapplied. Thus, Cyr61 represents another typical example of a stress-responsive gene. The level of the Nov mRNA was low in the stressed state, but increased in the relaxed state. This CCN gene therefore shows an inverted regulation relative to that of Cyr61 and CTGF. Inhibition of protein kinases by means of staurosporine suppressed the stress-induced expression of Cyr61 and CTGF. Elevated levels of cAMP induced by forskolin mimicked the effects of relaxation on the regulation of Cyr61, CTGF and Nov. Thus, adenylate cyclase as well as one or several protein kinases might be involved in the mechanoregulation of these CCN genes.

Mechanical stress is required for high-level expression of connective tissue growth factor.

We used gene array technology to analyze differences in gene expression between mechanically stressed and relaxed fibroblasts. A number of stress-responsive genes that showed a two- to sixfold difference in their relative expression were identified. Connective tissue growth factor (CTGF) was among those genes that showed the most striking up-regulation by mechanical stress. Its regulation occurred at the transcriptional level and was reversible. A new steady state level of CTGF mRNA was reached within less than 6 h after stress relaxation. Mechanical stress was absolutely required for sustained high-level expression; TGF-beta, which is also known to stimulate CTGF synthesis, was not sufficient on its own. Experiments with specific inhibitors suggested that a protein kinase and a tyrosine phosphatase were involved in the transduction of the mechanical stimulus to gene expression. Since CTGF controls the synthesis of several extracellular matrix proteins, it is likely that this growth factor is responsible for the increased synthesis of collagen I and other matrix proteins in stressed fibroblasts.