Application of a fast and cost-effective 'three-in-one' MMR ELISA as a tool for surveying anti-MMR humoral immunity: the Hungarian experience.

In Hungary, between February 2017 and July 2019, 70 confirmed measles cases were reported, raising questions about the adequacy of population-level immunity. Although the assumed vaccination coverage is ≥99%, in a recent study, we detected potential gaps in the anti-measles humoral immunity. In Hungary, according to a decree by the Ministry of Public Welfare, beginning from 2021, the healthcare provider should conduct a serosurvey of anti-measles protection levels of healthcare professionals. To facilitate the compliance with this requirement, we developed a quick 'three-in-one' or 'triple' MMR (measles, mumps and rubella) indirect ELISA (IgG); an assay format that is currently not available commercially. High throughput applicability of the 'three-in-one' ELISA was verified using 1736 sera from routine laboratory residual samples, using an automated platform (Siemens BEP 2000 Advance). Assay verification was performed by comparing the full antigen repertoire-based 'target' assay with in-house 'control' assays using recombinant viral antigen coatings, and by validated commercially available kits. Indirect immunofluorescence was used as an independent reference method. Data were analysed using OriginLab, IBM SPSS, RStudio and MedCalc. In case of measles, we combined our current results with previously published data (Ntotal measles = 3523). Evaluation of anti-mumps and anti-rubella humoral antibody levels was based on the measurement of 1736 samples. The lowest anti-measles seropositivity (79.3%) was detected in sera of individuals vaccinated between 1978 and 1987. Considering the antigen-specific seropositivity ratios of all samples measured, anti-measles, -mumps and -rubella IgG antibody titres were adequate in 89.84%, 91.82% and 92.28%, respectively. Based on the virus-specific herd immunity threshold (HIT) values (HITMeasles = 92-95%, HITMumps = 75-86%, HITRubella = 83-86), it can be stated that regarding anti-measles immunity, certain age clusters of the population may have inadequate levels of humoral immunity. Despite the potential gaps in herd immunity, the use of MMR vaccine remains an effective and low-cost approach for the prevention of measles, mumps and rubella infections.

Correlation of natural autoantibodies and cardiovascular disease-related anti-bacterial antibodies in pericardial fluid of cardiac surgery patients.

Our previous studies showed that anti-citrate synthase (anti-CS) immunoglobulin (Ig)M natural autoantibodies are present in healthy individuals without previous antigen stimulation, but no studies have investigated their presence in the pericardial fluid (PF). Therefore, we detected the natural anti-CS IgG/M autoantibody levels in plasma and PF of cardiac surgery patients and investigated their relationship with cardiovascular disease-associated bacterial pathogens. PF and blood samples of 22 coronary artery bypass graft (CABG) and 10 aortic valve replacement (AVR) patients were tested for total Ig levels, natural autoantibodies and infection-related antibodies using enzyme-linked immunosorbent assay (ELISA) and Luminex methods. The B cell subsets were measured by flow cytometry. The total Ig subclass levels were four to eight times lower in PF than in plasma, but the natural anti-CS IgM autoantibodies showed a relative increase in PF. The frequency of CD19+ B lymphocytes was significantly lower in PF than in blood (P = 0·01), with a significant relative increase of B1 cells (P = 0·005). Mycoplasma pneumoniae antibody-positive patients had significantly higher anti-CS IgM levels. In CABG patients we found a correlation between anti-CS IgG levels and M. pneumoniae, Chlamydia pneumoniae and Borrelia burgdorferi antibody titres. Our results provide the first evidence that natural autoantibodies are present in the PF, and they show a significant correlation with certain anti-bacterial antibody titres in a disease-specific manner.

Neural stem cell-mediated CE/CPT-11 enzyme/prodrug therapy in transgenic mouse model of intracerebellar medulloblastoma.

Medulloblastoma is a heterogeneous diffuse neoplasm that can be highly disseminated, and is the most common malignant childhood brain tumor. Although multimodal treatments have improved survival rates for patients with medulloblastoma, these tumors are associated with high morbidity and mortality. New treatment strategies are urgently needed to improve cure rates and, importantly, to spare normal brain tissue from neurotoxicity and patients from life-long cognitive and functional deficits associated with current therapies. In numerous preclinical brain tumor models, neural stem cells (NSCs) have shown great promise as delivery vehicles for therapeutic genes. Here, we have used an established, genetically modified human NSC line (HB1.F3.CD) to deliver carboxylesterase (CE) to cerebellar tumor foci and locally activate the prodrug camptothecin-11 (CPT-11) (Irinotecan) to the potent topoisomerase I inhibitor SN-38. HB1.F3.CD NSC tumor tropism, intratumoral distribution and therapeutic efficacy were investigated in clinically relevant experimental models. Magnetic resonance imaging was used for in vivo tracking of iron nanoparticle-labeled NSCs, and to assess the therapeutic efficacy of CE-expressing HB1.F3.CD cells. As compared with controls, a significant decrease in tumor growth rate was seen in mice that received both NSCs and CPT-11 as their treatment regimen. Thus, this study provides proof-of-concept for NSC-mediated CE/CPT-11 treatment of medulloblastoma, and serves as a foundation for further studies toward potential clinical application.

Stem and progenitor cell-mediated tumor selective gene therapy.

The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-beta, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

Expression of active caspase-3 in mitotic and postmitotic cells of the rat forebrain.

Active caspase-3 immunoreactivity was detected in the rat forebrain proliferative regions at birth and remained high in these areas for about 2 weeks, during which period labeled cells were present centroperipherally across the olfactory bulb. By the end of the third postnatal week, only a small number of immunolabeled cells remained in these forebrain structures. Active caspase-3 immunolabeling was localized mostly to cell nuclei and co-localized partially with TuJ1 and NeuN immunoreactivity, but not with glial fibrially acidic protein, OX-42, gamma-aminobutyric acid, or terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive labeling. Active caspase-3 and 5-bromo-2'-deoxyuridine (BrdU) double-labeled nuclei were seen in the proliferative regions after 2 hours and in the periglomerular region of the bulb after 7 days following BrdU injections. Examination of the cells with electron microscopy confirmed that the active caspase-3-containing nuclei in the proliferative regions often had infoldings and appeared to be undergoing division. Some of the cells with active caspase-3-labeled nuclei in the bulb had synapses on their somata or dendrites. Labeled dendritic spines and a few axon terminals were also observed in the olfactory bulb. Taken together, it appears that a wave of active caspase-3-positive cells are dividing in the proliferative zones and then migrating to the bulb as they differentiate into neurons. Therefore, active caspase-3 may play a role in cellular processes such as neuronal differentiation, migration, and plasticity, in addition to its role in cell death.

The aspirin metabolite sodium salicylate causes focal cerebral hemorrhage and cell death in rats with kainic acid-induced seizures.

Aspirin (acetylsalicylic acid), and its main metabolite sodium salicylate, have been shown to protect neurons from excitotoxic cell death in vitro. The objective of our study was to investigate the possible neuroprotective effects of sodium salicylate in vivo in rats with kainic acid-induced seizures, a model for temporal lobe epilepsy in human patients. Male Sprague-Dawley rats received intraperitoneal injections of kainic acid either alone, or with sodium salicylate given before and for 40h after kainic acid injections. The control group received either phosphate-buffered saline or sodium salicylate without co-administration of kainic acid. Animals developed status epilepticus, which was aborted 1.5-2h later with diazepam. On day 3 following kainic acid-induced seizures, animals received bromodeoxyuridine to measure cellular proliferation, and were killed under anesthesia 24h later. Brains were removed, sectioned, and analysed for gross histological changes, evidence of hemorrhage, DNA fragmentation, cellular proliferation, and microglial immunohistochemistry. We report that sodium salicylate did not protect neurons from seizure-induced cell death, and to the contrary, it caused focal hemorrhage and cell death in the hippocampal formation and the entorhinal/piriform cortex of rats with kainic acid-induced seizures. Hemorrhage was never observed in animals that received vehicle, kainic acid or sodium salicylate only, which indicated that sodium salicylate exerted its effect only in animals with seizures, and was confined to select regions of the brain that undergo seizure activity. Large numbers of cells displaying DNA fragmentation were detected in the hippocampal formation, entorhinal/piriform cortex and the dorsomedial thalamic nucleus of rats that received kainic acid or kainic acid in combination with sodium salicylate. Bromodeoxyuridine immunohistochemistry revealed large numbers of proliferating cells in and around the areas with most severe neural injury induced by kainic acid or kainic acid co-administered with sodium salicylate. These same brain regions displayed intense staining with a microglia-specific marker, an indication of microglial activation in response to brain damage. In all cases, the degree of cell death, cell proliferation and microglia staining was more severe in animals that received the combination of kainic acid and sodium salicylate when compared to animals that received kainic acid alone. We hypothesize that our findings are attributable to sodium salicylate-induced blockade of cellular mechanisms that protect cells from calcium-mediated injury. These initial observations may have important clinical implications for patients with epilepsy who take aspirin while affected by these conditions, and should promote further investigation of this relationship.

Modulation of cell-cell and cell-antigen interactions by 1,25-dihydroxyvitamin D3 and vitamin D3 sulfate in vitro: a study on pregnancy lymphocytes and hybridoma cells.

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was investigated in single cell cytotoxicity assays, using K-562 target cells. The action of vitamin D3 sulfate (VD3S) in natural cytotoxicity assays as well as its effect on the antigen-specific adherence of hybridoma cells has also been studied. In the single cell cytotoxicity assay 1,25(OH)2D3 dose-dependently and significantly increased the binding of PBMC to target, the number of lysed target cells and NK activity. RU486, a compound known as a potent blocker of progesterone and glucocorticoid receptors, suppressed the effect of 1,25(OH)2D3 in all systems. VD3S dose-dependently decreased the natural cytotoxicity of PBMC and the binding of hybridoma cells to antigen immobilized on plastic surfaces. The results suggest that both 1,25(OH)2D3 and VD3S are potent modulatory agents in cell-cell and cell-antigen interactions.

Olfactory experience modulates Bcl-2 expression in the developing olfactory bulb.

The effect of olfactory deprivation on cellular expression of the Bcl-2 gene in the olfactory bulb of young rats was investigated. Restriction of olfactory stimuli caused an overall increase in Bcl-2 mRNA expression, with increases seen in the lateral aspects of glomerular, external plexiform, mitral and granule cell layers, as well as the medial aspects of external plexiform layer. No differences were found in the unoperated control group. In addition, we found an inverse relationship between the incidence of apoptosis induced by olfactory deprivation and the magnitude of increase in Bcl-2 mRNA expression in the glomerular layer. These data raise the possibility that Bcl-2 may be involved in olfactory experience-related neural plasticity by regulating cell survival.

Olfactory experience modulated apoptosis in the developing olfactory bulb.

Early sensory stimulation plays a key role in shaping the structure and function of the developing olfactory system. Here, we provide the first direct evidence for apoptotic cell death in the olfactory bulbs of rat pups during normal development and we also demonstrate that olfactory deprivation by unilateral naris occlusion causes a dramatic increase in apoptotic cell death in the glomerular and granule cell layers of the deprived bulb. The accessory olfactory bulbs displayed a remarkably high basal level of apoptosis but the occluded accessory bulb did not differ in that regard from the control accessory bulb. These results suggest that apoptosis may be an important mechanism by which the olfactory system can adjust its cell numbers in response in sensory stimuli experienced in early life, thereby underlying one form of plasticity in the developing olfactory system.

Antigen-specific cell adherence assay: a new method for separation of antigen-specific hybridoma cells.

A new method for the detection and separation of antigen-specific antibody-producing cells on the basis of antibody-mediated recognition of solid-phase immobilized antigen molecules is described. Hybridoma cells are placed on microtiter plate wells coated with antigen molecules, and antigen-specific antibody-producing cells bind to the immobilized antigen molecules; antibody nonproducing or nonspecific antibody-producing cells can be easily separated from the bound cells by inverting the plate. Cells bound to solid-phase immobilized antigen molecules can readily be quantitated by counting under a light microscope, and the cells recovered can produce antibody in culture. Unspecific binding of cells in antigen-specific cell adherence assay (ASCAA) is optimally below 5%. Also, effect of drugs interfering with processes related to antibody production of antigen-specific cells can be detected and evaluated by ASCAA.

Diversity of methyl acceptor proteins in rat pheochromocytoma (PC12) cells revealed after treatment with adenosine dialdehyde.

Protein N-methylation is a widespread modification whose functions are poorly understood. To overcome the inherent technical difficulties in identification of N-methylated proteins, we cultured PC12 cells with a methylation inhibitor, in expectation that proteins would accumulate in a hypomethylated state. Cell extracts were then incubated with [methyl-3H]S-adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. Using two-dimensional gel electrophoresis we detected over 50 methyl acceptors, ranging from 18 to 120 kDa. Most had isoelectric points greater than 7.0. NG,NG-Dimethylarginine and NG-monomethylarginine accounted for about 90% of the methyl-3H-amino acids recovered after acid hydrolysis and thin-layer chromatography. The production of hypomethylated proteins should prove useful, not only in the identification of new methyl acceptors, but also in the isolation and characterization of new methyltransferases.

The effect of "facteur thymique serique" (FTS) on catecholamine and serotonin neurotransmission in discrete brain regions of mice.

The dose-related effect of Facteur Thymique Serique (FTS) on hypothalamic, mesencephalic and striatal neurotransmission were investigated after intracerebroventricular (icv) administration. FTS pretreatment with dose of 1 microgram (icv) increased the mesencephalic serotonin content, while failed to influence the hypothalamic and striatal serotonin levels. The nonapeptide in a dose of 0.1 microgram (icv) decreased the hypothalamic, while in a dose of 1 microgram the hypothalamic and also the mesencephalic dopamine content, but did not influence the striatal dopamine level. FTS in a dose of 1 microgram (icv) significantly decreased the hypothalamic noradrenaline level, but did not influence the noradrenaline content of the mesencephalon and striatum. These results suggest that FTS is able to modify central neurotransmission.

Non-specific peroxidase (donor: H2O2-oxidoreductase, EC 1.11.1.7) activity in multiple sclerosis.

The non-specific peroxidase (donor: H2O2-oxidoreductase, EC 1.11.1.7) activity of red blood cells in patients with multiple sclerosis, patients with other neurological diseases, and healthy control individuals was investigated. To this end, a simple method was developed. No significant difference was found in the non-specific peroxidase activity of red blood cells from patients with multiple sclerosis and controls.

Positive autoradiographic findings in brains of four MS patients.

Autoradiography of brain slices from 4 multiple sclerosis (MS) and 9 control patients was performed. After 6 weeks of exposure the exact picture of the white matter appeared on the X-ray films in all cases with MS, but only in one of the controls. The high level of autoradiographic signal from MS white matter suggests that an abnormal accumulation of radioactive trace elements takes place within the brains of MS victims.

Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.

A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.

Antigen-specific adherence of antibody-producing hybridoma cells: application to neural and nonneural antigens.

Antibody-producing hybridoma cells specifically bind to microgram quantities of antigen molecules adsorbed onto the surface of plastic microtiter plates. The binding of hybridoma cells to nonantigen is optimally below 5%, similar binding of non-antibody-producing cells is 4-7%, compared to the binding of the hybridomas to their antigen. There is a difference in the kinetics of binding hybridomas to antigen compared to nonantigen. The number of bound cells depends on the amount, i.e., the surface density, of the antigen molecules and shows typical saturation effects. Preincubation of hybridomas with excess free antigen and saturation of the antibody binding site on the surface with the hybridoma-produced antibody reduces binding of the hybridoma cells to the antigen. Treatment of cells with trypsin reduces binding to antigen-coated plastic surfaces. Drugs such as sodium azide, cytochalasin B, colchicine, vinpocetine, and vincristine sulfate reduce binding to the antigen. Hybridoma cells adhering to the antigen produce more antibody than nonadhering cells. The results reported in this paper show that antigen molecules adsorbed to include a plastic surface and hybridoma cells interact specifically. This system forms a suitable model to study the interaction of antigen with antigen-specific cells and may be useful as a separation method for specific antibody-producing cells.

Analysis of stable protein methylation in cultured cells.

It was demonstrated recently that substrates for protein N-methyltransferases (J. Najbauer and D. W. Aswad, 1990, J. Biol. Chem. 265, 12,717-12,721) and protein carboxyl methyltransferases (J. Najbauer, B. A. Johnson, and D. W. Aswad, 1991, Anal. Biochem. 197, 412-420) accumulate when rat PC12 cells are cultured in the presence of the methylation inhibitor, adenosine dialdehyde. In the present report, we have further characterized this phenomenon in PC12 cells and in two other, widely used cell types. Adenosine dialdehyde was found to increase the methyl-accepting capacity of proteins in human skin fibroblasts and mouse Sp2/0 myeloma cells. However, both the level of methyl incorporation in untreated cells and the amount of stimulation afforded by inhibitor treatment were substantially lower in these cells than in PC12 cells. All three cell lines accumulated methyl acceptor(s) at 17-21 kDa. The PC12 cells and the fibroblasts also exhibited stimulation of three apparently similar proteins in the 33- to 38-kDa region, where several arginine-methylated proteins involved in RNA processing would be expected. The optimal conditions for methylation of PC12 cell extracts with regard to pH, time of methylation, and S-[methyl-3H]adenosyl-L-methionine concentration were characterized. Increased methyl incorporation was detected after adenosine dialdehyde treatments as short as 2 h, and methylation of most substrates continued to increase as the time of treatment was extended to 72 h. The kinetics of accumulation varied from substrate to substrate. Fluorograms of two-dimensional gels of extracts from untreated PC12 cells incubated in the presence of S-[methyl-3H]adenosyl-L-methionine revealed patterns of methyl incorporation similar to those of treated cells, but longer exposure times were necessary (e.g., 35 days vs 7 days). These findings suggest that the inhibitor treatment works mainly by inhibiting the post- or cotranslational methylation of a "normal" array of cellular proteins.

Adherence of cells to myelin basic protein. I. Adherence of red and white blood cells from patients with multiple sclerosis to myelin basic protein.

Red blood cells (RBC) and white blood cells (WBC) of patients with multiple sclerosis (MS) show decreased adherence to myelin basic protein (MBP) immobilized on plastic surfaces compared to the binding of cells from patients with other neurological diseases (OND), or such other autoimmune diseases as psoriasis (PS), and to that of healthy controls (HC). No similar phenomenon occurred to basic and non-basic type proteins other than MBP, for example, to histone (HIS), lysozyme (LYS) and ovalbumin (OVA). Thus, decreased adherence of RBC and WBC in MS patients to MBP appears to be a unique feature of the disease if compared with OND or PS.

Adherence of cells to myelin basic protein. II. Adherence of red blood cells of SJL mice with chronic relapsing EAE.

Adherence of red blood cells from SJL mice suffering of chronic relapsing experimental allergic encephalomyelitis was studied to myelin basic protein coated microtiter plates. Control animals received either bovine serum albumine or "protein-antigen free" adjuvant using the same immunization protocol. Characteristic changes in adherence were found in bovine or human myelin basic protein injected animals compared to the bovine serum albumine immunized group. After a nonspecific increase in adherence between Days 2 to 6 observed in all 3 groups, in the encephalitogen challenged animals on Days 13-14 a marked decrease in red blood cell adherence was detected which maintained at this decreased level during the clinically active stage of the disease and reappeared with the relapse of EAE. No such decreased adherence of red blood cells was observed in BSA immunized animals or in adherence of cells from myelin basic protein injected animals to other basic type protein such as histone. Thus, decreased adherence of red blood cells in animals with EAE appears to be an interestingly unique measure of the disease activity.

Molecular aging of tubulin: accumulation of isoaspartyl sites in vitro and in vivo.

The formation of isoaspartyl sites during aging of rat tubulin in vitro and in vivo has been studied. When incubated in vitro at pH 7.4, 37 degrees C, purified rat brain tubulin accumulated isoaspartyl sites at a rate > or = 2.4 isoaspartyl sites per 100 tubulin subunits (50 kDa) per day for 30 days. Isoaspartate levels were estimated by the transfer of radiolabeled methyl groups from S-adenosyl-L-[methyl-3H]-methionine in a reaction catalyzed by protein-L-isoaspartyl methyltransferase. isoaspartate formation occurred in parallel with, but was not dependent upon, extensive cross-linking of tubulin via formation of intermolecular disulfide bonds. When rat PC12 cells were incubated for 24 or 72 h in the presence of adenosine dialdehyde, a potent methyltransferase inhibitor, a substantial and consistent increase in the isoaspartate content of tubulin was observed. This suggests that tubulin constantly undergoes isoaspartate formation in vivo, but that the levels are normally kept low by methylation-dependent repair. These findings support the hypothesis that protein-isoaspartyl methyltransferase plays a key role in countering spontaneous damage reactions to proteins associated with cell aging. These results also suggest that tubulin is an important target for protein-isoaspartyl methyltransferase in vivo.