Antititin antibody in early- and late-onset myasthenia gravis.

OBJECTIVES: Myasthenia gravis (MG) is an autoimmune disease caused by antibodies against neuromuscular junction proteins, 85% of patients have antibodies against acetylcholine receptor (AChR-MG). Antititin antibodies are present in a subset of patients with MG. We aimed to determine the value of antititin antibodies as severity markers and thymoma predictors in early- and late-onset MG. MATERIALS & METHODS: Two-hundred and ninety-five consecutive MG patients (188 F and 107 M) aged 12-89 years (mean 50y) were included. 164 patients had early-onset (EOMG, ≤50 years of age), 131 had late-onset MG (LOMG). Twenty-six patients had thymoma. symptoms, severity graded with MGFA scale, thymus histology, medications, and treatment results were analyzed. RESULTS: Antititin antibodies were present in 81 (27%) of all patients: 54% of thymoma MG, 0.6% of non-thymomatous EOMG, and 55% of LOMG, with proportion of titin-positive patients increasing linearly from 40% in the 6th to 88% in the 9th decade of life. Titin-positive patients had more bulbar symptoms (P = 0.003). Severity of MG, need for immunosuppression, myasthenic crisis risk or treatment results were not related to its presence. Antititin antibodies had 56% sensitivity, 99% specificity, 90% positive predictive value (PPV), and 95% negative predictive value (NPV) for thymoma diagnosis in EOMG, and 50% sensitivity, 75% specificity, 71% PPV and 55% NPV in LOMG. CONCLUSIONS: Antititin antibodies have high PPV and NPV for thymoma in EOMG. In MG without thymoma, antititin antibodies can be considered as markers of LOMG, but not of a severe course in our MG cohort.

[Kennedy's disease: expansion of the CAG trinucleotide]. / Choroba Kennedy'ego: ekspansja trójki nukleotydowej CAG.

Kennedy's disease is a rare X-linked spinal and bulbar muscular atrophy (SBMA). A degenerative process of the motor neurons is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor. Despite a distinctive clinical phenotype, SBMA can be misdiagnosed, usually due to the lack of clear family history. Accurate diagnosis is important for genetic counseling and because alternative diagnosis of amyotrophic lateral sclerosis usually means much worse prognosis. We report 2 unrelated patients with Kennedy's disease in whom the clinical diagnosis was confirmed by showing the CAG repeat expansion.

The connections of the endopiriform nucleus with the insular claustrum in the rat and rabbit.

The connections between two parts of the claustrum in the rat and rabbit were studied using the highly fluorescent lipophilic carbocyanine dye (Dil). After the application of Dil crystal into the endopiriform nucleus, labeled fibers in the insular claustrum were observed in its part directly neighboring the insular cortex and capsula externa. Additionally, numerous projections into the piriform, insular and entorhinal cortices were present. The presence of connections between the endopiriform nucleus and insular claustrum suggests its role concerned with the processes taking part in the allocortical regions as well as in the limbic system.

Effect of protein binding on renal extraction of 131I-OIH and 99mTc-labeled tubular agents.

UNLABELLED: The clearance of 99mTc-mercaptoacetyltriglycine (MAG3) is less than the clearances of o-131I-iodohippurate (OIH) and 99mTc-labeled DD- and LL-ethylenedicysteine (EC). This difference could be associated with the lower affinity of MAG3 for the tubular transport receptor, but MAG3 is more highly protein bound than OIH and the EC isomers; protein binding could also be an important factor governing tubular extraction. To separate the effects of protein binding from tubular receptor affinity, the extraction fractions (EFs) of MAG3, OIH, and the DD, LL, and DL isomers of 99mTc-EC were measured in an isolated perfused rat kidney model using a protein-free perfusate and perfusates containing bovine serum albumin. METHODS: The right kidney was removed from the rat and perfused with modified Krebs-Henseleit buffers containing 7.5 or 2.5 g/dL bovine serum albumin or a protein-free perfusate. OIH was coinjected into the renal artery with each of the 99mTc-tracers. Protein binding was measured in each of the perfusates, and the venous outflow was collected to determine the EF. RESULTS: The protein binding of MAG3 in the albumin perfusates ranged from 87% to 95%, significantly higher than the 20%-34% range of protein binding observed with the three EC complexes (P < 0.05). In the 2.5 g/dL albumin perfusate, the EF of MAG3 was 44%, significantly less than the 57%-77% EF of the three EC complexes; in the 7.5 g/dL perfusate, the MAG3 EF fell to 18% versus 39%-45% for the EC complexes (P < 0.05). However, in the protein-free perfusate, the EF of MAG3 was 64%, equal to or higher than the 46%-62% EF of the three EC complexes. CONCLUSION: Protein binding modulates the tubular extraction of renal tracers. Protein binding and receptor affinity must be considered in the design of future renal radiopharmaceuticals as well as radiopharmaceuticals targeting other receptors.

The piriform cortex and the endopiriform nucleus in the rat reveal generally similar pattern of connections.

The afferent and efferent connections of the piriform cortex and the endopiriform nucleus in the rat were studied by the method of axonal transport of two fluorescent tracers: Fluoro-Gold and Fluoro-Ruby. The results indicate that both structures possess not only the connections with the olfactory system, but also the reciprocal connections with the limbic system (entorhinal cortex, amygdaloid body), thalamus (mediodorsal and midline nuclei), extrapyramidal system (ventral part of the nucleus accumbens). The topographic differences in the organization of the association connections between the anterior and posterior parts of the piriform cortex are reported. Additionally, the reciprocal and relatively numerous connections between the endopiriform nucleus and the piriform cortex may result in their modulatory function, which in some pathological circumstances may have a critical significance in epileptogenesis.

The corticoclaustral connections in the rat studied by means of the fluorescent retrograde axonal transport method.

The corticoclaustral connections in the rat were investigated by means of the method of the retrograde axonal transport of the fluorescent tracer (Fluoro-Gold; FG). The material consisted of 20 adult Wistar rats. The fluorescent tracer was injected into the anterior, middle or posterior parts of the claustrum. The retrogradely labeled neurons were detected in the layer VI of the neocortex. Injections of the tracer into the anterior part of the claustrum resulted in labeling of the neurons in the motor cortex. After administration of the tracer into the middle part of the insular claustrum, labeled neurons were present both in the motor and somatosensory cortices, while the injections of the tracer into its posterior part resulted in labeling of neurons in the visual cortex. Administration of the fluorescent tracer into the insular claustrum of the rat resulted in labeling of the cortical neurons of the corresponding areas of both hemispheres, however, the contralateral projections seem to be less numerous than the ipsilateral. Our results confirm the existence of reciprocal connections of the claustrum with the neocortex and suggest its role in integration and modification of information reaching the neocortex.

Postnatal development of the rat striatum--a study using in situ DNA end labeling technique.

We have examined the development of rat striatum for evidence of cells dying in the process of physiological cell death. In present study we have indicated apoptotic cells in sections stained with cresyl violet (cell death characterized by pyknosis) or with DNA end labeling assay (TUNEL method). Our results demonstrated that cell loss during maturation of the rat striatum had the characteristics of apoptosis rather than necrosis. The greatest number of TUNEL-positive and pyknotic cells in the striatum were found during the first postnatal days; after 7th day of postnatal life a rapid decrease of its number was observed after the second postnatal week no TUNEL-positive cells were observed in the striatum. Our analysis suggests that apoptotic cell death occurring during the development of striatal neuronal population takes place during the first week of postnatal life.

Parvalbumin immunoreactivity changes in the thalamic reticular nucleus during the maturation of the rat's brain.

The thalamic reticular nucleus (Rt) is a thin lamina of cells, through which thalamocortical and corticothalamic fibers pass. It is interposed between the thalamic nuclei and the internal capsule and it is composed of GABA-ergic cells with synapses that receive impulses from both kinds of fibers. Rt takes part in the negative feed-back system of controlling the information transfer from the thalamus to the cerebral cortex and it is focused in the sleep-waking cycle. The pattern of parvalbumin reactivity during maturation of Rt becomes the main aim of our study. The study was performed on 36 rats on various postnatal days (P0, P1, P2, P4, P5, P7, P10, P14, P17, P21, P30 and P90). The animals were anesthetized, transcardially perfused, cut on cryostat into 30-microns-thick frontal sections, stained immunocytochemically using standard ABC method and a mouse monoclonal antibody against parvalbumin. A small amount of round and oval, parvalbumin immunopositive cells was detected at stage PO, predominantly in the intermediate part of Rt, whereas the cells in ventral and lateral part at the same time were only slightly immunopositive. At P10 the cells in the intermediate part became more fusiform or oval because of the appearance of dendrites. At P14 we were able to observe separate, punctuated structures interpreted as the axonal endings. There were plenty of them at the time of full maturation of the intermediate portion of reticular nucleus (stage P21). At this time, the dorsal and ventral parts had their first synapses, too, but their maturation ended a week later. At P30 multipolar neurons, with round and fusiform somata were distributed relatively homogeneously throughout Rt. We compared the stage with the parvalbumin reactivity of the adult rat and found no difference in the morphological pattern of PV neurons.

Comparison of technetium-99m-ll-EC isomers in rats and humans.

UNLABELLED: Technetium-99m-L,L-ethylenedicysteine (99mTc-LL-EC) is a new renal imaging agent with pharmacokinetic properties reported to be slightly superior to those of 99mTc-mercaptoacetyltriglycine (99mTc-MAG3); however, to better define the potential of the enantiomer 99mTc-DD-EC and the diastereomer 99mTc-DL-EC as renal imaging agents, we compared the three EC stereoisomers with 131I-orthoiodohippurate (OIH) in a series of rats and humans. METHODS: Each 99mTc-EC stereoisomer was coinjected with OIH in six Sprague-Dawley rats for measurements of clearance and extraction fraction. Each stereoisomer was also coinjected with OIH in three human volunteers followed by sequential imaging, plasma clearance measurements and timed urine collections. RESULTS: Technetium-99m-DD-EC had the highest clearance and extraction efficiency in rats (p < or = 0.02). In humans, image quality was good with all three agents. The clearance ratio (EC/OIH) was 82% +/- 8% for 99mTc-DD-EC compared to 70% +/- 3% and 40% +/- 5% for 99mTc-LL-EC and 99mTc-DL-EC, respectively. Technetium-99m-DD and 99mTc-LL-EC were excreted more rapidly than 99mTc-DL-EC. CONCLUSION: Technetium-99m-DD-EC has excellent imaging properties and the data suggest that its clearance may approach that of OIH more closely than any other 99mTc renal agent. A potential limitation is the fact that both 99mTc-DD and LL-EC exist in dianionic (80%) and monoanionic (20%) forms at physiological pH and it is unlikely that these two forms have the same clearance or protein binding affinity.

Design and development of a fiber-optic immunosensor utilizing near-infrared fluorophores.

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10-15 min.

Instrument to detect near-infrared fluorescence in solid-phase immunoassay.

The construction of a near-infrared (near-IR) fluorescence detector for measuring picomolar levels of near-IR laser dyes is described. The detector is designed for use in an immunoassay technique that employs antibodies labeled with near-IR polymethine cyanine dyes. These dyes possess spectral properties that are exclusive to the near-IR region (650-1100 nm). The instrumentation is characterized, including its hardware and data acquisition software components. The detector is capable of measuring fluorescence in both solution and solid-phase environments. Data on the detector's performance is presented.

Spectroscopic studies of a near-infrared absorbing pH sensitive aminodienone-carbocyanine dye system.

Synthetic red and near-infrared absorbing dyes may be used as probe molecules in a large number of applications. Dyes exhibiting spectral changes with hydrogen ion concentration are useful as pH probes. Those dyes which have their absorption and fluorescence maxima in the long wavelength region of the visible spectral region are specially valuable because of decreased interference and semiconductor laser applications. In this paper we have evaluated an aminodienone dyes 1 which demostrates pH dependent absorption and fluorescence spectra as well as solvent polarity dependence. In organic solvents the long wavelength absorption band of the dye is in the reduced interference region. The absorption maximum is at 535 nm in neutral or alkaline solutions in methanol. The absorption spectra undergo a strong bathochromic shift in the presence of acids (lambda(max) = 709 nm) with a concomitant change in the fluorescence spectra. This pH sensitive dye was found to be specially especially useful for organic solvents. The analytical utility of this and similar near-infrared absorbing dyes is discussed.

Comparison of covalent and noncovalent labeling with near-infrared dyes for the high-performance liquid chromatographic determination of human serum albumin.

Noncovalent and covalent methods of labeling protein with near-infrared polymethine cyanine dyes were compared for use in analyzing human serum albumin (HSA) by high-performance liquid chromatography (HPLC) with near-infrared absorbance detection. While noncovalent labeling was faster than covalent labeling and took place in the physiological pH range, covalent labeling was more stable under conditions encountered in many of the widely used types of HPLC. Covalently labeled HSA protein peaks indicated uniform labeling of amino groups at both hydrophilic and hydrophobic binding sites, while noncovalent labeling showed a preference for hydrophobic binding sites.