Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones.

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.

P2X receptor expression on astrocytes in the nucleus accumbens of rats.

Astrocytes express a variety of neurotransmitter receptors which render them capable of responding to extracellular stimuli, like ATP. Release of ATP, e.g. after brain injury, may initiate reactive gliosis via stimulation of purinergic P2X and P2Y receptors. In the present study, the expression and cellular localization of P2X receptor subtypes on astrocytes in the nucleus accumbens of rats under normal physiological conditions and after stab wound were investigated. Reverse transcription-polymerase chain reaction (RT-PCR) with specific P2X(1-7) primers, and double immunofluorescence with antibodies to glial fibrillary acidic protein (GFAP, a specific marker of fibrous astrocytes) and to different P2X receptor subtypes (P2X(1-4), P2X(7)) were used. The RT-PCR of tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all seven currently known P2X receptor subtype mRNAs indicating the presence of these receptors in this region. A double immunofluorescence approach with confocal laser scanning microscopy showed the localization of P2X(2-4) receptor subtypes on GFAP-labelled astrocytes in untreated rats. Labelling for P2X(1) and P2X(7) receptor subtypes was not found. After mechanical damage all P2X receptor subtypes studied (P2X(1-4), P2X(7)) were observed on the GFAP-labelled reactive astrocytes. A characteristic distribution of the P2X receptors on astrocytic processes and cell bodies as well as an up-regulation of the P2X-immunofluorescence was found. In conclusion, the data show the presence of P2X receptors on rat nucleus accumbens astrocytes and suggest that astrogliosis in vivo is associated with an up-regulation of distinct P2X receptor subtypes.

P2X(2), P2X(2-2) and P2X(5) receptor subunit expression and function in rat thoracolumbar sympathetic neurons.

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.

P2X7 receptors in Müller glial cells from the human retina.

ATP has been shown to be an important extracellular signaling molecule. There are two subgroups of receptors for ATP (and other purines and pyrimidines): the ionotropic P2X and the G-protein-coupled P2Y receptors. Different subtypes of these receptors have been identified by molecular biology, but little is known about their functional properties in the nervous system. Here we present data for the existence of P2 receptors in Müller (glial) cells of the human retina. The cells were studied by immunocytochemistry, electrophysiology, Ca(2+)-microfluorimetry, and molecular biology. They displayed both P2Y and P2X receptors. Freshly enzymatically isolated cells were used throughout the study. Although the [Ca(2+)](i) response to ATP was dominated by release from intracellular stores, there is multiple evidence that the ATP-induced membrane currents were caused by an activation of P2X(7) receptors. Immunocytochemistry and single-cell RT-PCR revealed the expression of P2X(7) receptors by Müller cells. In patch-clamp studies, we found that (1) benzoyl-benzoyl ATP (BzATP) was the most effective agonist to evoke large inward currents and (2) the currents were abolished by P2X antagonists; however, (3) long-lasting application of BzATP did not cause an opening of large pores in addition to the cationic channels. By microfluorimetry it was shown that the P2X receptors mediated a Ca(2+) influx that contributed a small component to the total [Ca(2+)](i) response. Activation of P2X receptors may modulate the uptake of neurotransmitters from the extracellular space by Müller cells in the retina.

Cytokine gene activation in synovial membrane, regional lymph nodes, and spleen during the course of rat adjuvant arthritis.

Cytokine gene activation was assessed during rat adjuvant arthritis (AA) in synovial membrane (SM), popliteal lymph node (popl-LN), and spleen, using semiquantitative, competitive RT-PCR. Changes in the popl-LN were considerably higher than in spleen or SM. In the preclinical phase (day 6), cytokine mRNA elevations occurred exclusively in the popl-LN and included IFN-gamma, IL-1beta, IL-5, IL-6, and IL-10. In the acute phase (days 13-16) all three organs became involved: (i) in the SM, significant elevations were limited to IL-1beta and IL-6, which, notably, correlated positively with the degree of arthritis; (ii) in the popl-LN, IFN-gamma, IL-1beta, IL-6, and IL-10 (but not IL-5) were still elevated, while IL-2 rose significantly; (iii) in the spleen, TNF-alpha peaked simultaneously with the arthritis score (day 16) and dramatically dropped thereafter. Upon transition into the chronic phase (day 20) the following phenomena were observed: (i) IL-1beta and IL-6 were still significantly increased in the SM; (ii) IFN-gamma, IL-1beta, IL-2, IL-6, and IL-10 were still elevated in the popl-LN; and (iii) there was a progressive rise of IL-5 mRNA in the spleen, positively correlated with the arthritis score. In conclusion, cytokines with pro- and anti-inflammatory functions overlap throughout disease, but in different organ-related patterns. Local (SM) and regional (popl-LN) IL-1beta and IL-6, elevated throughout the entire course of AA, may directly contribute to disease severity. While in AA spleen TNF-alpha appears to be a systemic marker of acute disease, spleen IL-5 may be involved in disease resolution.

Anti-inflammatory effects of systemic anti-tumour necrosis factor alpha treatment in human/murine SCID arthritis.

OBJECTIVES: To evaluate in vivo the contribution of tumour necrosis factor alpha (TNFalpha) to the chimeric transfer model of human rheumatoid arthritis synovial membrane into SCID mice (hu/mu SCID arthritis), systemic anti-TNFalpha treatment was performed and the clinical, serological, and histopathological effects of this treatment assessed. METHODS: Animals were treated with the rat-antimouse TNFalpha monoclonal antibody V1q, starting on day 1 after hu/mu engraftment, twice weekly for 12 weeks. Joint swelling, serum concentrations of human and murine interleukin 6 (IL6), and serum amyloid P (SAP) were measured. Histopathological and immunohistochemical analyses of the joints were also performed at the end of treatment. RESULTS: Neutralisation of murine TNFalpha induced the following effects: (a) reduction of extent and duration of the acute arthritis phase, with significant reduction of joint swelling at two weeks; (b) decrease of murine SAP concentrations after the first antibody administration; and (c) increase of murine IL6 in the serum. At the end of treatment, there was a significant reduction of the inflammatory infiltration in the engrafted joints. Because of the mild degree of joint erosion, no treatment effects could be demonstrated on the destructive process. CONCLUSION: In the lymphocyte independent hu/mu SCID arthritis, anti-TNFalpha treatment reduces local and systemic signs of inflammation.

Age-dependent levels of select immunological mediators in sera of healthy children.

Serum cytokine levels were measured in 275 healthy children of different ages (3 to 17 years). Interleukin-1 receptor antagonist (IL-1RA), soluble IL-2R (sIL-2R) (sCD25), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), soluble TNF receptor type II (sTNF-RII) (sCD120b), gamma interferon (IFN-gamma), soluble intercellular adhesion molecule 1 (sICAM-1) (sCD54), soluble E selectin (sE-selectin) (ELAM-1; sCD62E), sCD14, and neopterin were measured with commercial test kits. The mean levels of IL-1RA, sIL-2R, TNF-alpha, sICAM-1, sE-selectin, and sCD14 were higher than in healthy adults. In contrast, IFN-gamma and IL-8 were hardly detectable in children and thereby significantly lower than in adults. In the case of TNF-alpha, sICAM-1, sE selectin, and sCD14, there was a high interindividual variability, apparently unrelated to disease. The profiles of some cytokines, i.e., IL-1RA, IL-6, and TNF-alpha, showed age-related increases that overlapped with known patterns of physical growth. Of note, sIL-2R and sE-selectin instead declined with time. Because of the remarkable age-dependent variability in healthy pediatric subjects, disease-related changes, as well as therapy-dependent alterations, should be considered with caution.

[Quantitative assessment of amounts consumed using the combined methods of a food frequency questionnaire and a 3-day dietary protocol]. / Quantitative Einschätzung von Verzehrsmengen durch eine Methodenkombination von Häufigkeits- und 3-Tage-Ernährungs-Protokoll.

With this paper we attempt to present a possible way of making quantitative estimates of the quantities of selected, preventively important food stuff groups consumed using a food frequency questionnaire together with a 3-day dietary protocol. The 3-day dietary protocol used in the 1984/85 MONICA survey of the former GDR is used as the standard. For large nutritional/epidemiological studies (e.g., cancer or cardiovascular cohort studies) this combination of methods is suggested as a means of arriving at quantitative estimates for nutritional habits in relation to the product groups presented here. Further investigations regarding correction factors are necessary where the food frequency is either very low or very high to detect potential inaccuracy.

[Incidence and prognosis of stroke episodes in the East German population. Results from a stroke register 1985-1988]. / Häufigkeit und Prognose von Schlaganfallerkrankungen in der Bevölkerung Ostdeutschlands. Ergebnisse von Schlaganfallregistern in den Jahren 1985-1988.

OBJECTIVES: To determine the incidence rate of stroke, case-fatality within 28 days and diagnostic accuracy in stroke cases in the study population. METHODS: Prospective stroke register, including all cases of complete stroke which occurred during 1985-1988 in the population aged 25-64 and 25-74, of 14 districts in East Germany. Case-respectively finding, validation and uniform data collection were carried out by physicians trained to use the WHO-MONICA protocol. RESULTS: Annual age-standardized event rates were 113 per 100,000 for men and 68/100,000 for women aged 25-64, rates for first-ever stroke were 88/100,000 for men and 52/100,000 for women. For both sexes, incidence and event rates rose exponentially with increasing age. Before age 45 more women than men were affected. 34% of patients aged 25-64 had died by 28 days. Case-fatality in men and women aged 65-74 was higher than in those ten years younger (p < 0.01) and tended to be higher in women. 10% of patients aged 25-64 and 26% of those in the age of 65-74 were treated outside hospital. Before age 65, CT examination and angiography were done in only 18 and 20%, respectively; in elderly patients even less frequently. CONCLUSIONS: The findings suggest that since the 1970s there may have been an increase of stroke incidence in the population of Eastern Germany. The high case-fatality following stroke is confirmed. Evidently there are deficiencies in the medical care of elderly stroke patients.

Follow-up studies in a case of unusual congenital myopathy, suggestive of nemaline type.

A 20-month-old boy--offspring of consanguinous parents, whose mother presumably had subclinical myopathy--presented with clinical signs of congenital non-progressive myopathy, neurogenic-myogenic electromyographic findings and normal motor conduction velocity. Biopsy of quadriceps muscle showed fiber-type disproportion with hypotrophic type 1, hypertrophic 2A and absent 2B fibers. Subsarcolemmal segmental foci of abnormally, in part regularly arranged bundles of mostly thin myofilaments were found in 13% of hypotrophic type 1 fibers. Rods were seen in only 1 fiber out of 20 tissue blocks. Reexamination 6 years later revealed slightly increased muscle force, myopathic EMG pattern and borderline motor and sensory nerve conduction velocities. Biopsy specimen from deltoid muscle consisted of untypable fibers of varying diameters with jagged Z-lines and increased variability of myofibrillar diameters. Multiple rods were present in 1% of the fibers, the formerly seen segmental foci in 0.1% only. Several intramuscular nerves were normal. The case contributes some new features to the spectrum of congenital myopathies of the nemaline type and suggests different stages of arrested maturation of type 1 fibers at least in this particular case.

Intrathecal humoral immune reaction in zoster infections.

Intrathecal humoral immune reaction in 26 patients with a reactivation of varicella-zoster virus was analyzed. 11 suffered from ganglionitis, in 7 cases an additional affection of the lower motor neuron was demonstrable. In 8 patients, meningitis, myelitis or cerebral infarctions by zoster angiitis were diagnosed. Intrathecal immune reaction in ganglionitis was weak whereas an intense IgG synthesis became demonstrable in all meningomyelitis/cerebral infarction cases. As demonstrated by immunoblotting, in early stages of the disease immune reaction in serum and cerebrospinal fluid differed only quantitatively. In the further course, most intrathecally synthesized antibodies were directed against low molecular antigens whereas serum pattern did not change. In some patients, additional antibodies not detectable in serum were demonstrable. Though intensity markedly differed, no qualitative differences between the immune response in ganglionitis and more widespread zoster infections of the CNS were detectable.

[Acute paraparesis in chronic hepatic disease]. / Akute Paraparese bei chronischer Hepatopathie.

An acuta caude equina syndrome following portacaval shunt in a 64-year-old patient suffering from liver cirrhosis is reported. Diagnosis of a hepatic radiculopathy was confirmed by clinical and electrophysiological findings. A nearly complete remission of the neurological symptoms was achieved by treatment of the primary liver disease. Since there are only a few descriptions of severe hepatic polyneuro- and radiculopathies, the differential diagnosis of hepatic myelopathy is discussed.

[Diagnostic value of cerebrospinal fluid study in multiple sclerosis]. / Die diagnostische Wertigkeit der Liquoruntersuchung bei multipler Sklerose.

Multiple sclerosis ist a very frequent chronic inflammatory disease of the central nervous system. Since clinical and neuro-imaging findings often may be ambiguous, cerebrospinal fluid examination has received great importance for diagnosis. By that method it becomes possible to differentiate inflammatory from noninflammatory diseases. Furthermore, cerebrospinal fluid findings are discussed which seem to be characteristic for multiple sclerosis.

Differentiation of the humoral immune response in herpes simplex encephalitis.

Six patients with herpes simplex encephalitis were investigated. Antibody activities against different viral antigens were determined in serum and cerebrospinal fluid with an enzyme-linked immunosorbent assay after adjusting both fluids to identical immunoglobulin G concentrations. In serum, the strength of the antiviral reaction remained low and no qualitative changes became detectable. In cerebrospinal fluid, at first locally synthesized antibodies were directed against the same antigens as in serum. Ten days later, the intrathecal reaction increased with additional antibodies against at least two antigens. Once established, this expanded heterogeneity remained stable during the course of the disease.

[Diagnostic value of muscle biopsy in neuromuscular diseases]. / Diagnostische Wertigkeit der Muskelbiopsie bei neuromuskulären Erkrankungen.

Aside from clinical electrophysiology, muscle biopsy is an important method regarding the diagnosis of neuromuscular diseases. Functional disturbances like myasthenia, myotonia, or paramyotonia only lead to slight and unspecific morphological alterations of the muscle tissue. In most cases, progressive muscular dystrophies, myotonic dystrophy, and myositis of various etiologies each have their own bioptic and electrophysiological characteristics. The diagnosis of these diseases should be confirmed by both methods. Some inflammatory diseases, such as panarteriitis nodosa and muscular sarcoidosis, may be diagnosed on account of histologic findings only. The same is true for rhabdomyolysis and congenital myopathies.

A method to determine the end point of decalcification of hard tissue and bone.

A method for decalcification end point determination of mineralized tissue is described. The calcium content of the decalcification solution was determined colorimetrically with a "continuous automatic analyzer" with a high degree of accuracy. The end point method used has been tested on two decalcification methods, 5% nitric acid with or without ultrasonic treatment. The results suggest it is possible to quantitate the decalcification process.

Intrathecal synthesis of virus antibodies: a diagnostic test for multiple sclerosis.

Intrathecally synthesized antibodies against measles and rubella were determined in 221 patients with multiple sclerosis (MS) and in 476 control cases. A local production was detectable in more than 80% of the MS cases but in none of the control group. These results show that the demonstration of intrathecally synthesized antibodies against neurotropic viruses not related with respective infections may serve as helpful test in the diagnosis of MS.

Variation in coronary risk factor levels of men and women between the German-speaking MONICA centres.

The aim of this analysis was to compare levels of risk factors for coronary heart disease (CHD) in men and women aged 25-64 years between German-speaking MONICA collaborating centres, the German Democratic Republic (GDR), Augsburg - the Federal Republic of Germany (FRG)(Au), Bremen - FRG (Br), Heidelberg - FRG (He), and Vaud/Fribourg - Switzerland (CH, with a German-speaking minority). Prevalence of cigarette smoking in men showed little variation in four centres (34 to 40%) and was higher in BR men (49%), while it varied from 17% (GDR) to 33% (BR) in women. Mean total serum cholesterol values (mmol/L) were highest in GDR and CH men (both 6.2) and GDR women (6.1), and lowest in both He men (5.7) and He women (5.6). The proportion with cholesterol values greater than or equal to 6.7 mmol/L was largest in CH men (34%) and smallest in FRG (He) women (17%), while lowering the cut-off point from 6.7 to 6.5 mmol/L raised the prevalence of hypercholesterolaemia in all centres by 5 to 10%. Mean values (mmHg) of blood pressure (BP) were highest in both GDR men (140/88) and women (138/86), as was the prevalence of hypertensive BP values. In all centres, women aged 25-34 had BP values approximately 12/5 mmHg lower than age-matched men, but BP values similar to men at age 55-64, which indicates that age-parallel increase in BP was steeper in women than men.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of phospholipase A2 in human acute pancreatitis.

In a prospective clinical trial, 85 patients with acute pancreatitis, including 50 with acute interstitial-edematous pancreatitis and 35 with necrotizing pancreatitis, were recruited. Serum pancreatic immunoreactive phospholipase A2 (IR-PLA2), serum phospholipase A catalytic activity (CA-PLA), and serum phospholipase A2 catalytic activity (CA-PLA2) were determined daily between day 1 and day 10 after the onset of the disease. The serum course of IR-PLA2 values for patients with acute interstitial-edematous pancreatitis was comparable to that for patients with necrotizing pancreatitis. In contrast, the determination of CA-PLA and of CA-PLA2 specific activity in the serum revealed a high differentiation between patients with interstitial edematous and those with necrotizing pancreatitis. The overall accuracy for differentiating patients with necrotizing pancreatitis from those with the interstitial-edematous type was 79% for CA-PLA and 77% for CA-PLA2 (cut-off level: CA-PLA, 15 U/L, day 1-5; CA-PLA2, 3.5 U/L, day 1-5). Patients with pancreatitis-associated pulmonary complications showed significantly higher CA-PLA and CA-PLA2 values in the serum. This study demonstrates the role of serum catalytic phospholipase A2 in human acute pancreatitis where the development of pancreatic necrosis and pulmonary failure is concerned.

Involvement of oxygen radicals and phospholipase A2 in acute pancreatitis of the rat.

The purpose of this study was to assess the involvement of oxygen radicals and phospholipase A2 in acute pancreatitis. Acute pancreatitis was induced in rats by the CCK-analogue cerulein (5 micrograms/kg.h) for 30 min, 3.5 h, and 12 h. At the end of the infusion, serum enzymes and the lipid peroxidation products conjugated dienes and malondialdehyde in the tissue were measured. Moreover, tissue samples underwent lightmicroscopical examination. After 3.5 h cerulein, an interstitial edema and a beginning accumulation of granulocytes in the pancreatic gland is observed. These changes are aggravating within 12 h, leading to tissue necrosis and migration of the granulocytes into the tissue. Concomitantly amylase and lipase increased by 15 and 35 times, respectively. Conjugated dienes and malondialdehyde increase already after 30 min cerulein and reach their highest levels after 3.5 h cerulein. At the same time the tissue activity of phospholipase A2 is elevated three fold. Rats were treated with superoxide dismutase and catalase before cerulein infusion. Treatment significantly prevents tissue necrosis, granulocyte accumulation, and edema formation. The enhanced activity of phospholipase A2, however, is unaffected by the treatment. Oxygen radicals seem to be instrumental in the development of the disease.