A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.     

Genetic determinants: is there an "atherosclerosis gene"?

It is now clear that atherosclerotic disease is a chronic inflammatory disease triggered by a sequence of events initiated at sites with turbulent flow under normal conditions such as in the coronary arteries or at bifurcations or where normal laminar flow is replaced by turbulent flow because of vessel pathologies. Normally, laminar flow is protected by generation of NO by endothelial NO synthase (eNOS), which becomes activated via stretch activated channels. When the flow turns turbulent, such protective NO generation ceases, leading to endothelial cell activation and lipid deposition into the extra-cellular space. There, lipoproteins and specifically phospholipids become oxidized by cells of the monocytic-macrophage lineage. Only when the LDL-cholesterol level is high enough lipid peroxidation products are generated in sufficient amounts to perpetuate the disease by generating a feed forward loop of endothelial cell activation leading to an inflammatory response. That inflammatory response might also be added by bacterial or viral infections such as Chlamydia pneumoniae or viruses. The disease then progresses to a chronic inflammatory state, whereby the immune system seems to contribute significantly and markers of chronic inflammation such as fibrinogen, leukocytes, PAI-1 and CRP are found increased.     

Prostaglandin E2 synthesis in human monocyte-derived dendritic cells

Prostaglandin E2 (PGE2), which is generated by the enzymatic activity of cyclooxygenase-1 and -2 (COX-1/2), plays a central role in the maturation process of dendritic cells (DC). Since regulation of COX-1/2 expression in human DC is only partially understood, we addressed the expression and activity of COX-1/2 in these cells. Here we show that lipopolysaccharide (lps) induces COX-2 mRNA and protein synthesis as well as the release of PGE2 in human interleukin-4 and granulocyte/macrophage colony-stimulating factor-differentiated monocyte-derived DC cultivated in the presence of 1% human plasma. Moreover, we found that lps induces p38 stress-activated protein kinase (p38) in these cells and inhibitors of p38 blocked lps-induced COX-2 expression and activity. Our data indicate that during lps-induced maturation p38 regulates COX-2 expression and PGE2 synthesis in DC.     

Telecommunication and telepathology. New diagnostic routes for orthopaedics and orthopaedic pathology

In a pilot project of the Department of Bone Pathology of the University of Hamburg and the Orthopaedic Department of the University of Heidelberg, the cases of 121 patients with suspicion of a primary bone tumour have been discussed at weekly interdisciplinary conferences during the period from July 2001 to May 2002., The consequent differential diagnoses were made prior to the biopsy, the optimal location of the biopsy and the further strategy was determined according to the guidelines of the international bone tumour centres. The latter includes the decision if a conventional biopsy or a intraoperative pathology examination on frozen sections should be performed. In 27 cases an intraoperative pathology examination was performed and then assessed in Hamburg. In 24 cases this diagnosis was identical with the final diagnosis. In three cases no definitive diagnosis could be made from the frozen sections. Additionally the pathohistological diagnoses of the cases of the previous week have been discussed in the video-conferences. Through this a unusually close interdisciplinary cooperation over a large distance has evolved, that is highly appreciated especially by the young and less experienced colleagues at the department of bone pathology and the orthopaedic department in Heidelberg. The awareness of the potential and limitations of a medical subject leads to an improved safety in the diagnostic process for bone tumours. The interdisciplinary discussion of all aspects of the diseases may also optimise the therapy of bone tumours.     

Sleep and memory consolidation: The role of electrophysiological neuroimaging

Summary Memory consolidation involves a complex series of molecular, cellular and network-level processes that take place on time scales from millisecond to months. Evidence from a wide range of experimental observations supports the hypothesis that parts of these processes occur during sleep when the brain is not engaged in processing and encoding incoming information. Indeed, sleep seems to be favorable for brain plasticity. Experience-dependent cortical plasticity observed during sleep has been hypothesized to be part of the global process of memory consolidation. Thus, studying task-dependent, regionally specific reactivation of neuronal assemblies during posttraining sleep may make important contributions to elucidating the role of sleep in memory trace processing. A new methodology – low-resolution brain electromagnetic tomography (LORETA) – offers the possibility of localizing electrical activity produced by cortical neuronal generators under normal (undisturbed) sleeping conditions. The high time resolution of brain electrical data can be exploited to produce neuroimages for specific EEG spectral frequency bands (e.g. delta, theta, or spindle bands). This makes it possible to investigate, dependent on the type of memory, when – in which sleep stages (S2 sleep, SWS, REM sleep) – and where – in which cortical brain regions (primary sensory cortex, higher association cortex) – experience-dependent reactivation occurs.     

Localization of a highly specific neuronal protein,serotonin binding protein,in thyroid parafollicular cells

A highly specific serotonin binding protein (SBP) has been found in serotonergic neurons in both brain and gut. This protein has an extremely high affinity for serotonin and may be a storage protein. Serotonin is found in many endocrine cells, including parafollicular cells of the sheep thyroid, as well as in neurons. SBP is also present in sheep thyroid. The present study was done to localize the protein in the gland. Thyroid glands were divided into five segments. Concentrations of serotonin and SBP, as well as parafollicular cell volume were measured in each. Serotonin was assayed by enzymatic conversion to melatonin using tritiated S-adenosylmethionine. SBP was assayed by molecular sieve chromatography on sephadex G-50. The relative volume of parafollicular cells was obtained by stereological analysis of electron micrographs. Experiments were also done to demonstrate these cells by histofluorescence and radioautography following incubation with tritiated 5-hydroxytryptophan. Good correlations were found between serotonin and SBP concentrations, and parafollicular cell volume. These peaked in the rostro-central portion of the gland and were minimal at the poles. We conclude that thyroid SBP is probably localized in parafollicular cells.     

Dielectric properties of a combined main-chain/side-chain liquid-crystalline polymer

The dielectric relaxation properties of a combined main-chain/side-chain liquid-crystalline polymer were investigated. It was found that the rotation of the side chain about the main chain (δ-process) is not as strongly restricted as in side-chain liquid-crystalline polymers. This is attributed to the facts that the side chain is attached to the flexible spacer within the chain backbone and that the concentration of the side chains is comparatively small. Two low-temperature relaxation processes were observed to occur in the glassy smectic and the crystalline state. They are attributed to intramolecular motions with in the mesogenic groups.     

Urinary dopamine excretion in healthy volunteers: effect of sodium diet and acute water load

The present study was designed to investigate, in human subjects, urinary dopamine excretion under different conditions of sodium and water homeostasis. In a cross-over trial, ten healthy volunteers were subjected to low-salt (LS; dietary salt restriction, sodium chloride (NaCl) intake <5 g per day), normal-salt (NS; normal food ad libitum), and high-salt (HS; normal food plus NaCl 100 mg/kg per day) regimens for 8 days in a randomized order. On day 7, urine was collected for 24 h. The variations in urinary sodium excretion reflected the dietary salt intake (LS: 16.3+/-4.7; NS: 144.1+/-18.2; HS: 221.9+/-12.9 mmol 24 h(-1) 1.73 m(-2)), but were not accompanied by significant changes in urinary dopamine excretion. On day 8, clearance studies showed that an acute oral water load of 1500 ml did not alter glomerular filtration rate or renal plasma flow but significantly increased urinary flow rate without affecting dopamine excretion. Assuming that excreted dopamine is not metabolized or reabsorbed during the tubular passage, both the unchanged urinary dopamine output in spite of 14-fold variations in sodium excretion and its independence of an acute water load argue against the hypothesis that dopamine in the tubular lumen acts as a natriuretic and/or diuretic factor in humans.     

Pathologische Myophosphorylasereaktion bei der malignen Hyperthermie

Zusammenfassung Die Myophosphorylasereaktion (MPR) zeigt bei der malignen Hyperthermie (MH) charakteristische Veränderungen, die in Minutenschnelle entstehen: 1.) eine allgemein stark abgeschwächte Reaktion, 2.) zahlreiche negativ reagierende Fasern, 3.) häufig fleck- und/oder streifenförmig abgeschwächt oder negativ reagierende und streifenförmig verstärkt reagierende Faserabschnitte mit relativ engen Sarkomerenabständen (Zeichen der Hyperkontraktion). Diese morphologischen Befunde mit den kennzeichnenden Streifenfasern sind offenbar für das MH-Syndrom spezifisch! Wichtig ist, daß die derart veränderten Muskelfasern in der Regel bei den anderen Färbungen und Reaktionen unauffällig sind.Wir sahen diese pathologische MPR bei 5 verstorbenen Patienten, einmal unabhängig von einer Anästhesie nach einem Marsch, und bei 19 Schweinen, davon 18mal nach dem Halothantest und einmal bei einem Versuchstier mit gesicherter MH. Sie fehlte bei Schweinen mit negativer Halothanreaktivität und vor dem Halothantest und bei vielen, sehr verschiedenen gesunden und kranken Kontroll- und Vergleichsfällen aus dem bioptischen und autoptischen Untersuchungsgut. Die MPR erlaubt den sicheren Nachweis einer inszenierten oder abgelaufenen MH und damit eine Aufklärung rätselhafter oder scheinbar klarer Todesfälle während oder nach der Narkose oder allgemein nach Streß (human stress syndrome), von denen sicher viele der klinischen Aufmerksamkeit und den üblichen morphologischen Untersuchungsmethoden entgehen. Sie ist nicht geeignet, potentielle Opfer zu erkennen.Mit Unterstützung des Ministeriums f. Gesundheitswesen (HFR Schwangerschaft und frühkindliche Entwicklung, FR Genetische Defekte). Auszugsweise vorgetragen auf dem IV. Internationalen Myologie-Kolloquium in Jena 1984