Christa Liebenthal

Mechanisms of endotoxin tolerance in patients with alcoholic liver cirrhosis: role of interleukin 10, interleukin 1 receptor antagonist, and soluble tumour necrosis factor receptors as well as effector cell desensitisation

BACKGROUND In patients with alcoholic liver cirrhosis, endotoxaemia is a frequent finding. Unknown mechanisms, however, prevent typical clinical symptoms of endotoxaemia in many patients. METHODS We determined plasma levels of pro- and anti-inflammatory mediators, ex vivo cytokine secretion capacity, and expression of tumour necrosis factor (TNF) receptors on phagocytic blood cells in 49 patients with alcoholic cirrhosis and 41 age matched healthy controls. RESULTS In addition to increased levels of proinflammatory cytokines in cirrhotic patients, we observed consistent upregulation of the anti-inflammatory mediators interleukin 10 (IL-10) (plasma 15.75 (1.6) v6.6 (1.3) pg/ml (p<0.001); ex-vivo 108.4 (22.0)v 40.1 (7.4) pg/ml (p<0.05)), interleukin 1 receptor antagonist (plasma 527.1 (83) v331.4 (56) pg/ml (p<0.05); ex vivo 19.9 (3.4)v 10.2 (2.7) ng/ml (p<0.01)), and soluble TNF receptors (sTNF-R) in plasma (sTNF-RI 3157.2 (506.2)v 607.9 (300.3) pg/ml; sTNF-RII 3331.0 (506.2) v 1066.4 (225.1) pg/ml (p<0.001 for both)). Desensitisation at the target cell level was indicated by reduced expression of TNF receptor I on granulocytes (64.8 (6.5)v 40.1 (7.3)% positive cells; p<0.05) and unaltered plasma levels of soluble E-selectin. CONCLUSION In patients with alcoholic liver cirrhosis, upregulation of the pro- and anti-inflammatory cytokine system and simultaneous desensitisation of effector cells could explain the restricted systemic inflammatory response to chronic endotoxaemia. This alteration in immune status may lead to impairment of host defences against infections which are frequent complications of alcoholic cirrhosis.

Interleukin-6 and interleukin-8 concentrations as predictors of outcome in ventricular assist device patients before heart transplantation

ObjectiveTo determine whether the serum concentrations of some circulating cytokines (as highly sensitive markers of inflammation) are of value in predicting the outcome of patients with cardiogenic shock and end-stage heart disease, who undergo ventricular assist device implantation until heart transplantation. DesignCohort study. SettingUniversity teaching hospitals. PatientsTwenty patients with cardiogenic shock or end-stage heart disease were consecutively selected for this study, if assist device implantation was performed as a bridge to heart transplantation. Measurements and Main ResultsThe circulating concentrations of the cytokines inter-leukin (IL)-1 β, IL-6, IL-8 and tumor necrosis factor (TNF)-α were monitored from the beginning to the end of assist device support two to three times a week, using commercial enzyme-linked immunosorbent assays (ELISA). In all patients, circulating IL-6 and IL-8 values were increased shortly after assist device implantation. In patients with uncomplicated courses, IL-6 and IL-8 concentrations decreased after an initial increase and were low at the time of transplantation, whereas serum cytokine concentrations increased and remained increased in the nonsurvivors (survivors vs. nonsurvivors, p < .001). Circulating IL-1β and TNF-α concentrations were rarely detectable. ConclusionsMonitoring of IL-6 and IL-8 values during ventricular assist device support provides a means of early identification of highrisk patients that may allow optimization of antimicrobial therapy and selection of the appropriate time for transplantation. (Crit Care Med 1994; 22:448–454)

Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3-5) making the interpretation difficult. We modified the assay by the addition of interferon-alpha and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-alpha were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

Immortalization of magnetically separated human lymphocytes by electrofusion

Human lymphocytes from peripheral blood (MNC) were separated on magnetic beads for the presence of different surface markers. Cells from positive and negative fractions were successfully immortalized by electrofusion with the heteromyeloma line CB-Fu2. B cells, which were separated on anti-CD 19 coated beads, could be immortalized at a rate between 10(-5) and 10(-4) even if the fusion was conducted with just a few hundred thousand cells. Comparison of the frequency of Ig-positive hybridomas in B cell, T cell, and unseparated MNC fusions indicated that also non-B cells may give rise to HAT resistant hybridoma clones, although the fusion frequency was low.

Untersuchungen zur diagnostischen Wertigkeit des Lymphozytentransformationstestes bei Patienten mit Borreliose / Evaluation of the diagnostic significance of the lymphocyte proliferation test in patients with Lyme borreliosis

Zusammenfassung Borrelienspezifische Antikörper sind erst mehrere Wochen nach Infektion nachweisbar und allein kein Beweis für eine aktive Borreliose. Die Sensitivität von Kultur und PCR ist für den Nachweis bzw. Ausschluss einer Borreliose zu gering. Es bedarf einer Methode, die ausschließlich eine aktive Borrelieninfektion möglichst früh anzeigt. Es wurde dazu ein Lymphozytentransformationstest (LTT) mit drei Borrelienlysatantigenen (Borrelia B. sensu stricto, B. afzelii und B. garinii) sowie rekombinantem OspC entwickelt und mit Untersuchungen von seronegativen (n=100) und seropositiven Gesunden (n=36) sowie seropositiven Patienten mit klinischer Borreliose (n=44) validiert. Die Sensitivität des Borrelien-LTT für eine klinische Borreliose vor antibiotischer Behandlung wurde mit 91%, die Spezifität mit 94% ermittelt. Bei 820 Patienten mit klinischem Verdacht auf Borreliose wurde eine Übereinstimmung positiver und negativer serologischer und LTT-Ergebnisse von 77,3% gefunden. Serologisch positiv und im LTT negativ waren 165 Patienten (20,1%), überwiegend mit Borreliose nach antibiotischer Therapie. Serologisch negativ und im LTT positiv waren 21 Patienten (2,6%), davon sieben mit Erythema migrans. Nach antibiotischer Behandlung wird der Borrelien-LTT bei Patienten mit Frühmanifestationen der Borreliose (n=90) negativ bis grenzwertig, bei Spätmanifestationen (n=70) rückläufig mit verbleibender Restaktivität. Verlaufsuntersuchungen über ein Jahr von sechs Patienten mit Frühmanifestationen zeigten nur eine Reaktivierung. Bei acht von zehn Patienten mit Spätmanifestationen wurden häufig Reaktivierungen bzw. persistierend positive LTT-Reaktionen beobachtet. Als Indikation für den Borrelien-LTT wird deshalb besonders die Verlaufskontrolle bei disseminierten Borrelieninfektionen vorgeschlagen. Abstract Borrelia-specific antibodies are not detectable until several weeks after infection and their presence alone is not proof of an active infection. The sensitivity of culture methods and PCR for the confirmation or exclusion of Lyme borreliosis is too low. Therefore, a method is required that detects an active Borrelia infection as early as possible. For this purpose, a lymphocyte proliferation test (LPT) using three endogenous Borrelia antigens (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) and recombinant OspC was developed and validated by investigating seronegative (n=100) and seropositive (n=36) healthy individuals, as well as seropositive (n=44) patients with clinically overt borreliosis. The sensitivity of the Borrelia LPT in clinical borreliosis before the administration of antibiotic treatment was 91%, while the specificity was 94%. In 820 patients with clinical suspicion of borreliosis, positive and negative results by serology and LPT were in agreement in 77.3% of cases. Some 165 patients (20.1%) were serologically positive and negative by LPT. These were mainly patients with borreliosis after antibiotic therapy. Furthermore, 21 patients (2.6%) had negative serology and a positive LPT result, seven of whom had erythema migrans. Following antibiotic treatment, the LPT becomes negative or borderline in patients with early manifestations of borreliosis, whereas in patients with late symptoms it demonstrates a regression while remaining positive. Follow-up investigations over a period of 1 year yielded one reactivation among six patients with early manifestations, in contrast to eight out of ten patients with late symptomatology that exhibited frequent episodes of reactivation and/or persistently positive LPT reactions. Therefore, we propose follow-up monitoring of disseminated Borrelia infections as the main indication for the Borrelia LPT.