Volker von Baehr

Comparison of Monocyte Functions after LPS- or IL-10-Induced Reorientation: Importance in Clinical Immunoparalysis

Immunoparalysis is an acquired immunodeficiency which may occur in patients after major surgery, burns, polytrauma and sepsis. It is associated with a modified state of monocytes marked by their altered capacity to induce antigen-specific T cell stimulation and to release various cytokines. However, the pathogenesis of immunoparalysis may differ in various patient groups. It can develop in patients after systemic hyperinflammation induced by gastrointestinal translocation of endotoxin (lipopolysaccharide, LPS) or sepsis, as well as in patients without preceding systemic inflammation but primary anti-inflammation, for instance induced by sympathetic activation. To further elucidate the syndrome, we compared endotoxin tolerance as a model of immunoparalysis after systemic hyperinflammation versus interleukin-10 (IL-10) treatment as a model of primarily anti-inflammation-induced immunoparalysis. In vitro priming of peripheral blood mononuclear cells with either LPS or IL-10 for 24 h led to a strongly or moderately diminished LPS-induced tumor necrosis factor-α (TNF-α) production, compared to unprimed controls, respectively. Furthermore, LPS-induced reduction of TNF-α production capacity persisted over the following days whereas IL-10-primed monocytes rapidly recovered. Similarly, in contrast to persistently diminished MHC class II expression in LPS-treated monocytes, IL-10 only transiently downregulated these molecules. Consequently, in contrast to IL-10-primed monocytes, LPS-primed monocytes were greatly impaired in their capacity to induce antigen-specific T cell proliferation and IFN-γ production. These data indicate that LPS priming provokes a more profound modulation of monocyte function than IL-10 priming, raising the question of possible variations in the clinical course of immunoparalysis, dependent on its pathogenesis.

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.

Deficient EBV-Specific B- and T-Cell Response in Patients with Chronic Fatigue Syndrome

Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4+ and CD8+ T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.

Compound deletion of the rhoGAP C1 and V2 vasopressin receptor genes in a patient with nephrogenic diabetes insipidus

The function of small GTPases is fine‐tuned by a complex network of regulatory proteins such as GTPase‐activating proteins. The C1 gene at Xq28 encodes a protein assumed to function as a Rho GTPase‐activating protein (rhoGAP). Characterization of the molecular defect causing X‐linked nephrogenic diabetes insipidus (NDI) in a patient revealed a submicroscopic deletion of a 21.5‐kb genomic fragment encompassing the entire arginine‐vasopressin V2 receptor gene (AVPR2) and most of the C1 gene locus. In the absence of detailed information about the physiological relevance and specific functions of rhoGAP C1, a thorough clinical and laboratory investigation of the patient was performed. Besides clearly defined NDI symptoms caused by deletion of the AVPR2 gene, no major morphological abnormalities as determined by physical examination, radiography, ultrasound, and computed tomographic scan were detected. Extensive analysis of blood chemical, enzyme, and hormone values over a period of 16 years showed no deviations from normal ranges. On the basis of our observations, the rhoGAP C1 protein is not essential for normal development in the human. Because of a predominant expression pattern of the C1 gene in hematopoietic cells, we focused on immunologic and hematologic laboratory parameters of the affected boy and the mother who was found to be heterozygous. Differential white cell counts, including lymphocyte typing, determination of lymphokines, cytokines, and immunoglobulins, as well as numerous leukocyte function tests, showed no pathological findings. Therefore, we postulate that the loss of rhoGAP C1 function is most likely compensated by other members of the GAP family. Hum Mutat 14:163–174, 1999.

The Lymphocyte Transformation Test for Borrelia Detects Active Lyme Borreliosis and Verifies Effective Antibiotic Treatment

Borrelia-specific antibodies are not detectable until several weeks after infection and even if they are present, they are no proof of an active infection. Since the sensitivity of culture and PCR for the diagnosis or exclusion of borreliosis is too low, a method is required that detects an active Borrelia infection as early as possible. For this purpose, a lymphocyte transformation test (LTT) using lysate antigens of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii and recombinant OspC was developed and validated through investigations of seronegative and seropositive healthy individuals as well as of seropositive patients with clinically manifested borreliosis. The sensitivity of the LTT in clinical borreliosis before antibiotic treatment was determined as 89,4% while the specificity was 98,7%. In 1480 patients with clinically suspected borreliosis, results from serology and LTT were comparable in 79.8% of cases. 18% were serologically positive and LTT-negative. These were mainly patients with borreliosis after antibiotic therapy. 2.2% showed a negative serology and a positive LTT result. Half of them had an early erythema migrans. Following antibiotic treatment, the LTT became negative or borderline in patients with early manifestations of borreliosis, whereas in patients with late symptoms, it showed a regression while still remaining positive. Therefore, we propose the follow-up monitoring of dis-seminated Borrelia infections as the main indication for the Borrelia-LTT.

Clinical manifestation of mannose-binding lectin deficiency in adults independent of concomitant immunodeficiency.

Mannose-binding lectin (MBL) mediates important functions within the innate immune system, and its deficiency was associated with infectious complications. However, in adults without concomitant immunodeficiency the clinical relevance of MBL deficiency remains controversial. We analyzed the distribution of MBL deficiency and its association with concomitant immunodeficiency in 228 adult Caucasian patients with a history of recurrent and/or severe infections. Two hundred forty-one unrelated Caucasians without recurrent or severe infections served as control subjects. The frequency of severe MBL deficiency (plasma levels <or= 50 ng/ml) was significantly higher in patients with a history of recurrent and/or severe infections (p < 0.05, odds ratio 2.1, 95% confidence interval 1.1-4.1), and this association was independent of concomitant antibody or cellular immunodeficiency. Our data challenge the view that MBL deficiency in adulthood becomes relevant only in individuals who are immunocompromised for other reasons.

Hyperactivated Signaling Pathways of Chemokine RANTES/CCL5 in Osteopathies of Jawbone in Breast Cancer Patients—Case Report and Research

Background Hollow spaces in the jawbone have been defined as fatty degenerative osteonecrosis of jawbone (FDOJ) and have been linked with a dysregulated immune system. Little is known about the underlying relationship. Objectives Samples of FDOJ were analyzed to assess expression of cytokines which can play a role in the pathogenesis of breast cancer (MaCa). Material and Methods Samples of FDOJ extracted from 23 patients with MaCa and 19 healthy control jawbone samples were analyzed for 7 immune messengers. Results RANTES was found to be highly overexpressed in disease samples. No change was observed in expression levels of the other immune mediators. Discussion This data provides a compelling confirmation that FDOJ produces high levels of RANTES, a cytokine implicated in MaCa and metastasis. Levels detected in FDOJ are five-fold higher than that previously reported for MaCa tissue suggesting its role as a cytokine source in MaCa. Conclusion We thus hypothesize that FDOJ may serve as an expeditor of MaCa progression, through RANTES production.

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.

Impact of Endodontically Treated Teeth on Systemic Diseases

Background: This study compares the radiographic distribution of apical periodontitis (AP) in rootfilled and endodontically treated teeth among healthy controls and patients with systemic diseases; the incidence of AP was almost twice as high in the latter group.Objective: The question arises as to whether the biogenic amines (mercaptan/thioether/hydrogen sulfide) originating from endodontically treated teeth have systemic, subtoxic and immunological effects. Method: In order to determine this, local hydrogen sulfide measurements of endodontically treated teeth were combined with the laboratory serum analyses of modified proteins to assess the relationship of these compounds with type IV immune reactions.Results: It was found that 42.5% of the group with systemic diseases showed immunological disturbance as a result of root-filled teeth. Furthermore, the presence of AP was almost three times higher than in the control group (17.2% versus 5.9%, respectively).Conclusion: In summary, the data demonstrates that local pathologies caused by endodontically treated teeth may increase immunological and systemic dysfunction.

Aseptic-avascular osteonecrosis: local “silent inflammation” in the jawbone and RANTES/CCL5 overexpression

Of the definitions listed in the International Statistical Classification of Diseases and Related Health Problems, tenth revision (ICD-10), two disease descriptions can be found together: “idiopathic aseptic bone necrosis” and “avascular bone necrosis.” The relevant literature on both the conditions abbreviates both as “aseptic ischemic osteonecrosis in the jawbone” (AIOJ). To shed light on the clinical details of this condition, osteolytic jawbone samples of 24 patients with different systemic immunological diseases were examined using four steps: presurgical dental X-ray, postsurgical histology, polymerase chain reaction DNA analysis (PCR DNA) of bacteria, and RANTES/CCL5 (R/C) expression. These four steps showed that neither X-ray nor histology delivered unambiguous results with respect to inflammatory processes; furthermore, the PCR results did not show evidence of any microbial load within the jaw samples. However, there is a striking, coherent overexpression of chemokine R/C in the AIOJ samples. This study proved the aseptic existence of “silent inflammation” within the jawbone. The ICD-10 (AIOJ) definition, which is hard to interpret, can now be substantiated with clinical evidence, while the cytokine expressions described in this report can explain the systemic immunological effects observed within the group of examined patients.