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Johann Lechner,1 Volker von Baehr,2 Florian Notter,1 Fabian Schick1 1Clinic for Integrative Dentistry, Munich, Germany; 2Department of Immunology and Allergology, Institute for Medical Diagnostics, Berlin, GermanyCorrespondence: Johann Lechner, Gruenwalder Str. 10A, Munich, 81547, Germany, Tel +49 89 697 0129, Fax +49 89 692 5830, Email drlechner@aol.comIntroduction: Osteoimmunology recognizes the relationship between bone cells and immune cells. Chronic osteoimmune dysregulation is present in bone marrow defects of the jaw (BMDJ) as fatty-degenerative osteonecrosis (FDOJ). In comparison to samples from healthy jaw bone, the cytokine analysis of samples of BMDJ/FDOJ from 128 patients showed downregulated TNF-α and IL-6 expression and the singular overexpression of the chemokine RANTES/CCL5.Aim and Objectives: This paper raises the question of whether the osteoimmune defects due to incomplete wound healing in BMDJ/FDOJ in 128 patients are related to dysregulation of the Th1/Th2 ratio and regulatory T cell (T-reg) expression in a control group of 197 BMDJ/FDOJ patients, each presenting with BMDJ/FJOD and one of seven different immune disorders.Material and Methods: In the control group, serum concentrations of the cytokines IFN-y and IL-4 were determined after stimulated cytokine release and displayed as Th1/Th2 ratios.Results: Data show a shift in Th2 in more than 80% (n = 167) of the control cohort of 197 chronically ill patients with concomitant BMDJ/FDOJ. In these 167 subjects, the Th1/Th2 ratio was < 6.1 demonstrating impaired immune regulation. Forty-seven subjects or 30% showed not only a shift in Th2 but also excessive T-reg overactivation with levels of > 1.900 pg/mL, indicating strongly downregulated immune activity.Discussion: BMDJ/FDOJ is characterized by a lack of Th1 cytokines and an excessive expression of RANTES/CCL5 and IL-1ra and, thus, the inversion of an acute inflammatory cytokine pattern. In contrast, abdominal fat contains a very high proportion of regulatory Th1 cells and produces an inflammatory immune response through the high overexpression of TNF-α and IL-6. The lack of Th1 activation in BMDJ/FDOJ areas inhibits normal wound healing and supports the persistence of BMDJ/FDOJ.Conclusion: The Th1/Th2 ratio requires greater consideration, especially with respect to wound healing following dental surgical interventions, such as jaw surgery, implantation and augmentation, to avoid the emergence of the osteoimmune situation that is characteristic of BMDJ/FDOJ. Keywords: osteoimmunology, T-cells, Th1/Th2 shift, jawbone, osteoclastogenesis, RANTES/CCL5

Johann Lechner,1 Tilman Schulz,2 Beatrice Lejeune,3 Volker von Baehr4 1Clinic Integrative Dentistry Munich, Munich, Germany; 2Klinikum Bayreuth, Bayreuth, Germany; 3Labo’Life France, Nantes, France; 4Institute for Medical Diagnostics, Berlin, GermanyCorrespondence: Johann LechnerClinic Integrative Dentistry Munich, Praxisklinik Gruenwalder Str. 10A, Munich, 81547, GermanyTel +49 89 6970129Fax +49 89 6925830Email drlechner@aol.comBackground: The role of signaling pathways as part of the cell-cell communication within cancer progression becomes a crucial area. Chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted), also known as the chemokine C-C motif ligand 5 (CCL5) (R/C), is a protein on which cancer research focus due to its link with aggressive cancer development.Objective: Research on fatty-degenerative osteonecrosis in jawbone (FDOJ) shows striking overexpression of R/C in these areas. Here we try to elucidate a potential link between jawbone-derived R/C and breast cancer (BC) and compare these findings by immunohistochemical staining.Methods: Thirty-nine FDOJ samples extracted from 39 BC patients and samples from 19 healthy control were analyzed for R/C expression using bead-based Luminex® analysis. R/C levels from 5 BC patients were measured in serum before and after FDOJ surgery. Bone density, histology, R/C expression, and immunohistochemistry were analysed in 4 clinical case studies. The R/C staining of two FDOJ BC patients is compared with the immunohistochemical staining of BC cell preparations.Results: A high overexpression of R/C was seen in all FDOJ samples. R/C levels in serum were statistically downregulated after FDOJ surgery (p=0.0241).Discussion: R/C induced “silent inflammation” in BC is widely discussed in scientific papers along with R/C triggering of different signaling pathways, which might be a key point in the development of BC.Conclusion: Hypothesis that FDOJ may serve as a trigger of BC progression through R/C overexpression was set by the authors, who thus inspire clinicians to make aware of FDOJ throughout the dental and medical community in BC cases.Keywords: chemokine RANTES/CCL5, osteonecrosis of the jawbone, breast cancer, bead-based Luminex® analysis, hyperactivated signaling pathways

Johann Lechner,1 Marlene Schmidt,2 Volker von Baehr,3 Fabian Schick4 1Head of the Clinic for Integrative Dentistry, Munich, Germany; 2Data Scientist at STEYR Motorenwerke, Steyr, Austria; 3Head of the Department of Immunology and Allergology, Institute for Medical Diagnostics, Berlin, Germany; 4Dental Surgeon, Clinic for Integrative Dentistry, Munich, GermanyCorrespondence: Johann Lechner Tel +49-89-6970129Fax +49-89-6925830Email drlechner@aol.comBackground: Cytokines, especially chemokines, are of increasing interest in immunology. This study characterizes the little-known phenomenon of “bone marrow defects of the jawbone” (BMDJ) with known overexpression of the chemokine RANTES/CCL5 (R/C).Purpose: Our investigation clarifies why BMDJ and the intensity of local R/C overexpression are challenging to detect, as examined in patients with seven different systemic immunological diseases. Specifically, we investigate whether R/C overexpression is specific to certain disease groups or if it represents a type of signal disruption found in all systemic immunological diseases.Patients and Methods: In a total of 301 patients, BMDJ was surgically repaired during clinical practice to reduce “silent inflammation” associated with the presence of jaw-related pathologies. In each case of BMDJ, bone density was measured preoperatively (in Hounsfield units [HU]), while R/C expression was measured postoperatively. Each of the 301 patients suffered from allergies, atypical facial and trigeminal pain, or were diagnosed with neurodegenerative diseases, tumors, rheumatism, chronic fatigue syndrome, or parasympathetic disorders.Results: In all BMDJ cases, strongly negative HU values indicated decreased bone density or osteolysis. Consistently, all cases of BMDJ showed elevated R/C expression. These findings were consistently observed in every disease group.Discussion: BMDJ was confirmed in all patients, as verified by the HU measurements and laboratory results related to R/C expression. The hypothesis that a specific subset of the seven disease groups could be distinguished either based on the increased presence of BMDJ and by the overexpression of R/C could not be confirmed. A brief literature review confirms the importance of R/C in the etiology of each of the seven disease groups.Conclusion: In this research, the crucial role played by BMDJ and the chemokine R/C in inflammatory and immune diseases is discussed for seven groups of patients. Each specific immune disease can be influenced or propelled by BMDJ-derived R/C inflammatory signaling pathways.Keywords: bone marrow defects, jawbone, RANTES/CCL5 inflammatory signaling pathways, silent inflammation, systemic immune diseases

Johann Lechner,1 Volker von Baehr,2 Bernd Zimmermann3 1Clinic for Integrative Dentistry, Munich, Germany; 2Department of Immunology and Allergology, Institute for Medical Diagnostics, Berlin, Germany; 3QINNO, Wessling, GermanyCorrespondence: Johann LechnerClinic for Integrative Dentistry, Gruenwalder Str. 10A, Munich 81547, GermanyTel +49-89-6970129Fax +49-89-6925830Email drlechner@aol.comIntroduction: Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) is a complication of intravenous (IV) BP therapy. BP therapy locally affects the dentoalveolar area, while systemic effects are associated with parenteral/IV BP use. Despite numerous publications, the pathogenesis of BRONJ is not fully understood, as only some patients receiving IV BPs develop BRONJ.Purpose: Can impaired bone remodeling (found in aseptic-ischemic osteonecrosis of the jaw [AIOJ], bone marrow defects [BMD], or fatty-degenerative osteonecrosis of the jaw [FDOJ]) represent a risk factor for BRONJ formation?Patients and Methods: A literature search clarified the relationship between AIOJ, BMD, FDOJ, and BRONJ, in which common characteristics related to signal cascades, pathohistology, and diagnostics are explored and compared. A case description examining non-exposed BRONJ is presented.Discussion: Non-exposed BRONJ variants may represent one stage in undetected BMD development, and progression to BRONJ results from BPs.Conclusion: Unresolved wound healing at extraction sites, where wisdom teeth have been removed for example, may contribute to the pathogenesis of BRONJ. With IV BP administration, persisting AIOJ/BMD/FDOJ areas may be behind BRONJ development. Therapeutic recommendations include IV BP administration following AIOJ/BMD/FDOJ diagnosis and surgical removal of ischemic areas. BPs should not be regarded as the only cause of osteonecrosis.Keywords: bisphosphonates, bone marrow defects, osteonecrosis of the jaw, RANTES/CCL5, ultrasound sonography

Johann Lechner,1 Bernd Zimmermann,2 Marlene Schmidt,3 Volker von Baehr4 1Department of Clinical Research, Clinic Integrative Dentistry, Munich, Germany; 2Medical Devices, QINNO, Wessling, Germany; 3Department of Statistics, STEYR Motorenwerke, Steyr, Austria; 4Department of Immunology and Allergology, Institute for Medical Diagnostics Berlin, GermanyCorrespondence: Johann Lechner Tel +49-89-6970129Fax +49-89-6925830Email drlechner@aol.comIntroduction: The presently used impulse echo ultrasound examination is not suitable to provide relevant and reliable information about the jawbone, because ultrasound (US) almost completely reflects from the hard cortical jawbone. At the same time, “focal osteoporotic bone marrow defects” (BoneMarrowDefects = BMD) in jawbone are the subject of scientific presentations and discussions.Purpose: Can a newly developed trans-alveolar ultrasonic sonography (TAU-n) device locate and ascertain BMD?Patients and Methods: TAU-n consists of a two-part handpiece with an extraoral ultrasound transmitter and an intraoral ultrasound receiver. The TAU-n computer display shows the different jawbone densities with corresponding colour coding. The changes in jawbone density are also displayed numerically. The validation of TAU-n readings: A usual orthopantomogram (2D-OPG) on its own is not suitable for unequivocally determining jawbone density and has to be excluded from this validation. For validation, a 3D-digital volume tomogram/cone beam computer tomogram (DVT/CBCT) with the capacity to measure Hounsfield units (HU) and a TAU-n are used to determine the presence of preoperative BMD in 82 patient cases. Postoperatively, histology samples and multiplex analysis of RANTES/CCL5 (R/C) expression derived from surgically cleaned BMD areas are evaluated.Results: In all 82 bone samples, DVT-HU, TAU-n values and R/C expressions show the presence of BMD with chronic inflammatory character. However, five histology samples showed no evidence of BMD. All four evaluation criteria (DVT-HU, TAU-n, R/C, histology) confirm the presence of BMD in each of the 82 samples.Conclusion: The TAU-n method almost completely matches the diagnostic reliability of the other methods. The newly developed TAU-n scanner is a reliable and radiation-free option to detect BMD.Keywords: trans-alveolar ultrasonography, cone beam computed tomography, RANTES/CCL5, Hounsfield units, cavitational osteonecrosis of jawbone

Johann Lechner,1 Tatjana Rudi,2 Volker von Baehr3 1Clinic for Integrative Dentistry, Munich 81547, Germany; 2Institute for Epidemiological Studies, Berlin 10709, Germany; 3Department of Immunology and Allergology, Institute for Medical Diagnostics in MVZ GbR, Berlin 12247, Germany Background: The immune and bone systems are closely linked via cytokine cross-talk. This interdisciplinary field of research is referred to as osteoimmunology and pertains to inflammatory and osteoarticular diseases that feature the primary expression of tumor necrosis factor-alpha (TNF-α) and IL-6.Objective: Are there bone resorptive processes wherein chronic inflammatory conditions are not linked to TNF-α and IL-6 expression, but rather to the expression of other cytokines?Materials and methods: A comprehensive literature search was performed in PubMed Central.Discussion: Although all diseases with cytokines involved in bone resorption (TNF-α and IL-6) are at the forefront of destructive inflammatory processes, there is one exception in the literature: fatty oxide osteoporosis/osteolysis in the jawbone (FDOJ), which is associated with significant bone softening. However, it should be noted that TNF-α and IL-6 fall below the levels found in a healthy jawbone in this condition. Another conspicuous finding is that there is a nearly 35-fold overexpression of the chemokine RANTES/CCL5 (R/C) in all FDOJ cases studied thus far in the literature. Conclusion: FDOJ appears to represent a unique cytokine and inflammatory pattern from osteolysis in the body. R/C can be defined as the dominant carrier of a “maxillomandibular osteoimmunology”. Keywords: bone resorption, chemokine RANTES, CCL5, tumor necrosis factor-alpha, maxillomandibular osteoimmunology, maxillomandibular

Johann Lechner,1 Sabine Schuett,2 Volker von Baehr2 1Clinic for Integrative Dentistry, Munich, 2Department of Immunology and Allergology, Institute for Medical Diagnostics in MVZ GbR, Berlin, Germany Abstract: 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. Keywords: jawbone, osteonecrosis, RANTES, CCL5, silent inflammation, aseptic inflammation, PCR, ICD-10

Johann Lechner,1 Volker von Baehr2 1Clinic for Integrative Dentistry, Munich, Germany; 2Compartment of Immunology and Allergology, Institute for Medical Diagnostics, Berlin, Germany Abstract: Persistent microorganisms in endodontically treated teeth produce volatile sulfur compounds (VSC) such as methyl mercaptan, hydrogen sulfide, and thioether. In this retrospective study, we evaluated the ex vivo immune response of peripheral blood mononuclear cells to sulfur compounds in 354 patients with systemic diseases. These systemic findings are correlated with semiquantitative values of a VSC indicator applied directly on endodontically treated teeth. Data elucidate the role of VSC in patients with immunologic diseases and the role of a semiquantitative chairside test, like the VSC indicator presented here, in correlation to IFNg and IL-10 sensitization in peripheral blood mononuclear cells. The association between ex vivo-stimulated cytokines and endodontically derived sulfur components is supported by the fact that the number of interferon gamma- and/or interleukin-10-positive sensitized patients declined significantly 3–8 months after extraction of the corresponding teeth. Keywords: mercaptan, hydrogen sulfide, thioether, interferon gamma, interleukin 10

Johann Lechner,1 Volker von Baehr2 1Clinic for Integrative Dentistry, Munich, Germany; 2Compartment of Immunology and Allergology on Institute for Medical Diagnostics in MVZ GbR, Berlin, Germany Background: Jawbone cavitations (JC) are hollow dead spaces in jawbones with dying or dead bone marrow. These areas are defined as fatty degenerative osteonecrosis of the jawbone or neuralgia-inducing cavitational osteonecrosis and may produce facial pain. These afflictions have been linked to the immune system and chronic illnesses. Surgical debridement of JC is reported to lead to an improvement in immunological complaints, such as rheumatic, allergic, and other inflammatory diseases (ID). Little is known about the underlying cause/effect relationship. Objectives: JC bone samples were analyzed to assess the expression and quantification of immune modulators that can play a role in the pathogenesis of IDs. The study supports a potential mechanism where JC is a mediating link in IDs. Materials and methods: Samples of fatty softened bone taken from JCs were extracted from 31 patients. The specimens were analyzed by bead-based multiplex technology and tested for seven immune messengers. Results: Regulated upon activation, normal T-cell expressed, and secreted (RANTES) and fibroblast growth factor (FGF)-2 were found at high levels in the JCs tested. Other cytokines could not be detected at excessive levels. Discussion: The study confirms that JC is able to produce inflammatory messengers, primarily RANTES, and, secondarily, FGF-2. Both are implicated in many serious illnesses. The excessive levels of RANTES/FGF-2 in JC patients with amyotrophic lateral sclerosis, multiple sclerosis, rheumatoid arthritis, and breast cancer are compared to levels published in medical journals. Levels detected in JCs are higher than in the serum and cerebrospinal fluid of amyotrophic lateral sclerosis and multiple sclerosis patients and four-fold higher than in breast cancer tissue. Conclusion: This study suggests that JC might serve as a fundamental cause of IDs, through RANTES/FGF-2 production. Thus, JC and implicated immune messengers represent an integrative aspect of IDs and serve as a possible cause. Removing JCs may be a key to reversing IDs. There is a need to raise awareness about JC throughout medicine and dentistry. Keywords: RANTES/CCL5, fibroblast growth factor, FGF-2, bead-based Luminex analysis, osteolytic degenerated jaw bone, NICO, systemic signaling pathways

Background: This longitudinal prospective study aimed to assess orthodontic patients' immune system response to metal ion release in saliva. Methods: Thirty adult patients (18-35 years) were equally divided into three groups: groups at the end (G1) and beginning (G2) of multibracket appliances (MBA) treatment and a non-treated control group (G3). Participants were evaluated at four timepoints within 21 days, with saliva samples being analyzed for metal ion concentrations and blood for the lymphocyte transformation test (LTT). Results: There were no significant differences between groups or timepoints for saliva. LTT analyses revealed hypersensitivity in one-third of all patients and 50% of G2 for nickel, with three developing sensitizations after MBA insertion. All nickel-sensitized patients exhibited varying elevated saliva nickel concentrations. The most nickel-sensitized patients had low ion saliva loads. In borderline nickel-sensitization cases, saliva ion concentrations were up to 20 times higher than the reference. Hypersensitivity to palladium, gold, and mercury was also observed. Conclusions: These findings indicate that increased MBA ion release was not inherently linked to the immune response (Type-IV sensitization), as reactions occurred even with ion levels below thresholds. This underlines the need for a comprehensive evaluation of the immune response to metal ion release in orthodontic patients.

Objective: Bone marrow defects of the jaw (BMDJ) surrounding dental implants, in combination with impaired bone-to-implant contact (BIC), are difficult to detect in X-rays. This study evaluated BMDJ surrounding titanium (Ti-Impl) and ceramic (Cer-Impl) dental implants and incomplete BIC using a new trans-alveolar ultrasonography device (TAU) with numerical scaling for BIC., Methods: The titanium stimulation test (Ti-Stim) was used to detect immune overactivation in response to titanium. Bone density surrounding implants was measured using TAU. We also validated osteoimmune dysregulation., Results: TAU values showed reduced BIC and decreased osseointegration for Ti-Impl. Moreover, TAU values in the Cer-Impl group were more than twice those in the Ti-Impl cohort. The multiplex analysis of C-C motif chemokine 5 (CCL5, also known as RANTES) expression revealed a 20-fold increase in BMDJ surrounding Ti-Impl. Higher levels of CCL5 inflammation were present in the positive Ti-Stim group., Conclusions: Our data indicate that Cer-Impl have an osteoimmune advantage over Ti-Impl. The key determinant for osteoimmune sustainability appears to be the absence of inflammation at the implant site. We therefore recommend the use of TAU to assess the implant site prior to implantation., Competing Interests: Declaration of conflicting interestsThe TAU apparatus and associated software was provided without charge for the purposes of this study by CaviTAU® (Munich, Germany). The corresponding author (JL) is the holder of patents used in the TAU apparatus (CaviTAU®).

Due to rare but major adverse reactions to the AstraZeneca adenoviral ChAdOx1-S-nCoV-19 vaccine (ChAd), German health authorities recommended adults under 60 who received one dose of ChAd, to receive a second dose of the BioNTech mRNA BNT162b2 vaccine (BNT) as a booster. Studies in the general population suggest an enhanced efficacy of the heterologous (ChAd-BNT) compared to the homologous (BNT-BNT) vaccination regimen. However, an analysis of the efficacy in patient populations with a high risk of severe COVID-19 due to acquired immunodeficiency is still missing. We therefore compared both vaccination regimens in healthy controls, patients with gynecological tumors after chemotherapy, patients on dialysis and patients with rheumatic diseases concerning the humoral and cellular immune response. The humoral and cellular immune response differed substantially in healthy controls compared to patients with acquired immunodeficiency. Overall, the most significant differences between the two immunization regimens were found in neutralizing antibodies. These were always higher after a heterologous immunization. Healthy controls responded well to both vaccination regimens. However, the formation of neutralizing antibodies was more pronounced after a heterologous immunization. Dialysis patients, on the other hand, only developed an adequate humoral and particularly cellular immune response after a heterologous immunization. Tumor and rheumatic patients also - to a weaker extent compared to dialysis patients - benefited from a heterologous immunization. In conclusion, the heterologous COVID-19 vaccination regimens (ChAd-BNT) seem to have an advantage over the homologous vaccination regimens, especially in immunocompromised patients such as patients with end-stage kidney disease treated with hemodialysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chu, Schönbrunn, Fischer, Liu, Hocher, Weinerth, Klemm, von Baehr, Krämer, Elitok and Hocher.)

Background: The null hypotheses were tested that intraoral bone augmentation using two different allogeneic materials has no impact on the patient's blood levels of material-specific lymphocytes and on the immunohistochemical detection of pro-inflammatory cytokines IL-1α, IL1ß and TNF-α and T-cell markers CD4, CD8 in biopsies of the test groups., Methods: In this prospective RCT, 60 systemically healthy participants were randomly assigned to two allogeneic test groups (1: Maxgraft®, freeze-dried, multiple donors, and 2: Puros®, solvent-dehydrated, single donor) and an autologous control group (10 patients). Plasma samples were collected pre-(T1) and postoperatively (2 weeks (T2) and 4 months (T3)). The Lymphocyte Transformation Test (LTT) was used for analyzing levels of transformed lymphocytes for type IV immune reactions by 3H-thymidine activity. Bone biopsies were harvested at T3 and immunohistochemically analyzed for IL-1α, IL1ß, TNF-α, CD4, CD8 and correlated with the immunological and clinical findings., Results: A statistically significant difference between the tested materials was observed for LTT measurements at T3 (p = 0.033). Furthermore, three groups were identified: Group A (LTT negative T1-T3, n = 48), group B (LTT positive T1-T3, n = 7), group C (developing positive LTT at T2, n = 5). A highly significant elevation of IL-1α, IL1ß, TNF-α in patients of group C (p = 0.0001) and a significant elevation of CD4+ cells in patients of group B (p = 0.005) was shown., Conclusion: Our data show that following allogeneic bone grafting, local and systemic immunological reactions can be detected in some patients. These findings were statistically significant for the timepoint T3 between the tested materials as well as for the groups B and C correlated with group A for both tested materials. Therefore, the null hypotheses were rejected. A preoperative compatibility test for allogeneic materials in order to improve patient safety and the predictability of these materials would be desirable., Trial Registration: Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) as well as by the German Registry of Clinical Studies (DRKS00013010) on 30/07/2018 ( http://apps.who.int/trialsearch/ )., (© 2022. The Author(s).)

Vaccination against the SARS-CoV-2 virus or infection with SARS-CoV-2 will lead to the development of IgG antibodies against the S1 protein of the SARS-CoV-2 virus. However, even despite having high levels of IgG antibodies against the S1 protein of the SARS-CoV-2 virus, (re-)infection may occur. We thus examined 2994 consecutive blood samples of outpatients from the Berlin-Brandenburg area in Germany in which IgG antibodies against the S1 protein of the SARS-CoV-2 virus as well as neutralizing SARS-CoV-2 virus antibodies were determined from the same sample. When analyzing the entire study population (2994 outpatients), we saw that S1 IgG antibodies (women: 223.98 ± 3.81; men: 207.80 ± 4.59; p = 0.014) and neutralizing antibodies (women: 66.65 ± 0.82; men: 62.88 ± 1.01; p = 0.021) are slightly higher in women than in men. Curve fitting revealed a good non-linear relationship between S1 IgG and neutralizing SARS-CoV-2 antibodies. However, 51 out of the 2994 blood samples from individual subjects were positive with regard to the neutralizing antibodies and at the same time negative for S1 IgG antibodies, and 112 out of the 2994 blood samples from individual subjects were negative with regard to the neutralizing antibodies and at the same time positive for S1 IgG antibodies. In conclusion, our study shows that there is a relevant number of patients who, despite developing significant titers of S1 antibodies, do not have relevant amounts of neutralizing antibody titers and are probably at high risk of (re-)infection.

It was shown that hypertension delays SARS CoV-2 viral clearance and exacerbates airway hyperinflammation in the respiratory tract. However, it is unknown whether hypertension determines the long-term cellular and humoral response to SARS Cov2. Health care workers (HCWs) after an outbreak of SARS Cov-2 infections were analyzed. Infected HCWs were not vaccinated before blood collection. 5-14 months (median 7 months) after detection of SARS CoV-2 infection, blood was taken to analyze humoral response (S1 IgG and SARS CoV-2 neutralizing antibodies) and cellular (T cell responses to SARS-CoV-2 with Lymphocyte Transformation Test). To identify clinical factors that determine the immune response, a multivariate regression analysis was done considering age, BMI, sex, diabetes, hypertension, smoking, COPD, asthma and time between PCR positivity and blood collection as confounding factors. Infected hypertensive HCWs more often needed to be hospitalized than non-hypertensive HCWs, but were less likely to develop anosmia and myalgia. The long-term humoral and cellular immune response was significantly strengthened in hypertensive versus normotensive infected HCWs. Multivariate regression analysis revealed that hypertension was independently associated with the humoral response to SARS CoV-2 infection. Multivariate regression analysis using same confounding factors for the humoral response showed a clear trend for an association with the cellular response to SARS CoV-2 infection as well. In conclusion, SARS CoV-2 infection strengthened immune response to SARS CoV-2 infection in hypertensive HCWs independent of other risk factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chu, Schönbrunn, Klemm, von Baehr, Krämer, Elitok and Hocher.)

Both infection with and vaccination against SARS-CoV-2 trigger a complex B-cell and T-cell response. Methods for the analysis of the B-cell response are now well established. However, reliable methods for measuring the T-cell response are less well established and their usefulness in clinical settings still needs to be proven. Here, we have developed and validated a T-cell proliferation assay based on 3H thymidine incorporation. The assay is using SARS-CoV-2 derived peptide pools that cover the spike (S), the nucleocapsid (N) and the membrane (M) protein for stimulation. We have compared this novel SARS-CoV-2 lymphocyte transformation test (SARS-CoV-2 LTT) to an established ELISA assay detecting Immunoglobulin G (IgG) antibodies to the S1 subunit of the SARS-CoV-2 spike protein. The study was carried out using blood samples from both vaccinated and infected health care workers as well as from a non-infected control group. Our novel SARS-CoV-2 LTT shows excellent discrimination of infected and/or vaccinated individuals versus unexposed controls, with the ROC analysis showing an area under the curve (AUC) of > 0.95. No false positives were recorded as all unexposed controls had a negative LTT result. When using peptide pools not only representing the S protein (found in all currently approved vaccines) but also the N and M proteins (not contained in the vast majority of vaccines), the novel SARS-CoV-2 LTT can also discriminate T-cell responses resulting from vaccination against those induced by infection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In vitro studies indicate that 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits the synthesis of parathyroid hormone (PTH). The degree of PTH inhibition in humans by circulating 25(OH)D and 1,25(OH)2D may be different. Moreover, age and sex as well as confounding factors like calcium and phosphate may likewise affect the relationship between vitamin D and PTH in humans. However, this was not done so far in adequately powered studies. We investigated the relationship between 25(OH)D as well as 1,25(OH)2D and intact parathyroid hormone (iPTH) in 23,134 outpatients (age mean: 59.81 years) from the Berlin-Brandenburg area of Germany with normal serum creatinine considering confounding factors like age, sex, calcium and phosphate. 25(OH)D and iPTH were inversely correlated (r = -0.17, p < 0.0001). The inverse linear correlation was observed over the entire spectrum of 25(OH)D concentrations - from low 25(OH)D concentrations to very high 25(OH)D concentrations. Multiple linear regression analysis revealed that this correlation was independent of age, sex, creatinine, calcium and phosphate (unstandardized coefficients B: -0.16, p < 0.0001). However, 1,25(OH)2D was only positively associated with iPTH in women (r = 0.05, p = 0.033) and in the subgroup of patients with lower 25(OH)D (25(OH)D< 40 ng/ml) (r = 0.09, p < 0.0001), which was also presented in multiple linear regression analysis (unstandardized coefficients B: 0.20, p = 0.001). Circulating 1,25(OH)2D does not contribute substantially to the regulation of PTH in middle aged and vitamin D sufficient outpatients from the Berlin-Brandenburg area of Germany with normal kidney function. Presumably, serum 25(OH)D that is converted to 1,25(OH)2D after uptake in the parathyroid chief cells plays the critical role., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background. Local anesthetics (LAs) have potent anti-inflammatory properties. Inflammatory down-regulation is crucial in diseases with overactive immune reactions, such as acute respiratory distress syndrome (ARDS) and chronic inflammation. We investigated the influence of four LAs, procaine, lidocaine, mepivacaine, and bupivacaine, on the reduction of tumor necrosis factor-alpha (TNF-α) secretion in lipopolysaccharide (LPS)-activated human leucocytes. Methods. Blood samples of 28 individuals were stimulated with LPS. The reduction of TNF-α production by each of the four LAs added (0.5 mg/mL) was measured and correlated with biometric variables. A response was defined as reduction to <85% of initial levels. Results. All four LAs down-regulated the TNF-α secretion in 44−61%: Bupivacaine (44.4%), lidocaine (61.5%), mepivacaine (44.4%), and procaine (50% of the individuals, “responders”). The TNF-α secretion was reduced to 67.4, 68.0, 63.6, and 67.1% of the initial values in responders. The effects in both patients and healthy persons were the same. Interindividual responses to LAs were not correlated with the duration or type of complaints, basal TNF-α serum level, sex, BMI, or age of responders. Conclusions. Four clinically relevant LAs (amid-LA and ester-LA) attenuate the inflammatory response provoked by LPS. They are potential candidates for drug repositioning in treating overactive immune reactions and chronic inflammation.

Background: Animal studies suggested that vitamin D might decrease insulin resistance. Estrogen increased insulin sensitivity and glucose tolerance in rodents. However, sex-specific association of vitamin D with insulin resistance in humans remains unclear., Objectives: To investigate the sex-dependency of the association of insulin resistance and 25-hydroxyvitamin D [25(OH)D] in a large Caucasian population., Methods: Cross-sectional study from out-patients' blood samples with measurements of 25(OH)D and homeostatic model assessment of insulin resistance (HOMA-IR) drawn at exactly the same day (n = 1887). This cohort was divided into 3 groups: (1) group with vitamin D deficiency (n = 1190), (2) group with vitamin D sufficiency (n = 686), and (3) vitamin D excess groups (n = 11); the vitamin D excess group was excluded from further analysis due to the small size., Results: Analysis of the entire study population showed that serum 25(OH)D was inversely associated with HOMA-IR [Spearman correlation coefficient (rs) = -0.19, P < 0.0001]. When considering the vitamin D status, this association was only seen in the vitamin D deficiency group but not in the vitamin D sufficient group. The correlation was sex-dependent: HOMA-IR was inversely correlated with vitamin D in women with vitamin D deficiency (rs = -0.26, P < 0.0001) but not in men with vitamin D deficiency (rs = 0.01, P = 0.714). After multivariate linear regression analysis considering confounding factors, this relationship was again only seen in women., Conclusion: Vitamin D was inversely and independently associated with insulin resistance only in women with vitamin D deficiency. Based on our data, we suggest that in particular vitamin D deficient women might benefit from vitamin D substitution by improving insulin resistance. This, however, needs to be proven in adequately designed double-blind placebo-controlled clinical studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The role played by signaling pathways in the cell-cell communication associated with multiple sclerosis (MS) progression has become a critical area in research. Chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted), also named chemokine C-C motif ligand 5 (CCL5; R/C), is a protein that has been investigated in neuroinflammatory research due to its link to MS development., Objective: Research on bone marrow defects in the jawbone (BMDJ), which morphologically presents as fatty-degenerative osteonecrosis of the jawbone (FDOJ), presents overexpression of R/C signaling in affected areas. Here, we try to elucidate the potential link between jawbone-derived R/C and MS., Methods: Seventeen BMDJ/FDOJ samples extracted from 17 MS patients, as well as samples from 19 healthy controls, were analyzed for R/C expression using bead-based Luminex ® analysis. The serum R/C levels from 10 MS patients were examined. Further, bone density, histology, and R/C expression were analyzed in two clinical case studies., Results: High R/C overexpression was found in all BMDJ/FDOJ samples obtained from the MS group. Serum R/C levels were also upregulated in the MS group. R/C serum levels in the MS cohort were higher than in the healthy controls. In contrast, the histology of BMDJ/FDOJ samples showed no inflammatory cells., Discussion: R/C-induced "silent inflammation" in MS is widely discussed in the scientific literature, along with R/C triggering of inflammation in the central nervous system, which might be key in the development of MS., Conclusion: The authors suspect that BMDJ/FDOJ may serve as a trigger of MS progression via R/C overexpression. As such, the dental and medical communities should be made aware of BMDJ/FDOJ in cases of MS., Competing Interests: There are no conflicts of interest in this work., (© 2021 Lechner et al.)

Measurements of total 25-hydroxyvitamin D (t25(OH)D) are currently primarily used to assess the vitamin D status. The lipophilic cell membrane can only be passed by the un-bound form of 25-hydroxyvitamin D: free 25-hydroxyvitamin D (f25(OH)D). It is thought that f25(OH)D does reflect its biological actions better than t25(OH)D. However, as of today, there are no established guidelines for the clinical use of f25(OH)D. We analysed 5060 patients with simultaneous measurements of free and total 25(OH). Linear regression was used to study the relationship between free 25(OH)D and total 25(OH)D. We reviewed and used the established t25(OH)D reference values and determined the slope of the relationship between them to calculate reference values for f25(OH)D. F25(OH)D and t25(OH)D showed a strong positive linear (r = 0.8395, p < 0.0001) correlation. The slope of the relationship was 0.2833 ± 0.00257. The recommended threshold level of f25(OH)D is 8.499 pg/mL, corresponding to a target concentration for t25(OH)D of at least 30 ng/mL considered as sufficient in most of the international vitamin D guidelines. The upper limit for vitamin D is less clear in the guidelines. Most experts favour an upper limit for t25(OH)D of 100 ng/mL. This is equivalent to 28.330 pg/mL f25OHD. We established based on international guidelines for t25(OH)D reference values for f25(OH)D that are urgently needed for clinical use of f25(OH)D. However, clinical studies with f25(OH)D to confirm our suggestions are needed but will take time., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Fatty degenerative osteonecrosis in the medullary spaces of the jawbone (FDOJ) may be identified as a lesser known source of RANTES/CCL5 (R/C) overexpression. The chemokine R/C also interferes with bone metabolism leading to osteolysis in areas affected by FDOJ. Many dental surgeries require functioning repair mechanisms and these may be disrupted by R/C overexpression., Objective: To clarify the way in which R/C expression from adipocytes in FDOJ causes a disturbance in osteogenesis and impacts on medullary stem cells by investigating the detection of R/C expression with immunochemical staining., Materials and Methods: We examined the tissue samples of 449 patients with FDOJ to assess the level of the chemokine R/C using bead-based Luminex® analysis. In six clinical case studies of FDOJ, we compared bone density, histological findings, R/C expression, and immunohistochemical staining., Results: R/C is overexpressed by up to 30-fold in the 449 FDOJ cases when compared with healthy jawbone samples. The comparison of the six clinical cases consistently shows greatly reduced bone density, (i.e., osteolysis), but varies in terms of the level of agreement across the other three parameters., Discussion: R/C from FDOJ sources may be implicated in several immune responses and considered a key pathogenetic pathway for increased adipogenesis rather than desirable osteogenesis. Adipocytes pathogenetically act via R/C expression in local FDOJ and systemically on the immune system., Conclusion: R/C may be regarded as an important trigger for possible pathological developments in the fate of hematopoietic stem cells. FDOJ is not a rigidly uniform process but reflects changing stages of development. The absence of correlating findings should not be interpreted as a misdiagnosis. It seems appropriate to direct further research in the field of "maxillo-mandibular osteoimmunology" focusing on R/C overexpression in FDOJ areas. This may contribute to the development of personalized strategies in preventive medicine., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Background and Introduction: It is a well-known fact that titanium particles deriving from dental titanium implants (DTI) dissolve into the surrounding bone. Although titanium (TI) is regarded as a compatible implant material, increasing concern is coming up that the dissolved titanium particles induce inflammatory reactions around the implant. Specifically, the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is expressed in the adjacent bone. The transition from TNF-α-induced local inflammation following insertion of DTI surgery to a chronic stage of "silent inflammation" could be a neglected cause of unexplained medical conditions., Material and Methods: The signaling pathways involved in the induction of cytokine release were analyzed by multiplex analysis. We examined samples of jawbone (JB) for seven cytokines in two groups: specimens from 14 patients were analyzed in areas of DTI for particle-mediated release of cytokines. Each of the adjacent to DTI tissue samples showed clinically fatty degenerated and osteonecrotic medullary changes in the JB (FDOJ). Specimens from 19 patients were of healthy JB. In five cases, we measured the concentration of dissolved Ti particles by spectrometry ., Results: All DTI-FDOJ samples showed RANTES/CCL5 (R/C) as the only extremely overexpressed cytokine. DTI-FDOJ cohort showed a 30-fold mean overexpression of R/C as compared with a control cohort of 19 healthy JB samples. Concentration of dissolved Ti particles in DTI-FDOJ was 30-fold higher than an estimated maximum of 1.000 μg/kg., Discussion: As R/C is discussed in the literature as a possible contributor to inflammatory diseases, the here-presented research examines the question of whether common DTI may provoke the development of chronic inflammation in the jawbone in an impaired state of healing. Such changes in areas of the JB may lead to hyperactivated signaling pathways of TNF-α induced R/C overexpression, and result in unrecognized sources of silent inflammation. This may contribute to disease patterns like rheumatic arthritis, multiple sclerosis, and other systemic-inflammatory diseases, which is widely discussed in scientific papers., Conclusion: From a systemic perspective, we recommend that more attention be paid to the cytokine cross-talk that is provoked by dissolved Ti particles from DTI in medicine and dentistry. This may contribute to further development of personalized strategies in preventive medicine., Competing Interests: The authors declare that they have no competing interests.Not applicable.All the patients were informed about the purposes of the study and consequently have signed their “consent of the patient.” All investigations conformed to the principles outlined in the Declaration of Helsinki and were performed with permission by the responsible Ethics Committee of IMD-Berlin forensic accredited Institute DIN EN 15189/DIN EN 17025. This article does not contain any studies with animals performed by any of the authors.

This study elucidates the question of whether chronic inflammation in the jawbone contributes to the development of Chronic Fatigue Syndrome (CFS). Fatty degenerative osteonecrosis in jawbone (FDOJ) may contribute to CFS by induction of inflammatory mediators. We examined seven cytokines by multiplex analysis in jawbone samples from two groups of patients. In order to clarify neurological interrelations, specimens from 21 CFS patients were analyzed from areas of previous surgery in the retromolar wisdom tooth area. Each of the retromolar jawbone samples showed clinically fatty degenerated and osteonecrotic medullary changes. As control, healthy jawbone specimens from 19 healthy patients were analyzed. All fatty necrotic and osteolytic jawbone (FDOJ) samples showed high expression of RANTES and fibroblast growth factor (FGF)-2. FDOJ cohorts showed a 30-fold mean overexpression of RANTES and a 20-fold overexpressed level of FGF-2 when compared to healthy controls. As RANTES is discussed in the literature as a possible contributor to inflammatory diseases, we hypothesize that FDOJ in areas of improper and incomplete wound healing in the jawbone may hyperactivate signaling pathways. Constituting a hidden source of “silent inflammation” FDOJ may represent a hitherto unknown cause for the development of CFS.

Background: This research elucidates the question of whether common and widespread dental procedures (DP) like root filling (RF) and the removal of wisdom teeth (WT) contribute to chronic inflammation in the jawbone. Dentists, in carrying out these DP, can set off defective wound healing in the jawbone in ignorance of its connection to inflammatory mediators and the possibility of it being a hidden cause of chronic systemic diseases (SYD)., Materials and Methods: We examined samples of the jawbone for seven cytokines by multiplex analysis in three groups of jawbone areas. In order to clarify systemic interrelations, specimens from 16 patients were analyzed in areas of former surgery in the retromolar wisdom tooth area; specimens from 16 patients were analyzed in the jawbone, apically of teeth with RF; and specimens from 19 patients were of the healthy jawbone. Each of the retromolar and the apical jawbone samples showed clinically fatty degenerated and osteonecrotic medullary changes., Results: All fatty necrotic and osteolytic jawbone (FDOJ) samples showed regulated on activation, normal T-cell expressed and secreted (RANTES) and fibroblast growth factor (FGF)-2 as the only extremely overexpressed cytokines. FDOJ cohorts showed a 30-fold mean overexpression of RANTES and a 20-fold overexpressed level of FGF-2 when compared to healthy controls., Conclusions: As RANTES is discussed in the literature as a possible contributor to inflammatory diseases, and though it might have oncogenic effects, we hypothesize that FDOJ in areas of improper and incomplete wound healing in the jawbone might act as hyperactivated signaling pathways, while serving as an unknown source of "silent inflammation". Because of the wide range of RANTES in immune diseases, treating FDOJ can cover many potential prediction or prognosis of individual outcomes.

Background: Laboratory diagnosis of Lyme borreliosis follows a two-step algorithm, starting with a screening assay with maximum sensitivity followed by a confirmatory assay of positive results with respect to specificity. Line immunoassays with single recombinant antigen lines are the established confirmatory test method, although result interpretation is not standardized. The present study evaluates suitability of the multiplex test SeraSpot Anti-Borrelia IgG/IgM (SeraSpot) as confirmatory test in serodiagnosis of Lyme disease., Methods: For this retrospective study, serum samples from patients with suspicion of Lyme borreliosis were analyzed. For determination of specificity, blood donor samples as well as rheumatoid factor (RF), ANA, and EBV positive sera were investigated. The SeraSpot IgG/IgM results were compared with the recomBead Borrelia IgG/IgM (recomBead) test as alternative multiplex-based method., Results: In comparison to the recomBead test the sensitivity of SeraSpot was determined 95% for IgG/IgM detection. The analysis of blood donors revealed a specificity of ≥ 96.0% for SeraSpot IgG/IgM and of 97.3% for recomBead IgG/IgM. RF positive samples tested negative by both assays (specificity of 100%). ANA and EBV antibody positive samples resulted in specificities of 90% (SeraSpo IgG/IgM) and ≥ 94% (recomBead IgG/IgM) and ≥ 91% (SeraSpot IgG/IgM) and ≥ 91% (recomBead IgG/IgM). The intra- and interassay coefficient of variation (CV) and the lot-to-lot reproducibility of the SeraSpot assay ranged between 1 - 9% for the different Borrelia IgG and IgM antigens. Potentially interfering substances (bilirubin F, bilirubin C, hemoglobin, lipid factor, and rheumatoid factor) did not influence the SeraSpot assays., Conclusions: Our evaluation data confirm that new multiplex assay generations such as SeraSpot Anti-Borrelia IgG/IgM are reliable and robust test systems suitable for application as confirmatory tests for serodiagnosis of Lyme borreliosis.

Introduction. In this study, we elucidate the possible causative role of chronic subclinical inflammation in jawbone of patients with atypical facial pain (AFP) and trigeminal neuralgia (TRN) in the local overexpression of the chemokine regulated on activation and normal T-cell expressed and secreted (RANTES/C-C motif ligand 5 CCL5). Neurons contain opioid receptors that transmit antipain reactions in the peripheral and central nervous system. Proinflammatory chemokines like RANTES/CCL5 desensitize μ-opioid receptors in the periphery sensory neurons and it has been suggested that RANTES modifies the nociceptive reaction. Materials and Methods. In 15 patients with AFP/TRN, we examined fatty degenerated jawbone (FDOJ) samples for the expression of seven cytokines by multiplex analysis and compared these results with healthy jawbones. Results. Each of these medullary jawbone samples exhibited RANTES as the only highly overexpressed cytokine. The FDOJ cohort with AFP/TRN showed a mean 30-fold overexpression of RANTES compared to healthy jawbones. Conclusions. To the best of our knowledge, no other research has identified RANTES overexpression in silent inflamed jawbones as a possible cause for AFP/TRN. Thus, we hypothesize that the surgical clearing of FDOJ might diminish RANTES signaling pathways in neurons and contribute to resolving chronic neurological pain in AFP/TRN patients.

Bacterial infections of the residual dentin or infected pulp tissue are responsible for most cases of endodontic treatment failures. Persisting microorganisms in necrotic pulp tissue produce sulphur components such as methyl mercaptan and hydrogen sulfide as well as thioether derivatives. Although there is emerging evidence that these sulphur compounds stimulate immune cells and induce the inflammatory cascade, the immunological mechanisms of local and systemic inflammation have not been described. In this retrospective study we evaluated the ex-vivo immune response of peripheral blood mononuclear cells to sulphur compounds in 53 patients with clinical or radiologic endodontic treatment failure, 20 patients with clinical discomfort or radiological findings without previous endodontic treatment and a control group of 31 patients who had received successful endodontic treatment at least five years previously. Patients with endodontic abnormalities showed significantly higher ex-vivo sulphur compound-stimulated interferon-gamma (IFN-γ) and interleukin-10 (IL-10) levels as compared to the control group. The association between ex-vivo-stimulated cytokines and endodontically derived sulphur compounds was further substantiated by the fact that the number of IFN-γ and/or IL-10-positive patients decreased significantly 3-8 months after re-treatment of the root canal or tooth extraction. Furthermore, serum tumor necrosis factor-alpha (TNF-α) levels were higher in patients than in controls, and at the same time, the TNFA -308 G/A polymorphism was associated with endodontic treatment failure in our study population. We conclude that a cellular immune response to sulphur compounds contributes to the inflammatory process observed in relation to endodontic treatment failures.

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.

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.

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.

Synovial tissues in joints with prostheses display characteristic morphological changes in cases with aseptic failure, particularly macrophage infiltration. Since proliferation of the synovial lining cell layer represents a feature characteristic of autoimmune joint diseases, the possibility of morphological changes of the synovial lining cell layer in periprosthetic tissues was investigated. Synovial biopsies from five groups of morphologically well-defined lesions (osteoarthritis, rheumatoid arthritis, aseptic loosened metal-on-polyethylene and metal-on-metal arthroplasty and suggested metal hypersensitivity) were compared using a conventional staining method and immunohistochemistry. The synovial lining cell layer was substantially enlarged in both rheumatoid arthritis and cases suggestive of metal hypersensitivity. Macrophage infiltrates were apparent in rheumatoid arthritis and all specimens from retrieved hip arthroplasties. Although both synovial and subsynovial macrophages were positive for CD163 (indicating synovial M2 macrophages), the remaining fibroblast-like synoviocytes and scattered stromal fibroblasts showed a positive reaction with the D2-40 antibody (indicating fibroblast-like synoviocytes). Furthermore, in contrast to CD163-positive macrophages, the enlarged D2-40-positive fibroblast-like synoviocytes displayed cytoplasmatic tubular projections. Proliferation of the periprosthetic synovial lining cell layer occurred in cases with unexplained groin pain following metal-on-metal hip resurfacing arthroplasty, suggestive of hypersensitivity. Despite some important study limitations, the present observation adds to the evidence that metal hypersensitivity shares characteristic morphological features with autoimmune diseases of the joints.