• Generation of two human ISG15 knockout iPSC clones using CRISPR/Cas9 editing.

      Merkert, S; Jaboreck, M-C; Engels, L; Malik, M N H; Göhring, G; Pessler, F; Martin, U; Olmer, R; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Elsevier, 2020-12-22)
      Background and aims: T cells are the main mediators of allogeneic immune responses. Specific T cell clones can be tracked by their unique T cell receptor (TCR), but specificity and function remain elusive and have not been investigated in human liver biopsies thus far. Methods: TCR repertoire analysis of CD4+, CD8+ and regulatory T cells of the peripheral blood and liver graft was performed in seven liver transplant recipients with either stable course (non-rejector, NR), subclinical cellular rejection (SCR) or acute cellular rejection (ACR) during an observation period from pre-transplant to six years post-transplant. Furthermore, donor-reactive T cells, identified by their expression of CD154 and GARP after allogeneic activation, were tracked longitudinally in peripheral blood and within the liver allograft. Results: While overall clonality of the TCR repertoire did not increase in peripheral blood after liver transplantation, clonality of donor-reactive CD4+ and regulatory T cells increased and these clones accumulated within the liver graft. Surprisingly, the TCR repertoires between the liver graft and the periphery were distinct and showed only little overlap. Notably, during ACR TCR repertoires aligned suggesting either graft-specific homing or release of activated T cells from the graft. Conclusions: This is the first study comparing TCR repertoires between liver graft and blood in patients with NR, SCR and ACR. Moreover, we attribute specificity and function to a subgroup of intragraft T cell populations. Given the little overlap between peripheral blood and intragraft repertoires future studies investigating function and specificities of T cells after liver transplantation should focus on the intragraft immune response. For this purpose, protocol biopsies of patients with normal graft function and subclinical rejection have to be taken into account.
    • Targeted metabolomic profiling of cerebrospinal fluid from patients with progressive multifocal leukoencephalopathy.

      Luo, Yi; Möhn, Nora; Al-Mekhlafi, Amani; Schuchardt, Sven; Skripuletz, Thomas; Sühs, Wolfram; Pessler, Frank; Stangel, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2020-11-24)
      Progressive multifocal leukoencephalopathy (PML), caused by JC polyomavirus, is a demyelinating disease of the central nervous system that primarily affects oligodendrocytes. It can cause significant morbidity and mortality. An early diagnosis is of high relevance as timely immune reconstitution is essential. However, diagnosis can be challenging if virus detection via cerebrospinal fluid (CSF) PCR remains negative. Hence, identifying CSF biomarkers for this disease is of crucial importance. We applied a targeted metabolomic screen to CSF from 23 PML patients and eight normal pressure hydrocephalus (NPH) patients as controls. Out of 188 potentially detectable metabolites, 48 (13 amino acids, 4 biogenic amines, 1 acylcarnitine, 21 phosphatidylcholines, 8 sphingolipids, and the sum of hexoses) passed the quality screen and were included in the analyses. Even though there was a tendency towards lower concentrations in PML (mostly of phosphatidylcholines and sphingomyelins), none of the differences between PML and controls in individual metabolite concentrations reached statistical significance (lowest p = 0.104) and there were no potential diagnostic biomarkers (highest area under the ROC curve 0.68). Thus, CSF metabolite changes in PML are likely subtle and possibly larger group sizes and broader metabolite screens are needed to identify potential CSF metabolite biomarkers for PML.
    • Impact of Physical Exercise on Gut Microbiome, Inflammation, and the Pathobiology of Metabolic Disorders.

      Sohail, Muhammad U; Yassine, Hadi M; Sohail, Aaqib; Al Thani, Asmaa A; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Society for Biomedical Diabetes Research, 2019-08-04)
      Background: The gastrointestinal tract (GIT) harbors a complex and diverse microbial composition that outnumbers our own body cells and their gene contents. These microbes play a significant role in host metabolism and energy homeostasis. Emerging evidence suggests that the GIT microbiome significantly contributes to host health and that impairments in the microbiome may cause the development of metabolic diseases. The microbiome architecture is shaped by several genetic and environmental factors, including nutrition and physical activity. Physical exercise has preventive or therapeutic effects in respiratory, cardiovascular, neuroendocrine, and muscular diseases. Yet, we still have little information of the beneficial effects of physical exercise on GIT health and microbial composition. Furthermore, we are not aware whether exercise-derived benefits on microbiome diversity can beneficially influence other tissues and body organs. Objectives: The aim of this article is to review the available literature on exercise-induced microbiome changes and to explain how these changes may induce inflammatory, immune, and oxidative responses that may contribute to the improvement of metabolic disorders. Methods: A systemic and comprehensive search of the relevant literature using MEDLINE and Google Scholar databases was conducted during fall 2018 and spring 2019. The search identified sixty-two research and review articles that discussed exercise-induced microbiome changes. Results: The review of the relevant literature suggests that exercise-induced microbial changes affect the host's immune pathways and improve energy homeostasis. Microbes release certain neuroendocrine and immune-modulatory factors that may lower inflammatory and oxidative stress and relieve patients suffering from metabolic disorders. Conclusions: Exercise-induced changes in microbial diversity are able to improve tissue metabolism, cardiorespiratory fitness, and insulin resistance.
    • Human Lentiviral Gene Therapy Restores the Cellular Phenotype of Autosomal Recessive Complete IFN-γR1 Deficiency.

      Hahn, Katharina; Pollmann, Liart; Nowak, Juliette; Nguyen, Ariane Hai Ha; Haake, Kathrin; Neehus, Anna-Lena; Waqas, Syed F Hassnain; Pessler, Frank; Baumann, Ulrich; Hetzel, Miriam; et al. (Elsevier(Cell Press), 2020-04-11)
      Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency.
    • Presence of hepatitis B virus in synovium and its clinical significance in rheumatoid arthritis.

      Chen, Yu-Lan; Jing, Jun; Mo, Ying-Qian; Ma, Jian-Da; Yang, Li-Juan; Chen, Le-Feng; Zhang, Xiang; Yan, Tao; Zheng, Dong-Hui; Pessler, Frank; et al. (BMC, 2018-06-19)
      Previous studies have revealed that hepatitis B virus (HBV) infection may be related to rheumatoid arthritis (RA), but there are no studies on the presence of HBV antigens or nucleic acid in synovium from patients with RA with HBV infection. In the present study, we investigated the presence of HBV in the synovium and its clinical significance in RA. METHODS: Fifty-seven consecutive patients with active RA (Disease Activity Score 28-joint assessment based on C-reactive protein ≥ 2.6) and available synovial tissue who had completed 1 year of follow-up were recruited from a prospective cohort. The patients were divided into chronic HBV infection (CHB, n = 11) and non-CHB groups according to baseline HBV infection status. Clinical data were collected at baseline and at 1-, 3-, 6-, and 12-month follow-up. Radiographic changes of hand/wrist at baseline and month 12 were assessed with the Sharp/van der Heijde-modified Sharp score (mTSS). HBV in synovium was determined by immunohistochemical staining for hepatitis B virus surface antigen and hepatitis B virus core antigen (HBcAg) and by nested PCR for the HBV S gene. RESULTS: HBcAg was found in the synovium of patients with RA with CHB (7 of 11, 64%), which was confirmed by PCR for the HBV S gene. Compared with the non-CHB group, more CD68-positive macrophages, CD20-positive B cells, and CD15-positive neutrophils infiltrated the synovium in the CHB group (all p <  0.05). There were smaller improvements from baseline in most disease activity indicators mainly at month 12, and a significantly higher percentage of CHB patients experienced 1-year radiographic progression (ΔmTSS ≥ 0.5 unit/yr, 64% vs. 26%, p = 0.024). Multivariate logistic regression analysis showed that CHB status (OR 14.230, 95% CI 2.213-95.388; p = 0.006) and the density of synovial CD68-positive macrophages (OR 1.002, 95% CI 1.001-1.003; p = 0.003) were independently associated with 1-year radiographic progression. CONCLUSIONS: The presence of HBV in RA synovium may be involved in the pathogenesis of local lesions and exacerbate disease progression in RA.
    • Non-canonical Caspase-1 Signaling Drives RIP2-Dependent and TNF-α-Mediated Inflammation In Vivo.

      Reinke, Sören; Linge, Mary; Diebner, Hans H; Luksch, Hella; Glage, Silke; Gocht, Anne; Robertson, Avril A B; Cooper, Matthew A; Hofmann, Sigrun R; Naumann, Ronald; et al.
      Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1-/- mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo.
    • Phosphatidylcholine PC ae C44:6 in cerebrospinal fluid is a sensitive biomarker for bacterial meningitis.

      de Araujo, Leonardo Silva; Pessler, Kevin; Sühs, Kurt-Wolfram; Novoselova, Natalia; Klawonn, Frank; Kuhn, Maike; Kaever, Volkhard; Müller-Vahl, Kirsten; Trebst, Corinna; Skripuletz, Thomas; et al. (BioMed Central (BMC), 2020-01-07)
      BACKGROUND: The timely diagnosis of bacterial meningitis is of utmost importance due to the need to institute antibiotic treatment as early as possible. Moreover, the differentiation from other causes of meningitis/encephalitis is critical because of differences in management such as the need for antiviral or immunosuppressive treatments. Considering our previously reported association between free membrane phospholipids in cerebrospinal fluid (CSF) and CNS involvement in neuroinfections we evaluated phosphatidylcholine PC ae C44:6, an integral constituent of cell membranes, as diagnostic biomarker for bacterial meningitis. METHODS: We used tandem mass spectrometry to measure concentrations of PC ae C44:6 in cell-free CSF samples (n = 221) from patients with acute bacterial meningitis, neuroborreliosis, viral meningitis/encephalitis (herpes simplex virus, varicella zoster virus, enteroviruses), autoimmune neuroinflammation (anti-NMDA-receptor autoimmune encephalitis, multiple sclerosis), facial nerve and segmental herpes zoster (shingles), and noninflammatory CNS disorders (Bell's palsy, Tourette syndrome, normal pressure hydrocephalus). RESULTS: PC ae C44:6 concentrations were significantly higher in bacterial meningitis than in all other diagnostic groups, and were higher in patients with a classic bacterial meningitis pathogen (e.g. Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus) than in those with less virulent or opportunistic pathogens as causative agents (P = 0.026). PC ae C44:6 concentrations were only moderately associated with CSF cell count (Spearman's ρ = 0.45; P = 0.009), indicating that they do not merely reflect neuroinflammation. In receiver operating characteristic curve analysis, PC ae C44:6 equaled CSF cell count in the ability to distinguish bacterial meningitis from viral meningitis/encephalitis and autoimmune CNS disorders (AUC 0.93 both), but had higher sensitivity (91% vs. 41%) and negative predictive value (98% vs. 89%). A diagnostic algorithm comprising cell count, lactate and PC ae C44:6 had a sensitivity of 97% (specificity 87%) and negative predictive value of 99% (positive predictive value 61%) and correctly diagnosed three of four bacterial meningitis samples that were misclassified by cell count and lactate due to low values not suggestive of bacterial meningitis. CONCLUSIONS: Increased CSF PC ae C44:6 concentrations in bacterial meningitis likely reflect ongoing CNS cell membrane stress or damage and have potential as additional, sensitive biomarker to diagnose bacterial meningitis in patients with less pronounced neuroinflammation.
    • Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection.

      de Araujo, Leonardo Silva; Ribeiro-Alves, Marcelo; Leal-Calvo, Thyago; Leung, Janaína; Durán, Verónica; Samir, Mohamed; Talbot, Steven; Tallam, Aravind; Mello, Fernanda Carvalho de Queiroz; Geffers, Robert; et al. (America Society of Microbiology (ASM), 2019-12-03)
      In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.
    • Decreased plasma phospholipid concentrations and increased acid sphingomyelinase activity are accurate biomarkers for community-acquired pneumonia.

      Arshad, Haroon; Alfonso, Juan Carlos López; Franke, Raimo; Michaelis, Katina; Araujo, Leonardo; Habib, Aamna; Zboromyrska, Yuliya; Lücke, Eva; Strungaru, Emilia; Akmatov, Manas K; et al. (Wiley, 2019-11-11)
      BACKGROUND: There continues to be a great need for better biomarkers and host-directed treatment targets for community-acquired pneumonia (CAP). Alterations in phospholipid metabolism may constitute a source of small molecule biomarkers for acute infections including CAP. Evidence from animal models of pulmonary infections and sepsis suggests that inhibiting acid sphingomyelinase (which releases ceramides from sphingomyelins) may reduce end-organ damage. METHODS: We measured concentrations of 105 phospholipids, 40 acylcarnitines, and 4 ceramides, as well as acid sphingomyelinase activity, in plasma from patients with CAP (n = 29, sampled on admission and 4 subsequent time points), chronic obstructive pulmonary disease exacerbation with infection (COPD, n = 13) as a clinically important disease control, and 33 age- and sex-matched controls. RESULTS: Phospholipid concentrations were greatly decreased in CAP and normalized along clinical improvement. Greatest changes were seen in phosphatidylcholines, followed by lysophosphatidylcholines, sphingomyelins and ceramides (three of which were upregulated), and were least in acylcarnitines. Changes in COPD were less pronounced, but also differed qualitatively, e.g. by increases in selected sphingomyelins. We identified highly accurate biomarkers for CAP (AUC ≤ 0.97) and COPD (AUC ≤ 0.93) vs. Controls, and moderately accurate biomarkers for CAP vs. COPD (AUC ≤ 0.83), all of which were phospholipids. Phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins were also markedly decreased in S. aureus-infected human A549 and differentiated THP1 cells. Correlations with C-reactive protein and procalcitonin were predominantly negative but only of mild-to-moderate extent, suggesting that these markers reflect more than merely inflammation. Consistent with the increased ceramide concentrations, increased acid sphingomyelinase activity accurately distinguished CAP (fold change = 2.8, AUC = 0.94) and COPD (1.75, 0.88) from Controls and normalized with clinical resolution. CONCLUSIONS: The results underscore the high potential of plasma phospholipids as biomarkers for CAP, begin to reveal differences in lipid dysregulation between CAP and infection-associated COPD exacerbation, and suggest that the decreases in plasma concentrations are at least partially determined by changes in host target cells. Furthermore, they provide validation in clinical blood samples of acid sphingomyelinase as a potential treatment target to improve clinical outcome of CAP
    • Electro-Microbiology as a Promising Approach Towards Renewable Energy and Environmental Sustainability

      Ali, Jafar; Sohail, Aaqib; Wang, Lei; Rizwan Haider, Muhammad; Mulk, Shahi; Pan, Gang; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MDPI AG, 2018-07-12)
      Microbial electrochemical technologies provide sustainable wastewater treatment and energy production. Despite significant improvements in the power output of microbial fuel cells (MFCs), this technology is still far from practical applications. Extracting electrical energy and harvesting valuable products by electroactive bacteria (EAB) in bioelectrochemical systems (BESs) has emerged as an innovative approach to address energy and environmental challenges. Thus, maximizing power output and resource recovery is highly desirable for sustainable systems. Insights into the electrode-microbe interactions may help to optimize the performance of BESs for envisioned applications, and further validation by bioelectrochemical techniques is a prerequisite to completely understand the electro-microbiology. This review summarizes various extracellular electron transfer mechanisms involved in BESs. The significant role of characterization techniques in the advancement of the electro-microbiology field is discussed. Finally, diverse applications of BESs, such as resource recovery, and contributions to the pursuit of a more sustainable society are also highlighted.
    • Crystal structure of -aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis.

      Chen, Fangfang; Lukat, Peer; Iqbal, Azeem Ahmed; Saile, Kyrill; Kaever, Volkhard; van den Heuvel, Joop; Blankenfeldt, Wulf; Büssow, Konrad; Pessler, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (National Academy of Sciences, 2019-09-23)
      cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.
    • Self-reported diabetes and herpes zoster are associated with a weak humoral response to the seasonal influenza A H1N1 vaccine antigen among the elderly.

      Akmatov, Manas K; Riese, Peggy; Trittel, Stephanie; May, Marcus; Prokein, Jana; Illig, Thomas; Schindler, Christoph; Guzmán, Carlos A; Pessler, Frank; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (BioMedCentral, 2019-07-23)
      BACKGROUND: The immune response to seasonal influenza vaccines decreases with advancing age. Therefore, an adjuvanted inactivated trivalent influenza vaccine (Fluad®) exists for elderly individuals. Fluad® is more immunogenic and efficacious than conventional influenza vaccines. However, the immune response varies and may still result in high frequencies of poor responders. Therefore, we aimed to a) examine the prevalence of a weak response to Fluad® and b) identify potential risk factors. METHODS: A prospective population-based study among individuals 65-80 years old was conducted in 2015/2016 in Hannover, Germany (n = 200). Hemagglutination-inhibition titers 21 days after vaccination with Fluad® served as indicator of vaccine responsiveness. RESULTS: The percentage of vaccinees with an inadequate vaccine response varied depending on the influenza strain: it was lowest for H3N2 (13.5%; 95% CI, 9.4-18.9%), intermediate for B strain (37.0%; 30.6-43.9%), and highest for H1N1 (49.0%; 42.2-55.9%). The risk of a weak response to the influenza A H1N1 strain was independently associated with self-reported diabetes (AOR, 4.64; 95% CI, 1.16-18.54), a history of herpes zoster (2.27; 1.01-5.10) and, to a much lesser extent, increasing age (change per year, 1.08; 0.99-1.16). In addition, herpes zoster was the only risk factor for a weak response to the H3N2 antigen (AOR, 3.12; 1.18-8.23). We found no significant association between sex, Body Mass Index, cancer, hypertension, heart attack and CMV seropositivity and a weak response to these two influenza A antigens. Despite its occurence in over one third of vaccinees, none of the variables examined proved to be risk factors for a weak response to the B antigen. CONCLUSIONS: A considerable proportion of elderly individuals displayed a weak vaccine response to this adjuvanted seasonal influenza vaccine and further efforts are thus needed to improve immune responses to influenza vaccination among the elderly. Diabetes and herpes zoster were identified as potentially modifiable risk factors for a poor vaccine response against influenza A antigens, but the results also reveal the need for broader investigations to identify risk factors for inadequate responses to influenza B antigens.
    • Helicobacter pylori seropositivity: Prevalence, Associations, and the Impact on Incident Metabolic Diseases/Risk Factors in the Population-Based KORA Study.

      Wawro, Nina; Amann, Ute; Butt, Julia; Meisinger, Christa; Akmatov, Manas K; Pessler, Frank; Peters, Annette; Rathmann, Wolfgang; Kääb, Stefan; Waterboer, Tim; et al. (Frontiers, 2019-01-01)
      Introduction:Helicobacter pylori (H. pylori) is a common infection and known risk factor for gastric cancer. We assessed cross-sectional and longitudinal associations to study the impact of H. pylori seropositivity on metabolic diseases. Methods:Helicobacter pylori seropositivity in serum samples of the KORA study was analyzed by multiplex serology. We calculated sex-specific prevalence of H. pylori seropositivity for the year 2007 based on the first follow-up survey (termed F4) of the KORA study S4. We identified factors associated with H. pylori seropositivity in the F4 survey. Further, we assessed relative risks of incident metabolic diseases/risk factors at the time of the second follow-up survey of S4 (termed FF4) and H. pylori seropositivity at the F4 survey as a determinant. Models were adjusted for age, sex, overweight status, physical activity, smoking status, education level, alcohol intake, and other metabolic diseases. Results: Based on 3,037 persons aged 32 to 82 years, the H. pylori prevalence for 2007 was 30.2% in men (n = 1,465) and 28.1% in women (n = 1,572). Increasing age, current smoking, low education and no alcohol intake were significantly associated with H. pylori seropositivity in the F4 survey. However, no association between H. pylori seropositivity and BMI, metabolic diseases (type 2 diabetes, hypertension and dyslipidemia, gout or increased uric acid) and gastrointestinal diseases (gastritis, inflammatory bowel disease, and gastric or duodenal ulcer) was observed. No significant associations between H. pylori seropositivity and one of the five investigated incident metabolic diseases/risk factors were detected in the longitudinal analysis. Conclusion: We identified associations between age, smoking, education and alcohol intake and H. pylori seropositivity but no impact of H. pylori seropositivity on incident metabolic diseases/risk factors.
    • Kynurenine is a cerebrospinal fluid biomarker for bacterial and viral CNS infections.

      Sühs, Kurt-Wolfram; Novoselova, Natalia; Kuhn, Maike; Seegers, Lena; Kaever, Volkhard; Müller-Vahl, Kirsten; Trebst, Corinna; Skripuletz, Thomas; Stangel, Martin; Pessler, Frank; et al. (Oxford University Press, 2019-02-05)
      The tryptophan-kynurenine-NAD+ pathway is closely associated with regulation of immune cells toward less inflammatory phenotypes and may exert neuroprotective effects. Investigating its regulation in CNS infections would improve our understanding of pathophysiology and end-organ damage, and, furthermore, open doors to its evaluation as a source of diagnostic and/or prognostic biomarkers. We measured concentrations of kynurenine (Kyn) and tryptophan (Trp) in 220 cerebrospinal fluid samples from patients with bacterial and viral (herpes simplex, varicella zoster, enteroviruses) meningitis/encephalitis, neuroborreliosis, autoimmune neuroinflammation (anti-NMDA-R encephalitis, multiple sclerosis), and noninflamed controls (Bell's palsy, normal pressure hydrocephalus, Tourette syndrome). Kyn concentrations correlated strongly with CSF markers of neuroinflammation (leukocyte count, lactate, and blood-CSF-barrier dysfunction) and were highly increased in bacterial and viral CNS infections, but were low or undetectable in anti-NMDA-R encephalitis, multiple sclerosis, and controls. Trp was decreased mostly in viral CNS infections and neuroborreliosis. Multiple logistic regression analysis revealed combinations of Kyn, Trp and Kyn/Trp ratio with leukocyte count or lactate as accurate classifiers for the clinically important differentiation between neuroborreliosis, viral CNS infections, and autoimmune neuroinflammation. The Trp-Kyn-NAD+ pathway is activated in CNS infections and provides highly accurate CSF biomarkers, particularly when combined with standard CSF indices of neuroinflammation.
    • Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display.

      Ramli, Siti Roszilawati; Moreira, Gustavo M S G; Zantow, Jonas; Goris, Marga G A; Nguyen, Van Kinh; Novoselova, Natalia; Pessler, Frank; Hust, Michael; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (PLOS, 2019-01-01)
      Leptospirosis is the most common zoonotic disease worldwide. The diagnostic performance of a serological test for human leptospirosis is mainly influenced by the antigen used in the test assay. An ideal serological test should cover all serovars of pathogenic leptospires with high sensitivity and specificity and use reagents that are relatively inexpensive to produce and can be used in tropical climates. Peptide-based tests fulfil at least the latter two requirements, and ORFeome phage display has been successfully used to identify immunogenic peptides from other pathogens. Two ORFeome phage display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75). This analysis lends further support for using ORFeome phage display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen.
    • Identification of Cerebrospinal Fluid Metabolites as Biomarkers for Enterovirus Meningitis.

      Ratuszny, Dominica; Sühs, Kurt-Wolfram; Novoselova, Natalia; Kuhn, Maike; Kaever, Volkhard; Skripuletz, Thomas; Pessler, Frank; Stangel, Martin; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MPDI, 2019-01-15)
      Enteroviruses are among the most common causes of viral meningitis. Enteroviral meningitis continues to represent diagnostic challenges, as cerebrospinal fluid (CSF) cell numbers (a well validated diagnostic screening tool) may be normal in up to 15% of patients. We aimed to identify potential CSF biomarkers for enteroviral meningitis, particularly for cases with normal CSF cell count. Using targeted liquid chromatography-mass spectrometry, we determined metabolite profiles from patients with enteroviral meningitis (n = 10), and subdivided them into those with elevated (n = 5) and normal (n = 5) CSF leukocyte counts. Non-inflamed CSF samples from patients with Bell’s palsy and normal pressure hydrocephalus (n = 19) were used as controls. Analysis of 91 metabolites revealed considerable metabolic reprogramming in the meningitis samples. It identified phosphatidylcholine PC.ae.C36.3, asparagine, and glycine as an accurate (AUC, 0.92) combined classifier for enterovirus meningitis overall, and kynurenine as a perfect biomarker for enteroviral meningitis with an increased CSF cell count (AUC, 1.0). Remarkably, PC.ae.C36.3 alone emerged as a single accurate (AUC, 0.87) biomarker for enteroviral meningitis with normal cell count, and a combined classifier comprising PC.ae.C36.3, PC.ae.C36.5, and PC.ae.C38.5 achieved nearly perfect classification (AUC, 0.99). Taken together, this analysis reveals the potential of CSF metabolites as additional diagnostic tools for enteroviral meningitis, and likely other central nervous system (CNS) infections.
    • An Interferon Signature Discriminates Pneumococcal From Staphylococcal Pneumonia.

      Strehlitz, Anja; Goldmann, Oliver; Pils, Marina C; Pessler, Frank; Medina, Eva; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Frontiers, 2018-01-01)
      Streptococcus pneumoniae is the most common cause of community-acquired pneumonia (CAP). Despite the low prevalence of CAP caused by methicillin-resistant Staphylococcus aureus (MRSA), CAP patients often receive empirical antibiotic therapy providing coverage for MRSA such as vancomycin or linezolid. An early differentiation between S. pneumoniae and S. aureus pneumonia can help to reduce the use of unnecessary antibiotics. The objective of this study was to identify candidate biomarkers that can discriminate pneumococcal from staphylococcal pneumonia. A genome-wide transcriptional analysis of lung and peripheral blood performed in murine models of S. pneumoniae and S. aureus lung infection identified an interferon signature specifically associated with S. pneumoniae infection. Prediction models built using a support vector machine and Monte Carlo cross-validation, identified the combination of the interferon-induced chemokines CXCL9 and CXCL10 serum concentrations as the set of biomarkers with best sensitivity, specificity, and predictive power that enabled an accurate discrimination between S. pneumoniae and S. aureus pneumonia. The predictive performance of these biomarkers was further validated in an independent cohort of mice. This study highlights the potential of serum CXCL9 and CXCL10 biomarkers as an adjunctive diagnostic tool that could facilitate prompt and correct pathogen-targeted therapy in CAP patients.
    • Development of a Bead-Based Multiplex Assay for the Analysis of the Serological Response against the Six Pathogens HAV, HBV, HCV, CMV, T. gondii, and H. pylori.

      Filomena, Angela; Pessler, Frank; Akmatov, Manas K; Krause, Gérard; Duffy, Darragh; Gärtner, Barbara; Gerhard, Markus; Albert, Matthew L; Joos, Thomas O; Schneiderhan-Marra, Nicole; et al. (2017-10-30)
      The spread of infectious diseases and vaccination history are common subjects of epidemiological and immunological research studies. Multiplexed serological assays are useful tools for assessing both current and previous infections as well as vaccination efficacy. We developed a serological multi-pathogen assay for hepatitis A, B and C virus, cytomegalovirus (CMV),
    • NS Segment of a 1918 Influenza A Virus-Descendent Enhances Replication of H1N1pdm09 and Virus-Induced Cellular Immune Response in Mammalian and Avian Systems.

      Petersen, Henning; Mostafa, Ahmed; Tantawy, Mohamed A; Iqbal, Azeem A; Hoffmann, Donata; Tallam, Aravind; Selvakumar, Balachandar; Pessler, Frank; Beer, Martin; Rautenschlein, Silke; et al. (2018-01-01)
      The 2009 pandemic influenza A virus (IAV) H1N1 strain (H1N1pdm09) has widely spread and is circulating in humans and swine together with other human and avian IAVs. This fact raises the concern that reassortment between H1N1pdm09 and co-circulating viruses might lead to an increase of H1N1pdm09 pathogenicity in different susceptible host species. Herein, we explored the potential of different NS segments to enhance the replication dynamics, pathogenicity and host range of H1N1pdm09 strain A/Giessen/06/09 (Gi-wt). The NS segments were derived from (i) human H1N1- and H3N2 IAVs, (ii) highly pathogenic- (H5- or H7-subtypes) or (iii) low pathogenic avian influenza viruses (H7- or H9-subtypes). A significant increase of growth kinetics in A549 (human lung epithelia) and NPTr (porcine tracheal epithelia) cells was only noticed
    • Early Lymphocyte Loss and Increased Granulocyte/Lymphocyte Ratio Predict Systemic Spread of in a Mouse Model of Acute Skin Infection.

      Loof, Torsten G; Sohail, Aaqib; Bahgat, Mahmoud M; Tallam, Aravind; Arshad, Haroon; Akmatov, Manas K; Pils, Marina C; Heise, Ulrike; Beineke, Andreas; Pessler, Frank; et al.
      Group A streptococci may induce lymphopenia, but the value of lymphocyte loss as early biomarkers for systemic spread and severe infection has not been examined systematically. We evaluated peripheral blood cell indices as biomarkers for severity and spread of infection in a mouse model of skin infection, using two isolates of greatly differing virulence. Internal organs were examined histologically. After subcutaneous inoculation, strain AP1 disseminated rapidly to peripheral blood and internal organs, causing frank sepsis. In contrast, seeding of internal organs by 5448 was mild, this strain could not be isolated from blood, and infection remained mostly localized to skin. Histopathologic examination of liver revealed microvesicular fatty change (steatosis) in AP1 infection, and examination of spleen showed elevated apoptosis and blurring of the white pulp/red pulp border late (40 h post infection) in AP1 infection. Both strains caused profound lymphopenia, but lymphocyte loss was more rapid early in AP1 infection, and lymphocyte count at 6 h post infection was the most accurate early marker for AP1 infection (area under the receiver operator curve [AUC] = 0.93), followed by the granulocyte/lymphocyte ratio (AUC = 0.89). The results suggest that virulence of correlates with the degree of early lymphopenia and underscore the value of peripheral blood indices to predict severity of bacterial infections in mice. Early lymphopenia and elevated granulocyte/lymphocyte ratio merit further investigation as biomarkers for systemic spread of skin infections in humans and, possibly, related pyogenic streptococci in humans and animals.