The potential of heparin-induced extracorporeal LDL/fibrinogen precipitation (H.E.L.P.)-apheresis for patients with severe acute or chronic COVID-19.

Patients with long COVID and acute COVID should benefit from treatment with H.E.L.P. apheresis, which is in clinical use for 37 years. COVID-19 can cause a severe acute multi-organ illness and, subsequently, in many patients the chronic illness long-COVID/PASC. The alveolar tissue and adjacent capillaries show inflammatory and procoagulatory activation with cell necrosis, thrombi, and massive fibrinoid deposits, namely, unsolvable microthrombi, which results in an obstructed gas exchange. Heparin-induced extracorporeal LDL/fibrinogen precipitation (H.E.L.P.) apheresis solves these problems by helping the entire macro- and microcirculation extracorporeally. It uses unfractionated heparin, which binds the spike protein and thereby should remove the virus (debris). It dissolves the forming microthrombi without bleeding risk. It removes large amounts of fibrinogen (coagulation protein), which immediately improves the oxygen supply in the capillaries. In addition, it removes the precursors of both the procoagulatory and the fibrinolytic cascade, thus de-escalating the entire hemostaseological system. It increases myocardial, cerebral, and pulmonary blood flow rates, and coronary flow reserve, facilitating oxygen exchange in the capillaries, without bleeding risks. Another factor in COVID is the "cytokine storm" harming microcirculation in the lungs and other organs. Intervention by H.E.L.P. apheresis could prevent uncontrollable coagulation and inflammatory activity by removing cytokines such as interleukin (IL)-6, IL-8, and TNF-α, and reduces C-reactive protein, and eliminating endo- and ecto-toxins, without touching protective IgM/IgG antibodies, leukocyte, or platelet function. The therapy can be used safely in combination with antiviral drugs, antibiotics, anticoagulants, or antihypertensive drugs. Long-term clinical experience with H.E.L.P. apheresis shows it cannot inflict harm upon patients with COVID-19.

Antibiotic-Resistant E. coli in Uncomplicated Community-Acquired Urinary Tract Infection.

BACKGROUND: Routine urine culture testing is not recommended for uncomplicated urinary tract infections (UTIs). As a result, the antibiotic resistance patterns or the organisms causing UTIs are not adequately reflected in routine data. We studied the sensitivity of Escherichia coli (E. coli) to trimethoprim (TMP) and to cotrimoxazole (i.e., trimethoprim/sulfamethoxazole, TMP/SMX) in community-acquired UTI and compared the findings with the resistance data of the Antimicrobial Resistance Surveillance System (ARS). METHODS: General practitioners and internists in private practice prospectively recruited all of their adult patients with symptoms of a urinary tract infection from May 2015 to February 2016. Urine specimens from all patients were tested (including urine culture testing and antibiotic susceptibility) and infections were defined as uncomplicated or complicated UTIs. RESULTS: 1245 participants from 58 medical practices were enrolled in the study. Pathogenic organisms were found in the urine of 877 patients, of whom 74.5% had E. coli infections. Among the E.-coli-positive UTIs, 52.4% were classified as uncomplicated and 47.6% as complicated. The prevalence of E. coli that was resistant to TMP and to TMP/SMX in uncomplicated UTIs was 15.2% and 13.0%, respectively, compared to 25.3% and 24.4%, respectively, from all UTIs in ARS in 2015. Study participants who had previously taken antibiotics had the highest prevalence of E. coli resistance (30.9%), followed by those who had two or more UTIs within the past six months (28.9%). CONCLUSION: E. coli with resistance to TMP was significantly less prevalent among the study patients with uncomplicated UTIs than in the routine data of the ARS. Accordingly, TMP should still be considered as an option for the treatment of uncomplicated UTIs. TMP/SMX is considered the agent of second choice because of its side effects. Surveillance systems based on routine data do not yield a representative sample for the evaluation of the resistance situation in patients with uncomplicated UTIs.

Prevalence and spread of multidrug-resistant Escherichia coli including ST131 in different patient populations in the Euroregion Meuse-Rhine.

AIM: To investigate the prevalence and genetic background of Escherichia coli collected from different patient populations in the Euroregion Meuse-Rhine. MATERIALS & METHODS: Susceptibility testing was performed on 1651 E. coli isolates with broth microdilution. Their genetic background was determined using pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: The prevalence of resistance varied significantly between the populations. Approximately 10% of the E. coli isolates were multidrug-resistant and/or a ß-lactamase producer. The most prevalent extended-spectrum ß-lactamase type was CTX-M-15 and ST131 was the most prevalent multilocus sequence typing type. RESULTS from pulsed-field gel electrophoresis of the ST131 isolates indicate the spread of these isolates in the Euroregion. CONCLUSION: E. coli ST131 was the most prevalent sequence type in our Euroregional study. It is essential to control the spread of these resistant strains (e.g., with infection-control policies, antibiotic stewardship programs and antibiotic resistance surveillance). In this way we could observe shifts in the prevalence of resistance of the E. coli population and act accordingly.

Antimicrobial resistance and spread of multi drug resistant Escherichia coli isolates collected from nine urology services in the Euregion Meuse-Rhine.

We determined the prevalence and spread of antibiotic resistance and the characteristics of ESBL producing and/or multi drug resistant (MDR) Escherichia coli isolates collected from urine samples from urology services in the Euregio Meuse-Rhine, the border region of the Netherlands (n=176), Belgium (n=126) and Germany (n=119). Significant differences in resistance between the three regions were observed. Amoxicillin-clavulanic acid resistance ranged from 24% in the Netherlands to 39% in Belgium (p=0.018), from 20% to 40% (p<0.004) for the fluoroquinolones and from 20% to 40% (p=0.018) for the folate antagonists. Resistance to nitrofurantoin was less than 5%. The prevalence of ESBL producing isolates varied from 2% among the Dutch isolates to 8% among the German ones (p=0.012) and were mainly CTX-M 15. The prevalence of MDR isolates among the Dutch, German and Belgian isolates was 11%, 17% and 27%, respectively (p< =0.001 for the Belgian compared with the Dutch isolates). The majority of the MDR and ESBL producing isolates belonged to ST131. This study indicates that most antibiotics used as first choice oral empiric treatment for UTIs (amoxicillin-clavulanic acid, fluoroquinolones and folate antagonists) are not appropriate for this purpose and that MDR strains such as CTX-M producing ST131 have spread in the entire Euregion. Our data stress the importance of ward specific surveillance to optimize empiric treatment. Also, prudent use of antibiotics and further research to alternative agents are warranted.

Anti-inflammatory arene--chromium complexes acting as specific inhibitors of NOD2 signalling.

Inflammation is a hallmark of microbial infection in mammals and is the result of a pathogen-induced release of inflammatory effectors. In humans a variety of germ-line encoded receptors, so-called pattern-recognition receptors, respond to conserved signatures on invading pathogens, which results in the transcriptional activation of pro-inflammatory responses. Inflammation is often detrimental to the host and leads to tissue damage and/or systemic dysfunctions. Thus, specific inhibitors of these pathways are desirable for medical interventions. Herein we report on the synthesis and use of some chromium-containing compounds (arene--Cr(CO)3 complexes) with a core structure related to anti-inflammatory diterpenes produced by the sea whip Pseudopterogorgia elisabethae. By using cell-based reporter assays we identified complexes with a potent inhibitory activity on tumour necrosis factor (TNF), Toll-like receptor (TLR), and nucleotide binding domain, leucine-rich repeat-containing receptor (NLR) pathways. Moreover, we found one complex to be a specific inhibitor of inflammatory responses mediated by the NLR protein NOD2, a pivotal innate immune receptor involved in bacterial recognition. Synthesis and characterisation of a set of derivatives of this substance revealed structural requirements for NOD2 specificity. Taken together, our studies suggest this type of arene--Cr(CO)3 complex as a potential lead for the development of antiphlogistica and pharmacologically relevant NOD2 inhibitors.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells.

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

The modulation of adaptive immune responses by bacterial zwitterionic polysaccharides.

The detection of pathogen-derived molecules as foreign particles by adaptive immune cells triggers T and B lymphocytes to mount protective cellular and humoral responses, respectively. Recent immunological advances elucidated that proteins and some lipids are the principle biological molecules that induce protective T cell responses during microbial infections. Polysaccharides are important components of microbial pathogens and many vaccines. However, research concerning the activation of the adaptive immune system by polysaccharides gained interest only recently. Traditionally, polysaccharides were considered to be T cell-independent antigens that did not directly activate T cells or induce protective immune responses. Here, we review several recent advances in "carbohydrate immunobiology". A group of bacterial polysaccharides that are known as "zwitterionic polysaccharides (ZPSs)" were recently identified as potent immune modulators. The immunomodulatory effect of ZPSs required antigen processing and presentation by antigen presenting cells, the activation of CD4 T cells and subpopulations of CD8 T cells and the modulation of host cytokine responses. In this review, we also discuss the potential use of these unique immunomodulatory ZPSs in new vaccination strategies against chronic inflammatory conditions, autoimmunity, infectious diseases, allergies and asthmatic conditions.

Interleukin-6 is essential for zwitterionic polysaccharide-mediated abscess formation.

Abscess formation associated with secondary peritonitis causes severe morbidity and can be fatal. Formation of abscesses requires the presence of CD4+ T-cells. Zwitterionic polysaccharides (ZPSs) represent a novel class of immunomodulatory bacterial antigens that stimulate CD4+ T-cells in a major histocompatibility complex (MHC) class II-dependent manner. The capsular polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 possesses a zwitterionic charge with free amino groups and promotes T-cell-dependent abscess formation in an experimental mouse model. So far, nothing is known about the function of Interleukin (IL)-6 in intraperitoneal abscess formation. Here, we demonstrate that macrophages and dendritic cells (DCs), the most prevalent professional antigen-presenting cells involved in the formation of abscesses, secrete Interleukin (IL)-6 and are incorporated in the abscess capsule. Sp1 inhibits apoptosis of CD4+ T-cells and causes IL-17 expression by CD4+ T-cells in an IL-6-dependent manner. Abrogation of the Sp1-induced pleiotropic effects of IL-6 in IL-6-deficient mice and mice treated with an IL-6-specific neutralizing antibody results in significant inhibition of abscess formation. The data delineate the essential role of IL-6 in the linkage of innate and adaptive immunity in polysaccharide-mediated abscess formation.

Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking.

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an alpha-helix configuration. In this paper we look at the immune response of CD8(+) T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8(+)CD28(-) T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8(+)CD28(-) T lymphocytes. The Sp1-induced CD8(+)CD28(-) T lymphocytes are CD122(low)CTLA-4(+)CD39(+). They synthesize IL-10 and TGF-beta. The Sp1-induced CD8(+)CD28(-) T cells exhibit immunosuppressive properties on CD4(+) T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8(+) T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8(+) T cell activation by enhancing crosslinking of TCR. The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8(+)CD28(-) T lymphocytes in vivo and in vitro. The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

Airway infection with a novel Cupriavidus species in persons with cystic fibrosis.

We describe the recovery and identification of a bacterium that represents a new species of the genus Cupriavidus from cultures of respiratory tract specimens from two patients with cystic fibrosis (CF). The elucidation of the role of this species in CF lung disease will require an evaluation of a greater number of cases.

Oligoclonal CD4+ T cells promote host memory immune responses to Zwitterionic polysaccharide of Streptococcus pneumoniae.

Zwitterionic polysaccharides of the normal flora bacteria represent a novel class of antigens in that they correct systemic CD4(+) T-cell deficiencies and direct lymphoid organogenesis during colonization of the host. Presentation of these polysaccharides to CD4(+) T cells depends on major histocompatibility complex class II- and DM-dependent retrograde transport from lysosomes to the cell surface. Yet the phenotype and clonality of the immune response to the polysaccharide in the mature host immune system have not been studied. Using the zwitterionic capsular polysaccharide Sp1 of Streptococcus pneumoniae, a transient member of the bacterial flora, in an experimental mouse model of cellular immunity, we demonstrated the accumulation of TH1- and TH17-polarized CD4(+) CD44(high) CD62(low) CD25(-) memory T cells. Subcutaneous immunization with Sp1 resulted in an increase of serum immunoglobulin G (IgG), predominantly of the IgG1 subclass, and suggested the presence of a humoral memory response to the polysaccharide. CD4(+) T cells stimulated with polysaccharide in vitro and in vivo showed a nonrestricted pattern for the T-cell receptor (TCR) beta-chain variable region, as demonstrated by semiquantitative reverse transcription-PCR and flow cytometry. Clonotype mapping of in vivo and in vitro polysaccharide-activated CD4(+) T cells revealed clonotypic TCR transcripts. Taken together, the data show the induction of clonal expansion of CD4(+) T cells by polysaccharides of commensal bacteria. Cellular and humoral memory host responses imply the ability of these polysaccharides to mediate the expansion of T cells via recognition within the CDR3 region of the TCR.

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors.

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c(+) CD11b(+) DCs derived from murine CD34-positive precursors. DCs derived from CD34(+) cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34(+) cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.

HLA-DR alpha 2 mediates negative signalling via binding to Tirc7 leading to anti-inflammatory and apoptotic effects in lymphocytes in vitro and in vivo.

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRalpha2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-zeta chain & ZAP70, and inhibition of IFN-gamma and FasL expression. HLA-DRalpha2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRalpha2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway.

Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules.

Bacterial capsular polysaccharides are virulence factors and are considered T cell-independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+) T cells in a major histocompatibility complex (MHC) class II-dependent manner. The mechanism of carbohydrate presentation to CD4(+) T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell-dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide-carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

Modulation of surgical fibrosis by microbial zwitterionic polysaccharides.

Bacterial carbohydrates have long been considered T cell-independent antigens that primarily induce humoral immune responses. Recently, it has been demonstrated that bacterial capsules that possess a zwitterionic charge motif can activate CD4(+) T cells after processing and presentation by antigen-presenting cells. Here we show that these zwitterionic polysaccharides can prevent T helper 1-mediated fibrosis by signaling for the release of IL-10 from CD4(+) T cells in vivo. IL-10 production by these T cells and their ability to prevent fibrosis is controlled by the inducible costimulator (ICOS)-ICOS ligand pathway. These data demonstrate that the interaction of the zwitterionic polysaccharides with T cells results in modulation of surgical fibrosis in vivo and suggest a previously undescribed approach to "harnessing" T cell function to prevent inflammatory tissue disorders in humans.

Effect of B7-2 and CD40 signals from activated antigen-presenting cells on the ability of zwitterionic polysaccharides to induce T-Cell stimulation.

Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria elicit potent CD4+-T-cell responses in vivo and in vitro. We demonstrated that HLA-DR on professional antigen-presenting cells (APCs) is required for ZPS-induced T-cell proliferation in vitro (15). Recently, it was shown that ZPSs are processed to low-molecular-weight carbohydrates by a nitric oxide-mediated mechanism in endosomes and locate in the major histocompatibility complex class II pathway (5, 15). The effect of the ZPS-mediated expression of HLA-DR and costimulatory molecules on the APC and T-cell engagement and subsequent T-cell activation has not been elucidated. Herein, we report that ZPS-mediated induction of HLA-DR-surface expression and T-cell proliferation are maximally enhanced after incubation of APCs for 8 h with ZPS. Treatment of APCs with bafilomycin A inhibits the up-regulation of ZPS-mediated HLA-DR surface expression and leads to inhibition of T-cell proliferation. Monoclonal antibodies (MAbs) to the costimulatory molecules B7-2 and CD40L specifically block ZPS-mediated T-cell activation, while a MAb to B7-1 does not. Surface expression of B7-2 and B7-1 but not of CD40 is maximally enhanced at 8 to 16 h of treatment of APCs with ZPS. The results demonstrate that the cellular immune response to ZPS depends on the translocation of HLA-DR to the cell surface and requires costimulation via B7-2 and CD40 on activated APCs. The implication is that activation of ZPS-specific T cells requires an orchestrated arrangement of both presenting and costimulatory molecules to form an immunological synapse.

Zwitterionic polysaccharides stimulate T cells by MHC class II-dependent interactions.

Polysaccharides of pathogenic extracellular bacteria commonly have negatively charged groups or no charged groups at all. These molecules have been considered classic T cell-independent Ags that do not elicit cell-mediated immune responses in mice. However, bacterial polysaccharides with a zwitterionic charge motif (ZPSs), such as the capsular polysaccharides of many strains of Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae type 1 elicit potent CD4(+) T cell responses in vivo and in vitro. The cell-mediated response to ZPS depends on the presence of both positively charged and negatively charged groups on each repeating unit of the polysaccharide. In this study, we define some of the requirements for the presentation of ZPS to CD4(+) T cells. We provide evidence that direct interactions of T cells with APCs are essential for T cell activation by ZPS. Monocytes, dendritic cells, and B cells are all able to serve as APCs for ZPS-mediated T cell activation. APCs lacking MHC class II molecules do not support this activity. Furthermore, mAb to HLA-DR specifically blocks ZPS-mediated T cell activation, while mAbs to other MHC class II and class I molecules do not. Immunoprecipitation of lysates of MHC class II-expressing cells following incubation with ZPS shows binding of ZPS and HLA-DR. Electron microscopy reveals colocalization of ZPS with HLA-DR on the cell surface and in compartments of the endocytic pathway. These results indicate that MHC class II molecules expressing HLA-DR on professional APCs are required for ZPS-induced T cell activation. The implication is that binding of ZPS to HLA-DR may be required for T cell activation.