Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery.

Introduction: Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery. Materials and Methods: In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus. Results: Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P. Discussion: In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.

Case Report: Interferon-γ Restores Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) in Severe COVID-19 With Acquired Immunosuppression Syndrome.

Background: The major histocompatibility complex (MHC) class II characterized by monocytes CD14+ expression of human leukocyte antigen receptors (HLA-DR), is essential for the synapse between innate and adaptive immune response in infectious disease. Its reduced expression is associated with a high risk of secondary infections in septic patients and can be safely corrected by Interferon-y (IFNy) injection. Coronavirus disease (COVID-19) induces an alteration of Interferon (IFN) genes expression potentially responsible for the observed low HLA-DR expression in circulating monocytes (mHLA-DR). Methods: We report a case of one-time INFy injection (100 mcg s.c.) in a superinfected 61-year-old man with COVID-19-associated acute respiratory distress syndrome (ARDS), with monitoring of mHLA-DR expression and clinical tolerance. Observations: Low mHLA-DR pretreatment expression (26.7%) was observed. IFNy therapy leading to a rapid increase in mHLA-DR expression (83.1%). Conclusions: Severe ARDS in a COVID-19 patient has a deep reduction in mHLA-DR expression concomitantly with secondary infections. The unique IFNy injection was safe and led to a sharp increase in the expression of mHLA-DR. Based on immune and infection monitoring, more cases of severe COVID-19 patients with low mHLA-DR should be treated by IFNy to test the clinical effectiveness.

Contact sensitizations to disinfectants containing alcohols or quaternary ammonium compounds are rarely of clinical relevance.

BACKGROUND: The use of disinfectants is part of the everyday life of people, especially in the medical profession. During the coronavirus disease 2019 (COVID-19) pandemic, the use of disinfectants continues to increase and is of fundamental importance in infection control. OBJECTIVES: To determine the frequency of sensitization and the value of patch testing to didecyldimethylammonium chloride (DDAC) and the alcohols ethanol, 1-propanol, and isopropanol. METHODS: Clinical patch test data of 145 patients with suspected contact allergy to disinfectants were retrospective analysed. RESULTS: Among the 145 patients patch tested with the different alcohols, only one nurse was detected with a possible allergy to 1-propanol. Additional patch testing in 84 patients with DDAC 0.05% resulted in five patients with weakly positive reactions only, without clinical relevance. Patch testing with DDAC 0.03% showed no positive reactions at all on day 3 readings. CONCLUSIONS: DDAC and alcohols are rarely responsible for allergic contact dermatitis. The accused products of the patients should be checked for other allergens and further additives with skin-irritating properties. Individual susceptibility and mishandling of the disinfectants should be considered.

Improving povidone-iodine and iodine preparations for patch testing.

BACKGROUND: Allergy evaluation by patch testing with povidone-iodine (PVP-I) or iodine remains challenging, because current patch test preparations frequently lead to false-positive or irritant skin reactions. OBJECTIVES: To investigate different preparations for iodine patch tests and to assess their clinical relevance with repeated open application tests (ROATs). METHODS: We monocentrically analyzed 95 patients with suspected allergy to disinfectants in retrospect who underwent parallel iodine patch testing with four preparations: PVP-I 2% aq., 5% aq., 10% aq., and iodine 0.5% pet. RESULTS: In 27 of 95 patients (28.4%), we found positive reactions to one of the four test preparations. After ROATs in 22 of these 27 positively tested individuals, only one patient was diagnosed with iodine allergy. In contrast, 31 of 95 patients (32.6%) showed irritant or questionable patch test reactions on day 2 (D2) and/or D3 and/or D7 to one or more test preparations. Testing with PVP-I 2% aq. resulted in the lowest number of doubtful skin reactions while detecting the single allergic patient. CONCLUSION: PVP-I 2% aq. was found to be the optimal patch test preparation. In general, iodine allergy appears to be substantially overestimated, and positive patch test responses to iodine should prompt an urgent ROAT for confirmation before diagnosing iodine allergy.

Modulating Tumor Cell Functions by Tunable Nanopatterned Ligand Presentation.

Cancer comprises a large group of complex diseases which arise from the misrouted interplay of mutated cells with other cells and the extracellular matrix. The extracellular matrix is a highly dynamic structure providing biochemical and biophysical cues that regulate tumor cell behavior. While the relevance of biochemical signals has been appreciated, the complex input of biophysical properties like the variation of ligand density and distribution is a relatively new field in cancer research. Nanotechnology has become a very promising tool to mimic the physiological dimension of biophysical signals and their positive (i.e., growth-promoting) and negative (i.e., anti-tumoral or cytotoxic) effects on cellular functions. Here, we review tumor-associated cellular functions such as proliferation, epithelial-mesenchymal transition (EMT), invasion, and phenotype switch that are regulated by biophysical parameters such as ligand density or substrate elasticity. We also address the question of how such factors exert inhibitory or even toxic effects upon tumor cells. We describe three principles of nanostructured model systems based on block copolymer nanolithography, electron beam lithography, and DNA origami that have contributed to our understanding of how biophysical signals direct cancer cell fate.

Analyses of sphingosine-1-phosphate in the context of transfusion: how much is in stored blood products and in patient blood?

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bloodborne lipid that regulates vascular tone and endothelial permeability. S1P concentrations are reduced in critically ill patients. As hematopoietic cells produce S1P, this study intends to investigate S1P concentrations in blood products during storage and in patient plasma after blood transfusion. STUDY DESIGN AND METHODS: S1P concentrations were measured in 83 red blood cell (RBC) units and 73 platelet concentrates (PCs) before and after storage. In addition, 26 critically ill patients who received one or two RBC units were recruited to measure S1P plasma levels before and three times within 24 hours after transfusion. RESULTS: The highest S1P concentrations were found in fresh PCs. S1P concentrations in PCs are reduced by 60% when stored at room temperature for 4 days, whereas in RBCs S1P concentrations remained stable when stored at 4°C within 35 days. S1P concentrations in PCs and RBCc were 2.5 to 6 times higher compared to patient plasma. Plasma S1P levels in critically ill patients, however, transiently decreased after transfusion of RBCs and recover to pretransfusion values within the following 24 hours. CONCLUSION: S1P concentrations in blood products are significantly higher compared to human plasma S1P levels, even though plasma S1P levels decreased after RBC transfusion in critically ill patients and reached pretransfusion values within 24 hours.

Morphological Plasticity of Human Melanoma Cells Is Determined by Nanoscopic Patterns of E- and N-Cadherin Interactions.

Loss of E-cadherin and concomitant upregulation of N-cadherin is known as the cadherin switch, and has been implicated in melanoma progression. Mechanistically, homophilic ligation of N-cadherin-expressing melanoma cells with N-cadherin presented within the microenvironment is thought to facilitate invasion. However, the biophysical aspects governing molecular specificity and function of such interactions remain unclear. By using precisely defined nano-patterns of N- or E-cadherin (with densities tunable by more than one order of magnitude from 78 to 1,128 ligands/µm2), we analyzed adhesion and spreading of six different human melanoma cell lines with distinct constitutive cadherin expression patterns. Cadherin-mediated homophilic cell interactions (N/N and E/E) with cadherin-functionalized nano-matrices revealed an unexpected functional dichotomy inasmuch as melanoma cell adhesion was cadherin density-dependent, while spreading and lamellipodia formation were independent of cadherin density. Surprisingly, E-cadherin-expressing melanoma cells also interacted with N-cadherin-presenting nano-matrices, suggesting heterophilic (N/E) interactions. However, cellular spreading in these cases occurred only at high densities of N-cadherin (i.e., >285 ligands/µm2). Overall, our approach using nano-patterned biomimetic surfaces provides a platform to further refine the roles of cadherins in tumor cell behavior and it revealed an intriguing flexibility of mutually compensating N- and E-cadherin interactions relevant for melanoma progression.

c-Rel is a cell cycle modulator in human melanoma cells.

Melanoma progression and resistance to therapy are associated with faulty regulation of signalling molecules including the central transcription factor NF-κB. Increased expression of the c-Rel subunit of NF-κB has been described in progressing melanoma, though mechanistic implications of this upregulation remain unclear. To elucidate the functional role of c-Rel in melanoma biology, we have assessed its expression in human melanoma as well as in melanoma cell lines. Suppression of c-Rel expression in four melanoma cell lines resulted in reduced growth and altered cell cycle regulation, namely G2/M and polyploid phase induction. Moreover, mitotic spindle morphology was profoundly altered in three of the cell lines with a predominance of monopolar structures. These findings suggest that c-Rel is involved in G2/M phase regulation, prevention of polyploidy and, consequently, chromosomal stability. Our results highlight a novel tumor-promoting function of c-Rel in human melanoma cells through governing cell cycle regulation.

Nanoscale Tuning of VCAM-1 Determines VLA-4-Dependent Melanoma Cell Plasticity on RGD Motifs.

The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/µm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor-matrix interactions.Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528-42. ©2017 AACR.

The IKKα-dependent non-canonical pathway of NF-κB activation is constitutively active and modulates progression-related functions in a subset of human melanomas.

Owing to activation of several resistance-mediating pathways including NF-κB signaling, metastasized melanoma is almost universally resistant against chemotherapy. Given that blocking of NF-κB either by proteasome-, pan-IKK- or selective IKKß-inhibitors may increase the susceptibility of melanoma cells to chemotherapy, we have assessed the role of the second kinase within the IKK complex, IKKα. While expression of IKKα and overall activation of NF-κB were heterogeneous, the IKKα-specific p100/p52 processing was detected in a small subset of melanomas (1/9 primary and 1/12 metastatic melanomas) as well as in 1/8 melanoma cell lines. Down-modulation of IKKα by siRNA resulted in diminution of doxorubicin-induced NF-κB activation, constitutive and TNFα-stimulated expression of CXCL8 and ICAM-1, and cell migration. In contrast, overexpression of IKKα in melanoma cells did not significantly affect progression-related functions. Thus, IKKα may be a worthwhile target only in selected individualized therapies but not in general melanoma therapy.

Nanoscale integrin ligand patterns determine melanoma cell behavior.

Cells use integrin receptors to adhere onto surfaces by binding to ligands such as the arginine-glycine-aspartic acid (RGD) motif. Cancer cells make use of this adhesion process, which has motivated the development of integrin-directed drugs. However, those drugs may exert paradoxical effects on tumor progression, which raises the question of how integrin function is governed in tumor cells on the nanoscale. We have utilized precisely defined and tunable RGD ligand site densities spanning 1 order of magnitude, i.e., 103 to 1145 ligand sites/µm(2), by using RGD-functionalized gold nanoparticle patterns immobilized on glass by block copolymer (micellar) nanolithography. In an αVß3 integrin-dependent fashion, human melanoma cells spread, formed focal contacts, and reorganized cytoskeletal fibers on a physiologically relevant RGD density of 349 sites/µm(2). Intriguingly, low doses of solute RGD "shifted" the optimal densities of immobilized ligand along with corresponding melanoma cell integrin clusters and cytoskeletal changes toward those typical for "intermediate" ligand presentation. Consequently, melanoma cells were forced into a "permissive" state, optimizing interactions with suboptimal nanostructured biomimetic surfaces, thus providing an explanation for the seemingly paradoxical effects on tumor progression and a potential clue for individualized antitumoral therapies.

Adhesion maturation of neutrophils on nanoscopically presented platelet glycoprotein Ibα.

Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates via the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). In vivo, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic in vitro system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such in vitro studies can be useful to determine optimum therapeutic ranges for targeting this interaction.

Atypical location of lymphocytoma cutis in a child.

Lyme borreliosis is a common infectious disease that can affect myocardial muscle or the central nervous system if not treated at an early stage. Here we report a unique case of an atypical location of lymphocytoma cutis in a 3-year-old boy. Histologic and immunohistochemical analysis revealed the correct diagnosis.

NF-kappaB inhibition through proteasome inhibition or IKKbeta blockade increases the susceptibility of melanoma cells to cytostatic treatment through distinct pathways.

Metastasized melanoma is almost universally resistant to chemotherapy. Given that constitutive or drug-induced upregulation of NF-kappaB activity is associated with this chemoresistance, NF-kappaB inhibition may increase the susceptibility to antitumoral therapy. On the cellular level, two principles of NF-kappaB inhibition, proteasome inhibition by bortezomib and IkappaB kinase-beta (IKKbeta) inhibition by the kinase inhibitor of NF-kappaB-1 (KINK-1), significantly increased the antitumoral efficacy of camptothecin. When combined with camptothecin, either of the two NF-kappaB-inhibiting principles synergistically influenced progression-related in vitro functions, including cell growth, apoptosis, and invasion through an artificial basement membrane. In addition, when C57BL/6 mice were intravenously injected with B16F10 melanoma cells, the combination of cytostatic treatment with either of the NF-kappaB-inhibiting compounds revealed significantly reduced pulmonary metastasis compared to either treatment alone. However, on the molecular level, nuclear translocation of p65, cell cycle analysis, and expression of NF-kappaB-dependent gene products disclosed distinctly different molecular mechanisms, resulting in the same functional effect. That proteasome inhibition and IKKbeta inhibition affect distinct molecular pathways downstream of NF-kappaB, both leading to increased chemosensitivity, is previously unreported. Thus, it is conceivable that switching the two principles of NF-kappaB inhibition, once resistance to one of the agents occurs, will improve future treatment regimens.

KINK-1, a novel small-molecule inhibitor of IKKbeta, and the susceptibility of melanoma cells to antitumoral treatment.

BACKGROUND: Increasing the efficacy of chemotherapeutics by reducing chemoresistance may be a useful strategy in cancer therapy. Constitutive activation of nuclear factor-kappa B (NF-kappaB) is a hallmark of various cancers, including melanoma, which is almost universally resistant to chemotherapy. NF-kappaB is regulated by inhibitory kappaB (IkappaB) proteins, which are in turn phosphorylated by the IkappaB kinase (IKK) complex. METHODS: The effect on NF-kappaB activity of a novel small-molecule inhibitor of the beta subunit of IKK (KINK-1; kinase inhibitor of nuclear factor-kappaB-1) was assessed by measuring phosphorylation of the alpha subunit of IkappaB by immunoblotting, DNA binding by electrophoretic mobility shift assays, and nuclear translocation of NF-kappaB using immunofluorescence. Regulation of NF-kappaB-dependent gene expression was determined by microarray analysis, real-time and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses. The effects of KINK-1 (alone and in combination with cytostatic agents) on melanoma cells were characterized by assessing proliferation, soft agar colony formation, and markers of apoptosis. The antitumoral efficacy of KINK-1 in combination with the cytostatic agents doxorubicin or camptothecin (all injected intraperitoneally) was tested in vivo by measuring lung weight and counting metastases in C57BL6 mice (groups of six) bearing metastases of melanoma cells. All statistical tests were two-sided. Results KINK-1 strongly suppressed both constitutive and induced NF-kappaB activity in melanoma cells. It reduced the expression of NF-kappaB-dependent gene products that regulate proliferation, cytokine production, and antiapoptotic responses but exhibited little antiproliferative or proapoptotic activity at the cellular level. However, KINK-1 markedly increased the activities of some cytostatic agents in vitro and abrogated doxorubicin-induced NF-kappaB activation. Combined treatment of C57BL6 mice that had been injected with melanoma cells with KINK-1 and doxorubicin or camptothecin reduced metastases and pulmonary tumor mass compared with either treatment alone (mean lung weight 19 days after injection of melanoma cells of mice treated with 3 mg/kg KINK-1 alone, 1 mg/kg doxorubicin alone, and 1 mg/kg doxorubicin plus 3 mg/kg KINK-1 = 260 mg, 95% confidence interval (CI) = 216 to 305 mg; 268 mg, 95% CI = 224 to 313 mg; and 181 mg, 95% CI = 171 to 192 mg, respectively, P < .001 from t tests comparing mean lung weight of double-treated mice to that in mice treated with either compound alone). CONCLUSION: Inhibition of constitutive and induced IKKbeta-activity through treatment with KINK-1 might increase tumor susceptibility to chemotherapy.