Infrared Spectroscopy for Rapid Triage of Cancer Using Blood Derivatives: A Reality Check.

Infrared (IR) spectroscopy of serum/plasma represents an alluring molecular diagnostic tool, especially for cancer, as it can provide a molecular fingerprint of clinical samples based on vibrational modes of chemical bonds. However, despite the superior performance, the routine adoption of this technique for clinical settings has remained elusive. This is due to the potential confounding factors that are often overlooked and pose a significant barrier to clinical translation. In this Perspective, we summarize the concerns associated with various confounding factors, such as fluid sampling, optical effects, hemolysis, abnormal cardiovascular and/or hepatic functions, infections, alcoholism, diet style, age, and gender of a patient or normal control cohort, and improper selection of numerical methods that ultimately would lead to improper spectral diagnosis. We also propose some precautionary measures to overcome the challenges associated with these confounding factors.

Lipophilic derivatives of EGCG as potent α-amylase and α-glucosidase inhibitors ameliorating oxidative stress and inflammation.

Uncontrolled hyperglycemia leads to increased oxidative stress, chronic inflammation, and insulin resistance, rendering diabetes management harder to accomplish. To tackle these myriads of challenges, researchers strive to explore innovative multifaceted treatment strategies, including inhibiting carbohydrate hydrolases. Herein, we report alkyl-ether EGCG derivatives as potent α-amylase and α-glucosidase inhibitors that could simultaneously ameliorate oxidative stress and inflammation. 4″-C18 EGCG, the most promising compound, showed multifold improvement in glycaemic management compared to acarbose, with 230-fold greater inhibition (competitive) of α-glucosidase (IC50 0.81 µM) and 3-fold better inhibition of α-amylase (IC50 3.74 µM). All derivatives showed stronger antioxidant activity (IC50 6.16-15.76 µM) than vitamin C, while acarbose showed none. 4″-C18 EGCG also downregulated pro-inflammatory cytokines and showed no significant cytotoxicity up to 50 µM in primary human peripheral blood mononuclear cells (PBMC), non-cancerous cell line, 3T3-L1 and HEK 293. The in silico binding affinity analysis of 4″-C18 EGCG with α-amylase and α-glucosidase was found to exhibit a good extent of interaction as compared to acarbose. In comparison to EGCG, 4″-Cn EGCG derivatives were found to remain stable in the physiological conditions even after 24 h. Together, the reported molecules demonstrated multifaceted antidiabetic potential inhibiting carbohydrate hydrolases, reducing oxidative stress, and inflammation, which are known to aggravate diabetes.

Comparative Analysis of the Aspergillus fumigatus Cell Wall Modification and Ensuing Human Dendritic Cell Responses by ß-(1,3)-Glucan Synthase Inhibitors-Caspofungin and Enfumafungin.

Caspofungin, a lipopeptide, is an antifungal drug that belong to the class of echinocandin. It inhibits fungal cell wall ß-(1,3)-glucan synthase activity and is the second-line of drug for invasive aspergillosis, a fatal infection caused mainly by Aspergillus fumigatus. On the other hand, Enfumafungin is a natural triterpene glycoside also with a ß-(1,3)-glucan synthase inhibitory activity and reported to have antifungal potential. In the present study, we compared the growth as well as modifications in the A. fumigatus cell wall upon treatment with Caspofungin or Enfumafungin, consequentially their immunomodulatory capacity on human dendritic cells. Caspofungin initially inhibited the growth of A. fumigatus, but the effect was lost over time. By contrast, Enfumafungin inhibited this fungal growth for the duration investigated. Both Caspofungin and Enfumafungin caused a decrease in the cell wall ß-(1,3)-glucan content with a compensatory increase in the chitin, and to a minor extent they also affected cell wall galactose content. Treatment with these two antifungals did not result in the exposure of ß-(1,3)-glucan on A. fumigatus mycelial surface. Enzymatic digestion suggested a modification of ß-(1,3)-glucan structure, specifically its branching, upon Enfumafungin treatment. While there was no difference in the immunostimulatory capacity of antifungal treated A. fumigatus conidia, alkali soluble-fractions from Caspofungin treated mycelia weakly stimulated the dendritic cells, possibly due to an increased content of immunosuppressive polysaccharide galactosaminogalactan. Overall, we demonstrate a novel mechanism that Enfumafungin not only inhibits ß-(1,3)-glucan synthase activity, but also causes modifications in the structure of ß-(1,3)-glucan in the A. fumigatus cell wall.

Induction of antiviral and cell mediated immune responses significantly reduce viral load in an acute foot-and-mouth disease virus infection in cattle.

Foot-and-mouth disease virus (FMDV) causes a severe infection in ruminant animals. Here we present an in-depth transcriptional analysis of soft-palate tissue from cattle experimentally infected with FMDV. The differentially expressed genes from two Indian cattle (Bos indicus) breeds (Malnad Gidda and Hallikar) and Holstein Friesian (HF) crossbred calves, highlighted the activation of metabolic processes, mitochondrial functions and significant enrichment of innate antiviral immune response pathways in the indigenous calves. The results of RT-qPCR based validation of 12 genes was in alignment with the transcriptome data. The indigenous calves showing lesser virus load, elicited early neutralizing antibodies and IFN-γ immune responses. This study revealed that induction of potent innate antiviral response and cell mediated immunity in indigenous cattle, especially Malnad Gidda, significantly restricted FMDV replication during acute infection. These data highlighting the molecular processes associated with host-pathogen interactions, could aid in the conception of novel strategies to prevent and control FMDV infection in cattle.

Structural and evolutionary exploration of the IL-3 family and its alpha subunit receptors.

Interleukin-3 (IL-3) is a cytokine belonging to the family of common ß (ßc) and is involved in various biological systems. Its activity is mediated by the interaction with its receptor (IL-3R), a heterodimer composed of two distinct subunits: IL-3Rα and ßc. IL-3 and its receptor, especially IL-3Rα, play a crucial role in pathologies like inflammatory diseases and therefore are interesting therapeutic targets. Here, we have performed an analysis of these proteins and their interaction based on structural and evolutionary information. We highlighted that IL-3 and IL-3Rα structural architectures are conserved across evolution and shared with other proteins belonging to the same ßc family interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The IL-3Rα/IL-3 interaction is mediated by a large interface in which most residues are surprisingly not conserved during evolution and across family members. In spite of this high variability, we suggested small regions constituted by few residues conserved during the evolution in both proteins that could be important for the binding affinity.

Impact of Autoantibodies to Complement Components on the Disease Activity in SLE.

INTRODUCTION: Systemic Lupus Erythematosus (SLE) is a chronic multi-system autoimmune disease with varied clinical presentations. Complement components are the major players in disease pathogenesis. This retrospective cross-sectional study was aimed at assessing the role of autoantibodies to these complement components and their association disease activity in newly diagnosed SLE patients from India. METHOD: Clinically diagnosed SLE patients (n=57) classified as per 2015 ACR/SLICC revised criteria were enrolled between November 2016 to April 2017. Patients' sera were tested for C3 and C4 by nephelometry, while serum levels of factor H, factor P (properdin) as well as autoantibodies to C3, C4, factor H and factor P were detected by ELISA. GraphPad Prism Version 6.01 was used for statistical analysis. Mean, SD, SEM were calculated. Mann Whittney U-test, ANOVA, Chi-square test, Odd's Ratio were calculated. Pearson's correlation was used to study relativeness of the study parameters. RESULTS: Among the 57 SLE patients, low C3 were seen in 51% patients, low C4 in 49%, low factor H in 19% and low factor P in 49% patients. Positivity for autoantibodies against complement components, anti-C3 were seen in 42% patients, anti-C4 in 7%, anti-factor H in 19% and anti-factor P in 28% patients. Serum levels of C3 (p=0.0009), C4 (p=0.0031) and anti-C3 autoantibodies (p=0.0029) were significantly associated with ACR/SLICC 2015 scores. CONCLUSION: Hypocomplementemia was found to be associated with higher disease damage score in newly diagnosed SLE patients. This study adds novel arguments for the importance of the anti-C3 autoantibodies as a marker of SLE.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Intravenous immunoglobulin protects from experimental allergic bronchopulmonary aspergillosis via a sialylation-dependent mechanism.

Intravenous immunoglobuin (IVIG) exerts protective effects in experimental allergic bronchopulmonary aspergillosis (ABPA) via a sialylation-dependent mechanism. The protection was associated with reduced recruitment of eosinophils, diminished goblet cell hyperplasia, suppressed Th2 and Th17 responses and reciprocally enhanced regulatory T cells and IL-10, and decreased IgE levels in the circulation.

Stimulation with FITC-labeled antigens confers B cells with regulatory properties.

B cells with regulatory properties (Bregs) were identified in human and in mice among different B-cell subsets. Their regulatory properties rely mainly on the production of anti-inflammatory cytokines, in particular IL10, IL-35 and TGFß, and were extensively studied in mouse models of autoimmune and inflammatory diseases. However, the exact nature of the stimulatory signals conferring regulatory properties to B cells is still not clear. We serendipitously observed that fluorescein isothiocyanate (FITC) binds to a significant proportion of naïve mouse B cells. Binding of FITC to the B-cell surface implicated at least in part the B-cell receptor. It triggered IL-10 production and allowed the endocytosis of FITC-coupled antigens followed by their presentation to CD4+ T cells. In particular, B cells incubated with FITC-OVA polarized OTII T cells towards a Tr1/Th2 phenotype in vitro. Further, the adoptive transfer of B cells incubated with FITC-labeled myelin oligodendrocyte glycoprotein peptide protected mice from experimental autoimmune encephalomyelitis, a T-cell-dependent autoimmune model. Together, the data show that FITC-stimulated B cells polarize immune responses towards Tr1/Th2 and acquire immuno-modulatory properties.

Circulating Normal IgG as Stimulator of Regulatory T Cells: Lessons from Intravenous Immunoglobulin.

Intravenous immunoglobulin (IVIG), a pooled normal IgG formulation prepared from thousands of healthy donors' plasma, is extensively used for the immunotherapy of autoimmune and inflammatory disorders. Recent reports demonstrate that IVIG exerts anti-inflammatory actions by stimulating the activation and expansion of regulatory T (Treg) cells by multiple mechanisms via antigen-presenting cells (APCs).

Passive Serum Therapy to Immunomodulation by IVIG: A Fascinating Journey of Antibodies.

The immunoregulatory and anti-infective properties of normal circulating polyclonal Abs have been exploited for the therapeutic purposes in the form of IVIG as well as several hyperimmune globulins. Current knowledge on the therapeutic use of normal Igs is based on the discoveries made by several pioneers of the field. In this paper, we review the evolution of IVIG over the years. More importantly, the process started as an s.c. replacement in γ globulin-deficient patients, underwent metamorphosis into i.m. Ig, was followed by IVIG, and is now back to s.c. forms. Following successful use of IVIG in immune thrombocytopenic purpura, there has been an explosion in the therapeutic applications of IVIG in diverse autoimmune and inflammatory conditions. In addition to clinically approved pathological conditions, IVIG has been used as an off-label drug in more than 100 different indications. The current worldwide consumption of IVIG is over 100 tons per year.

Intravenous immunoglobulin induces IL-4 in human basophils by signaling through surface-bound IgE.

BACKGROUND: Therapeutic normal IgG or intravenous immunoglobulin (IVIG) exerts anti-inflammatory effects through several mutually nonexclusive mechanisms. Recent data in mouse models of autoimmune disease suggest that IVIG induces IL-4 in basophils by enhancing IL-33 in SIGN-related 1-positive innate cells. However, translational insight on these data is lacking. OBJECTIVE: We sought to investigate the effect of IVIG on human basophil functions. METHODS: Isolated circulating basophils from healthy donors were cultured in the presence of IL-3, IL-33, GM-CSF, thymic stromal lymphopoietin, or IL-25. The effect of IVIG and F(ab')2 and Fc IVIG fragments was examined based on expression of various surface molecules, phosphorylation of spleen tyrosine kinase, induction of cytokines, and histamine release. Basophil phenotypes were also analyzed from IVIG-treated patients with myopathy. Approaches, such as depletion of anti-IgE reactivity from IVIG, blocking antibodies, or inhibitors, were used to investigate the mechanisms. RESULTS: We report that IVIG directly induces activation of IL-3-primed human basophils, but IL-33 and other cytokines were dispensable for this effect. Activation of basophils by IVIG led to enhanced expression of CD69 and secretion of IL-4, IL-6, and IL-8. IVIG-treated patients with myopathy displayed enhanced expression of CD69 on basophils. The spleen tyrosine kinase pathway is implicated in these functions of IVIG and were mediated by F(ab')2 fragments. Mechanistically, IVIG induced IL-4 in human basophils by interacting with basophil surface-bound IgE but independent of FcγRII, type II Fc receptors, C-type lectin receptors, and sialic acid-binding immunoglobulin-like lectins. CONCLUSION: These results uncovered a pathway of promoting the TH2 response by IVIG through direct interaction of IgG with human basophils.

Acid Stripping of Surface IgE Antibodies Bound to FcεRI is Unsuitable for the Functional Assays that Require Long-Term Culture of Basophils and Entire Removal of Surface IgE.

Basophils are rare granulocytes and dysregulated functions of these cells are associated with several atopic and non-atopic allergic diseases of skin, respiratory system and gastrointestinal tract. Both cytokines and immunoglobulin E (IgE) are implicated in mediating the basophil activation and pathogenesis of these disorders. Several reports have shown that healthy individuals, and patients with allergic disorders display IgG autoantibodies to IgE and hence functional characterization of these anti-IgE IgG autoantibodies is critical. In general, anti-IgE IgG autoantibodies modulate basophil activation irrespective of allergen specificity by interacting with constant domains of IgE. Therefore, an ideal solution to prove the functions of such anti-IgE IgG autoantibodies would be to completely eliminate type I high affinity immunoglobulin E receptor (FcɛRI)-bound IgE from the surface of basophils and to demonstrate in an unequivocal manner the role of anti-IgE IgG autoantibodies. In line with previous reports, our data show that FcɛRI on peripheral blood basophils are almost saturated with IgE. Further, acetic acid buffer (pH 4) efficiently removes these FcɛRI-bound IgE. Although immediately following acetic acid-elution of IgE had no repercussion on the viability of basophils, following 24 hours culture with interleukin-3 (IL-3), the viability and yield of basophils were drastically reduced in acid-treated cells and had repercussion on the induction of activation markers. Lactic acid treatment on the other hand though had no adverse effects on the viability of basophils and IL-3-induced activation, it removed only a small fraction of the cell surface bound IgE. Thus, our results show that acid buffers could be used for the elution of FcɛRI-bound IgE on the basophil surface for the biochemical characterization of IgE antibodies or for the immediate use of basophils to determine their sensitivity to undergo degranulation by specific allergens. However, these methods are not utile for the functional assays of basophils that require longer duration of culture and entire removal of surface IgE to validate the role of anti-IgE IgG autoantibodies that interact with FcɛRI-bound IgE irrespective of allergen specificity.

Fungal melanin stimulates surfactant protein D-mediated opsonization of and host immune response to Aspergillus fumigatus spores.

Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus, but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus, galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D-opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D-/- mice challenged intranasally with wildtype conidia or melanin ghosts (i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.

Intravenous Immunoglobulin Therapy Eliminates Candida albicans and Maintains Intestinal Homeostasis in a Murine Model of Dextran Sulfate Sodium-Induced Colitis.

Intravenous immunoglobulin (IVIg) therapy has diverse anti-inflammatory and immunomodulatory effects and has been employed successfully in autoimmune and inflammatory diseases. The role of IVIg therapy in the modulation of intestinal inflammation and fungal elimination has not been yet investigated. We studied IVIg therapy in a murine model of dextran sulfate sodium (DSS)-induced colitis. Mice received a single oral inoculum of Candida albicans and were exposed to DSS treatment for 2 weeks to induce colitis. All mice received daily IVIg therapy starting on day 1 for 7 days. IVIg therapy not only prevented a loss of body weight caused by the development of colitis but also reduced the severity of intestinal inflammation, as determined by clinical and histological scores. IVIg treatment significantly reduced the Escherichia coli, Enterococcus faecalis, and C. albicans populations in mice. The beneficial effects of IVIg were associated with the suppression of inflammatory cytokine interleukin (IL)-6 and enhancement of IL-10 in the gut. IVIg therapy also led to an increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), while toll-like receptor 4 (TLR-4) expression was reduced. IVIg treatment reduces intestinal inflammation in mice and eliminates C. albicans overgrowth from the gut in association with down-regulation of pro-inflammatory mediators combined with up-regulation of anti-inflammatory cytokines.

Correction: Saha, C., et al. Differential Effects of Viscum album Preparations on the Maturation and Activation of Human Dendritic Cells and CD4+ T Cell Responses. Molecules 2016, 21, 912.

The authors wish to make the following corrections to this paper [...].

Rapalog combined with CCR4 antagonist improves anticancer vaccines efficacy.

mTOR pathway inhibitors such as rapalogs represent a promising tool to induce functional memory CD8 T cells. In our study, we investigated the combination of temsirolimus with anticancer vaccines. Using various designs of cancer vaccines (short and long peptides or the B subunit of Shiga toxin as an antigen delivery vector) and tumor models (melanoma, lung and colon cancer), we showed that the administration of temsirolimus efficiently decreased tumor growth and enhanced tumor-specific CD8 T-cell responses induced by vaccination. Furthermore, tumor-specific CD8 T cells induced by the bi-therapy (vaccine + temsirolimus) exhibit phenotypic characteristics of central memory (CD127+ CD62L+ ) CD8 T cells compared to vaccination alone. We demonstrated that regulatory CD4 T cells (Tregs ) expansion in vivo limits the efficacy of the bi-therapy by altering the antitumor CD8 T-cell responses. Finally, the use of a small molecule CCR4 antagonist to prevent Tregs induction considerably improved the efficacy of the bi-therapy by enhancing CD8 T cells-mediated antitumor immunity. Taken together, our study highlights the potential interest of combining cancer vaccines with drugs that promote memory CD8 T cells and inhibit Tregs .

IVIG-mediated effector functions in autoimmune and inflammatory diseases.

Intravenous immunoglobulin (IVIG) is a pooled preparation of normal IgG obtained from several thousand healthy donors. It is widely used in the immunotherapy of a large number of autoimmune and inflammatory diseases. The mechanisms of action of IVIG are complex and, as discussed in this review, experimental and clinical data provide an indicator that the therapeutic benefit of IVIG therapy is due to several mutually non-exclusive mechanisms affecting soluble mediators as well as cellular components of the immune system. These mechanisms depend on Fc and/or F(ab')2 fragments. A better understanding of the effector functions of IVIG should help in identification of biomarkers of responses to IVIG in autoimmune patients.