Multiplexed Quantitative Analysis of Antibody-Drug Conjugates with Labile CBI-Dimer Payloads In Vivo Using Immunoaffinity LC-MS/MS.

Quantitative analysis of antibody-drug conjugates (ADCs) involves cleavage of ADCs into smaller analytes representing different components and subsequent measurements from multiple assays for a more comprehensive pharmacokinetic (PK) assessment. Multiple PK analytes including the drug remaining conjugated to the antibody (or antibody-conjugated drug, acDrug) and total antibody can be accessed simultaneously using a multiplex assay by proteolytic digestion of an ADC, if the sites of conjugation are homogeneous for an ADC and the linker drug is stable to proteases. Herein, a multiplexed immunoaffinity liquid chromatography-mass spectrometry (LC-MS)/MS PK assay is described involving immunoaffinity enrichment, enzymatic conversion of prodrug, trypsin digestion, and LC-MS/MS as applied to next-generation ADCs constructed from linker drugs bearing dimeric cyclopropabenzindole (CBI) payloads (duocarmycin analogues). The cytotoxic payload is chemically labile, requiring extensive optimization in sample preparation steps to stabilize the drug without ex vivo modification and to convert the prodrug into a single active form of the drug. The qualification data for this assay format showed that this approach provides robust acDrug and total antibody data and can be extended to ADCs with different monoclonal antibody frameworks and linker chemistries. Applications of this multiplexed assay to support preclinical studies are presented.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

Conjugation Site Influences Antibody-Conjugated Drug PK Assays: Case Studies for Disulfide-Linked, Self-Immolating Next-Generation Antibody Drug Conjugates.

Immunoaffinity (IA) LC-MS/MS pharmacokinetic (PK) assays are widely used in the field for antibody drug conjugates (ADCs) containing peptide linkers that are enzymatically cleavable, such as MC-ValCit-PAB. Conjugate PK assay strategies for these ADCs involve cleavage with cathepsin B or papain to release and measure the antibody-conjugated drug (acDrug) concentration. However, robust acDrug PK methods for disulfide-linked self-immolating ADCs are lacking as they are a different conjugation modality. We developed acDrug PK assays for next-generation disulfide-linked ADCs involving immunoaffinity capture, chemical cleavage, and LC-MS/MS. Disulfide-linked ADCs captured from plasma were chemically reduced at basic pH to release the linker-drug, followed by self-immolation to liberate the active drug, and quantified by MRM LC-MS/MS. Herein, we detail the development and optimization of this chemical cleavage acDrug PK assay, resulting in robust accuracy and precision (±20%). The conjugation site of the linker-drug on the antibody was found to affect the kinetics of drug release. Multiple biophysical and chemical characteristics, such as tertiary structure, fractional solvent accessibility, pKa of the conjugation site, surrounding residue's pI, and electrostatic charge, may directly impact the drug release kinetics. Similar site-specific stability has been previously reported for ADCs in vivo. The assay development and qualification data for this original assay format are presented along with its application to multiple in vitro and in vivo studies across species.

Automated, Generic Reagent and Ultratargeted 2D-LC-MS/MS Enabling Quantification of Biotherapeutics and Soluble Targets down to pg/mL Range in Serum.

Mass spectrometry has recently emerged as a powerful analytical tool for the assessment of pharmacokinetics and biomarkers in drug development. Compared with ligand binding assays, a major advantage of mass spectrometry-based assays is that they are less dependent on high quality binding reagents, while a key limitation is the relatively lower sensitivity. To address the sensitivity issue, we have developed a generic reagent, ultratargeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method which combines commercially available protein A affinity capture, targeted analyte isolation by 2D-LC, and targeted detection by multiple reaction monitoring (MRM). A targeted-2D-with-dilution configuration was designed to automate 2D-LC-MS/MS. This method was systematically evaluated using an anti-CD22 monoclonal antibody spiked into monkey and human serum, where lower limits of quantification (LLOQ) of 0.78 and 1.56 ng/mL were achieved, respectively. This represents an over 100-fold improvement in assay sensitivity compared to the conventional LC-MS/MS method. The performance of the method was further confirmed by analyzing another monoclonal antibody, bevacizumab, as well as a soluble antigen, circulating PD-L1. The results indicate that our method enables quantification of antibody therapeutics and antigen biomarkers in both clinical and nonclinical samples in the pg/mL to low ng/mL range. Protein A affinity capture was employed as a universal sample preparation procedure applicable to both full-length antibody therapeutics and antibody-antigen complexes. This novel method is also fully automated and proven to be highly robust for routine bioanalysis in drug development.

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

Method development of a novel PK assay for antibody-conjugated drug measurement of ADCs using peptide-linker drug analyte.

Pharmacokinetic analysis of antibody-drug conjugates (ADCs) requires characterization and quantification of both the antibody-conjugated cytotoxic drug molecule (acDrug) as well as the antibody vehicle, among other analytes, in order to assess the safety and efficacy of ADCs. Due to the complexity of biological matrices, immunoaffinity capture is widely used for enrichment of the biotherapeutic, followed by enzymatic or chemical release of the drug and LC-MS/MS analysis to provide the concentration of acDrug. This bioanalytical strategy has been used successfully with ADCs, but is limited to ADCs having cleavable linkers. Herein, we developed a sensitive and specific method that involved subjecting the ADC to tryptic digestion, and measured a peptide that included cysteine conjugated to the drug to provide quantification of acDrug. Using this method for a THIOMAB™ antibody-drug conjugate (TDC) conjugated to MMAE via a cleavable linker, valine-citrulline, we compared peptide-linker MMAE data from the new assay format with earlier MMAE data for acDrug. This showed that the new assay format provides robust acDrug as well as total antibody concentration to study in vitro stability of the TDC in multiple matrices and in vivo pharmacokinetic models of TDC in rat and mouse. The data from the two orthogonal modes of acDrug analysis showed good agreement with each other, allowing us to successfully quantify acDrug to study the stability in vitro and the pharmacokinetic parameters in vivo. This new assay strategy allows acDrug quantification for ADCs with non-cleavable linkers where the resulting acDrug analyte is a peptide-linker drug.

Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification.

Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.

Formation of a nanobubble and its effect on the structural ordering of water in a CH4-N2-CO2-H2O mixture.

The replacement of methane (CH4) from its hydrate by a mixture of nitrogen (N2) and carbon dioxide (CO2) involves the dissociation of methane hydrate leading to the formation of a CH4-N2-CO2-H2O mixture that can significantly influence the subsequent steps of the replacement process. In the present work, we study the evolution of dissolved gas molecules in this mixture by applying classical molecular dynamics simulations. Our study shows that a higher CO2 : N2 ratio in the mixture enhances the formation of nanobubbles composed of N2, CH4 and CO2 molecules. To understand how the CO2 : N2 ratio affects nanobubble nucleation, the distribution of molecules in the bubble formed is examined. It is observed that unlike N2 and CH4, the density of CO2 in the bubble reaches a maximum at the surface of the bubble. The accumulation of CO2 molecules at the surface makes the bubble more stable by decreasing the excess pressure inside the bubble as well as surface tension at its interface with water. It is found that a frequent exchange of gas molecules takes place between the bubble and the surrounding liquid and an increase in concentration of CO2 in the mixture leads to a decrease in the number of such exchanges. The effect of nanobubbles on the structural ordering of water molecules is examined by determining the number of water rings formed per unit volume in the mixture. The role of nanobubbles in water structuring is correlated to the dynamic nature of the bubble arising from the exchange of gas molecules between the bubble and the liquid.

High-Resolution Accurate-Mass Mass Spectrometry Enabling In-Depth Characterization of in Vivo Biotransformations for Intact Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules and therefore have complex molecular structures. In vivo biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated in vivo biotransformations of multiple site-specific THIOMAB antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety in vivo.

Microbial keratinases: industrial enzymes with waste management potential.

Proteases are ubiquitous enzymes that occur in various biological systems ranging from microorganisms to higher organisms. Microbial proteases are largely utilized in various established industrial processes. Despite their numerous industrial applications, they are not efficient in hydrolysis of recalcitrant, protein-rich keratinous wastes which result in environmental pollution and health hazards. This paved the way for the search of keratinolytic microorganisms having the ability to hydrolyze "hard to degrade" keratinous wastes. This new class of proteases is known as "keratinases". Due to their specificity, keratinases have an advantage over normal proteases and have replaced them in many industrial applications, such as nematicidal agents, nitrogenous fertilizer production from keratinous waste, animal feed and biofuel production. Keratinases have also replaced the normal proteases in the leather industry and detergent additive application due to their better performance. They have also been proved efficient in prion protein degradation. Above all, one of the major hurdles of enzyme industrial applications (cost effective production) can be achieved by using keratinous waste biomass, such as chicken feathers and hairs as fermentation substrate. Use of these low cost waste materials serves dual purposes: to reduce the fermentation cost for enzyme production as well as reducing the environmental waste load. The advent of keratinases has given new direction for waste management with industrial applications giving rise to green technology for sustainable development.

Effectiveness and cost-effectiveness of mindfulness-based cognitive therapy compared with maintenance antidepressant treatment in the prevention of depressive relapse or recurrence (PREVENT): a randomised controlled trial.

BACKGROUND: Individuals with a history of recurrent depression have a high risk of repeated depressive relapse or recurrence. Maintenance antidepressants for at least 2 years is the current recommended treatment, but many individuals are interested in alternatives to medication. Mindfulness-based cognitive therapy (MBCT) has been shown to reduce risk of relapse or recurrence compared with usual care, but has not yet been compared with maintenance antidepressant treatment in a definitive trial. We aimed to see whether MBCT with support to taper or discontinue antidepressant treatment (MBCT-TS) was superior to maintenance antidepressants for prevention of depressive relapse or recurrence over 24 months. METHODS: In this single-blind, parallel, group randomised controlled trial (PREVENT), we recruited adult patients with three or more previous major depressive episodes and on a therapeutic dose of maintenance antidepressants, from primary care general practices in urban and rural settings in the UK. Participants were randomly assigned to either MBCT-TS or maintenance antidepressants (in a 1:1 ratio) with a computer-generated random number sequence with stratification by centre and symptomatic status. Participants were aware of treatment allocation and research assessors were masked to treatment allocation. The primary outcome was time to relapse or recurrence of depression, with patients followed up at five separate intervals during the 24-month study period. The primary analysis was based on the principle of intention to treat. The trial is registered with Current Controlled Trials, ISRCTN26666654. FINDINGS: Between March 23, 2010, and Oct 21, 2011, we assessed 2188 participants for eligibility and recruited 424 patients from 95 general practices. 212 patients were randomly assigned to MBCT-TS and 212 to maintenance antidepressants. The time to relapse or recurrence of depression did not differ between MBCT-TS and maintenance antidepressants over 24 months (hazard ratio 0·89, 95% CI 0·67-1·18; p=0·43), nor did the number of serious adverse events. Five adverse events were reported, including two deaths, in each of the MBCT-TS and maintenance antidepressants groups. No adverse events were attributable to the interventions or the trial. INTERPRETATION: We found no evidence that MBCT-TS is superior to maintenance antidepressant treatment for the prevention of depressive relapse in individuals at risk for depressive relapse or recurrence. Both treatments were associated with enduring positive outcomes in terms of relapse or recurrence, residual depressive symptoms, and quality of life. FUNDING: National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme, and NIHR Collaboration for Leadership in Applied Health Research and Care South West Peninsula.

Custom-Designed Affinity Capture LC-MS F(ab')2 Assay for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates.

Affinity capture liquid chromatography-mass spectrometry (LC-MS) intact antibody assay has been widely used for direct drug-to-antibody ratio (DAR) and catabolite characterization of antibody-drug conjugates (ADCs). However, the intact mass spectra of new ADCs, which incorporate new types of linkers and payloads other than maytansines and auristatins, are more complex than those examined previously. The current method has showed some limitations in elucidating certain structural modifications. Herein, we report an alternative analytical approach for ADCs, such as THIOMAB antibody-drug conjugates (TDCs), where the linker drugs are site-specifically conjugated in the Fab region. The newly developed affinity capture LC-MS F(ab')2 assay incorporates affinity capture of human IgGs via binding to the Fab region, followed by on-bead IdeS digestion to remove the Fc domain specifically and uniformly. The resulting F(ab')2 (∼100 kDa) fragments contain the key ADC biotransformation information, such as drug-to-antibody ratio and drug metabolism and are more readily analyzed by electrospray ionization LC-MS than the intact ADC (∼150 kDa). The reduced size of analytes results in improved mass spectral sensitivity and resolution. In addition, the reduced and optimized sample preparation time, for example, rapid removal of the Fc fragment by IdeS digestion, minimizes assay artifacts of drug metabolism and skewed DAR profiles that may result from the prolonged incubation times (e.g., overnight enzymatic treatment for Fc deglycosylation). The affinity capture LC-MS F(ab')2 assay provides more detailed and accurate information on ADC biotransformations in vivo, enabling analysis of low-dose, labile, and complex site-specific ADCs with linker-drug conjugated in the Fab region.

AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia.

Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at <5 nM or <150 nM in enzyme and cell assays, respectively. AZD1208 inhibited the growth of 5 of 14 acute myeloid leukemia (AML) cell lines tested, and sensitivity correlates with Pim-1 expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2 antagonist of cell death, 4EBP1, p70S6K, and S6, as well as increases in cleaved caspase 3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML.

Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.

We provide evidence that type I IFN-induced STAT activation is diminished in cells with targeted disruption of the Rictor gene, whose protein product is a key element of mTOR complex 2. Our studies show that transient or stable knockdown of Rictor or Sin1 results in defects in activation of elements of the STAT pathway and reduced STAT-DNA binding complexes. This leads to decreased expression of several IFN-inducible genes that mediate important biological functions. Our studies also demonstrate that Rictor and Sin1 play essential roles in the generation of the suppressive effects of IFNα on malignant erythroid precursors from patients with myeloproliferative neoplasms. Altogether, these findings provide evidence for critical functions for Rictor/Sin1 complexes in type I IFN signaling and the generation of type I IFN antineoplastic responses.

Recent trends in biological extraction of chitin from marine shell wastes: a review.

The natural biopolymer chitin and its deacetylated product chitosan are widely used in innumerable applications ranging from biomedicine, pharmaceuticals, food, agriculture and personal care products to environmental sector. The abundant and renewable marine processing wastes are commercially exploited for the extraction of chitin. However, the traditional chitin extraction processes employ harsh chemicals at elevated temperatures for a prolonged time which can harm its physico-chemical properties and are also held responsible for the deterioration of environmental health. In view of this, green extraction methods are increasingly gaining popularity due to their environmentally friendly nature. The bioextraction of chitin from crustacean shell wastes has been increasingly researched at the laboratory scale. However, the bioextraction of chitin is not currently exploited to its maximum potential on the commercial level. Bioextraction of chitin is emerging as a green, cleaner, eco-friendly and economical process. Specifically in the chitin extraction, microorganisms-mediated fermentation processes are highly desirable due to easy handling, simplicity, rapidity, controllability through optimization of process parameters, ambient temperature and negligible solvent consumption, thus reducing environmental impact and costs. Although, chitin production from crustacean shell waste through biological means is still at its early stage of development, it is undergoing rapid progress in recent years and showing a promising prospect. Driven by reduced energy, wastewater or solvent, advances in biological extraction of chitin along with valuable by-products will have high economic and environmental impact.

Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.

IFNs transduce signals by binding to cell surface receptors and activating cellular pathways and regulatory networks that control transcription of IFN-stimulated genes (ISGs) and mRNA translation, leading to generation of protein products that mediate biological responses. Previous studies have shown that type I IFN receptor-engaged pathways downstream of AKT and mammalian target of rapamycin complex (mTORC) 1 play important roles in mRNA translation of ISGs and the generation of IFN responses, but the roles of mTORC2 complexes in IFN signaling are unknown. We provide evidence that mTORC2 complexes control IFN-induced phosphorylation of AKT on serine 473 and their function is ultimately required for IFN-dependent gene transcription via interferon-stimulated response elements. We also demonstrate that such complexes exhibit regulatory effects on other IFN-dependent mammalian target of rapamycin-mediated signaling events, likely via engagement of the AKT/mTORC1 axis, including IFN-induced phosphorylation of S6 kinase and its effector rpS6, as well as phosphorylation of the translational repressor 4E-binding protein 1. We also show that induction of ISG protein expression and the generation of antiviral responses are defective in Rictor and mLST8-KO cells. Together, our data provide evidence for unique functions of mTORC2 complexes in the induction of type I IFN responses and suggest a critical role for mTORC2-mediated signals in IFN signaling.

Induction of human plasmablasts during infection with antibiotic-resistant nosocomial bacteria.

OBJECTIVES: Nosocomial pathogens such as Acinetobacter baumannii are a growing public health threat, due in part to their increasing resistance to antibiotics. Since some strains are resistant to all available antibiotics, novel therapies are urgently needed. Plasmablasts are short-lived B cells found in the blood that can be collected and harnessed to produce therapeutic antibodies. We set out to determine whether plasmablasts are induced during infection with A. baumannii and other nosocomial pathogens. METHODS: We obtained blood samples from patients infected with antibiotic-resistant nosocomial pathogens, and analysed their plasmablast response by flow cytometry. RESULTS: We observed a strong induction of plasmablasts in patients with antibiotic-resistant A. baumannii infection. Furthermore, plasmablasts were also induced in response to other drug-resistant nosocomial pathogens. CONCLUSIONS: These data suggest that plasmablasts may be broadly harnessed to develop therapeutic antibodies to combat otherwise untreatable antibiotic-resistant infections.

The versatile biopolymer chitosan: potential sources, evaluation of extraction methods and applications.

Among the biopolymers, chitin and its derivative chitosan (CTS) have been receiving increasing attention. Both are composed of randomly distributed ß-(1-4)-linked d-glucosamine and N-acetyl glucosamine units. On commercial scale, CTS is mainly obtained from the crustacean shells. The chemical methods employed for extraction of CTS from crustacean shells are laden with many disadvantages. Waste fungal biomass represents a potential biological source of CTS, in fact with superior physico-chemical properties, such as high degree of deacetylation, low molecular weight, devoid of protein contamination and high bioactivity. Researchers around the globe are attempting to commercialize CTS production and extraction from fungal sources. Fungi are promising and environmentally benign source of CTS and they have the potential to completely replace crustacean-derived CTS. Waste fungal biomass resulting from various pharmaceutical and biotechnological industries is grown on inexpensive agro-industrial wastes and its by-products are a rich and inexpensive source of CTS. CTS is emerging as an important natural polymer having broad range of applications in different fields. In this context, the present review discusses the potential sources of CTS and their advantages and disadvantages. This review also deals with potential applications of CTS in different fields. Finally, the various attributes of CTS sought in different applications are discussed.

Th17 cells in multiple sclerosis express higher levels of JAK2, which increases their surface expression of IFN-γR2.

IFN-ß inhibits the expansion of Th17 cells in active multiple sclerosis (AMS), and this might contribute to improve the clinical symptoms. The effectiveness of this inhibition, however, requires intact IFN-γ signaling in T cells. In this study, we report that both mRNA and cell surface expression of the signaling chain of the IFN-γ receptor (IFN-γR2) and its cognate tyrosine kinase JAK2 are enhanced in peripheral blood Th17 cells and clones from patients with AMS compared with those with inactive multiple sclerosis (IMS) or healthy subjects (HS). IFN-γ decreased the frequency of Th17 peripheral cells and proliferation of Th17 clones from AMS patients. Stimulation of PBMCs from HS in Th17-polarizing conditions resulted in the enhancement of JAK2 expression and accumulation of cell surface IFN-γR2. The role of JAK2 in the modulation of IFN-γR2 was demonstrated as its transduction prevented rapid internalization and degradation of IFN-γR2 in JAK2-deficient γ2A cells. In conclusion, these data identify JAK2 as a critical factor that stabilizes IFN-γR2 surface expression in Th17 cells from AMS patients, making them sensitive to IFN-γ. These data may have clinical implications for a better use of IFNs in multiple sclerosis and possibly other inflammatory diseases.