Assessment of the antimicrobial and immunomodulatory activity of QS-CATH, a promising therapeutic agent isolated from the Chinese spiny frogs (Quasipaa spinosa).

Cathelicidins are important antimicrobial peptides in various vertebrate species where they are crucial parts of the innate immune system. The current understanding of amphibian cathelicidins is limited, particularly with regard to their immunomodulatory effects. To address this knowledge gap, we produced the cDNA sequence of the cathelicidin gene from a skin transcriptome of the Chinese spiny frog Quasipaa spinosa. The amino acid sequence of the Quasipaa spinosa cathelicidin (QS-CATH) was predicted to consist of a signal peptide, a cathelin domain, and a mature peptide. Comparative analysis of the QS-CATH amino acid sequence with that of other amphibian cathelicidins revealed high variability in the functional mature peptide among amphibians, whereas the cathelin domain was conserved. The QS-CATH gene was expressed in several tissues, with the highest level of expression in the spleen. Upregulation of QS-CATH after Aeromonas hydrophila infection occurred in the kidney, gut, spleen, skin, and liver. Chemically synthesized QS-CATH exhibited pronounced antibacterial activity against Shigella flexneri, Staphylococcus warneri, Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Furthermore, QS-CATH disrupted the cell membrane integrity of S. flexneri, as evidenced by a lactate dehydrogenase release assay, and it hydrolyzed the genomic DNA of S. flexneri. Additionally, QS-CATH elicited chemotaxis and modulated the expression of inflammatory cytokine genes in RAW264.7 mouse leukemic monocyte/macrophage cells. These findings confirm the antimicrobial effects of amphibian cathelicidin and its ability to influence immune cell function. This will expedite the potential utilization of amphibian antimicrobial peptides as therapeutic agents.

Preclinical risk assessment strategy to mitigate the T-cell dependent immunogenicity of protein biotherapeutics: State of the art, challenges and future perspectives.

Protein therapeutics hold a prominent role and have brought significant diversity in efficacious medicinal products. Not just monoclonal antibodies and different antibody formats (pegylated antigen-binding fragments, bispecifics, antibody-drug conjugates, single chain variable fragments, nanobodies, dia-, tria- and tetrabodies), but also purified blood products, growth factors, recombinant cytokines, enzyme replacement factors, fusion proteins are all good instances of therapeutic proteins that have been developed in the past decades and approved for their value in oncology, immune-oncology, and autoimmune diseases discovery programs. Although there was an ingrained belief that fully humanized proteins were expected to have limited immunogenicity, adverse effects associated with immune responses to biological therapies raised some concern in biotech companies. Consequently, drug developers are designing strategies to assess potential immune responses to protein therapeutics during both the preclinical and clinical phases of development. Despite the many factors that can contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) immunogenicity seems to play a crucial role in the development of anti-drug antibodies (ADAs) to biologics. A broad range of methodologies to predict and rationally assess Td immune responses to protein drugs has been developed. This review aims to briefly summarize the preclinical immunogenicity risk assessment strategy to mitigate the risk of potential immunogenic candidates coming towards clinical phases, discussing the advantages and limitations of these technologies, and suggesting a rational approach for assessing and mitigating Td immunogenicity.

Assessment the efficacy of some various treatment methods, in vitro and in vivo, against Aeromonas hydrophila infection in fish with regard to side effects and residues.

Aeromonas hydrophila is an opportunistic bacteria with an overwhelming impact on fish farming industry especially with upraising of drug resistant mutants. This study aimed to evaluate and compare the therapeutic and side effects of levofloxacin (LEV), chitosan-nanoparticles (CNPs), and fructooligosaccharides (FOS) in control of this infection in tilapia. A total of 160 Nile-tilapia divided into 8-groups; G1: negative-control, G2: infected-control, G3: non-infected-(levofloxacin (LEV) 10 mg/kg bwt), G4: non-infected-(chitosan-nanoparticles (CNPs) 1 g/kg ration), G5: non-infected-(fructooligosaccharides (FOS) 20 g/kg ration), G6: infected-LEV, G7: infected-CNPs and G8: infected-FOS for 7 days. MICs were (0.125 µg/ml and 1.25 mg/ml) for LEV and CNPs respectively. No mortalities or significant adverse effects were recorded in non-infected treated-groups while infected were (20%) LEV, (30%) CNPs, (40%) FOS and (70%) G2. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) decreased by LEV and CNPs than FOS while all increased total protein (TP) and albumin than G2. Malondialdehyde (MDA) significantly decreased and superoxide dismutase (SOD) and reduced glutathione (GSH) increased in all infected-treated groups than G2 in various degrees. Urea and creatinine descending order were FOS, LEV then CNPs decreased significantly than G2. LEV musculature residues, using HPLC, decreased gradually till the 5th day; 621.00 ± 0.66, 270.00 ± 0.48 then 64.00 ± 0.40, and 471.00 ± 0.79, 175.00 ± 0.52 ppb then not detected at 1st, 3rd, and 5th days of withdrawal in non-infected and infected groups respectively. Finally, LEV and CNPs were superior as bactericidal, decreasing mortalities and enzyme activities while CNPs and FOS increased performance, non-specific immunity, and antioxidant biomarkers.

A safety review of biologic therapies for the management of hidradenitis suppurativa and unmet needs.

INTRODUCTION: Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disorder characterized by nodules, abscesses, fistulae, and significant scarring in intertriginous areas rich in apocrine glands. Immunomodulator drugs, including biologics, are a mainstay of treatment for this disease. AREAS COVERED: This review details the safety profiles of various biologic therapies currently available commercially that have been tried for HS as assessed in clinical trials and observational studies. As the only Food and Drug Administration (FDA)-approved medication for the treatment of moderate-to-severe HS, adalimumab is discussed in the most detail. Additional biologic medications, including tumor necrosis factor α (TNFα) inhibitors, interleukin 1 (IL-1) inhibitors, IL-12 and IL-23 inhibitors, IL-17 inhibitors, and IL-23 inhibitors, are discussed as well. Safety concerns in special populations, including pregnant women and children, are outlined. EXPERT OPINION: Existing data support excellent short-term and long-term safety profiles for adalimumab, although caution must be taken with use in high-risk patient populations, including those with chronic infections or increased risk of malignancy. Based on their safety data for other indications, additional biologic agents appear safe in HS as well. However, further research is needed to fully understand the safety profiles of these medications in the HS population.

Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei.

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

18F-AraG PET for CD8 Profiling of Tumors and Assessment of Immunomodulation by Chemotherapy.

Most clinical trials exploring various combinations of chemo- and immunotherapy rely on serial biopsy to provide information on immune response. The aim of this study was to assess the value of 18F-arabinosyl guanine (18F-AraG) as a noninvasive tool that profiles tumors on the basis of the key player in adaptive antitumor response, CD8+ cells, and evaluates the immunomodulatory effects of chemotherapy. Methods: To evaluate the ability of 18F-AraG to report on the presence of CD8+ cells within the tumor microenvironment, we imaged a panel of syngeneic tumor models (MC38, CT26, LLC, A9F1, 4T1, and B16F10) and correlated the signal intensity with the number of lymphocytes found in the tumors. The capacity of 18F-AraG to detect immunomodulatory effects of chemotherapy was determined by longitudinal imaging of tumor-bearing mice (MC38 and A9F1) undergoing 2 types of chemotherapy: oxaliplatin/cyclophosphamide, shown to induce immunogenic cell death, and paclitaxel/carboplatin, reported to cause immunogenically silent tumor cell death. Results: In the tumor panel, 18F-AraG revealed strikingly different uptake patterns resembling cancer-immune phenotypes observed in the clinic. A statistically significant correlation was found between the 18F-AraG signal and the number of PD-1-positive CD8+ cells isolated from the tumors (r2 = 0.528, P < 0.0001). In the MC38 model, paclitaxel/carboplatin did not result in an appreciable change in signal after therapy (1.69 ± 0.25 vs. 1.50 ± 0.33 percentage injected dose per gram), but oxaliplatin/cyclophosphamide treatment led to close to a 2.4-fold higher 18F-AraG signal (1.20 ± 0.31 vs. 2.84 ± 0.93 percentage injected dose per gram). The statistically significant increase in signal after oxaliplatin/cyclophosphamide was also observed in the A9F1 model (0.95 ± 0.36 vs. 1.99 ± 0.54 percentage injected dose per gram). Conclusion: The ability of 18F-AraG PET to assess the location and function of CD8+ cells, as well immune activity within tumors after immune priming therapy, warrants further investigation into its utility for patient selection, evaluation of optimal time to deliver immunotherapies, and assessment of combinatorial therapies.

Practical management of inflammatory bowel disease patients during the COVID-19 pandemic: expert commentary from the Gastroenterological Society of Australia Inflammatory Bowel Disease faculty.

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has emerged as a public health emergency and challenged healthcare systems globally. In a minority of patients, SARS-CoV-2 manifests with a severe acute respiratory illness and currently there is insufficient data regarding the virulence of COVID-19 in inflammatory bowel disease patients taking immunosuppressive therapy. This review aims to summarise the current literature and provide guidance on the management of inflammatory bowel disease patients in the context of the COVID-19 pandemic in the Australasian setting.

Profiling individual clinical responses by high-frequency serum neurofilament assessment in MS.

Objective: To evaluate individual neurofilament light chain (NfL) variation over the time of disease course and the potential of NfL measurement to predict treatment response in patients with MS. Methods: We investigated 15 patients with MS after immune reconstitution treatment with alemtuzumab (ATZ). Monthly serum NfL (sNFL) measurements were correlated with Expanded Disability Status Scale (EDSS), MRI, and relapse activity over an observational period of up to 102 months. Results: Before ATZ, sNfL was significantly increased in correlation with previous relapse/MRI activity. After ATZ, sNfL decreased quickly within the first 6 months. In patients classified as NEDA-3, sNfL declined and persisted at an individual low steady-state level of <8 pg/mL. During follow-up, 34 sNfL peaks with a >20 fold increase could be detected, which were associated with clinical or MRI disease activity. Even patient-reported relapse-suspicious symptoms, which have not been confirmed because relapses were accompanied by sNfL, increase, proposing sNfL assessment as a marker for relapse activity. sNfL started to increase earliest 5 months before, peaked at clinical onset, and recovered within 4-5 months. sNfL presented at higher levels in active patients requiring ATZ retreatment compared with responder patients. During 2 documented pregnancies, sNfL was at a low level, whereas a postpartum transient sNfL increase was seen without any signs of activity. Conclusions: This study applied a long-term high-frequency sNfL assessment in an ATZ-treated cohort, allowing a holistic profiling on the individual level and highlighted that sNfL can eminently complement the individual clinical and MRI monitoring in clinical practice.

A Comprehensive Assessment of Apigenin as an Antiproliferative, Proapoptotic, Antiangiogenic and Immunomodulatory Phytocompound.

Apigenin (4',5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley. This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release. Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 µM and 60 µM. Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining. The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production. The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api. Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%. Api elicited antiangiogenic properties in a dose-dependent manner. Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.

Systematic review and network meta-analysis comparing ocrelizumab with other treatments for relapsing multiple sclerosis.

BACKGROUND: Ocrelizumab was approved for the treatment of relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS) by the US Food and Drug Administration in March 2017 and by the European Medicines Agency in January 2018. These approvals were based on two pivotal randomized controlled trials (RCTs), OPERA I and OPERA II, comparing ocrelizumab 600 mg with an active comparator, interferon ß-1a 44 µg (Rebif), and the first trial with positive results in patients with PPMS, which compared ocrelizumab with placebo. However, direct evidence of the efficacy and safety of ocrelizumab in RMS compared with other disease-modifying therapies (DMTs) approved for RMS is not available from RCTs. In the absence of such RCTs, network meta-analyses (NMAs) were conducted to compare indirectly the relative efficacy and safety of ocrelizumab with all other approved DMTs for the treatment of RMS. METHODS: Systematic literature searches were conducted in MEDLINE, Embase, the Cochrane Library, trial registers, relevant conference websites and health technology assessment agency websites. Eligible RCTs evaluated approved treatments for multiple sclerosis (MS) in which more than 75% of patients had a relapsing form of MS. NMAs were conducted for four efficacy and three safety outcomes, and treatment hierarchies were generated for each outcome using surface under the cumulative ranking curve (SUCRA) values. RESULTS: Results suggest that ocrelizumab has superior efficacy to 10 of the 17 treatments in the 12-week confirmed disability progression network and 12 of the 17 treatments in the annualized relapse rate network (both including placebo). The efficacy of ocrelizumab was comparable with the other treatments in both networks. In the serious adverse events and discontinuation due to adverse events networks, ocrelizumab demonstrated a safety profile comparable with all other treatments (including placebo). SUCRA values consistently ranked ocrelizumab among the most effective or tolerable treatments across all outcomes. CONCLUSIONS: Results suggest that ocrelizumab has an efficacy superior to or comparable with all other currently approved DMTs across all endpoints analyzed, and a similar safety profile, indicating it offers a valuable package for the treatment of patients with RMS.

Nonclinical safety assessment of a human interleukin-22FC IG fusion protein demonstrates in vitro to in vivo and cross-species translatability.

Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross-species translatability of the biological response in activating the IL-22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on-target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.

Intravenous immunoglobulins in dermatology. Part 1: biological mechanisms and methods of administration.

Intravenous immunoglobulin (IVIg) is a solution of human IgG, salt, sugars and solvents, which is used to treat a multitude of diseases. Although IVIg has been known to treat many diseases safely and successfully, there are relatively few supporting randomized controlled trials. In this article, we review the biological mechanisms of IVIg in dermatological disorders and the practicalities of its use, including its mechanism of action, dosing, availability, costs and adverse effects.

Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia.

Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 µg/mL, respectively, whereas IC50 of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 µg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.

Reverse Translation for Assessment of Confidence in Animal Models of Multiple Sclerosis for Drug Discovery.

The poor predictive quality of currently used animal models in preclinical research is an important cause of the high attrition of promising drug candidates for human autoimmune disease in clinical trials. Examples from own work in a primate multiple sclerosis (MS) model illustrate that important lessons can be learned from a critical reassessment of failed drugs in the animal model, which can help improve the animal model and better understand the targeted disease.

Assessment of structural and functional similarity of biosimilar products: Rituximab as a case study.

Biosimilars are products that are similar in terms of quality, safety, and efficacy to an already licensed reference/ innovator product and are expected to offer improved affordability. The most significant source of reduction in the cost of development of a biosimilar is the reduced clinical examination that it is expected to undergo as compared to the innovator product. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. As a result, establishing analytical similarity is arguably the most important step towards successful development of a biosimilar. Here, we present results from an analytical similarity exercise that was performed with five biosimilars of rituximab (Ristova®, Roche), a chimeric mouse/ human monoclonal antibody biotherapeutic, that are available on the Indian market. The results show that, while the biosimilars exhibited similarity with respect to protein structure and function, there were significant differences with respect to size heterogeneity, charge heterogeneity and glycosylation pattern.

Preliminary assessment on potentials of probiotic B. subtilis RX7 and B. methylotrophicus C14 strains as an immune modulator in Salmonella-challenged weaned pigs.

A total of 40 crossbred weaned piglets (28 days old; [Landrace × Yorkshire] × Duroc) were used for preliminary assessment on potentials of Bacillus-based probiotics as an immune modulator in a Salmonella Typhimurium challenge model in a 3-week experiment. Pigs were randomly allotted to four experimental diets according to their initial body weight (9.21 ± 1.1 kg) and sex (10 pigs per treatment; 5 barrows and 5 gilts). The dietary treatments were basal diet (CON), basal diet + oral administration of Salmonella enterica ser. Typhimurium at the dosage of 1 mL containing 1 × 1011 cfu/mL of viable cell concentrations at day 21 (SC), SC + Bacillus subtilis (BS), and SC+ Bacillus methylotrophicus (BM). After 12 h of Salmonella challenge, the red blood cell (RBC), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations were reduced (P < 0.05) whereas haptoglobin and cortisol levels were greater (P < 0.05) in SC compared with CON. However, the concentrations of RBC, IgG, and IgM were increased whereas haptoglobin and cortisol levels were reduced in BS and BM compared with SC. The probiotic-treated groups showed reduced (P < 0.05) IgM levels and increased (P < 0.05) WBC and cortisol levels compared with CON. The supplementation of probiotics showed increased (P < 0.05) fecal Lactobacillus counts and reduced Escherichia coli and Salmonella counts in piglets though there was no biological relevance compared with SC. Thus, in our preliminary study, Bacillus-based probiotic has shown some positive immunomodulatory effects in Salmonella-challenged pigs which provided a base for further studies.

Immunoregulatory effect assessment of a novel melanin and its carboxymethyl derivative.

A novel melanin with low molecular weight (LIM205, 522Da) was isolated from the fermentation broth of Lachnum sp. and its carboxymethyl derivative (CLIM205) was prepared. The immunoregulatory effects of LIM205 and CLIM205 in immuno-compromised mice induced by cyclophosphamide were investigated. The results demonstrated that both LIM205 and CLIM205 could significantly increase the thymus and spleen indices, specific and nonspecific (including carbon clearance ability) immunity, humoral and cellular immunity of mice. Treatment with LIM205 and CLIM205 could increase activities of SOD, GSH-PX, CAT and decrease content of MDA in the mice. Furthermore, for all animal tests, the immunoregulatory activities of CLIM205 were more prominent than that of LIM205. In conclusion, our findings suggested that the natural products LIM205, as well as its carboxymethyl derivative CLIM205, had significant immunoregulatory activities, which might be a promising source of immunoregulator in healthcare field.