A novel targeted multifunctional nanoplatform for visual chemo-hyperthermia synergy therapy on metastatic lymph nodes via lymphatic delivery.

BACKGROUND: Distant metastasis to vital organs is the major contributor to breast cancer mortality, and regional lymph node metastasis is an important facilitator of distant metastasis and recurrence in this cancer. The early diagnosis and precise treatment of lymph node metastasis are crucial for staging and prognosis in breast cancer. Herein, we report a visualized precision medicine nanoplatform of metastatic lymph nodes for ultrasonic/photoacoustic (US/PA) dual modal imaging-guided in situ targeted hyperthermia-combined chemotherapy. RESULTS: Carbon nanoparticles (CNs), approved by the China Food and Drug Administration, were loaded with docetaxel and rationally combined with anti-hypoxia-inducible factor 1α antibody-modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles to achieve the combination of passive targeting at the lymph nodes and intracellular targeting at HIF 1α factor. The accumulation and retention of nanoparticles in metastatic lymph nodes via lymphatic delivery were enhanced. Docetaxel could be effectively offloaded by CNs that have active carbon nanoparticles, and the PLGA membrane prevented drug leakage. The nanoparticles exhibited excellent photothermal performance with a photothermal conversion efficiency of 28.9%, killing tumor cells in metastatic lymph nodes through hyperthermia. In vitro and in vivo systematic evaluations revealed that hyperpyrexia triggered the rupture of nanoparticles caused by the phase transition of perfluorohexane, resulting in docetaxel release for achieving in situ hyperthermia-combined chemotherapy. CONCLUSIONS: The laser-triggered highly efficient in situ chemotherapy nanosystem achieves targeted synergistic chemo-hyperthermia treatment of metastatic lymph nodes, and lymphatic delivery represents a strategy to avoid additional injury caused by drugs entering the blood circulation.

How to Prevent and Mitigate Hypersensitivity Reactions to Biologicals Induced by Anti-Drug Antibodies?

Biologicals are widely used therapeutic agents for rheumatologic diseases, cancers, and other chronic inflammatory diseases. They are characterized by complex structures and content of variable amounts of foreign regions, which may lead to anti-drug antibodies (ADA) development. ADA onset may limit the clinical usage of biologicals because they may decrease their safety. In fact they are mainly associated with immediate hypersensitivity reactions (HSRs). Development of ADAs is reduced by concomitant immunosuppressive treatment, while it is increased by longer intervals between drug administrations; thus, regular infusion regimens should be preferred to reduce HSRs. Once ADAs have formed, some procedures can be implemented to reduce the risk of HSRs. ADAs may belong to different isotype; the detection of IgE ADA is advisable to be assessed when high and early ADAs are detected, in order to reduce the risk of severe HRs. In patients who need to reintroduce the biological culprit, as alternative therapies are not available, drug desensitization (DD) may be applied. Desensitization should be conceptually dedicated to patients with an IgE-mediated HSR; however, it can be performed also in patients who had developed non-IgE-mediated HSRs. Although the underlying mechanisms behind successful DD has not been fully clarified, the DD procedure is associated with the inhibition of mast cell degranulation and cytokine production. Additionally, some data are emerging about the inhibition of drug-specific immune responses during DD.

Targeting small GTPases and their downstream pathways with intracellular macromolecule binders to define alternative therapeutic strategies in cancer.

The RAS superfamily of small GTPases regulates major physiological cellular processes. Mutation or deregulation of these small GTPases, their regulators and/or their effectors are associated with many diseases including cancer. Hence, targeting these classes of proteins is an important therapeutic strategy in cancer. This has been recently achieved with the approval of the first KRASG12C covalent inhibitors for the clinic. However, many other mutants and small GTPases are still considered as 'undruggable' with small molecule inhibitors because of a lack of well-defined pocket(s) at their surface. Therefore, alternative therapeutic strategies have been developed to target these proteins. In this review, we discuss the use of intracellular antibodies and derivatives - reagents that bind their antigen inside the cells - for the discovery of novel inhibitory mechanisms, targetable features and therapeutic strategies to inhibit small GTPases and their downstream pathways. These reagents are also versatile tools used to better understand the biological mechanisms regulated by small GTPases and to accelerate the drug discovery process.

Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies.

Biotin interference in streptavidin/biotin-based immunoassays has been recently recognized as a confounding factor in clinical settings. Depending on the nature of the assay, the presence of excess biotin in patient samples can cause falsely high or low results. One of the platforms known to be affected, Roche Cobas, is widely used in anti-doping laboratories to test for intact chorionic gonadotropin (hCG) in urine. While biotin levels in blood have been well studied, less is known about urinary biotin due to its limited clinical significance. Having analyzed over 4,000 urine samples, we have established a reference range for urinary biotin with a median concentration of approximately 12 ng/ml. However, a significant number of samples contain much higher amounts, with a maximum approaching 10 µg/ml, suggesting biotin supplementation. Consequently, the tolerance of hCG STAT assay towards biotin was investigated over a wide concentration range. The apparent hCG concentration was found to decrease almost linearly as biotin increased from 100 to 1,000 ng/ml, with only 10% of the expected value reported by the assay as biotin reached 1,000 ng/ml. Further increase of biotin resulted in a progressive, albeit more moderate, decline in measured hCG concentration. To avoid a false negative result in the context of anti-doping analysis, it is highly recommended to monitor biotin in urine and perform diafiltration before hCG measurement in samples with elevated biotin to remove the interference.

Chitosan functionalized graphene oxide nanocomposites for fluorescence imaging of apoptotic processes and targeted anti-inflammation study.

This work reports novel chitosan functionalized graphene oxide (GO) nanocomposites combined fluorescence imaging and therapeutic functions in one agent, which can serve as a promising alternative to alleviate related diseases caused hyperinflammation. Briefly, GO was designed to be conjugated with chitosan, fluorescein-labeled peptide, toll-like receptor 4 antibody and hydroxycamptothecin/aloe emodin. We have demonstrated that such nanocomposites could effectively achieve active targeted delivery of pro-apoptotic and anti-inflammatory drugs into inflammatory cells and cause cells apoptosis by acid-responsive drug release. Moreover, confocal fluorescence imaging confirms that the drug-induced inflammatory cells apoptosis could be visualized the light-up fluorescence of fluorescein activated by caspase-3. Meanwhile, inflammatory-related biomarkers have down-regulated after the nanocomposites' treatment in both vitro and vivo experiments consistent with the results in histological sections. In summary, the bifunctional nanocomposites that possess anti-inflammation and fluorescence imaging could serve as a promising therapeutic agent for reducing hyperinflammation caused by numerous diseases.

Preparation of the peroxisome proliferator-activated receptor α polyclonal antibody: Its application in fatty liver hemorrhagic syndrome.

Peroxisome proliferator-activated receptor α (PPARα) play a key role in the regulation of metabolic homeostasis, inflammation, cellular growth, and differentiation. To further explore the potential role of PPARα in the energy homeostasis of fatty liver hemorrhagic syndrome (FLHS), we reported the prokaryotic expression and purification of chicken PPARα subunit protein, and successfully prepared a polyclonal antibody against PPARα recombinant protein. The 987 bp PPARα subunit genes were cloned into the pEASY-T3 clone vector. Then the plasmid PCR products encoding 329 amino acids were ligated to pEASY-Blunt E2 vector and transformed into BL21 to induce expression. The recombinant PPARα subunit protein, containing His-tag, was purified by affinity column chromatography using Ni-NTA affinity column. Rabbit antiserum was generated by using the concentration of recombinant PPARα subunit protein as the antigen. The results of western blotting showed that the antiserum can specifically recognize chicken endogenous PPARα protein. Immunohistochemistry and immunofluorescence showed that the PPARα mainly existed in the nucleus of hepatocytes, renal epithelial cells and hypothalamic endocrine nerve cells. More importantly, western blotting and real-time quantitative PCR indicated that FLHS significantly decreased the expression of PPARα.

Statistical methods of screening cut point determination in immunogenicity studies.

Background: Currently, screening cut point (CP) calculated from an assay validation with replicates are applied to an immunogenicity study with nonreplicates, for which the antidrug antibodies rate is determined. IID treats the replicate of a sample as coming from another independent sample. AVE uses average results from each sample across runs but inter-assay variability is reduced. Therefore, we propose a random effect model (REM) for calculating CP. Materials & method: We investigate impact of noncompatibility design between validation and immunogenicity studies on CP and compare these methods. Conclusion: IID may not fit for use when replicates' variability dominates all sources of uncertainty. REM considers covariance structure of repeated measurements. CP by REM is smaller than that by IID but larger than that by AVE.

Gastric Enzyme Supplementation Inhibits Food Allergy in a BALB/c Mouse Model.

Impaired gastric digestion due to suppressed gastric acidity enhances the risk for food allergy development. In the current study, we aimed to evaluate the impact of a supported gastric digestion via application of a pharmaceutical gastric enzyme solution (GES) on food allergy development and allergic reactions in a BALB/c mouse model. The ability of the GES to restore hypoacidic conditions was tested in mice treated with gastric acid suppression medication. To evaluate the impact on allergic symptoms, mice were orally sensitized with ovalbumin (OVA) under gastric acid suppression and subjected to oral challenges with or without GES. The immune response was evaluated by measurement of antibody titers, cytokine levels, mucosal allergy effector cell influx and regulatory T-cell counts. Clinical response was objectified by core body temperature measurements after oral OVA challenge. Supplementation of GES transiently restored physiological pH levels in the stomach after pharmaceutical gastric acid suppression. During oral sensitization, supplementation of gastric enzymes significantly reduced systemic IgE, IgG1 and IgG2a levels and allergic symptoms. In food allergic mice, clinical symptoms were reduced by co-administration of the gastric enzyme solution. Support of gastric digestion efficiently prevents food allergy induction and alleviates clinical symptoms in our food allergy model.

Cloning and prokaryotic expression of the chicken liver kinase B1 (LKB1) and its localization in liver, heart and hypothalamus.

Liver kinase B1 (LKB1) is a member of the serine/threonine kinase family, which plays an indispensable role in the organism of animals. In the current study, the chicken LKB1 protein gene was amplified by PCR based on the primers and cDNA templates. Then, the cloning vector was constructed and the target gene was cloned. After that, the target gene was inserted into the expression vector to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) host cells and the LKB1 recombinant proteins were successfully expressed by using Isopropyl-ß-D-thiogalactopyranoside (IPTG). Finally, purified LKB1 proteins were used as antigen and the rabbit-derived antiserums were collected. The antiserum titer determined by ELISA was not less than 1:128000. The results of Western blot suggested that the polyclonal antibody is highly specific to chicken LKB1 protein. Immunofluorescence indicated that the LKB1 protein is mainly expressed in the cytoplasm of liver, heart and hypothalamus cells of chicken. Our study showed that the LKB1 polyclonal antibodies produced by this method are effective and can be used to further study the role of LKB1 in the pathogenesis of chicken disease.

Industry experiences with immune-mediated findings in biotherapeutic nonclinical toxicology studies.

With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between. In addition, an increasing number of oncology therapeutics are being developed that enhance or directly stimulate immune responses by a variety of mechanisms, which could increase the risk of autoreactivity and an autoimmune-like syndrome in animals and humans. When evaluating the risk of biotherapeutics prior to entering the clinic, the nonclinical safety data may include any of these responses and it is critical to understand whether they represent a safety liability for humans. The DruSafe Leadership group of the IQ Consortium conducted a survey of industry to understand sponsors' experiences with these immune reactions in nonclinical studies related to both immunogenicity and pharmacologically-mediated immune perturbations. The survey covered what pathways were affected, how the immune responses were presented, how the company and health authorities interpreted the data and whether the immune responses were observed in the clinic. Additionally, the survey gathered information on association of these findings with anti-drug antibodies as well as sponsor's use of immunogenicity predictive tools. The data suggests that the ability of a biotherapeutic to activate the immune system, intended or not, plays a significant role on characteristics of the response and whether theys are translatable.

Oral Immunotherapy Using Probiotic Ice Cream Containing Recombinant Food-Grade Lactococcus lactis Which Inhibited Allergic Responses in a BALB/c Mouse Model.

This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20 µl of functional ice cream with 1012 CFU/ml of r-L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN-γ and TGF-ß cytokines increased in the 20 µl ice cream treatment group as well as 40 µg/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20 µl ice cream with 1012 CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses.

Near-infrared fluorescence imaging in immunotherapy.

Near-infrared (NIR) light possesses many suitable optophysical properties for medical imaging including low autofluorescence, deep tissue penetration, and minimal light scattering, which together allow for high-resolution imaging of biological tissue. NIR imaging has proven to be a noninvasive and effective real-time imaging methodology that provides a high signal-to-background ratio compared to other potential optical imaging modalities. In response to this, the use of NIR imaging has been extensively explored in the field of immunotherapy. To date, NIR fluorescence imaging has successfully offered reliable monitoring of the localization, dynamics, and function of immune responses, which are vital in assessing not only the efficacy but also the safety of treatments to design immunotherapies optimally. This review aims to provide an overview of the current research on NIR imaging of the immune response. We expect that the use of NIR imaging will expand further in response to the recent success in cancer immunotherapy. We will also offer our insights on how this technology will meet rapidly growing expectations in the future.

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDE-modified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

Reversion of arterial calcification by elastin-targeted DTPA-HSA nanoparticles.

Generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE) are characterized by pathologic calcifications in the media of large- and medium sized arteries. GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. Different treatment approaches including bisphosphonates and orally administered pyrophosphate (PPi) were investigated in recent years, but reversion of calcification could not be achieved. With this study, we pursued the idea of a combination of controlled drug delivery through nanoparticles and active targeting via antibody conjugation to develop a treatment for GACI and PXE. To establish a suitable drug delivery system, the chelating drug diethylenetriamine pentaacetic acid (DTPA) was conjugated to nanoparticles composed of human serum albumin (HSA) as biodegradable and non-toxic particle matrix. To accomplish an active targeting of the elastic fibers exposed through calcification of the affected areas, the nanoparticle surface was functionalized with an anti-elastin antibody. Cytotoxicity and cell interaction studies revealed favorable preconditions for the intended i.v. application. The chelating ability was evaluated in vitro and ex vivo on aortic ring culture isolated from two mouse models of GACI and PXE. The positive results led to the conclusion that the produced nanoparticles might be a promising therapy in the treatment of GACI and PXE.

A new strategy for the preparation of antibody against natural glycoside: With astragaloside IV as an example.

A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6"-CH2OH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6"-COOH, which was subsequently condensed with -NH2 on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC50, and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO4) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO4 oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies.

Screening protocol for the identification of modulators by immunofluorescent cell-based assay.

High-throughput assays are a common strategy for the identification of compounds able to modulate a certain cellular activity. Here, we show an automatized analysis platform for the quantification of nuclear foci as inhibitory effect of compounds on a target protein labeled by fluorescent antibodies. Our experience led us to a fast analysis platform that combines cell-based assays, high-content screening, and confocal microscopy, with an automatic and user-friendly statistical analysis of plate-based assays including positive and negative controls, able to identify inhibitory effect of compounds tested together with the Z-prime and Window of individual plate-based assays to assess the reliability of the results. The platform integrates a web-based tool implemented in Pipeline Pilot and R, and allows computing the inhibition values of different parameters obtained from the high-content screening and confocal microscopy analysis. This facilitates the exploration of the results using the different parameters, providing information at different levels as the number of foci observed, the sum of intensity of foci, area of foci, etc, the detection and filtering of outliers over the assay plate, and finally providing a set of statistics of the parameters studied together with a set of plots that we believe significantly helps to the interpretation of the assay results.

Molecular mimicry between Larrea divaricata Cav. plant and a reference strain of Pseudomonas aeruginosa.

Currently, the world health sector faces a big problem due to the increase of bacterial strains resistant to antibiotics. In 2017, the World Health Organization reported a list of resistant bacteria, among which Pseudomonas aeruginosa was present. This opportunistic pathogen is associated to nosocomial infections, and no effective vaccines against this bacterium have been found. Larrea divaricata Cav. (jarilla) is a shrub highly distributed in America and widely used in folk medicine. In our laboratory, cross-reactivity of antibodies obtained from the recognition of jarilla proteins against proteins from gram-negative bacteria has been demonstrated. The objective of this study was to study the cross-reactivity of anti-L. divaricata antibodies with P. aeruginosa extracellular proteins in order to find an innocuous prophylactic therapy against this nosocomial pathogen. We observed that antibodies generated by proteins from jarilla crude extract recognized antigenic determinants present in extracellular proteins of P. aeruginosa. However, further studies are needed to investigate the neutralizing capacity of these antibodies on the specific enzymatic proteins involved in the pathogenicity of this bacterium.

Immunization to rituximab is more frequent in systemic autoimmune diseases than in rheumatoid arthritis: ofatumumab as alternative therapy.

OBJECTIVES: The frequency and consequences of anti-drug antibodies to rituximab (RTX-ADA) are not well known in RA and even less in other systemic auto-immune diseases (sAID). We aimed to evaluate the frequency, consequences and predictive factors of RTX-ADA in RA and sAID. METHODS: All patients presenting with RA or other sAID treated with RTX from 2012 to 2017 in our tertiary reference centre for sAID were retrospectively studied. Patients who were tested for RTX-ADA were identified. RESULTS: One hundred and ninety-nine patients were treated with RTX (RA: 124, other sAID: 75). Among 62/199 (31.1%) tested for RTX-ADA, 14 were positive: 3/35 RA (8.6%) and 11/27 (40.7%) other sAID, (P = 0.0047). Among the whole RTX-treated populations, the frequency of RTX-ADA was 2.4% and 14.7% (P = 0.0026) in RA and sAID, respectively. Most of the immunized patients had infusion reactions to second or subsequent RTX cycles (11/14) and loss of efficacy (2/14). Predictive factors of immunization were sAID vs RA (78.6% vs 21.4%, P = 0.026, adjusted odds ratio (OR) = 5.35[1.43-54.75]) and African ethnicity (57.1% vs 4.2%, P < 0.001, adjusted OR = 9.25 [5.08-302.12]). Associated immunosuppressive therapy did not protect against immunization. Three patients with pSS immunized against RTX were treated with ofatumumab with complete remission of their disease. CONCLUSION: Immunization against RTX is more frequent in other sAID than in RA. Testing for RTX-ADA must be performed in patients with infusion reactions or loss of efficacy especially if they are of African origin. Immunized patients might be treated efficiently and safely with ofatumumab. This alternative should be further evaluated for sAID.