Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages.

The current chapter focuses on the use of filamentous phages to display and modify biologically active cytokines, with special emphasis on directed evolution of novel variants showing improved receptor binding. Cytokines are essential protein mediators involved in cell-to-cell communication. Their functional importance and the complexity of their interactions with multichain receptors make cytokine engineering a promising tool for the discovery and optimization of therapeutic molecules. Protocols used at the laboratory are illustrated through examples of manipulation of interleukin-2 and interleukin-6, two members of the family of alpha-helix-bundle cytokines playing pivotal roles in immunity and inflammation.

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

Diagnóstico de la Enfermedad de Chagas congénito: Aspectos relevantes

Introducción: El presente artículo describe aspectos relevantes entorno de la Enfermedad de Chagas congénita, tales como epidemiología, sintomatología, revisión de casos clínicos y las técnicas diagnósticas. Métodos: Se realizó una revisión de la literatura por medio de bases de datos bibliográficas como PubMed, Science direct, Scopus, Plos One, SciELO, teniendo como criterio de inclusión las publicaciones artículos o comprendidos entre enero de 2013 y enero del año 2022 en idioma español e inglés. Resultados: Se determinó que la prevalencia de la Enfermedad de Chagas congénita aún es un problema de salud pública en áreas endémicas y no endémicas, siendo la serología materna indispensable para dar seguimiento oportuno a los casos. Conclusiones: Los seguimientos diagnósticos actuales difieren en los países endémicos y se están aplicando tamizajes en zonas no endémicas donde migran mujeres procedentes de áreas de trasmisión activa de la Enfermedad Chagásica.

Introduction: This article describes relevant aspects of congenital Chagas disease, such as epidemiology, symptoms, review of clinical cases, and diagnostic techniques. Methods: A review of the literature was carried out through bibliographic databases such as PubMed, Science direct, Scopus, Plos One, SciELO, having as inclusion criteria articles or publications between January 2013 and January 2022 in Spanish and English. Results: It was determined that the prevalence of congenital Chagas disease is still a public health problem in endemic and non-endemic areas, and maternal serology is essential for timely monitoring of cases. Conclusions: Current diagnostic follow-ups differ in endemic countries and screening is being applied in non-endemic areas where women from areas of active transmission of Chagasic disease migrate.

Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies.

Background: Antitumor therapies targeting HER1/EGFR and HER2, such as monoclonal antibodies (MAbs) and tyrosine-kinase inhibitors (TKIs), have demonstrated a significant clinical benefit, but the emergence of resistance limits long-term efficacy. While secondary HER1 mutations confer tolerance to TKI, compensatory upregulation of HER2 drives resistance to anti-HER1 MAbs, which identifies MAb combinations targeting both receptors as an attractive therapeutic strategy. Nevertheless, toxicity hampers the clinical validation of this approach. Alternatively, cancer vaccines may induce antibodies directed against several antigens with less concern about induced toxicity. Methods: Polyclonal antibodies (PAbs) targeting HER1 and HER2 were induced in mice or rabbits through immunization. Recognition of different epitopes on targets by PAbs was validated by phage-display technology. Receptor downregulation was evaluated by flow cytometry, immunofluorescence, and Western blot. MTT assays assessed cytotoxicity, while the antitumor effect of PAbs was assayed in nude mice. Results: PAbs promoted degradation of HER1 and HER2 regarding clinical MAbs or their combinations. As a result, inhibition of cytotoxicity on tumor cell lines was improved, even in the presence of oncogenic mutations in HER1, as well as in cetuximab-insensitive cells. Accordingly, the antitumor effect of vaccination-induced PAbs was observed in lung tumor lines representative of sensitivity or resistance to HER1 targeting therapies. Conclusions: Immunization against HER1 and HER2 receptors offers an alternative to passive administration of combinations of MAbs, since vaccination-induced PAbs promote the downregulation of both receptors and they have a higher impact on the survival of tumor cells.

Domain-level epitope mapping of polyclonal antibodies against HER-1 and HER-2 receptors using phage display technology.

HER-1 and HER-2 are tumor-associated antigens overexpressed in several epithelial tumors, and successfully targeted by therapeutic approaches against cancer. Vaccination with their recombinant extracellular domains has had encouraging results in the pre-clinical setting. As complex humoral responses targeting multiple epitopes within each antigen are the ultimate goal of such active immunotherapy strategies, molecular dissection of the mixture of antibody specificities is required. The current work exploits phage display of antigenic versions of HER-1 and HER-2 domains to accomplish domain-level epitope mapping. Recognition of domains I, III and IV of both antigens by antibodies of immunized mice was shown, indicating diverse responses covering a broad range of antigenic regions. The combination of phage display and site-directed mutagenesis allowed mutational screening of antigen surface, showing polyclonal antibodies' recognition of mutated receptor escape variants known to arise in patients under the selective pressure of the anti-HER-1 antibody cetuximab. Phage-displayed HER domains have thus the potential to contribute to fine specificity characterization of humoral responses during future development of anti-cancer vaccines.

Impact of Change in Allocation Score Methodology on Post Kidney Transplant Average Length of Stay.

Background: In December 2014, a new Kidney Allocation System (KAS) was implemented nationwide to improve access and quality of care to historically disadvantaged patients. However, no study to date has examined the relationship between the KAS and potential changes in hospital length of stay (LOS). This study aimed to examine the relationship between the KAS implemented in December 2014 and potential changes in hospital LOS. Methods: We used data from the Florida Agency for Health Care Administration on kidney transplant surgeries completed between 2011 and 2018. A cross-sectional cohort study design included seven hospitals that performed kidney transplants for the duration of the study. A propensity score matching approach was used to examine the relationship between KAS and LOS. All acute general medical and surgical hospitals in Florida that performed kidney transplant surgery were included in the analysis. Results: We included 7,795 patients, 6,119 discharged to home, and 1,676 discharged to home with home health services after transplant. The average LOS prior to KAS was 6.52 days and 6.08 days post KAS. Propensity matched results show that patients transferred to home experienced a decrease in the LOS (coefficient (ß) = -0.68; 95% confidence interval (CI): -0.95, -0.42) after the new allocation score was implemented. Similarly, patients transferred to home with home health experienced a decrease in the LOS (ß = -1.90; 95% CI: -2.69, -1.11) after the new allocation was implemented. Conclusion: In conclusion, results indicate that KAS implementation did not add a burden on the health system by increasing LOS when considering patients with similar characteristics before and after KAS implementation. KAS is an important policy change that appears to not negatively affect the LOS when sicker patients could receive a kidney transplant. Our findings improve our understanding of the KAS policy and its influence on the health system.

Patients seeking stem cell therapies-a prospective qualitative analysis from a Regenerative Medicine Consult Service.

Despite patient demand for stem cell therapies (SCTs) for musculoskeletal conditions, there remains limited research on why patients seek SCTs or their sources of information. We employ three questions into a consult intake form: (1) Why are you interested in stem cell treatment for your condition? (2) How did you find out about stem cell treatment for your condition? (3) Have you contacted a stem cell clinic? Responses analyzed, using a qualitative content analysis approach to identify themes reveal many patients seek SCTs to treat pain or delay surgery which may align with some current clinical evidence while other patients express motivations as expected outcomes (e.g., SCTs are better than standard of care or can regenerate tissue) which are not supported by current medical evidence. These differences suggests that patient-centered counseling may help patients by addressing misconceptions and increasing health literacy about expected outcomes of SCTs for treating musculoskeletal conditions.

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI-MS spectrum.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.

Opioid Use in Fibromyalgia Continues Despite Guidelines That Do Not Support Its Efficacy or Risk.

BACKGROUND/OBJECTIVE: The aim of this cross-sectional study is to determine the prevalence of opioid use in a large sample of fibromyalgia (FM) patients and examine the factors associated with opioid prescription/use despite multiple clinical guidelines that do not recommend opioid use in this population. METHODS: Data were collected from a convenience sample of 698 patients admitted from August 2017 to May 2019 into an intensive 2-day Fibromyalgia Treatment Program at a tertiary medical center in the United States after FM diagnosis. Patients were administered the Fibromyalgia Impact Questionnaire-Revised, the Center for Epidemiologic Study of Depression Scale, and the Pain Catastrophizing Scale upon admission to the program. Demographic information and opioid use were self-reported. Logistic regression analysis was utilized to determine associations between patient-related variables and opioid use in this prospective study. RESULTS: Of 698 patients, 27.1% (n = 189) were taking opioids at intake. Extended duration of symptoms (>3 years), increased age, higher degree of functional impairment, and increased pain catastrophizing were significantly associated with opioid use. CONCLUSIONS: Opioids are not recommended for the treatment of FM under current guidelines. Greater burden of illness appeared to be associated with the prescription and use of opioids in this population. These findings suggest that some providers may not be aware of current recommendations that have been found to be effective in the management of FM that are contained in guidelines. Alternative approaches to the management of FM that do not involve opioids are reviewed in an effort to improve care.

Innate immunity in vertebrates: an overview.

Innate immunity is a semi-specific and widely distributed form of immunity, which represents the first line of defence against pathogens. This type of immunity is critical to maintain homeostasis and prevent microbe invasion, eliminating a great variety of pathogens and contributing with the activation of the adaptive immune response. The components of innate immunity include physical and chemical barriers, humoral and cell-mediated components, which are present in all jawed vertebrates. The understanding of innate defence mechanisms in non-mammalian vertebrates is the key to comprehend the general picture of vertebrate innate immunity and its evolutionary history. This is also essential for the identification of new molecules with applications in immunopharmacology and immunotherapy. In this review, we describe and discuss the main elements of vertebrate innate immunity, presenting core findings in this field and identifying areas that need further investigation.