Almost 50 Years of Monomeric Extracellular Ubiquitin (eUb).

Monomeric ubiquitin (Ub) is a 76-amino-acid highly conserved protein found in eukaryotes. The biological activity of Ub first described in the 1970s was extracellular, but it quickly gained relevance due to its intracellular role, i.e., post-translational modification of intracellular proteins (ubiquitination) that regulate numerous eukaryotic cellular processes. In the following years, the extracellular role of Ub was relegated to the background, until a correlation between higher survival rate and increased serum Ub concentrations in patients with sepsis and burns was observed. Although the mechanism of action (MoA) of extracellular ubiquitin (eUb) is not yet well understood, further studies have shown that it may ameliorate the inflammatory response in tissue injury and multiple sclerosis diseases. These observations, compounded with the high stability and low immunogenicity of eUb due to its high conservation in eukaryotes, have made this small protein a relevant candidate for biotherapeutic development. Here, we review the in vitro and in vivo effects of eUb on immunologic, cardiovascular, and nervous systems, and discuss the potential MoAs of eUb as an anti-inflammatory, antimicrobial, and cardio- and brain-protective agent.

Efficacy, Pharmacokinetics, and Toxicity Profiles of a Broad Anti-SARS-CoV-2 Neutralizing Antibody.

We recently reported the isolation and characterization of an anti-SARS-CoV-2 antibody, called IgG-A7, that protects transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from an infection with SARS-CoV-2 Wuhan. We show here that IgG-A7 protected 100% of the transgenic mice infected with Delta (B.1.617.2) and Omicron (B.1.1.529) at doses of 0.5 and 5 mg/kg, respectively. In addition, we studied the pharmacokinetic (PK) profile and toxicology (Tox) of IgG-A7 in CD-1 mice at single doses of 100 and 200 mg/kg. The PK parameters at these high doses were proportional to the doses, with serum half-life of ~10.5 days. IgG-A7 was well tolerated with no signs of toxicity in urine and blood samples, nor in histopathology analyses. Tissue cross-reactivity (TCR) with a panel of mouse and human tissues showed no evidence of IgG-A7 interaction with the tissues of these species, supporting the PK/Tox results and suggesting that, while IgG-A7 has a broad efficacy profile, it is not toxic in humans. Thus, the information generated in the CD-1 mice as a PK/Tox model complemented with the mouse and human TCR, could be of relevance as an alternative to Non-Human Primates (NHPs) in rapidly emerging viral diseases and/or quickly evolving viruses such as SARS-CoV-2.

Discovery and Optimization of Neutralizing SARS-CoV-2 Antibodies Using ALTHEA Gold Plus Libraries™.

We recently reported the isolation and characterization of anti-SARS-CoV-2 antibodies from a phage display library built with the VH repertoire of a convalescent COVID-19 patient, paired with four naïve synthetic VL libraries. One of the antibodies, called IgG-A7, neutralized the Wuhan, Delta (B.1.617.2) and Omicron (B.1.1.529) strains in authentic neutralization tests (PRNT). It also protected 100% transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from SARS-CoV-2 infection. In this study, the four synthetic VL libraries were combined with the semi-synthetic VH repertoire of ALTHEA Gold Libraries™ to generate a set of fully naïve, general-purpose, libraries called ALTHEA Gold Plus Libraries™. Three out of 24 specific clones for the RBD isolated from the libraries, with affinity in the low nanomolar range and sub-optimal in vitro neutralization in PRNT, were affinity optimized via a method called "Rapid Affinity Maturation" (RAM). The final molecules reached sub-nanomolar neutralization potency, slightly superior to IgG-A7, while the developability profile over the parental molecules was improved. These results demonstrate that general-purpose libraries are a valuable source of potent neutralizing antibodies. Importantly, since general-purpose libraries are "ready-to-use", it could expedite isolation of antibodies for rapidly evolving viruses such as SARS-CoV-2.

Brucella abortus induces mast cell activation through TLR-2 and TLR-4.

The Gram-negative bacteria Brucella abortus is a major cause of brucellosis in animals and humans. The host innate immune response to B. abortus is mainly associated with phagocytic cells such as dendritic cells, neutrophils, and macrophages. However, as mast cells naturally reside in the main bacterial entry sites they may be involved in bacterial recognition. At present, little is known about the role of mast cells during B. abortus infection. The role of the innate immune receptors TLR2 and TLR4 in activation of mast cells by B. abortus (strain RB51) infection was analyzed in this study. The results showed that B. abortus did not induce mast cell degranulation, but did induce the synthesis of the cytokines IL-1ß, IL-6, TNF-α, CCL3, CCL4, and CCL5. Furthermore, B. abortus stimulated key cell signaling molecules involved in mast cell activation such as p38 and NF-κB. Blockade of the receptors TLR2 and TLR4 decreased TNF-α and IL-6 release by mast cells in response to B. abortus. Taken together, our results demonstrate that mast cells are activated by B. abortus and may play a role in inducing an inflammatory response during the initial phase of the infection.

Role of CD36 in cancer progression, stemness, and targeting.

CD36 is highly expressed in diverse tumor types and its expression correlates with advanced stages, poor prognosis, and reduced survival. In cancer cells, CD36: 1) increases fatty acid uptake, reprogramming lipid metabolism; 2) favors cancer cell proliferation, and 3) promotes epithelial-mesenchymal transition. Furthermore, CD36 expression correlates with the expression of cancer stem cell markers and CD36+ cancer cells display increased stemness functional properties, including clonogenicity, chemo- and radioresistance, and metastasis-initiating capability, suggesting CD36 is a marker of the cancer stem cell population. Thus, CD36 has been pointed as a potential therapeutic target in cancer. At present, at least three different types of molecules have been developed for reducing CD36-mediated functions: blocking monoclonal antibodies, small-molecule inhibitors, and compounds that knock-down CD36 expression. Herein, we review the role of CD36 in cancer progression, its participation in stemness control, as well as the efficacy of reported CD36 inhibitors in cancer cell cultures and animal models. Overall, the evidence compiled points that CD36 is a valid target for the development of new anti-cancer therapies.

In Vitro and In Vivo Characterization of a Broadly Neutralizing Anti-SARS-CoV-2 Antibody Isolated from a Semi-Immune Phage Display Library.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 have shown reduced efficacy or have failed to neutralize the variants of concern (VOCs), particularly B.1.1.529 (Omicron). Previously, we reported the discovery and characterization of antibodies with high affinity for SARS-CoV-2 RBD Wuhan (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) strains. One of the antibodies, called IgG-A7, also blocked the interaction of human angiotensin-converting enzyme 2 (hACE2) with the RBDs of the three strains, suggesting it may be a broadly SARS-CoV-2 neutralizing antibody. Herein, we show that IgG-A7 efficiently neutralizes all the three SARS-CoV-2 strains in plaque reduction neutralization tests (PRNTs). In addition, we demonstrate that IgG-A7 fully protects K18-hACE2 transgenic mice infected with SARS-CoV-2 WT. Taken together, our findings indicate that IgG-A7 could be a suitable candidate for development of antibody-based drugs to treat and/or prevent SARS-CoV-2 VOCs infection.

Valproic acid restricts mast cell activation by Listeria monocytogenes.

Mast cells (MC) play a central role in the early containment of bacterial infections, such as that caused by Listeria monocytogenes (L.m). The mechanisms of MC activation induced by L.m infection are well known, so it is possible to evaluate whether they are susceptible to targeting and modulation by different drugs. Recent evidence indicates that valproic acid (VPA) inhibits the immune response which favors L.m pathogenesis in vivo. Herein, we examined the immunomodulatory effect of VPA on L.m-mediated MC activation. To this end, bone marrow-derived mast cells (BMMC) were pre-incubated with VPA and then stimulated with L.m. We found that VPA reduced MC degranulation and cytokine release induced by L.m. MC activation during L.m infection relies on Toll-Like Receptor 2 (TLR2) engagement, however VPA treatment did not affect MC TLR2 cell surface expression. Moreover, VPA was able to decrease MC activation by the classic TLR2 ligands, peptidoglycan and lipopeptide Pam3CSK4. VPA also reduced cytokine production in response to Listeriolysin O (LLO), which activates MC by a TLR2-independent mechanism. In addition, VPA decreased the activation of critical events on MC signaling cascades, such as the increase on intracellular Ca2+ and phosphorylation of p38, ERK1/2 and -p65 subunit of NF-κB. Altogether, our data demonstrate that VPA affects key cell signaling events that regulate MC activation following L.m infection. These results indicate that VPA can modulate the functional activity of different immune cells that participate in the control of L.m infection.

Evolution of Anti-SARS-CoV-2 Therapeutic Antibodies.

Since the first COVID-19 reports back in December of 2019, this viral infection caused by SARS-CoV-2 has claimed millions of lives. To control the COVID-19 pandemic, the Food and Drug Administration (FDA) and/or European Agency of Medicines (EMA) have granted Emergency Use Authorization (EUA) to nine therapeutic antibodies. Nonetheless, the natural evolution of SARS-CoV-2 has generated numerous variants of concern (VOCs) that have challenged the efficacy of the EUA antibodies. Here, we review the most relevant characteristics of these therapeutic antibodies, including timeline of approval, neutralization profile against the VOCs, selection methods of their variable regions, somatic mutations, HCDR3 and LCDR3 features, isotype, Fc modifications used in the therapeutic format, and epitope recognized on the receptor-binding domain (RBD) of SARS-CoV-2. One of the conclusions of the review is that the EUA therapeutic antibodies that still retain efficacy against new VOCs bind an epitope formed by conserved residues that seem to be evolutionarily conserved as thus, critical for the RBD:hACE-2 interaction. The information reviewed here should help to design new and more efficacious antibodies to prevent and/or treat COVID-19, as well as other infectious diseases.

Anti-SARS-CoV-2 Omicron Antibodies Isolated from a SARS-CoV-2 Delta Semi-Immune Phage Display Library.

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile.

Development and Evaluation of a Set of Spike and Receptor Binding Domain-Based Enzyme-Linked Immunosorbent Assays for SARS-CoV-2 Serological Testing.

The implementation and validation of anti-SARS-CoV-2 IgG serological assays are reported in this paper. S1 and RBD proteins were used to coat ELISA plates, and several secondary antibodies served as reporters. The assays were initially validated with 50 RT-PCR positive COVID-19 sera, which showed high IgG titers of mainly IgG1 isotype, followed by IgG3. Low or no IgG2 and IgG4 titers were detected. Then, the RBD/IgG assay was further validated with 887 serum samples from RT-PCR positive COVID-19 individuals collected at different times, including 7, 14, 21, and 40 days after the onset of symptoms. Most of the sera were IgG positive at day 40, with seroconversion happening after 14-21 days. A third party conducted an additional performance test of the RBD/IgG assay with 406 sera, including 149 RT-PCR positive COVID-19 samples, 229 RT-PCR negative COVID-19 individuals, and 28 sera from individuals with other viral infections not related to SARS-CoV-2. The sensitivity of the assay was 99.33%, with a specificity of 97.82%. All the sera collected from individuals with infectious diseases other than COVID-19 were negative. Given the robustness of this RBD/IgG assay, it received approval from the sanitary authority in Mexico (COFEPRIS) for production and commercialization under the name UDISTEST-V2G®.

TLR2 Regulates Mast Cell IL-6 and IL-13 Production During Listeria monocytogenes Infection.

Listeria monocytogenes (L.m) is efficiently controlled by several cells of the innate immunity, including the Mast Cell (MC). MC is activated by L.m inducing its degranulation, cytokine production and microbicidal mechanisms. TLR2 is required for the optimal control of L.m infection by different cells of the immune system. However, little is known about the MC receptors involved in recognizing this bacterium and whether these interactions mediate MC activation. In this study, we analyzed whether TLR2 is involved in mediating different MC activation responses during L.m infection. We found that despite MC were infected with L.m, they were able to clear the bacterial load. In addition, MC degranulated and produced ROS, TNF-α, IL-1ß, IL-6, IL-13 and MCP-1 in response to bacterial infection. Interestingly, L.m induced the activation of signaling proteins: ERK, p38 and NF-κB. When TLR2 was blocked, L.m endocytosis, bactericidal activity, ROS production and mast cell degranulation were not affected. Interestingly, only IL-6 and IL-13 production were affected when TLR2 was inhibited in response to L.m infection. Furthermore, p38 activation depended on TLR2, but not ERK or NF-κB activation. These results indicate that TLR2 mediates only some MC activation pathways during L.m infection, mainly those related to IL-6 and IL-13 production.

Severe COVID-19 is marked by dysregulated serum levels of carboxypeptidase A3 and serotonin.

The immune response plays a critical role in the pathophysiology of SARS-CoV-2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS-CoV-2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID-19 patients. We noticed that SARS-CoV-2-infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS-CoV-2-negative patients. CPA3 levels correlated with C-reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID-19 patients, whereas serotonin was a good predictor of SARS-CoV-2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS-CoV-2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID-19 and may represent targets for therapeutic intervention.

LPA1 Receptor Promotes Progesterone Receptor Phosphorylation through PKCα in Human Glioblastoma Cells.

Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA1 receptor is expressed in GBM cells and its signaling pathways activate protein kinases C (PKCs). A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.

Quality attributes of partially hydrolyzed collagen in a liquid formulation used for skin care.

BACKGROUND: The deterioration of the skin accentuates over time, affecting its aesthetic appearance. This is characterized by the weakening of the mechanisms involved in the regeneration and repair of the dermal matrix. Consequently, the skin losses elasticity and smoothness resulting in the formation of wrinkles. The alternatives for facial rejuvenation include surgery, injection of botulinum toxin, and the application of masks. Topic products are less invasive, can be self-applied, and have an increased benefit/risk relationship. AIM: We developed a liquid formulation containing collagen hydrolyzed and evaluated the product by cutting-edge technology in order to define proper its quality attributes. METHODS: We employed nuclear magnetic resonance (NMR), size-exclusion chromatography (SEC), and mass spectrometry (MS). Additionally, we analyzed its cosmetical effect in five volunteers and we demonstrate the product safety. RESULTS: Our results demonstrate the following: (a) a stable secondary structure identity associated to the known triple helix arrangement in liquid and solid states; (b) a typical conformational flexibility depending on its hydration state; (c) thermal stability confirmed by liquid- and solid-state nuclear magnetic resonance schemes; and (d) a molecular mass distribution of peptides between 0.5 and 19.5 kDa. The product faded wrinkles in the forehead, an effect that remained after removing the mask. The formula was non-irritating and hypoallergenic. CONCLUSION: We characterized, using state-of-the-art methodologies, the quality attributes that are critical for the safety and beneficial effect of a new collagen-containing formula.

Antibody-drug conjugates: the new generation of biotechnological therapies against cancer.

Therapeutic antibodies are recombinant proteins used in the treatment of cancer. There is a new generation of monoclonal antibodies with activity against cancer cells, known as antibody-drug conjugates. These molecules are made up of three elements: a monoclonal antibody, a highly potent cytotoxic drug, and a chemical linker that binds them together. The antibody recognizes tumor antigens, thereby allowing targeted delivery of the cytotoxic agent to cancer cells. After recognizing its antigen, the antibody-drug conjugate is endocytosed by the target cells, where the protein fraction is degraded into lysosomes, releasing the cytotoxic drug. This article reviews antibody-drug conjugates general characteristics and describes the clinical evidence of efficacy and safety of the first four approved by regulatory agencies in the United States and Europe.

Los anticuerpos terapéuticos son proteínas recombinantes empleadas en el tratamiento del cáncer. Existe una nueva ­generación de anticuerpos monoclonales con actividad contra las células cancerosas, conocidos como anticuerpos conjugados a fármacos. Estas moléculas están integradas por tres elementos: un anticuerpo monoclonal, un fármaco citotóxico con alta potencia y un enlazador químico que los une. El anticuerpo reconoce antígenos tumorales, por lo que permite la entrega dirigida del agente citotóxico hacia las células cancerosas. Tras el reconocimiento de su antígeno, el anticuerpo conjugado a fármaco es endocitado por las células blanco, donde se induce la degradación lisosomal de la fracción proteica y se libera el fármaco citotóxico. En el presente artículo se revisan las características generales de los anticuerpos conjugados a fármacos y se describe la evidencia clínica de la eficacia y seguridad de los primeros cuatro aprobados por las agencias reguladoras de Estados Unidos y Europa.

Pranlukast Antagonizes CD49f and Reduces Stemness in Triple-Negative Breast Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. MATERIALS AND METHODS: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. RESULTS: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with ß1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. CONCLUSION: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

Anticuerpos conjugados a fármaco: la nueva generación de terapias biotecnológicas contra el cáncer / Antibody-drug conjugates: the new generation of biotechnological therapies against cancer

Resumen Los anticuerpos terapéuticos son proteínas recombinantes empleadas en el tratamiento del cáncer. Existe una nueva generación de anticuerpos monoclonales con actividad contra las células cancerosas, conocidos como anticuerpos conjugados a fármacos. Estas moléculas están integradas por tres elementos: un anticuerpo monoclonal, un fármaco citotóxico con alta potencia y un enlazador químico que los une. El anticuerpo reconoce antígenos tumorales, por lo que permite la entrega dirigida del agente citotóxico hacia las células cancerosas. Tras el reconocimiento de su antígeno, el anticuerpo conjugado a fármaco es endocitado por las células blanco, donde se induce la degradación lisosomal de la fracción proteica y se libera el fármaco citotóxico. En el presente artículo se revisan las características generales de los anticuerpos conjugados a fármacos y se describe la evidencia clínica de la eficacia y seguridad de los primeros cuatro aprobados por las agencias reguladoras de Estados Unidos y Europa.

Abstract Therapeutic antibodies are recombinant proteins used in the treatment of cancer. There is a new generation of monoclonal antibodies with activity against cancer cells, known as antibody-drug conjugates. These molecules are made up of three elements: a monoclonal antibody, a highly potent cytotoxic drug, and a chemical linker that binds them together. The antibody recognizes tumor antigens, thereby allowing targeted delivery of the cytotoxic agent to cancer cells. After recognizing its antigen, the antibody-drug conjugate is endocytosed by the target cells, where the protein fraction is degradated into lysosomes, releasing the cytotoxic drug. This article reviews antibody-drug conjugates general characteristics and describes the clinical evidence of efficacy and safety of the first four approved by regulatory agencies in the United States and Europe.

Heptapeptide HP3 acts as a potent inhibitor of experimental imiquimod­induced murine psoriasis and impedes the trans­endothelial migration of mononuclear cells.

During the progression of psoriatic lesions, abundant cellular infiltration of myeloid cells, such as macrophages and activated dendritic cells, occurs in the skin and the infiltrating cells interact with naive lymphoid cells to generate a T helper (Th)1 and Th17 environment. Therapies to treat psoriasis include phototherapy, non­steroidal and steroidal drugs, as well as antibodies to block tumor necrosis factor­α, interleukin (IL)­17­A and IL­12/IL­23, which all focus on decreasing the proinflammatory hallmark of psoriasis. The present study obtained the heptapeptide HP3 derived from phage display technology that blocks mononuclear cell adhesion to endothelial cells and inhibits trans­endothelial migration in vitro. The activity of the heptapeptide in a murine model of psoriasis was also assessed, which indicated that early administration inhibited the development of psoriatic lesions. Therefore, the results suggested that HP3 may serve as a potential therapeutic target for psoriasis.

Identity Profiling of Complex Mixtures of Peptide Products by Structural and Mass Mobility Orthogonal Analysis.

Identity is a critical quality attribute that must be determined before releasing batches of medicinal and dietary products. However, the identities of peptide-derived products composed of a large number of diverse molecules is challenging since most analytical techniques cannot analyze multiple molecules simultaneously. Here, we proposed the determination of the weight-average molecular weight (Mw) and polydispersity index (PDI) by mass spectrometry for control quality for the batch release of complex products, namely, glatiramer acetate (Copaxone), collagen hydrolysate (Colagenart), and a human dialyzable leucocyte extract (Transferon). The Mw and PDI values were orthogonally determined by PFG-STE-H2O(presaturation)-DOSY-NMR analysis. To the best of our knowledge, this is the first time that MS and NMR spectra have been combined to determine the PDI of complex products derived from protein hydrolysis that are not monodisperse. The performance of each method was evaluated by comparing the obtained results to those reported for glatiramer acetate using MALLS, the technique commonly employed to determine PDI. This combined approach demonstrates the ability of these techniques to separate peptide populations from complex mixtures to establish their identity through their mass distribution profiles.

CD80 Expression Correlates with IL-6 Production in THP-1-Like Macrophages Costimulated with LPS and Dialyzable Leukocyte Extract (Transferon®).

Transferon® is a complex drug based on a mixture of low molecular weight peptides. This biotherapeutic is employed as a coadjuvant in clinical trials of several diseases, including viral infections and allergies. Given that macrophages play key roles in pathogen recognition, phagocytosis, processing, and antigen presentation, we evaluated the effect of Transferon® on phenotype and function of macrophage-like cells derived from THP-1 monocytes. We determined the surface expression of CD80 and CD86 by flow cytometry and IL-1ß, TNF-α, and IL-6 levels by ELISA. Transferon® alone did not alter the steady state of PMA-differentiated macrophage-like THP-1 cells. On the contrary, simultaneous stimulation of cells with Transferon® and LPS elicited a significant increase in CD80 (P ≤ 0.001) and CD86 (P ≤ 0.001) expression, as well as in IL-6 production (P ≤ 0.05) compared to the LPS control. CD80 expression and IL-6 production exhibited a positive correlation (r = 0.6, P ≤ 0.05) in cells exposed to Transferon® and LPS. Our results suggest that the administration of Transferon® induces the expression of costimulatory molecules and the secretion of cytokines in LPS-activated macrophages. Further studies are necessary to determine the implication of these findings in the therapeutic properties of Transferon®.