Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge.

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCV-Sf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

Crystal structure of propane-1,3-diaminium squarate dihydrate.

Propane-1,3-diaminium squarate dihydrate, C3H12N2 2+·C4O4 2-·2H2O, results from the proton-transfer reaction of propane-1,3-di-amine with squaric acid and subsequent crystallization from aqueous medium. The title compound crystallizes in the tetra-gonal crystal system (space group P4bm) with Z = 2. The squarate dianion belongs to the point group D 4h and contains a crystallographic fourfold axis. The propane-1,3-diaminium dication exhibits a C 2v -symmetric all-anti conformation and resides on a special position with mm2 site symmetry. The orientation of the propane-1,3-diaminium ions makes the crystal structure polar in the c-axis direction. The solid-state supra-molecular structure features a triperiodic network of strong hydrogen bonds of the N-H⋯O and O-H⋯O types.

Study on cellulose nanofibrils/copolymacrolactone based nano-composites with hydrophobic behaviour, self-healing ability and antioxidant activity.

The multiple uses of cellulose nanofibrils (CNFs) originate from their availability from renewable resources, and are due to their physico-chemical properties, biodegradability and biocompatibility. At the same time, reducing sensitivity to humidity, increasing interfacial adhesion and hydrophobic modification of the CNF surface to diversify applications and improve operation, are current targets pursued. This study focuses on the preparation of a novel gel structure using cellulose nanofibrils (CNFs) and poly(ethylene brassylate-co-squaric acid) (PEBSA50/50), a bio-based copolymacrolactone. The primary goal is to achieve the gel with reduced sensitivity to humidity and enhanced hydrophobic behaviour. The new system was characterized in comparison to its constituent components using various techniques, such as Fourier transform infrared spectroscopy, thermal analysis, X-ray diffraction, and NIR - chemical imaging. Rheological tests demonstrated the formation of the CNF_PEBSA50/50 gel as a result of physical interactions between the two polymeric partners and revealed self-healing abilities for the prepared gels. Determination of the contact angle, surface free energy, as well as dynamic measurements of the vapour sorption of the CNF_PEBSA50/50 system, confirmed the achievement of the study's aim. Furthermore, the CNF_PEBSA50/50 network was utilized to encapsulate citric acid, resulting in the creation of a new bioactive composite with both antioxidant and antimicrobial activity.

Synthesis and evaluation of druglike parameters via in silico techniques for a series of heterocyclic monosquarate-amide derivatives as potential carboxylic acid bioisosteres.

Herein, we present a synthetic compound library comprising of 13 structurally diverse heterocyclic monosquarate-amide derivatives. The compounds featured in this library were designed as potential bioisosteric replacements carboxylic acid moiety's. A good selection of the compounds presented exhibit unique molecular architecture and have shown promising results following in silico evaluation of 'druglike properties' using Swiss ADME. The research presented in this work focuses on the preparation of derivatives of 3,4-dihydroxycyclobut-3-ene-1,2-dione, a known carboxylic acid bioisostere.

Modification of the Human Amniotic Membrane Using Different Cross-Linking Agents as a Promising Tool for Regenerative Medicine.

Human amniotic membranes (hAMs) obtained during cesarean sections have proven to be clinically useful as an interesting biomaterial in a wide range of tissue engineering applications such as ocular surface reconstruction, burn treatments, chronic wounds, or bedsore ulcers. It presents antimicrobial properties, promotes epithelization, reduces inflammation and angiogenesis, contains growth factors, and constitutes the reservoir of stem cells. However, variability in hAM stiffness and its fast degradation offers an explanation for the poor clinical applications and reproducibility. In addition, the preparatory method of hAM for clinical use can affect its mechanical properties, and these differences can influence its application. As a directly applied biomaterial, the hAM should be available in a ready-to-use manner in clinical settings. In the present study, we performed an analysis to improve the mechanical properties of hAM by the addition of various reagents used as protein cross-linkers: EDC/NHS, PEG-dialdehyde, PEG-NHS, dialdehyde starch, and squaric acid. The effect of hAM modification using different cross-linking agents was determined via infrared spectroscopy, thermal analyses, mechanical properties analyses, enzymatic degradation, and cytotoxicity tests. The use of PEG-dialdehyde, PEG-NHS, dialdehyde starch, and squaric acid increases the mechanical strength and elongation at the breaking point of hAM, while the addition of EDC/NHS results in material stiffening and shrinkage. Also, the thermal stability and degradation resistance were evaluated, demonstrating higher values after cross-linking. Overall, these results suggest that modification of human amniotic membrane by various reagents used as protein cross-linkers may make it easier to use hAM in clinical applications, and the presented study is a step forward in the standardization of the hAM preparation method.

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

Preparation of an Antioxidant Assembly Based on a Copolymacrolactone Structure and Erythritol following an Eco-Friendly Strategy.

The study presents the achievement of a new assembly with antioxidant behaviour based on a copolymacrolactone structure that encapsulates erythritol (Eryt). Poly(ethylene brassylate-co-squaric acid) (PEBSA) was synthesised in environmentally friendly conditions, respectively, through a process in suspension in water by opening the cycle of ethylene brassylate macrolactone, followed by condensation with squaric acid. The compound synthesised in suspension was characterised by comparison with the polymer obtained by polymerisation in solution. The investigations revealed that, with the exception of the molecular masses, the compounds generated by the two synthetic procedures present similar properties, including good thermal stability, with a Tpeak of 456 °C, and the capacity for network formation. In addition, the investigation by dynamic light scattering techniques evidenced a mean diameter for PEBSA particles of around 596 nm and a zeta potential of -25 mV, which attests to their stability. The bio-based copolymacrolactone was used as a matrix for erythritol encapsulation. The new PEBSA-Eryt compound presented an increased sorption/desorption process, compared with the PEBSA matrix, and a crystalline morphology confirmed by X-ray diffraction analysis. The bioactive compound was also characterised in terms of its biocompatibility and antioxidant behaviour.

Development of a new polymer network system carrier of essential oils.

The study presents the development of a new copolymacrolactone structure based on ethylene brassylate (EB) and squaric acid (SA) with different ratios between comonomers. The new system was tested as a network for essential oils encapsulation. The structure of the copolymers was confirmed by spectroscopic investigations and correlated in interdependence with the comonomers content. The interfacial characteristics of the poly(ethylene brassylate-co-squaric acid) copolymers were determined, and the transition from a moderate hydrophilic surface towards a hydrophobic region by increasing the molar content of SA comonomer was highlighted. The affinity for hydrophobic substances of the synthesised macromolecular compounds was used in a process of encapsulation of thymol (Thy) and carvacrol (CC). The newly prepared bioactive compounds were characterised by in vivo biocompatibility tests, and antimicrobial activity, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).

Use of Contact Immunotherapy in the Treatment of Skin Diseases Other than Alopecia Areata.

For decades, contact immunotherapy with dinitrochlorobenzene, diphencyprone, and squaric acid dibutylester has played an important role in both clinical practice and scientific research. It is listed as the first-line treatment for extensive alopecia areata and was more recently approved for melanoma treatment as an orphan drug in the USA. Moreover, owing to the relative low cost and safety, topical immunotherapy has also been used in many infectious, neoplastic, and inflammatory dermatological diseases. It is especially valuable in vulnerable groups, for cosmetic/pain sensitive areas, or for multiple lesions. In this review, we summarize the current evidence supporting the use of contact immunotherapy for treatment of skin diseases, from articles collected from PubMed database. Owing to space limitation and already numerous studies focusing on alopecia areata, we include only skin diseases other than alopecia areata. In addition to diseases that have been reported to be treated by contact immunotherapy, the hypothesized mechanism, prognosis prediction, efficacy, and safety of these topical agents are discussed.

From Automated Synthesis to In Vivo Application in Multiple Types of Cancer-Clinical Results with [68Ga]Ga-DATA5m.SA.FAPi.

Radiolabeled FAPI (fibroblast activation protein inhibitors) recently gained attention as widely applicable imaging and potential therapeutic compounds targeting CAF (cancer-associated fibroblasts) or DAF (disease-associated fibroblasts in benign disorders). Moreover, the use of FAPI has distinct advantages compared to FDG (e.g., increased sensitivity in regions with high glucose metabolism, no need for fasting, and rapid imaging). In this study, we wanted to evaluate the radiochemical synthesis and the clinical properties of the new CAF-targeting tracer [68Ga]Ga-DATA5m.SA.FAPi. The compound consists of a (radio)chemically easy to use hybrid chelate DATA.SA, which can be labeled at low temperatures, making it an interesting molecule for 'instant kit-type' labeling, and a squaric acid moiety that provides distinct advantages for synthesis and radiolabeling. Our work demonstrates that automatic synthesis of the FAP inhibitor [68Ga]Ga-DATA5m.SA.FAPi is feasible and reproducible, providing convenient access to this new hybrid chelator-based tracer. Our studies demonstrated the diagnostic usability of [68Ga]Ga-DATA5m.SA.FAPi for the unambiguous detection of cancer-associated fibroblasts of various carcinomas and their metastases (NSCLC, liposarcoma, parotid tumors, prostate cancer, and pancreas adenocarcinoma), while physiological uptake in brain, liver, intestine, bone, and lungs was very low.

New Cryogels Based on Poly(vinyl alcohol) and a Copolymacrolactone System: I-Synthesis and Characterization.

Physical cryogels were obtained using the successive freeze-thaw technique of poly(vinyl alcohol) (PVA)/poly(ethylene brassylate-co-squaric acid) (PEBSA) solutions. The cryogel systems were prepared by using two different molecular weights of PVA and PEBSA with three different ratios between the ethylene brassylate (EB) and squaric acid (SA) comonomers. The presence of interactions, the thermal properties and the morphology were investigated using Fourier Transform Infrared Spectroscopy (FT-IR), thermogravimetry (TGA and DTG) and scanning electron microscopy (SEM), respectively. The influence of the composition on the degree of swelling in a physiological environment was demonstrated. The study highlighted improvements in terms of new network flexibility due to the intermolecular chains interactions brought by the introduction of PEBSA in the cryogel structure. We also concluded that the presence of PEBSA in the PVA/PEBSA cryogel network improved the loading capacity of the new system with specific hydrophobic agents, for example essential oils, which (due to their antimicrobial character) can lead to the use of new systems obtained for various applications.

Making a mouse out of a molehill: how precision modeling repurposes drugs for congenital giant nevi.

Patients with congenital giant nevi (CGN), which can compromise quality of life and progress to melanoma, have limited treatment options. Choi et al. have demonstrated that topical application of a proinflammatory hapten for alopecia treatment [squaric acid dibutylester (SADBE)] caused nevus regression and prevented melanoma in an Nras mouse CGN model. Their results demonstrate the promise of repurposing drugs through precision modeling.

Piperazine squaric acid diamides, a novel class of allosteric P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) stands out among the purinergic receptors due to its strong involvement in the regulation of tumor growth and metastasis formation as well as in innate immune responses and afferent signal transmission. Numerous studies have pointed out the beneficial effects of P2X7R antagonism for the treatment of a variety of cancer types, inflammatory diseases, and chronic pain. Herein we describe the development of novel P2X7R antagonists, incorporating piperazine squaric diamides as a central element. Besides improving the antagonists' potency from pIC50 values of 5.7-7.6, ADME properties (logD7.4 value, plasma protein binding, in vitro metabolic stability) of the generated compounds were investigated and optimized to provide novel P2X7R antagonists with drug-like properties. Furthermore, docking studies revealed the antagonists binding to the allosteric binding pocket in two distinct binding poses, depending on the substitution of the central piperazine moiety.

Therapeutic approach with squaric acid dibutylester for steroid resistant-alopecia areata incognita: A pilot study of a single center.

Topical immunotherapy is widely used in the treatment of alopecia areata (AA). Alopecia areata incognita (AAI) is a relatively common disorder, predominantly affecting females, characterized by widespread hair thinning in the absence of typical alopecic patches. AAI can have a chronic relapsing course and in some cases can be resistant to current standard treatments. Topical immunotherapy has been used in the management of AA with encouraging results, but to date there are no literature studies reporting the efficacy of topical immunotherapy with squaric acid dibutylester (SADBE) in AAI. The aim of our study is to evaluate the efficacy and tolerance of topical immunotherapy with SADBE in AAI not responding to conventional steroid therapy. A total of 12 patients were enrolled in our Hair Disease Outpatient Service, with a proved histological diagnosis of AAI, and resistant to classical steroid therapy. Each patient underwent global photography, pull test, and trichoscopy at beginning and during the follow-ups. The efficacy of topical immunotherapy with SADBE was assessed by evaluating the changes of clinical and trichoscopic signs. Complete regrowth was achieved in 66.7% of cases (8/12), three patients remained unchanged on clinical evaluation but showed subclinical improvement on trichoscopy, whereas one patient progressed and worsened both on clinical and trichoscopic examination. All patients reported scalp diffuse mild erythema and itching the day after the application of SADBE, which were well tolerated. Three patients developed reactive cervical lymphoadenomegaly. No other side effects were observed. Topical immunotherapy with SADBE is widely used in the management of patchy AA and can be considered an effective alternative in resistant AAI, providing visible clinical and trichoscopic improvement in the majority of cases. Further studies are warranted to confirm and validate our findings.

Synthesis of Poly(Ethylene Brassylate-Co-squaric Acid) as Potential Essential Oil Carrier.

Bio-based compounds are a leading direction in the context of the increased demand for these materials due to the numerous advantages associated with their use over conventional materials, which hardly degrade in the environment. At the same time, the use of essential oils and their components is generated mainly by finding alternative solutions to antibiotics and synthetic preservatives due to their bioactive characteristics, but also to their synergistic capacity during the manifestation of different biological properties. The present study is devoted to poly(ethylene brassylate-co-squaric acid) (PEBSA), synthesis and its use for thymol encapsulation and antibacterial system formation. The synthesized copolymer, performed through ethylene brassylate macrolactone ring-opening and copolymerization with squaric acid, was physicochemical characterized. Its amphiphilic character allowed the entrapment of thymol (Ty), a natural monoterpenoid phenol found in oil of thyme, a compound with strong antiseptic properties. The copolymer chemical structure was confirmed by spectroscopic analyses. Thermal analysis evidenced a good thermal stability for the copolymer. Additionally, the antimicrobial activity of PEBSA_Ty complex was investigated against eight different reference strains namely: bacterial strains-Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Enterococcus faecalis ATCC 29212, Klebsiella pneumonie ATCC 10031 and Salmonella typhimurium ATCC 14028, yeast strains represented by Candida albicans ATCC10231 and Candida glabrata ATCC 2001, and the fungal strain Aspergillus brasiliensis ATCC9642.

Application of Topical Immunotherapy in the Treatment of Alopecia Areata: A Review and Update.

Treatment of extensive or recalcitrant alopecia areata (AA) is a major clinical challenge. Even after thorough investigation of several medications, its treatment outcomes have remained unsatisfactory. While there is no US Food and Drug Administration-approved medication for AA yet, topical immunotherapy has been a well-documented treatment option. Dinitrochlorobenzene, squaric acid dibutylester, and diphenylcyclopropenone are three substances that have demonstrated efficacy in the treatment of extensive or recalcitrant AA. Despite being commonly used, the mechanism underlying topical immunotherapy is not well-elucidated and a wide range of clinical efficacies have been reported in the literature. The aim of this review was to summarize and update the pharmacology, mechanism of action, therapeutic efficacy, and tolerability of topical immunotherapy in the treatment of AA.

Examining the Impact of Squaric Acid as a Crosslinking Agent on the Properties of Chitosan-Based Films.

Hydrogels based on chitosan are very versatile materials which can be used for tissue engineering as well as in controlled drug delivery systems. One of the methods for obtaining a chitosan-based hydrogel is crosslinking by applying different components. The objective of the present study was to obtain a series of new crosslinked chitosan-based films by means of solvent casting method. Squaric acid-3,4-dihydroxy-3-cyclobutene-1,2-dione-was used as a safe crosslinking agent. The effect of the squaric acid on the structural, mechanical, thermal, and swelling properties of the formed films was determined. It was established that the addition of the squaric acid significantly improved Young's modulus, tensile strength, and thermal stability of the obtained materials. Moreover, it should be stressed that the samples consisting of chitosan and squaric acid were characterized by a higher swelling than pure chitosan. The detailed characterization proved that squaric acid could be used as a new effective crosslinking agent.

AAZTA5-squaramide ester competing with DOTA-, DTPA- and CHX-A″-DTPA-analogues: Promising tool for 177Lu-labeling of monoclonal antibodies under mild conditions.

BACKGROUND: Combining the advantages of both cyclic and acyclic chelator systems, AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) is well suited for complexation of various diagnostic and therapeutic radiometals such as gallium-68, scandium-44 and lutetium-177 under mild conditions. Due to its specificity for primary amines and pH dependent binding properties, squaric acid (SA) represents an excellent tool for selective coupling of the appropriate chelator to different target vectors. Therefore, the aim of this study was to evaluate radiolabeling properties of the novel bifunctional AAZTA5-SA being coupled to a model antibody (bevacizumab) in comparison to DOTA-SA, DTPA-p-Bn-SA and CHX-A″-DTPA-p-Bn-SA using the therapeutic nuclide lutetium-177. METHODS AND RESULTS: As proof-of-concept, bevacizumab was first functionalized with AAZTA5-SA, DOTA-SA, DTPA-p-Bn-SA or CHX-A″-DTPA-p-Bn-SA. After purification via fractionated size exclusion chromatography (SEC), the corresponding immunoconjugates were subsequently radiolabeled with lutetium-177 at pH 7 and room temperature (RT) as well as 37 °C. After 90 min, labeling of AAZTA5-SA-mAb resulted in almost quantitative radiochemical yields (RCY) of >98% and >99%, respectively. Formation of [177Lu]Lu-DTPA-p-Bn-SA-mAb indicated rapid labeling kinetics reaching similar yields at RT already after 30 min. Fast but incomplete radiolabeling of the CHX-A″-analogue could be observed with a yield of 74% after 10 min and no further significant increase. In contrast, 177Lu-labeling of DOTA-SA-mAb showed negligible radiochemical yields of <2% both at room temperature and 37 °C. In vitro complex stability measurements of [177Lu]Lu-AAZTA5-SA-mAb at 37 °C indicated >94% protein bound activity in human serum and >92% in phosphate buffered saline (PBS), respectively within 15 days. [177Lu]Lu-DTPA-p-Bn-SA-mAb and [177Lu]Lu-CHX-A″-DTPA-p-Bn-SA-mAb revealed similar to even slightly higher in vitro stability in both media. CONCLUSION: Coupling of AAZTA5-SA to the monoclonal antibody bevacizumab allowed for 177Lu-labeling with almost quantitative radiochemical yields both at room temperature and 37 °C. Within 15 days, the resulting radioconjugate indicated very high in vitro complex stability both in human serum and PBS. Therefore, AAZTA5-SA is a promising tool for 177Lu-labeling of sensitive biomolecules such as antibodies for theranostic applications.

Comparative Study of Gelatin Hydrogels Modified by Various Cross-Linking Agents.

Gelatin is a natural biopolymer derived from collagen. Due to its many advantages, such as swelling capacity, biodegradability, biocompatibility, and commercial availability, gelatin is widely used in the field of pharmacy, medicine, and the food industry. Gelatin solutions easily form hydrogels during cooling, however, the materials are mechanically poor. To improve their properties, they are often chemically crosslinked. The cross-linking agents are divided into two groups: Zero-length and non-zero-length cross-linkers. In this study, gelatin was cross-linked by three different cross-linking agents: EDC-NHS, as a typically used cross-linker, and also squaric acid (SQ) and dialdehyde starch (DAS), as representatives of a second group of cross-linkers. For all prepared gelatin hydrogels, mechanical strength tests, thermal analysis, infrared spectroscopy, swelling ability, and SEM images were performed. The results indicate that the dialdehyde starch is a better cross-linking agent for gelatin than EDC-NHS. Meanwhile, the use of squaric acid does not give beneficial changes to the properties of the hydrogel.