Low Molecular Size Research Articles

Micro(nano)plastics from synthetic oligomers persisting in Mediterranean seawater: Comprehensive NMR analysis, concerns and origins

The presence in seawater of low-molecular-weight polyethylene (PE) and polydimethylsiloxane (PDMS), synthetic polymers with high chemical resistance, has been demonstrated in this study for the first time by developing a novel methodology for their recovery and quantification from surface seawater. These synthetic polymer debris (SPD) with very low molecular weights and sizes in the nano- and micro-metre range have escaped conventional analytical methods. SPD have been easily recovered from water samples (2 L) through filtration with a nitrocellulose membrane filter with a pore size of 0.45 μm. Dissolving the filter in acetone allowed the isolation of the particulates by centrifugation followed by drying. The isolated SPD were analysed by 1H nuclear magnetic resonance spectroscopy (1H NMR), identifying PE and PDMS. These polymers are thus persisting on seawater because of their low density and the ponderal concentrations were quantified in mg/m3. This method was used in an actual case study in which 120 surface seawater samples were collected during two sampling campaigns in the Mediterranean Sea (from the Gulf of Salerno to the Gulf of Policastro in South Italy). The developed analytical protocol allowed achieving unprecedented simplicity, rapidity and sensitivity. The 1H and 13C NMR structural analysis of the PE debris indicates the presence of oxidised polymer chains with very low molecular weights. Additionally, the origin of those low molecular weight polymers was investigated by analysing influents and effluents from a wastewater treatment plant (WWTP) in Salerno as a hot spot for the release of SPD: the analysis indicates the presence of low molecular weight polymers compatible with wax-PE, widely used for coating applications, food industry, cosmetics and detergents. Moreover, the origin of PDMS debris found in surface seawater can be ascribed to silicone-based antifoamers and emulsifiers.

Structure characterization and gelling properties of RG-I-enriched pectins extracted from citrus peels using four different methods

To facilitate the application of rhamnogalacturonan-I (RG-I)-enriched pectins (RGPs) as novel, healthy, and gelling food additives, this study compared the structural characteristics and gelling properties of RGPs extracted from citrus peel via four methods (alkali: AK, high-temperature/pressure: TP, citric acid: CA, and enzyme-assisted: EA extractions). AK and CA yielded pectins with the highest RG-I proportions (54.8 % and 51.9 %, respectively) by disrupting the homogalacturonan region; TP and EA increased the RG-I proportions by ~10 %. Among the four methods, AK induced the lowest degree of esterification (DE) (6.7 %) and longer side chains that form strong entanglement, contributing to its highest gel hardness. The relatively low DE (18.5 %) of CA RGP facilitated stable gel formation. Notably, its highly branched RG-I region afforded more intramolecular hydrophobic interactions, making a more highly cross-linked gel network of better gel resilience. In contrast, TP induced the highest DE (57 %) and curved molecular chains; it inhibited Ca2+ binding, entanglement, and intramolecular hydrophobic interactions, and thus no gel formed. EA RGP was associated with the lowest molecular size, rendering it more difficult for Ca2+ to form links, which resulted no gel. These findings offer insights into the relationship among the extraction methods, molecular structures, and gelling properties of RGPs.

A metabolomics perspective on the effect of environmental micro and nanoplastics on living organisms: A review

The increasing trend regarding the use of plastics has arisen an exponential concern on the fate of their derived products to the environment. Among these derivatives, microplastics and nanoplastics (MNPs) have been featured for their associated environmental impact due to their low molecular size and high surface area, which has prompted their ubiquitous transference among all environmental interfaces. Due to the heterogenous chemical composition of MNPs, the study of these particles has focused a high number of studies, as a result of the myriad of associated physicochemical properties that contribute to the co-transference of a wide range of contaminants, thus becoming a major challenge for the scientific community. In this sense, both primary and secondary MNPs are well-known to be adscribed to industrial and urbanized areas, from which they are massively released to the environment through a multiscale level, involving the atmosphere, hydrosphere, and lithosphere. Consequently, much research has been conducted on the understanding of the interconnection between those interfaces, that motivate the spread of these contaminants to biological systems, being mostly represented by the biosphere, especially phytosphere and, finally, the anthroposphere. These findings have highlighted the potential hazardous risk for human health through different mechanisms from the environment, requiring a much deeper approach to define the real risk of MNPs exposure. As a result, there is a gap of knowledge regarding the environmental impact of MNPs from a high-throughput perspective. In this review, a metabolomics-based overview on the impact of MNPs to all environmental interfaces was proposed, considering this technology a highly valuable tool to decipher the real impact of MNPs on biological systems, thus opening a novel perspective on the study of these contaminants.

Evaluation of free radical‐induced structural changes and their effect on antioxidant and anti‐inflammatory activities of UV/H2O2‐degraded dextrans

AbstractAlthough ultraviolet (UV)/H2O2 treatment could effectively improve the bioactivities of polysaccharides, the relationship between free radical‐induced structural changes and the bioactivities of products remains unknown. Therefore, this work investigated the changes in the chemical characteristics and chain conformations of degraded dextrans by the UV/H2O2 system as well as their antioxidant and anti‐inflammatory activities. The apparent degradation efficiencies of dextrans under different UV/H2O2 parameters were first investigated. UV/H2O2 treatment effectively destroyed the pyranose rings and glycosidic bonds of dextrans and significantly improved their uronic acid and carboxy contents. Furthermore, conformational studies revealed that besides the rapid decrease in the molecular weight, the compact spherical conformation of dextrans was gradually depolymerized into the flexible random coil conformation. Moreover, the significant antioxidant and anti‐inflammatory activities of the degraded dextrans were mainly attributed to their more extended random coil chains, increased quantity of reducing groups, and lower molecular sizes. This study contributes to investigating the structure–activity relationship of polysaccharides prepared by free radicals‐mediated degradation.

Production and characterization of novel thermostable CotA-laccase from Bacillus altitudinis SL7 and its application for lignin degradation

Laccases are multi-copper oxidases and found in ligninolytic bacteria catalyzing the oxidation of both phenolic and non-phenolic compounds, however its application in lignin degradation suffers due to low oxidation rate, which have intensified the search for new laccases. In the present study, spore coat A protein (CotA) encoding gene having laccase like activity from Bacillus altitudinis SL7 (CotA-SL7) was cloned and expressed in Escherichia coli. The purified CotA-SL7 was active at wide range of temperature and pH with optimum activity at 55 °C and pH 5.0. The kinetic parameters of CotA-SL7 was determined with Km, Vmax, and kcat values 0.4 mM, 2777 μmol/min/mg, and 5194 s−1, respectively. Molecular docking revealed the presence of Pro, Phe, Asp, Asn, His, and Ile residues at the active site taking part in the oxidation of ABTS. The purified CotA-SL7 reduced lignin contents by 31 % and changes in lignin structure were analyzed through fourier transformed infrared spectroscopy (FTIR), scanning electron microsscopy (SEM) and gas chromatography mass-spectrometry (GC-MS). The appearance of low molecular size compounds clearly indicates the cleavage of lignin polymer and opening of the benzene ring by purified CotA-SL7. Thus, high catalytic efficiency of CotA-SL7 makes it a suitable bio-catalyst for remediation of lignin contaminated wastewater from pulp and paper industries with clear insights into lignin degradation at molecular level.

Nanobody-Based EGFR-Targeting Immunotoxins for Colorectal Cancer Treatment.

Immunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (VHH 7D12) as the targeting domain. First, we generated a nanoITX, combining VHH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (VHHEGFRαS). Then, we incorporated a trimerization domain (TIEXVIII) into the construct, obtaining a trimeric nanoITX (TriVHHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the VHH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness.

PHYSICAL AND CHEMICAL MUTAGENESIS IN CATHARANTHUS ROSEUS PLANT USING SCOT MARKERS

The current study comprising molecular identification of the mutagenic effects of gamma rays and sodium azide on Catharanthus roseus using scot markers took place at the Department of Biology, University of Kufa, Iraq. The seeds of two cultivars, i.e., ‘victory carmine’ and a local variety (pink variety) of Catharanthus roseus L (carmine var.) received irradiation with two doses of gamma rays (50 Gray and 75 Gray). Afterward, these got soaked in sodium azide with four concentrations (0.2%, 0.4%, 0.6%, and 0.8%) to induce the genetic variation in the cultivar. The non-treated seeds served as control treatments for comparison with treated ones. Accomplishing irradiation was at an average of 18 Gy/h using Cobalt-60. The effects of gamma rays and sodium azide treatments’ evaluation were through 10 start codon targeted (SCoT) DNA markers. The higher number of distinctive fingerprints was three produced by primers SCoT-26, while primer SCoT-60 provided a distinct fingerprint for only one treatment. However, primer SCoT-40 failed to recognize any treatment with a distinctive fingerprint. The highest molecular size (2488 bp) came from primer SCoT-54, whereas the lower molecular size (143 bp) resulted in primer SCoT-12. Primer SCoT-33 provided the highest value for chief and unique bands, reaching 33 and 18, respectively. The highest number of amplified bands was 77 bands in primer SCoT-54. Primer SCoT-60 produced the highest value for monomorphic bands, primer SCoT-12 gave the highest value for polymorphic bands, polymorphism, and discriminatory (15, 68.18, and 20.83, respectively), and primer SCoT-9 produced the highest value for efficiency.

Novel Design of Citral-Thiourea Derivatives for Enhancing Antifungal Potential against Colletotrichum gloeosporioides.

Although much progress has been made in developing botanical fungicides to combat fungal diseases in crops, there remains a great need to improve the efficiency and long-term safety of these fungicides. This study proposes a novel strategy for designing citral-thiourea derivatives that feature such desirable properties. The motivation of the work herein was to enhance the antifungal activity of citral against C. gloeosprioides by exploiting the synergistic effect that arises from combining citral and thiourea compounds, thereby producing citral-thiourea derivatives that exhibit good long-term safety. The results revealed that the generated compounds e1, e3, e6, e18, and g showed remarkable antifungal activities against C. gloeosprioides, with corresponding EC50 values reaching 0.16, 1.66, 1.37, 4.76, and 4.60 mg/L, respectively, showing that the compounds significantly outperformed both the positive control kresoxim-methyl and the commercially available fungicide carbendazim. Furthermore, compound g showed stronger protective efficacy against C. gloeosprioides than carbendazim on mango fruit at 25 mg/L. Investigating the preliminary structure-activity relationship (SAR) of the compounds also revealed that the citral-thiourea derivatives exhibited higher antifungal activities against C. gloeosprioides compared to citral and thiourea compounds. This reinforcement of antifungal activity observed in the derivatives was found to be attributable to the two characteristics of low molecular size and the presence of a fluorine atom in the meta-position of the benzene ring. Beyond this, it was determined from QSAR that two molecular descriptors (the Kier-Hall index (order 3) and Tot dipole of the molecules) were negatively related to the antifungal activity of the citral-thiourea derivatives, while one other (the maximum resonance energy of a C-H bond) was positively related to their antifungal activity. More importantly, the citral-thiourea derivatives with high antifungal activities (i.e., compounds e1, e3, e6, e14, e15, e18, and g) exhibited negligible cytotoxicity to LO2 and HEK293T cell lines. The antifungal mechanism of the generated citral-thiourea derivatives was investigated by scanning electron microscopy (SEM) and relative conductivity. The derivatives were found to affect mycelial morphology and increase fungal cell membrane permeability, thereby resulting in the destruction of fungal cell membranes. These promising results provide novel insights into the study and potential application value of citral-thiourea derivatives as high-efficiency antifungal agents against C. gloeosprioides.

Methodological Studies of the Mechanism of Anion Insertion in Nanometer-Sized Carbon Micropores.

Dual-ion hybrid capacitors (DIHCs) are a promising class of electrochemical energy storage devices intermediate between batteries and supercapacitors, exhibiting both high energy and power density, and generalizable across wide chemistries beyond lithium. In this study, a model carbon framework material with a periodic structure containing exclusively 1.2 nm width pores, zeolite-templated carbon (ZTC), was investigated as the positive electrode for the storage of a range of anions relevant to DIHC chemistries. Screening experiments were carried out across 21 electrolyte compositions within a common stable potential window of 3.0-4.0 V vs. Li/Li+ to determine trends in capacity as a function of anion and solvent properties. To achieve fast rate capability, a binary solvent balancing a high dielectric constant with a low viscosity and small molecular size was used; optimized full-cells based on LiPF6 in binary electrolyte exhibited 146 Wh kg-1 and >4000 W kg-1 energy and power densities, respectively.

Dextran-enhanced CEST MRI reveals the size effect of BBB dysfunction associated with neuroinflammation

INTRODUCTION: The blood-brain barrier (BBB) is compromised in multiple central nervous system (CNS) disorders associated with neuroinflammation, including multiple sclerosis (MS). Currently available magnetic resonance imaging (MRI) methods, however, are only able to measure BBB leakage in the lower molecular size range with the use of small molecular tracers, i.e., gadolinium (Gd) agents (<1 kDa)1,2 and water (18 Da).3,4 The goal of this study is to adopt a dextran-based chemical exchange saturation transfer (CEST) MRI approach for assessing BBB leakage in the larger size range and studying the size characteristics of BBB dysfunction.
 METHODS: All animal experiments will be approved by the Animal Care and Use Committee of Johns Hopkins University. EAE MS mouse model: C57Bl/6 mice (F/6-10w), were injected s.c. with myelin peptide (MOG35-55, 200 μL, 0.5 mg/mL) emulsified in incomplete Freund's adjuvant supplemented with M. tuberculosis H37Ra (5 mg/mL) and i.p. with 300 ng of pertussis toxin on days 0 and 2. Mice were observed daily for signs of paralysis using a 0-5 rating system. Fluorescent imaging. EAE mice (n=3) were injected with the combination of fixable Dex40-TRITC and Dex3-FITC (i.v.) at the dose of 80 mg/kg, and sacrificed at 30 min after injection (without perfusion) to collect brains. Fluorescence microscopy was then performed on tissue sections. MRI: all in vivo MRI was acquired using a Biospec 11.7 T horizontal MRI scanner (Bruker, Ettlingen, Germany). According to our previously reported protocol,5 CEST MRI was performed before and after the i.v. injection of 200 µL dex40 saline solution (750 mg/kg b.w), using parameters: B1=1.8 µT, Tsat=3 s, Δω=-3 to +3 ppm with a step size of 0.2 ppm. MTRasym=(S-Δω–S+Δω)/S0 was computed after the B0 correction using the WASSR method. ΔMTRasym (1 ppm) at each time point was calculated by MTRasym (t)- MTRasym (pre).
 RESULTS: 1. The size-dependent BBB disruption in MS can be detected by fluorescent dextran-tracers of different sizes: Immunofluorescent results show dextrans of smaller sizes (e.g., 3 kDa) penetrated the brain parenchyma deeper than larger sizes (e.g., 40 kDa). Our study proves the feasibility to use dextrans as a group of tracers with different sizes for probing the size effect of BBB dysfunction. 2. Dex-enhanced CEST MRI: As shown in Figure 1, mice with high clinical disability scores have BBB impairment in the mouse brain, confirmed with Gd-enhanced MRI (Figure 1B). Dex-enhanced MRI results (Figure 1C) showed substantial contrast enhancement in the corresponding brain regions. Interestingly, while the size of Dex (40 kDa) is larger than the size of Gd-DOTA (559 Da), the area showing enhanced Dex-CEST signal is slightly larger than that of Gd-enhancement, suggesting that, besides size, other particle properties such as shape and surface properties of a given agent/particle may also contribute to the permeation across BBB.
 CONCLUSIONS: We have established a dextran-based imaging protocol for assessing the biodistribution of dextrans in the brains of EAE mice. We will continue studying the size effect of dextrans and determining the optimal dextran size for accurately mentoring the disease progression.

Geochemical distortion on shale oil maturity caused by oil migration: Insights from the non-hydrocarbons revealed by FT-ICR MS

Oil migration may influence the reliability of molecular maturity parameters. Sample extracts from a shaly sequence subjected to intrasource migration, were characterized using FT-ICR MS and GC–MS techniques. The hydrocarbon maturity parameters including sterane isomer ratios and methylphenanthrene indices (MPI) are relatively higher in siltstones than in shales. In contrast, the non-hydrocarbons indicate a lower maturity in siltstones as revealed by the smaller size of molecular cores, the larger length of alkyl side chains, and the more enrichment of Ox species. It is concluded that this discrepancy is attributable to petroleum migration. The thermal-stable isomers of hydrocarbons, used as numerators in the ratios, have stronger mobility consequently leading to a faster migration into siltstones. In contrast, the molecules of non-hydrocarbons with low aromatization and molecular size, and higher alkylation degrees, easily migrate into the siltstones due to relatively lower polarity, precipitability, and absorptivity. Additionally, the hydrophilic matrix surface and high porosity of the strata also facilitate the accumulation of non-hydrocarbons in the siltstones during the early diagenesis. Such findings contribute to a better understanding of oil molecular migration behavior, and suggest a caution for using geochemical indices as maturity parameters when involving petroleum migration.

Corn arabinoxylan has a repeating structure of subunits of high branch complexity with slow gut microbiota fermentation

Corn arabinoxylan (CAX), a cell wall-derived dietary fiber, was extracted with alkali, partially purified, and treated with hydrolytic enzymes in order to investigate the relationship of fine structure and fermentability by the human gut microbiota. Glycosyl composition and linkage analysis of CAX and two hydrolysates, coupled with molecular size analysis, indicated an organized structural feature of the native polymer, which consists of a repeating structural subunit containing complex branching patterns along the xylan backbone and flanked by regions of less complexity. The two lengths of the highly branched subunit were isolated and were shown to have enhanced slow fermentation property compared to the native structure (3.3 vs. 5.9 mL gas, 4 h), that was related to increasing complexity of the branched structures. Lower molecular size structures with higher branch complexity fermented slower, contrary to a conventional view that small fiber structures approaching the oligosaccharide level are necessarily more rapidly fermented.

In-Vivo study of nano chitosan as therapeutic agent for toxic metal implant

Chromium (Cr6+) toxicity is a well-known problem in environmental health and safety Cr6+ can cause respiratory disorders, skin irritation, cancer, and kidney disorders. During implantation, cases of biocorrosion and metallosis have occurred due to Cr6+ contact and exposure. Biosorption materials such as chitosan are a promising therapeutic agent to adsorb Cr6+ toxins in the body, as well as aiding the healing process for damaged internal organs. In-vivo studies between relationship of Cr6+ toxicity and chitosan concentration are investigated to recovery progress. We exhibited that Cr6+ can caused severe toxicity and induce death in rats due to organ damage and failure. Meanwhile, nano chitosan was able to adsorb Cr6+ via detection of blood. Recovery of internal organ damage was boosted through therapeutic low molecular size (385 Da) nano chitosan, which we were able to confirm from X-ray images within six weeks of treatment. Average particle size and particles distribution of nano chitosan were influenced by the concentration of nano chitosan powder and dilution solvent. However, we were unable to obtain linear correlations due to the in-vivo condition.

Temporal changes in hydrochemistry and DOM characteristics of the Brahmaputra River: implication to the seasonality of water quality.

Fluorescent dissolved organic matter (fDOM) in the Brahmaputra River water was characterized using excitation-emission matrix fluorescence spectroscopy (EEM) and parallel factor analysis (PARAFAC) model. EEM and PARAFAC model identified five fluorophores (Peak A, C, M, T, Tuv) and four fDOM components (two humic-, tryptophan-, and tyrosine-like) in the Brahmaputra River water. DOC varied between 0.8 and 3.9mg/L and along with the intensities of the fDOM components showed higher concentration in the pre-monsoon and monsoon than post-monsoon. Higher biological oxygen demand (BOD) and chemical oxygen demand (COD) confirmed the presence of a high amount of organic pollutants in the Brahmaputra River of Bangladesh. Cations and anions concentrations were comparatively lower in the monsoon and pre-monsoon compared to post-monsoon. Mg2+, Na+, and HCO3- ions were predominant; catchments were carbonate mineral-dominated; and the abundance of Na+ and SO42- ions described the presence of uncommon dissolution in the Brahmaputra River. Optical indices described that fDOM components were less aromatic, had low molecular size and weight, terrestrial and biological derived, and were largely affected by microbial decomposition. The Brahmaputra River water was in the higher microbial risk at the pre-monsoon and monsoon than the post-monsoon of the year. Entropy and Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS) methods based water quality index (WQI) was developed using PARAFAC components matrix of DOM. Newly derived WQI showed high seasonal variability of water quality in the Brahmaputra River due to the changes in local hydro-climate.

Fate of dissolved organic matter substructure in a full-scale wastewater treatment plant by using size exclusion chromatography multi-excitation-emission matrix analysis

Dissolved organic matter (DOM) in wastewater treatment plant (WWTP) effluent threats aquatic environment and challenges engineered aquatic systems. In order to predict its environmental risk and guide the selection of suitable strategy and technology, understanding the removal efficiency and the fate of DOM along the treatment is helpful. Herein, to qualitatively and quantitatively estimate the composition of DOM in a full-scale WWTP in northern China, a combined approach of chromatography and spectroscopy was employed, and the contribution of size-dependent substructure to the global feature of DOM was discussed simultaneously. The results suggest that the WWTP could remove the free proteins of low molecular size by physical treatment with removal ratios of 46.4% and 41.0% for tyrosine and tryptophan, respectively, whereas humic-like and humic-protein complex accumulate in the effluent of treatment with a ratio of 40.6%. Biotreatment is of importance for changing the proportion of different types of DOM, especially for anaerobic tank and the anoxic tank, which results in the increase of humification. Size exclusive chromatography (SEC) coupled with multi emission maps identifies six DOM substructures. Two protein-like DOM were removed by the WWTP, except the composition with the molecular weight ranging from 0.75 to 0.25 kDa, whereas humic-like DOM with whole size range accumulated to varying extent. The standard deviation analysis states that the structure variation of bulk DOM in the WWTP is primarily contributed by the decrease of small-sized proteins and the increase of humic-like DOM. This study presents insight into the fate and behavior of DOM components during the full-scale WWTP treatment and provides guidelines for altering DOM-treating technologies. Besides, innovative strategies suggest using DOM as a resource based on the critical review of previous research.

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Empirical Analysis Revealing Privileged Chemical Space of Cosmetic Preservatives

Most cosmetic products require preservation to prevent microbial contamination and to ensure consumer safety. Due to regulatory restrictions and rejection by consumers, preservative options have become limited and the development of novel solutions is needed. This search can be guided by knowledge about favorable chemical space for cosmetic preservatives. Therefore, we used preservatives allowed in the EU as training set and calculated various molecular properties. Empirical analysis revealed two separated areas of privileged chemical space with the net charge as distinctive property. The first area comprises the group of neutral and anionic preservatives and is characterized by low molecular size as well as limited hydrogen-bonding capacity, polarity, and flexibility. The second area includes cationic preservatives, which are rather diffusely distributed regarding molecular weight and hydrogen-bonding, however, all members share high flexibility. Both groups significantly differ from antibiotics, reflecting the specific requirement of cosmetic preservation. The molecular properties defining the privileged chemical space are easy to calculate, and thus, can provide guidance for the development of novel preservatives.

Linking oral microbial proteolysis to aMMP-8 PoC diagnostics along with the stage and grade of periodontitis: A cross-sectional study.

Linking oral microbial proteolysis to aMMP-8 PoC diagnostics along with the stage and grade of periodontitis: A cross-sectional study.

Periodontitis and peri-implantitis tissue levels of Treponema denticola-CTLP and its MMP-8 activating ability

Background and AimsChymotrypsin-like-proteinase of Treponema denticola (Td-CTLP) can stimulate the protein expression and activation of matrix metalloproteinase (MMP)-8 (or collagenase-2), a potent tissue destructive enzyme from gingival cells in vitro. The aims of this study were 1) to demonstrate the proMMP-8 (or latent MMP-8) activation by Td-CTLP in vitro and 2) to detect Td-CTLP and MMP-8 protein levels in the tissue samples of peri-implantitis and periodontitis patients. Materials and MethodsproMMP-8 activation by Td-CTLP was analyzed by immunoblots. Tissue specimens were collected from 38 systemically healthy and non-smoking patients; 14 of whom had moderate to severe periodontitis, 10 of whom were suffering from peri-implantitis, and finally 14 of whom showed no sign of periodontal inflammation nor radiological bone decay (control group). The immune-expression levels of MMP-8 and Td-CTLP in the epithelium and the connective tissue were analyzed immunohistochemically. A pixel color-intensity analyze was performed with ImageJ software (version 1.46c; Rasband WS, National Institutes of Health, Bethesda, MD, USA) to obtain a comparable numeral score for each patient’s epithelium and connective tissue MMP-8 and Td-CTLP enzyme level. ResultsTd-CTLP activated proMMP-8 in vitro by converting the 70−75 kDa proMMP-8 to 65 kDa active MMP-8. Also, lower molecular size 25−50 kDa parts of MMP-8 were formed. There was no statistically significant difference between the study groups in terms of their MMP-8 and Td-CTLP levels in the epithelium or in the connective tissue. ConclusionRegarding the limits of this study, it can thus be said that the Td-CTLP enzyme can activate the host proMMP-8 enzyme. Tissue protein levels of MMP-8 and Td-CTLP do not seem to be changed in peri-implantitis and in periodontitis.

Abstract 1855: Potent anti-tumor activity against patient CLL, MM and AML by LAVA-051, a bispecific Vγ9Vδ2-T and type 1 NKT cell engager targeting CD1d

Abstract Bispecific antibodies that target tumors by engaging innate-like semi-invariant T cell subsets with inherent anti-tumor activity, such as Vγ9Vδ2-T cells and type 1 natural killer T (NKT) cells, may combine high therapeutic efficacy with limited off-tumor toxicity. Type 1 NKT cells respond to self and foreign (glyco)lipid antigens presented in the context of the MHC class I like molecule CD1d which is expressed on various (hematological) malignancies. Vγ9Vδ2-T cells respond to intracellular accumulation of phosphoantigens in cancer cells by sensing conformational alterations in the butyrophilin (BTN)2A1-3A1 complex. By linking a CD1d-specific single domain antibody (VHH) to a Vδ2-TCR specific VHH, we created a bispecific VHH, termed LAVA-051. Besides targeting Vγ9Vδ2-T cells to CD1d-expressing tumor cells, LAVA-051 has the unique ability to also trigger strong activation of type 1 NKT cells in a CD1d-restricted fashion, resulting in potent killing of CD1d-expressing tumor cells by engagement of Vγ9Vδ2-T cells and/or type 1 NKT cells (EC50 1 pM for Vγ9Vδ2-T cells and 216 pM for type 1 NKT cells; >85% target cell lysis in overnight assays at low effector:target ratios of 1:2). LAVA-051 triggered strong activation, pro-inflammatory cytokine production, and proliferation of type 1 NKT and Vγ9Vδ2-T cells and exerted substantial antitumor activity against human primary acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells that express CD1d. Moreover, improved survival was observed in in vivo AML and MM mouse models. Due to lack of cross-reactivity of LAVA-051 with non-human primate (NHP) CD1d and Vγ9Vδ2-T cells, a cross-reactive surrogate bispecific engager was generated to assess safety, pharmacokinetic (PK) and pharmacodynamic (PD) parameters. Multiple dose studies in NHP (7 daily doses up to 1 mg/kg i.v.) showed clear signs of Vγ9Vδ2-T cell engagement but no clinical, laboratory, or histopathological toxicity. Reflecting the low molecular size of this bispecific engager, PK studies revealed a short plasma half-life which was however compensated for by the prolonged (up to 5 days) binding of the bispecific engager to peripheral blood Vγ9Vδ2-T cells allowing intermittent dosing. Although VHHs have low inherent immunogenicity LAVA-051 was humanized and engineered to avoid binding to pre-existing anti-VHH antibodies that have been reported in ±15% of human individuals and that may limit therapeutic activity. Based on the here reported strong activity against CD1d-expresssing tumors in in vitro, ex vivo and in vivo models and the favorable safety profile of the surrogate engager in NHP, LAVA-051 has been selected for a first-in-human clinical phase 1-2 study in patients with CD1d-expressing, therapy refractory, CLL, MM, or AML. Initiation of this clinical program is scheduled for Q1 2021. Citation Format: Roeland Lameris, Jurjen M. Ruben, Iris de Weerdt, Rob C. Roovers, Arnon P. Kater, Thilo A. Riedl, Victoria Iglesias, Ton Adang, Tanja D. de Gruijl, Paul W.H.I Parren, Hans J. van der Vliet. Potent anti-tumor activity against patient CLL, MM and AML by LAVA-051, a bispecific Vγ9Vδ2-T and type 1 NKT cell engager targeting CD1d [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1855.