How Do Molecular Tweezers Bind to Proteins? Lessons from X-ray Crystallography.

To understand the biological relevance and mode of action of artificial protein ligands, crystal structures with their protein targets are essential. Here, we describe and investigate all known crystal structures that contain a so-called "molecular tweezer" or one of its derivatives with an attached natural ligand on the respective target protein. The aromatic ring system of these compounds is able to include lysine and arginine side chains, supported by one or two phosphate groups that are attached to the half-moon-shaped molecule. Due to their marked preference for basic amino acids and the fully reversible binding mode, molecular tweezers are able to counteract pathologic protein aggregation and are currently being developed as disease-modifying therapies against neurodegenerative diseases such as Alzheimer's and Parkinson's disease. We analyzed the corresponding crystal structures with 14-3-3 proteins in complex with mono- and diphosphate tweezers. Furthermore, we solved crystal structures of two different tweezer variants in complex with the enzyme Δ1-Pyrroline-5-carboxyl-dehydrogenase (P5CDH) and found that the tweezers are bound to a lysine and methionine side chain, respectively. The different binding modes and their implications for affinity and specificity are discussed, as well as the general problems in crystallizing protein complexes with artificial ligands.

Rate of severe and fatal infections in a cohort of patients with interstitial lung disease associated with rheumatoid arthritis: a multicenter prospective study.

Objective: To describe severe infection, foci of infection, microorganisms, associated factors, and impact on mortality in patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Patients and methods: The study was based on a multicenter prospective cohort of patients with RA-ILD followed up from 2015 to 2023. The main outcome measures were incident severe infection and fatal infection. We evaluated infectious foci, etiologic agents, vaccination status, variables associated with lung function, and clinical-therapeutic variables in RA. The incidence rate (IR) for infection and mortality was calculated per 100 person-years, and 3 multivariate models were constructed to explore factors associated with infection. Results: We followed up 148 patients with RA-ILD for a median 56.7 months (699.3 person-years). During this period, 142 patients (96%) had at least 1 infection. A total of 368 infectious episodes were recorded, with an IR of 52.6 per 100 person-years. Of the 48 patients who died, 65% did so from infection. Respiratory infections were the most common first infection (74%), infection overall (74%), and fatal infection (80%) and were caused mostly by SARS CoV-2, Streptococcus pneumoniae, Pseudomonas aeruginosa, and influenza A virus. The factors associated with an increased risk of infection and death in patients with RA-ILD were age, inflammatory activity, and therapy with corticosteroids and immunosuppressants. Conclusion: Patients with RA-ILD have a high risk of serious infection, especially respiratory infection. Infection develops early, is recurrent, and is frequently fatal. The presence of associated factors such as advanced age, joint inflammation, and treatment highlight the importance of integrated and preventive medical care.

Functional impact and molecular binding modes of drugs that target the PI3K isoform p110δ.

Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.

LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1.

Microenvironmental bystander cells are essential for the progression of chronic lymphocytic leukemia (CLL). We have discovered previously that LYN kinase promotes the formation of a microenvironmental niche for CLL. Here we provide mechanistic evidence that LYN regulates the polarization of stromal fibroblasts to support leukemic progression. LYN is overexpressed in fibroblasts of lymph nodes of CLL patients. LYN-deficient stromal cells reduce CLL growth in vivo. LYN-deficient fibroblasts show markedly reduced leukemia feeding capacity in vitro. Multi-omics profiling reveals that LYN regulates the polarization of fibroblasts towards an inflammatory cancer-associated phenotype through modulation of cytokine secretion and extracellular matrix composition. Mechanistically, LYN deletion reduces inflammatory signaling including reduction of c-JUN expression, which in turn augments the expression of Thrombospondin-1, which binds to CD47 thereby impairing CLL viability. Together, our findings suggest that LYN is essential for rewiring fibroblasts towards a leukemia-supportive phenotype.

Analysis of comorbidity in rheumatoid arthritis-associated interstitial lung disease: a nested case-cohort study.

OBJECTIVES: To describe comorbid conditions in patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD) and to analyze factors associated with multimorbidity. METHODS: Nested case-cohort study of 2 prospective cohorts: one with RA-ILD (cases) and another with RA but not ILD (controls). The cohorts were matched for age, sex, and time since diagnosis. Multimorbidity was defined as the co-occurrence of 2 or more chronic diseases, in addition to RA and ILD. We evaluated the comorbid conditions included in the Charlson Comorbidity Index, cardiovascular risk factors, neuropsychiatric conditions, and other frequent conditions in RA. We also recorded clinical-laboratory variables, inflammatory activity according to the 28-joint Disease Activity Score, C-reactive protein (CRP), physical function, and pulmonary function. We performed 2 multivariate analyses to identify factors associated with multimorbidity in RA and RA-ILD. RESULTS: The final study population comprised 110 cases and 104 controls. Multimorbidity was more frequent among cases than controls (80 [72.7] vs 60 [57.7]; p = 0.021). In both groups, multimorbidity was associated with ILD (OR [95% CI] 1.92 [1.03-3.59]; p = 0.039), age (OR [95% CI] 1.05 [1.01-1.08]; p = 0.004), CRP (OR [95% CI] 1.16 [1.05-1.29]; p = 0.003), and erosions (OR [95% CI] 1.05 [1.01-1.08]; p = 0.004); in the cases, it was associated with CRP (OR [95% CI] 1.17 [1.01-1.35]; p = 0.027), anti-citrullinated peptide antibody (OR [95% CI] 1.23 [1.14-13.02]; p = 0.049), and forced vital capacity (OR [95% CI] 0.79 [0.96-0.99]; p = 0.036). CONCLUSION: In patients with RA, multimorbidity was associated with ILD, systemic inflammation, and advanced age.

HPIPred: Host-pathogen interactome prediction with phenotypic scoring.

Protein-protein interactions (PPIs) are involved in most cellular processes. Unfortunately, current knowledge of host-pathogen interactomes is still very limited. Experimental methods used to detect PPIs have several limitations, including increasing complexity and economic cost in large-scale screenings. Hence, computational methods are commonly used to support experimental data, although they generally suffer from high false-positive rates. To address this issue, we have created HPIPred, a host-pathogen PPI prediction tool based on numerical encoding of physicochemical properties. Unlike other available methods, HPIPred integrates phenotypic data to prioritize biologically meaningful results. We used HPIPred to screen the entire Homo sapiens and Pseudomonas aeruginosa PAO1 proteomes to generate a host-pathogen interactome with 763 interactions displaying a highly connected network topology. Our predictive model can be used to prioritize protein-protein interactions as potential targets for antibacterial drug development. Available at: https://github.com/SysBioUAB/hpi_predictor.

Evaluación antimicrobiana y coagulante de extractos de cinco vegetales de interés etnobotánico / Antimicrobial and coagulating evaluation of extracts of five vegetables of ethnobotanical interest

Resumen Objetivo: Evaluar la actividad antibacteriana de los extractos de Mimosa tenuiflora, Equisetum arvense, Syzygium aromaticum, Lippia graveolens y Aloe vera contra cepas bacterianas de S. mutans (ATCC700611) y S. sobrinus (ATCC33478) comparado con clorhexidina a 1200 µg/mL (0.12%) y la actividad coagulante en sangre humana. Materiales y métodos: Estudio comparativo, abierto, experimental, prospectivo y transversal in vitro. Se realizaron diluciones a 500 y 1000 µg/mL de cinco extractos y se probaron por triplicado contra microorganismos orales por medio de técnica de pozo en agar y en la evaluación de la actividad coagulante se probaron los cinco extractos por triplicado en sangre humana evaluando TP (tiempo de protrombina) y TTPa (tiempo de tromboplastina parcial activado) mediante coagulómetro. Resultados: El extracto de Lippia graveolens a 500 y 1000 µg/mL mostró un promedio de halos de inhibición sobre S. mutans de 26mm con respecto a clorhexidina a 1200 µg/mL que mostró un promedio de 15mm. Contra cepas de S. sobrinus mostraron un promedio de 19mm a 500 µg/mL y 23mm a 1000 µg/mL con respecto a 15mm de clorhexidina. El valor de TP (tiempo de protrombina) de la muestra de sangre fue 12.27 segundos, al aplicarle E. arvense y S. aromaticum ambos a 1000 µg/mL presentaron tiempos de 13.37 segundos. En cuanto al tiempo de tromboplastina parcial activada (TTPa) el valor de la muestra sin extracto fue 32.63 segundos, al aplicar M. tenuiflora a 500 µg/mL se aumentó el tiempo a 39.17 segundos. Conclusiones: Se concluye que Lippia graveolens tiene mejor efecto antibacteriano contra micrrorganismos orales y M. tenuiflora fue el extracto que aumentó por más tiempo el valor de TTPa.

Abstract Objective: To evaluate the antimicrobial and coagulating activity from five vegetables of ethnobotanical interest extracts (Mimosa tenuiflora, Equisetum arvense, Syzygium aromaticum, Lippia graveolens and Aloe vera). Materials and methods: It was a Comparative, open, experimental, prospective and cross-sectional study through antimicrobial evaluation of the five extracts against bacterial strains of S. mutans (ATCC700611) and S. sobrinus (ATCC33478) by means of agar well technique and an evaluation of coagulating activity by measuring TP (prothrombin time) and APTT (activated partial thromboplastin time) using a coagulometer and comparing the results with those of a healthy patient. Results: It was found that the antimicrobial activity of the extracts on S. mutans at 500 and 1000ppm is statistically significant in the extracts of E. arvense and L. graveolens (p= 0.0057) and (p= 0.0000) respectively and on strains of S. sobrinus from the extracts of A. vera (p= 0.0011) and L. graveolens (p= 0.0089) in both concentrations, which show an antimicrobial effect superior to chlorhexidine. The PT patient's (prothrombin time) value was 12.27 seconds, no statistical difference was observed with a value of (p<0.05), however, E. arvense and S. aromaticum, both at 1000ppm, presented times of 13.37 seconds and at the activated partial thromboplastin time (PTPA) the value of the patient was 32.63 seconds, highlighting M. tenuiflora at 500 ppm, which presented times of 39.17 seconds. Conclusions: The extracts described above contain chemical compounds that are valuable alternatives against microorganisms and oral treatments, and it is also very important that research suggests materials and medications that are effective in the treatment of patients and that do not represent a health risk.

Amoebicidal and trichomonicidal capacity of polymeric nanoparticles loaded with extracts of the plants Curcuma longa (Zingiberaceae) and Berberis vulgaris (Berberidaceae) / Capacidad amebicida y tricomonicida de nanopartículas poliméricas cargadas con extractos de las plantas Curcuma longa (Zingiberaceae) y Berberis vulgaris (Berberidaceae)

Abstract Introduction: Pathogenic protozoans, like Entamoeba histolytica and Trichomonas vaginalis, represent a major health problem in tropical countries; and polymeric nanoparticles could be used to apply plant extracts against those parasites. Objective: To test Curcuma longa ethanolic extract and Berberis vulgaris methanolic extracts, and their main constituents, against two species of protozoans. Methods: We tested the extracts, as well as their main constituents, curcumin (Cur) and berberine (Ber), both non-encapsulated and encapsulated in polymeric nanoparticles (NPs), in vitro. We also determined nanoparticle characteristics by photon correlation spectroscopy and scanning electron microscopy, and hemolytic capacity by hemolysis in healthy erythrocytes. Results: C. longa consisted mainly of tannins, phenols, and flavonoids; and B. vulgaris in alkaloids. Encapsulated particles were more effective (P < 0.001); however, curcumin and berberine nanoparticles were the most effective treatments. CurNPs had IC50 values (µg/mL) of 9.48 and 4.25, against E. histolytica and T. vaginalis, respectively, and BerNPs 0.24 and 0.71. The particle size and encapsulation percentage for CurNPs and BerNPs were 66.5 and 73.4 nm, and 83.59 and 76.48 %, respectively. The NPs were spherical and significantly reduced hemolysis when compared to non-encapsulated extracts. Conclusions: NPs represent a useful and novel bioactive compound delivery system for therapy in diseases caused by protozoans.

Resumen Introducción: Los protozoos patógenos, como Entamoeba histolytica y Trichomonas vaginalis, representan un importante problema de salud en los países tropicales; y se podrían usar nanopartículas poliméricas para aplicar extractos de plantas contra esos parásitos. Objetivo: Probar los extractos etanólicos de Curcuma longa y Berberis vulgaris, y sus principales constituyentes, contra dos especies de protozoos. Métodos: Probamos los extractos, así como sus principales constituyentes, curcumina (Cur) y berberina (Ber), tanto no encapsulados como encapsulados en nanopartículas poliméricas (NPs), in vitro. También determinamos las características de las nanopartículas por espectroscopía de correlación de fotones y microscopía electrónica de barrido, y la capacidad hemolítica por hemólisis en eritrocitos sanos. Resultados: C. longa tenía principalmente: taninos, fenoles y flavonoides; y B. vulgaris, alcaloides. Las partículas encapsuladas fueron más efectivas (P < 0.001); sin embargo, las nanopartículas de curcumina y berberina fueron los tratamientos más efectivos. CurNPs tenía valores IC50 (µg/mL) de 9.48 y 4.25, contra E. histolytica y T. vaginalis, respectivamente, y BerNPs 0.24 y 0.71. El tamaño de partícula y el porcentaje de encapsulación para CurNPs y BerNPs fueron: 66.5 y 73.4 nm, y 83.59 y 76.48 %, respectivamente. Los NP son esféricos y redujeron significativamente la hemólisis en comparación con los extractos no encapsulados. Conclusiones: Las NP representan un sistema de administración de compuestos bioactivos útil y novedoso para la terapia enfermedades causadas por protozoos.

Two Ways to Achieve the Same Goal-Two Validated Quantitative NMR Strategies for a Low-Abundance Natural Product in Standardized Extracts: The Case of Hepatodamianol in Turnera diffusa.

The quantification of low-abundance secondary metabolites in plant extracts is an analytical problem that can be addressed by different analytical platforms, the most common being those based on chromatographic methods coupled to a high-sensitivity detection system. However, in recent years nuclear magnetic resonance (NMR) has become an analytical tool of primary choice for this type of problem because of its reliability, inherent simplicity in sample preparation, reduced analysis time, and low solvent consumption. The versatility of strategies based on quantitative NMR (qNMR), such as internal and external standards and electronic references, among others, and the need to develop validated analytical methods make it essential to compare procedures that must rigorously satisfy the analytical well-established acceptance criteria for method validation. In this work, two qNMR methods were developed for the quantification of hepatodamianol, a bioactive component of T. diffusa. The first method was based on a conventional external standard calibration, and the second one was based on the pulse length-based concentration determination (PULCON) method using the ERETIC2 module as a quantitation tool available in TopSpin software. The results show that both procedures allow the content of the analyte of interest in a complex matrix to be determined in a satisfactory way, under strict analytical criteria. In addition, ERETIC2 offers additional advantages such as a reduction in experimental time, reagent consumption, and waste generated.

Safety and Effectiveness of Abatacept in a Prospective Cohort of Patients with Rheumatoid Arthritis-Associated Interstitial Lung Disease.

OBJECTIVE: To prospectively evaluate the safety and efficacy profile of abatacept in patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD). METHODS: We performed a prospective observational multicenter study of a cohort of patients with RA-ILD treated with abatacept between 2015 and 2021. Patients were evaluated using high-resolution computed tomography and pulmonary function tests at initiation, 12 months, and the end of follow-up. The effectiveness of abatacept was evaluated based on whether ILD improved, stabilized, progressed, or was fatal. We also evaluated factors such as infection, hospitalization, and inflammatory activity using the 28-joint Disease Activity Score with the erythrocyte sedimentation rate (DAS28-ESR). Cox regression analysis was performed to identify factors associated with progression of lung disease. RESULTS: The study population comprised 57 patients with RA-ILD treated with abatacept for a median (IQR) of 27.3 (12.2-42.8) months. Lung disease had progressed before starting abatacept in 45.6% of patients. At the end of follow-up, lung disease had improved or stabilized in 41 patients (71.9%) and worsened in 13 (22.8%); 3 patients (5.3%) died. No significant decreases were observed in forced vital capacity (FVC) or in the diffusing capacity of the lung for carbon monoxide (DLCO).The factors associated with progression of RA-ILD were baseline DAS28-ESR (OR [95% CI], 2.52 [1.03-3.12]; p = 0.041), FVC (OR [95% CI], 0.82 [0.70-0.96]; p = 0.019), and DLCO (OR [95% CI], 0.83 [0.72-0.96]; p = 0.018). Only 10.5% of patients experienced severe adverse effects. CONCLUSION: Pulmonary function and joint inflammation stabilized in 71% of patients with RA-ILD treated with abatacept. Abatacept had a favorable safety profile.

Preliminary Study of the Antimicrobial, Anticoagulant, Antioxidant, Cytotoxic, and Anti-Inflammatory Activity of Five Selected Plants with Therapeutic Application in Dentistry.

The usefulness of traditional plants in Mexico to treat human ailments has been known since ancient times. This work evaluated the antimicrobial, anticoagulant, antioxidant, cytotoxic, and anti-inflammatory potential of ethanolic extracts of Aloe vera, Equisetum arvense, Mimosa tenuiflora, Lippia graveolens, and Syzygium aromaticum. The antimicrobial activity of the extracts was evaluated against Streptococcus mutans and Streptococcus sorbinus; a significant inhibitory effect of the L. graveolens extract on both bacteria was observed at concentration levels of 250 µg/mL and greater. The anticoagulant activity was evaluated in terms of prothrombin time (PT) and activated partial thromboplastin time (APTT), A. vera and M. tenuiflora extracts showed no significant difference (p ˂ 0.05) in PT compared with the control, and for APTT the extracts of A. vera, L. graveolens, and S. aromaticum decreased the APTT significantly (p ˂ 0.05) compared with the control. The antioxidant potential by DPPH assay indicated that the E. arvense extract behaved statistically the same as the control. The cytotoxic activity was evaluated in HGF-1 cells using the fluorometric microculture cytotoxicity assay technique, and none of the extracts was toxic at 125 and 250 µg/mL concentrations. Finally, the anti-inflammatory activity was evaluated using ELISA, where the A. vera extract showed the best anti-inflammatory capacity. Further research on the search for bioactive metabolites and elucidation of action mechanisms of the most promising extracts will be carried out.

Importance of Vaccination against SARS-CoV-2 in Patients with Interstitial Lung Disease Associated with Systemic Autoimmune Disease.

Objectives: To describe the frequency of COVID-19 and the effect of vaccination in patients with interstitial lung disease and systemic autoimmune disease (ILD-SAD) and to identify factors associated with infection and severity of COVID-19. Methods: We performed a cross-sectional multicenter study of patients with ILD-SAD followed between June and October 2021. The main variable was COVID-19 infection confirmed by a positive polymerase chain reaction (PCR) result for SARS-CoV-2. The secondary variables included severity of COVID-19, if the patient had to be admitted to hospital or died of the disease, and vaccination status. Other variables included clinical and treatment characteristics, pulmonary function and high-resolution computed tomography. Two logistic regression was performed to explore factors associated with "COVID-19" and "severe COVID-19". Results: We included 176 patients with ILD-SAD: 105 (59.7%) had rheumatoid arthritis, 49 (27.8%) systemic sclerosis, and 22 (12.54%) inflammatory myopathies. We recorded 22/179 (12.5%) SARS-CoV-2 infections, 7/22 (31.8%) of them were severe and 3/22 (13.22%) died. As to the vaccination, 163/176 (92.6%) patients received the complete doses. The factors associated with SARS-CoV-2 infection were FVC (OR (95% CI), 0.971 (0.946−0.989); p = 0.040), vaccination (OR (95% CI), 0.169 (0.030−0.570); p = 0.004), and rituximab (OR (95% CI), 3.490 (1.129−6.100); p = 0.029). The factors associated with severe COVID-19 were the protective effect of the vaccine (OR (95% CI), 0.024 (0.004−0.170); p < 0.001) and diabetes mellitus (OR (95% CI), 4.923 (1.508−19.097); p = 0.018). Conclusions: Around 13% of patients with ILD-SAD had SARS-CoV-2 infection, which was severe in approximately one-third. Most patients with severe infection were not fully vaccinated.

Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Efficacy and Safety of Rituximab in Autoimmune Disease-Associated Interstitial Lung Disease: A Prospective Cohort Study.

OBJECTIVES: To analyze the efficacy and safety of rituximab (RTX) in connective tissue disease associated with interstitial lung disease (CTD-ILD). METHODS: We performed a multicenter, prospective, observational study of patients with CTD-ILD receiving rituximab between 2015 and 2020. The patients were assessed using high-resolution computed tomography and pulmonary function tests at baseline, at 12 months, and at the end of follow-up. The main outcome measure at the end of follow-up was forced vital capacity (FVC) > 10% or diffusing capacity of the lungs for carbon monoxide (DLCO) > 15% and radiological progression or death. We recorded clinical characteristics, time to initiation of RTX, concomitant treatment, infections, and hospitalization. A Cox regression analysis was performed to identify factors associated with worsening ILD. RESULTS: We included 37 patients with CTD-ILD treated with RTX for a median (IQR) of 38.2 (17.7-69.0) months. At the end of the follow-up, disease had improved or stabilized in 23 patients (62.1%) and worsened in seven (18.9%); seven patients (18.9%) died. No significant decline was observed in median FVC (72.2 vs. 70.8; p = 0.530) or DLCO (55.9 vs. 52.2; p = 0.100). The multivariate analysis showed the independent predictors for worsening of CTD-ILD to be baseline DLCO (OR (95% CI), 0.904 (0.8-0.9); p = 0.015), time to initiation of RTX (1.01 (1.001-1.02); p = 0.029), and mycophenolate (0.202 (0.04-0.8); p = 0.034). Only 28 of the 37 patients (75.6%) were still undergoing treatment with RTX: two patients (5.4%) stopped treatment due to adverse events and seven patients (18.9%) died owing to progression of ILD and superinfection. CONCLUSION: Lung function improved or stabilized in more than half of patients with CTD-ILD treated with RTX. Early treatment and combination with mycophenolate could reduce the risk of progression of ILD.

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

Introduction: In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods: We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results: Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion: Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

In-Vitro Effect of Kalanchoe daigremontiana and Its Main Component, Quercetin against Entamoeba histolytica and Trichomonas vaginalis.

BACKGROUND: Parasitic infections represent one of the main public health problems in humans according to the WHO. Therefore, the need has arisen to find new treatments that can be used as an alternative cure to parasitosis. We aimed to investigate the in-vitro effects of the methanolic extract of Kalanchoe daigremontiana as well as its main component, quercetin against Entamoeba histolytica and Trichomonas vaginalis. METHODS: For this purpose, the in-vitro activity of the methanol extract of K. daigremontiana also its main component, quercetin, against trophozoites of E. histolytica and T. vaginalis was evaluated, using the microassay technique. Furthermore, the antioxidant activity was determined. Finally, the cytotoxic and cytoprotective capacity was determined using the hemolysis technique. RESULTS: The IC50 indicated that quercetin significantly (P < 0.05) inhibited the growth rate of the trophozoite stage of E. histolytica and T. vaginalis in comparison to the methanolic extract of K. daigremontiana (KalL). Also, quercetin significantly (P < 0.05) was a better antioxidant as compared with the positive control. In the evaluation of cytotoxicity effects, it could be observed that KalL as compared with quercetin exhibited more cytotoxicity against human erythrocytes. Quercetin significantly (P < 0.001) exhibited better cytoprotective activity compared to KalL. CONCLUSION: Both K. daigremontiana methanolic extract and quercetin alone demonstrated high antiparasitic activity against E. histolytica and T. vaginalis. However, the in-vivo efficacy of K. daigremontiana and quercetin also requires to be evaluated using an animal model.

Characteristics and Predictors of Progression Interstitial Lung Disease in Rheumatoid Arthritis Compared with Other Autoimmune Disease: A Retrospective Cohort Study.

OBJECTIVES: To describe the characteristics and progression of interstitial lung disease in patients with associated systemic autoimmune disease (ILD-SAI) and to identify factors associated with progression and mortality. PATIENTS AND METHODS: We performed a multicenter, retrospective, observational study of patients with ILD-SAI followed between 2015 and 2020. We collected clinical data and performed pulmonary function testing and high-resolution computed tomography at diagnosis and at the final visit. The main outcome measure at the end of follow-up was forced vital capacity (FVC) >10% or diffusing capacity of the lungs for carbon monoxide >15% and radiological progression or death. Cox regression analysis was performed to identify factors associated with worsening of ILD. RESULTS: We included 204 patients with ILD-SAI: 123 (60.3%) had rheumatoid arthritis (RA), 58 had (28.4%) systemic sclerosis, and 23 (11.3%) had inflammatory myopathy. After a median (IQR) period of 56 (29.8-93.3) months, lung disease had stabilized in 98 patients (48%), improved in 33 (16.1%), and worsened in 44 (21.5%). A total of 29 patients (14.2%) died. Progression and hospitalization were more frequent in patients with RA (p = 0.010). The multivariate analysis showed the independent predictors for worsening of ILD-SAI to be RA (HR, 1.9 [95% CI, 1.3-2.7]), usual interstitial pneumonia pattern (HR, 1.7 [95% CI, 1.0-2.9]), FVC (%) (HR, 2.3 [95% CI, 1.4-3.9]), and smoking (HR, 2.7 [95%CI, 1.6-4.7]). CONCLUSION: Disease stabilizes or improves after a median of 5 years in more than half of patients with ILD-SAI, although more than one-third die. Data on subgroups and risk factors could help us to predict poorer outcomes.

In Vitro Antischistosomal Activity of the Argemone mexicana Methanolic Extract and Its Main Component Berberine.

BACKGROUND: Schistosomiasis has been identified as a major public health problem in tropical countries. The present study aimed to investigate the schistosomicidal effects of the methanolic extract of Argemone mexicana L. and its active component, berberine against Schistosoma mansoni on in-vitro experiments. METHODS: S. mansoni adults were used. Various concentrations of the methanolic extract (10 - 200 µg/ml) and berberine (2.5 - 50 µM) were tested from 24 to 72 h. The viability of S. mansoni was confirmed with an invertoscope-microscope. Furthermore, cytotoxic (Hemolysis test), and antioxidant (DPPH radical scavenging assay) capacities were determined. RESULTS: The viability tests on S. mansoni showed that A. mexicana at 50 µg/mL is lethal at 48 h and berberine at 10 µM is lethal at 24 h. The hemolytic activity at 1,000 µg/mL was 2.9% for A. mexicana and 90.2% for berberine. The antioxidant capacities shown by A. mexicana and berberine, were EC50 156.3 and 84.1 µg/mL, respectively. CONCLUSION: The extract of A. mexicana and berberine demonstrated high antischistosomal activities in low concentration and short exposure time on the in-vitro model.

A new RP-HPLC method as an auxiliary tool for optimization of sample preparation procedures for tracing of PPCPs of different hydrophilicities.

Recently, pharmaceutical and personal care products (PPCPs) have received considerable attention because of their increasing use. Analysis of PPCPs presents a significant analytical challenge, with high-performance liquid chromatography (HPLC) in reversed-phase mode, as the most widely used analytical technique. To facilitate the optimization of the procedures that are applied in the early stages of sample preparation, a simple and fast HPLC method is proposed in this work for the separation of some PPCPs with a wide range of hydrophilicity. Two columns were evaluated (Atlantis dC18 and Discovery HS F5); as for mobile phases: a formate buffer (40 mmol L-1, pH 4) and methanol were tested in a gradient mode. The fluorinated column allowed better separation in a shorter time and better resolution for all analytes (Rs > 1). The proposed method delivered good performance for the tracing of PPCPs and is a suitable alternative to traditional C18-based HPLC methods.

Berberine: A nematocidal alkaloid from Argemone mexicana against Strongyloides venezuelensis.

Strongyloidiasis is a parasitosis that represents a public health problem, in tropical regions. The present study aimed to investigate the anthelmintic effects of several extracts of Argemone mexicana, as well as its main component berberine (Ber) against the third-stage larvae (L3) of Strongyloides venezuelensis in-vitro experiments. Also, the anti-hemolytic activity of the extract, fractions, and Ber were tested in human erythrocytes. A dose-response anthelminthic bioassay demonstrated Ber as the most effective component, followed by methanolic subfraction (Fr3) and finally the crude extract of A. mexicana (Am) showing LC50 response values of 1.6, 19.5, and 92.1 µg/mL, at 96 h respectively. Also, Am, Fr3, and Ber did not produce significant hemolysis against human erythrocytes (p ≤ 0.05). Am and Fr3 showed erythrocyte protection effect capacity at the membrane level (p ≤ 0.05). Furthermore, Ber was found to have an antioxidant activity of 168.18 µg/mL. According to the results, the Fr3 of A. mexicana, and particularly Ber, exhibited potent in-vitro effects against L3 of S. venezuelensis, without hemolytic activity against human erythrocytes and presented good antioxidant capacity. In conclusion, the extracts of A. mexicana and the main component have activity against S. venezuelensis, nevertheless, further studies are required to elucidate the mechanism of action.