Daratumumab-based therapies in transplant-ineligible patients with untreated multiple myeloma and hepatic dysfunction: A systematic review of subgroup analyses.

INTRODUCTION: There is subgroup analysis suggesting a lack of benefit of daratumumab use in multiple myeloma (MM) and hepatic disease (HD). The objectives of this study were to conduct a systematic review and interpretation of daratumumab-based regimen efficacy in transplant-ineligible patients with untreated MM and HD. METHODS: A systematic search in Pubmed® database about randomized clinical trials (RCTs) with subgroup analysis regarding hepatic function for overall survival (OS) or progression-free survival (PFS) were developed. Two methodologies were applied. One of them considered statistical interaction, prespecification, biological support and consistency of subgroup results. Second methodology was two-part validated tool: preliminary questions to reject subset analysis without minimal relevance, and a checklist relating a recommendation for applicability in clinical practice. RESULTS: It was included three records. About first methodology, statistical interaction among subgroups was found for PFS in one RCT. Subsets were prespecified in all RCTs. Biological support of efficacy differences could be reasonable. Inconsistent results were found. Second methology directly rejected applicability of subset analysis in two records. Checklist recommended "null" application of results in the remaining RCT. CONCLUSIONS: No consistent heterogeneity for daratumumab-based regimen efficacy was observed among subgroups regarding hepatic function in transplant-ineligible patients with untreated MM. Patients with normal hepatic function and HD could benefit from these treatments.

Madurastatins with Imidazolidinone Rings: Natural Products or Side-Reaction Products from Extraction Solvents?

Madurastatins are a group of pentapeptides containing an oxazoline moiety, and, in a few cases, an imidazolidinone ring as an additional structural feature. In our search for new potential antiparasitic metabolites from natural sources, we studied the acetone extracts from a culture of Actinomadura sp. CA-135719. The LC/HRMS analysis of this extract identified the presence of the known madurastatins C1 (1), D1 (4), and D2 (5) together with additional members of the family that were identified as the new madurastatins H2 (2) and 33-epi-D1 (3) after isolation and spectroscopic analysis. The planar structures of the new compounds were established by HRMS, ESI-qTOF-MS/MS, and 1D and 2D NMR data, and their absolute configuration was proposed using Marfey's and bioinformatic analyses of the biosynthetic gene cluster (BGC). A revision of the absolute configuration of madurastatins D1 and D2 is proposed. Additionally, madurastatins containing imidazolidinone rings are proved to be artifacts originating during acetone extraction of the bacterial cultures.

Clinical and pharmacoeconomic impact of subgroup analysis in onco-hematological patients.

Subgroup analysis evaluates a health intervention in subpopulations according to a characteristic or factor. It can be useful for generating new hypotheses or conducting new studies. However, subgroup analysis presents several limitations and it should be considered cautiously. The development of new onco-hematological drugs is accelerating in recent years and the impact of subgroup analysis on clinical decision-making is increasing. The interpretation of subgroup analyses can be controversial in some cases, negatively affecting patients and healthcare systems. This work is a review of the clinical and pharmacoeconomic impact of subgroup analysis in onco-hematological patients. The study describes some illustrative examples of inadequate interpretations about subset analysis: combination of pembrolizumab plus chemotherapy in lung cancer, inhibitors of cyclin-dependent kinases in breast cancer, daratumumab-based regimens in newly diagnosed multiple myeloma, combination of nivolumab with ipilimumab in melanoma and docetaxel in prostate cancer. Subgroup analysis can have a significant impact on the data selection for the development of studies; efficacy, safety, and convenience of treatments in onco-hematological patients; efficiency of therapies in health systems; and therapeutic positioning of antineoplastic drugs. There is a strong need to establish homogeneous criteria for the assessment of subgroup analysis and to develop new tools for its consideration.

Pentaminomycins F and G, Nonribosomal Peptides Containing 2-Pyridylalanine.

Pentaminomycins F-H (1-3), a group of three new hydroxyarginine-containing cyclic pentapeptides, were isolated from cultures of a Streptomyces cacaoi subsp. cacaoi strain along with the known pentaminomycins A-E. The structures of the new peptides were determined by a combination of mass spectrometry, NMR, and Marfey's analyses. Pentaminomycins F (1) and G (2) were shown to contain the rare amino acid 3-(2-pyridyl)-alanine. This finding represents the first reported examples of nonribosomal peptides containing this residue. The ldlld chiral sequence found for the three compounds was in agreement with that reported for previously isolated pentaminomycins and consistent with the epimerization domains present in the putative nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster.

Use of docetaxel in low- and high-burden metastatic hormone-sensitive prostate cancer: A systematic review and assessment of subgroup analyses.

BACKGROUND: Use of docetaxel in low- and high-burden metastatic hormone-sensitive prostate cancer presents considerable controversy. There is literature suggesting lack of benefit for low-volume of metastases. OBJECTIVE: The study aims to develop a systematic review and methodological assessment of subset analysis about use of docetaxel in metastatic hormone-sensitive prostate cancer regarding volume of metastatic disease. METHODS: A systematic review in the Pubmed® database was conducted up to 25 September 2020. A reference tracking was also developed. Randomised clinical trials with subgroup analysis according volume of metastatic disease for overall survival were selected. Two methodologies were used. One of them considered statistical interaction of subsets (p(i) < 0.1), pre-specification, biological plausibility and consistency among subset results of similar randomised clinical trials. The second methodology was a two-part validated tool: preliminary questions to discard subset analysis without minimal relevance and a checklist The checklist provides recommendations for applicability of subgroup analysis in clinical practice. RESULTS: A total of 31 results were found in systematic reviews in the Pubmed® database. One result was identified in the reference tracking. Of the total of 32 results, four randomised clinical trials were included in the study. About first methodology, statistical interaction among subgroups was obtained in one randomised clinical trial. Subgroup analysis was pre-specified in two randomised clinical trials. Biological plausibility was reasonable. No external consistency among results of subgroup analyses in randomised clinical trials was observed. Preliminary questions of second methodology rejected applicability of subgroup analysis in three randomised clinical trials. A 'null' recommendation for applicability of subset results was obtained in the remaining randomised clinical trial. CONCLUSIONS: Patients with low- and high-burden metastatic hormone-sensitive prostate cancer would benefit from docetaxel therapy. No consistent differences for overall survival were observed in subgroup analyses regarding volume of metastatic disease.

Polyphenolic Extract (PE) from Olive Oil Exerts a Potent Immunomodulatory Effect and Prevents Graft-versus-Host Disease in a Mouse Model.

Polyphenols are a group of chemical substances found in plants, with immunomodulatory, antiproliferative, and anti-inflammatory properties that might be useful in the prophylaxis and treatment of graft-versus-host disease (GVHD). Polyphenolic extract (PE) obtained from extra virgin olive oil (EVOO) decreased the activation and proliferation of activated T cells. In addition, a decreased production of proinflammatory cytokines was observed upon exposure to PE. Western blot assays showed a marked inhibition of Akt phosphorylation and nuclear translocation of NF-κB in activated T cells. In a murine model of acute GVHD, we observed that mice that received a diet supplemented in PE (600 ppm) presented a higher survival rate and lower risk of developing GVHD when compared with the group that received a control diet. Histopathologic examination showed a significantly lower gut involvement in mice receiving PE, with a decrease in proinflammatory cytokines (IL-2, IL-17, and TNF-α) in serum and the reestablishment of butyrate concentration in the gut. In conclusion, PE obtained from EVOO exerted a potent immunomodulatory effect, reducing the activation and proliferation of activated T cells and the production of proinflammatory cytokines. In a murine model of acute GVHD, a PE-supplemented diet reduced the incidence and severity of the disease and increased survival after transplantation.

Checklist for clinical applicability of subgroup analysis.

WHAT IS KNOWN AND OBJECTIVE: Subgroup analysis plays an important role in clinical decision-making. Correct management of subgroup analysis is necessary to optimize effectiveness, safety and efficiency of treatments. No homogeneous criteria have been developed for interpretation of subgroup analysis. In this study, the researcher develops a checklist to evaluate the reliability and applicability of the results of subset analyses. METHODS: With a review of previous literature, three main criteria were included in the checklist: statistical association, biological plausibility and consistency among studies. Statistical association considered interaction probability, prespecification of analysis, number of subgroups analysed, sample size and positive/negative result in global analysis. Each item was given an indicative score. Total score was related to a level of applicability for the results in clinical practice. Checklist validation included interinvestigator concordance and assessment about utility. Three drug examples were used to validate the tool. RESULTS AND DISCUSSION: Twenty-six evaluators showed adequate interinvestigator concordance (kappa 0.79, 1 and 0.83 for each drug example regarding applicability). Kappa values increased to 0.94, 1 and 1 after group discussion. Checklist utility score was greater than 4.7/5 in three drug examples. In pre-analysis, inter-researcher agreement on global applicability recommendation of subgroup results to practice was 92.3% (ramucirumab), 96% (nivolumab) and 100% (mepolizumab). In post-analysis, inter-researcher agreement on applicability recommendation of subgroup results was 100%, 94.45% and 100%, respectively. The checklist validation shows a high interindividual agreement of the results, both with respect to the evaluation of the applicability of subgroup analysis and concerning clinical decision-making. WHAT IS NEW AND CONCLUSION: We have developed the first validated tool for interpretation of subgroup analyses. The checklist contributes to the adoption of homogeneous criteria for subgroup analyses, thereby allowing discussion and evaluation of the effects of a health intervention.

Identification, Cloning and Heterologous Expression of the Gene Cluster Directing RES-701-3, -4 Lasso Peptides Biosynthesis from a Marine Streptomyces Strain.

RES-701-3 and RES-701-4 are two class II lasso peptides originally identified in the fermentation broth of Streptomyces sp. RE-896, which have been described as selective endothelin type B receptor antagonists. These two lasso peptides only differ in the identity of the C-terminal residue (tryptophan in RES-701-3, 7-hydroxy-tryptophan in RES-701-4), thus raising an intriguing question about the mechanism behind the modification of the tryptophan residue. In this study, we describe the identification of their biosynthetic gene cluster through the genome mining of the marine actinomycete Streptomyces caniferus CA-271066, its cloning and heterologous expression, and show that the seven open reading frames (ORFs) encoded within the gene cluster are sufficient for the biosynthesis of both lasso peptides. We propose that ResE, a protein lacking known putatively conserved domains, is likely to play a key role in the post-translational modification of the C-terminal tryptophan of RES-701-3 that affords RES-701-4. A BLASTP search with the ResE amino acid sequence shows the presence of homologues of this protein in the genomes of eight other Streptomyces strains, which also harbour the genes encoding the RES-701-3, -4 precursor peptide, split-B proteins and ATP-dependent lactam synthetase required for the biosynthesis of these compounds.

Cacaoidin, First Member of the New Lanthidin RiPP Family.

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d-amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.

Polyphenolic extract from extra virgin olive oil inhibits the inflammatory response in IL-1ß-activated synovial fibroblasts.

The polyphenolic extract (PE) from extra virgin olive oil (EVOO) has been shown to possess important anti-inflammatory and joint protective properties in murine models of rheumatoid arthritis (RA). This study was designed to evaluate the effects of PE on IL-1ß-activated human synovial fibroblasts SW982 cell line. PE from EVOO treatment inhibited IL-1ß-induced matrix metalloproteases (P<0·001), TNF-α and IL-6 production (P<0·001). Similarly, IL-1ß-induced cyclo-oxygenase-2 and microsomal PGE synthase-1 up-regulations were down-regulated by PE (P<0·001). Moreover, IL-1ß-induced mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation were ameliorated by PE (P<0·001). These results suggest that PE from EVOO reduces the production of proinflammatory mediators in human synovial fibroblasts; particularly, these protective effects could be related to the inhibition of MAPK and NF-κB signalling pathways. Taken together, PE from EVOO probably could provide an attractive complement in management of diseases associated with over-activation of synovial fibroblasts, such as RA.

Structure elucidation and biosynthetic gene cluster analysis of caniferolides A-D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066.

Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D (1-4). Their connectivity was determined by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochemistry of each stereocluster in these compounds was established using NOE analysis, the universal database method and J-based configuration analysis, further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochemical outcome of their biosynthesis, confirming the relative stereochemistry of each stereocluster already determined by NMR and establishing their stereochemical relationship, ultimately rendering the absolute configuration of all chiral centers. Furthermore, based on our results and already published data, it has been possible to derive the complete absolute configuration of the related macrolides PM100117 and PM100118, astolides A and B, and deplelides A and B. Caniferolides A-D have shown pronounced antifungal activity against Candida albicans and Aspergillus fumigatus alongside antiproliferative activity against five human tumoral cell lines.

Quercus ilex Extract Ameliorates Acute TNBS-Induced Colitis in Rats.

Quercus ilex L. (Fagaceae) is one of the most commonly used plants in folk medicine in the Mediterranean region to treat gastrointestinal disorders. The aim of the present study was to evaluate the effects of a polyphenol extract from mature leaves of Q. ilex on acute 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats. A polyphenol extract from mature leaves of Q. ilex (250 and 500 mg/kg/day) was administered by gavage 48, 24, and 1 h prior to the induction of colitis with 2,4,6-trinitrobenzene sulfonic acid as well as 24 h later. The inflammation response was assessed by histology, myeloperoxidase activity, and Th1 proinflammatory cytokine production. The protein expression of cyclooxygenase-2 and inducible nitric oxide synthase, and signaling pathways were studied by Western blotting in the colon tissues. The polyphenol extract from mature leaves of Q. ilex decreased neutrophil infiltration, interleukin-1ß and TNF-α production, and proinflammatory proteins cyclooxygenase-2 and inducible nitric oxide synthase overexpression. Also, the polyphenol extract from mature leaves of Q. ilex was capable of blocking the activation of mitogen-activated protein kinases and nuclear transcription factor-kappa B. Furthermore, the reduction of inflammation by polyphenol extract from mature leaves of Q. ilex treatment was accompanied by a recovery of Nrf2 and heme oxygenase-1 protein expression levels. In conclusion, this study demonstrates that a polyphenol extract from mature leaves of Q. ilex improves 2,4,6-trinitrobenzene sulfonic acid-induced colitis, probably through mitogen-activated protein kinase/nuclear transcription factor-kappa B inhibition and Nrf2/heme oxygenase-1 activation signaling pathways, thus reducing the production of Th1 proinflammatory cytokines and cyclooxygenase-2 and inducible nitric oxide synthase overexpression.

Unveiling Concealed Functions of Endosymbiotic Bacteria Harbored in the Ascomycete Stachylidium bicolor.

Among the plethora of unusual secondary metabolites isolated from Stachylidium bicolor are the tetrapeptidic endolides A and B. Both tetrapeptides contain 3-(3-furyl)-alanine residues, previously proposed to originate from bacterial metabolism. Inspired by this observation, we aimed to identify the presence of endosymbiotic bacteria in S. bicolor and to discover the true producer of the endolides. The endobacterium Burkholderia contaminans was initially detected by 16S rRNA gene amplicon sequencing from the fungal metagenome and was subsequently isolated. It was confirmed that the tetrapeptides were produced by the axenic B. contaminans only when in latency. Fungal colonies unable to produce conidia and the tetrapeptides were isolated and confirmed to be free of B. contaminans A second endosymbiont identified as related to Sphingomonas leidyi was also isolated. In situ imaging of the mycelium supported an endosymbiotic relationship between S. bicolor and the two endobacteria. Besides the technical novelty, our in situ analyses revealed that the two endobacteria are compartmentalized in defined fungal cells, prevailing mostly in latency when in symbiosis. Within the emerging field of intracellular bacterial symbioses, fungi are the least studied eukaryotic hosts. Our study further supports the Fungi as a valuable model for understanding endobacterial symbioses in eukaryotes.IMPORTANCE The discovery of two bacterial endosymbionts harbored in Stachylidium bicolor mycelium, Burkholderia contaminans and Sphingomonas leidyi, is described here. Production of tetrapeptides inside the mycelium is ensured by B. contaminans, and fungal sporulation is influenced by the endosymbionts. Here, we illustrate the bacterial endosymbiotic origin of secondary metabolites in an Ascomycota host.

Virgin olive oil and its phenol fraction modulate monocyte/macrophage functionality: a potential therapeutic strategy in the treatment of systemic lupus erythematosus.

Monocytes and macrophages are critical effectors and regulators of inflammation and innate immune response, which appear altered in different autoimmune diseases such as systemic lupus erythematosus (SLE). Recent studies suggested that virgin olive oil (VOO) and particularly its phenol compounds might possess preventive effects on different immune-inflammatory diseases, including SLE. Here, we evaluated the effects of VOO (and sunflower oil) on lipopolysaccharide (LPS)-activated peritoneal macrophages from a model of pristane-induced SLE in BALB/c mice, as well as those of the phenol fraction (PF) from VOO on the immune-inflammatory activity and plasticity in monocytes and monocyte-derived macrophages from healthy volunteers. The release of nitrite and inflammatory cytokines was lower in LPS-treated peritoneal macrophages from pristane-SLE mice fed the VOO diet when compared with the sunflower oil diet. PF from VOO similarly decreased the secretion of nitrite and inflammatory cytokines and expression of inducible nitric oxide, PPARγ and Toll-like receptor 4 in LPS-treated human monocytes. PF from VOO also prevented the deregulation of human monocyte subset distribution by LPS and blocked the genetic signature of M1 macrophages while favouring the phenotype of M2 macrophages upon canonical polarisation of naïve human macrophages. For the first time, our study provides several lines of in vivo and in vitro evidence that VOO and PF from VOO target and counteract inflammatory pathways in the monocyte-macrophage lineage of mice with pristane-induced SLE and of healthy subjects, which is a meaningful foundation for further development and application in preclinical and clinical use of PF from VOO in patients with SLE.

An update on diet and nutritional factors in systemic lupus erythematosus management.

Systemic lupus erythematosus (SLE) is a chronic inflammatory and autoimmune disease characterised by multiple organ involvement and a large number of complications. SLE management remains complicated owing to the biological heterogeneity between patients and the lack of safe and specific targeted therapies. There is evidence that dietary factors can contribute to the geoepidemiology of autoimmune diseases such as SLE. Thus, diet therapy could be a promising approach in SLE owing to both its potential prophylactic effects, without the side effects of classical pharmacology, and its contribution to reducing co-morbidities and improving quality of life in patients with SLE. However, the question arises as to whether nutrients could ameliorate or exacerbate SLE and how they could modulate inflammation and immune function at a molecular level. The present review summarises preclinical and clinical experiences to provide the reader with an update of the positive and negative aspects of macro- and micronutrients and other nutritional factors, including dietary phenols, on SLE, focusing on the mechanisms of action involved.

Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK.

B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.

Dietary extra-virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen-induced arthritis.

PURPOSE: Current experimental studies support a beneficial role of extra-virgin olive oil (EVOO) in several inflammatory diseases. The present study was designed to evaluate the effects of dietary EVOO on type II collagen-induced arthritis (CIA) in mice. METHODS: DBA-1/J mice were randomized in four experimental groups (10 or 15 animals per group): (1) Sham sunflower diet (SO-Sham), (2) CIA sunflower diet (SO-CIA), (3) Sham EVOO diet (EVOO-Sham) and (4) CIA EVOO diet (EVOO-CIA) group. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrified 42 days after first immunization. In addition to macroscopic and histological analyses, serum levels of cartilage olimeric matrix protein (COMP), metalloproteinase-3 (MMP-3) and pro-inflammatory cytokines levels were evaluated by ELISA. The expressions of heme oxygenase-1 (HO-1), nuclear factor E2-related factor 2 (Nrf2), mitogen-activated protein kinases (MAPKs), Janus kinase-signal transducer and activator of transcription (JAK/STAT) and nuclear transcription factor-kappa B (NF-κB) pathways were studied by western blotting. RESULTS: EVOO diet significantly reduced joint edema and cartilage destruction, preventing the arthritis development. Dietary EVOO significantly decreased serum COMP and MMP-3 levels, as well as, the pro-inflammatory cytokines levels (TNF-α, IL-1ß and IL-17). Moreover, the activation of JAK/STAT, MAPKs and NF-κB pathways was drastically ameliorated. According to Nrf2 and HO-1, the protein expressions were up-regulated in those mice fed with EVOO. CONCLUSION: These results support the interest of EVOO as a beneficial functional food to prevent the development of the rheumatoid arthritis (RA).

Genome Mining of Streptomyces sp. Tü 6176: Characterization of the Nataxazole Biosynthesis Pathway.

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary-metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O-methyltransferase gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5-Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross-talk between the nataxazole biosynthesis pathway and an unknown pathway.

Identification in CLL of circulating intraclonal subgroups with varying B-cell receptor expression and function.

Chronic lymphocytic leukemia (CLL) is a tumor of circulating B cells, variably stimulated and anergized following exposure to antigen in lymphoid tissues. Down-modulation of surface IgM (sIgM) occurs, but expression and signal capacity can recover in vitro and apparently in vivo during recirculation. We have now dissected individual circulating clones of CLL cases according to sIgM expression level by differential binding to bead-bound anti-IgM. Four clear subgroups (SG1-4) with increasing sIgM were identified in 37/37 cases. Engagement of sIgM induced phosphorylation of PLCγ2 and ERK1/2 at levels ranging from very low in SG1 to high in SG4. Phosphorylation was suppressed by the BTK inhibitor ibrutinib. Expression of CXCR4 also increased from SG1 to SG4, but markers of previous activation and proliferation were dominant in SG1. Incubation of whole CLL populations in vitro led to striking increases in CXCR4 expression as well as recovery of sIgM. Clonal analysis reveals dynamic SGs following presumed antigen stimulation in tissues. SG4 represents a fully recovered, potentially dangerous population equipped to migrate to tissue and receive a proliferative stimulus. SG1 likely represents a postmitotic unresponsive "resting" population. The effect of ibrutinib on the small SG4 population may be the critical factor in therapeutic success.

Functional features of the exopolysaccharide extracts produced by Lactiplantibacillus strains isolated from table olives.

This study evaluates the functional characteristics of the exopolysaccharide (EPS) extracts produced by various strains of Lactiplantibacillus pentosus (LPG1, 119, 13B4, and Lp13) and Lactiplantibacillus plantarum (Lp15) isolated from table olives. None of the EPS crude extracts showed cytotoxicity when administered to THP-1 human macrophage cells at dosages ranging from 6.25 to 50 µg mL-1. Many exhibited anti-inflammatory properties (reduction of pro-inflammatory cytokines TNF-α and IL-6 production) and antioxidant activity (reduction of ROS%) when macrophages were stimulated with Escherichia coli lipopolysaccharide. Notably, the EPS extract produced by the L. pentosus LPG1 strain had the best results corroborated by western blot immune analysis for differential expression of COX-2, Nrf-2, and HO-1 proteins, with the most significant antioxidant and anti-inflammatory response observed at a dosage of 50 µg mL-1. Chemical analysis revealed that the EPS extract produced by this strain contains a heteropolymer composed of mannose (35.45%), glucose (32.99%), arabinose (17.93%), xylose (7.48%), galactose (4.03%), rhamnose (1.34%), and fucose (0.77%). Finally, we conducted response surface methodology to model the EPS extract production by L. pentosus LPG1 considering pH (3.48-8.52), temperature (16.59-33.41 °C) and salt concentration (0.03-8.77% NaCl) as independent variables. The model identified linear effects of salt and pH and quadratic effects of salt as significant terms. The maximum EPS extract production (566 mg L-1) in a synthetic culture medium (MRS) was achieved at pH 7.5, salt 7.0%, and a temperature of 20 °C. These findings suggest the potential for novel applications for the EPS produced by L. pentosus LPG1 as nutraceutical candidates for use in human diets.