Resolving therapy resistance mechanisms in multiple myeloma by multiomics subclone analysis.

Intratumor heterogeneity as a clinical challenge becomes most evident after several treatment lines, when multidrug-resistant subclones accumulate. To address this challenge, the characterization of resistance mechanisms at the subclonal level is key to identify common vulnerabilities. In this study, we integrate whole-genome sequencing, single-cell (sc) transcriptomics (scRNA sequencing), and chromatin accessibility (scATAC sequencing) together with mitochondrial DNA mutations to define subclonal architecture and evolution for longitudinal samples from 15 patients with relapsed or refractory multiple myeloma. We assess transcriptomic and epigenomic changes to resolve the multifactorial nature of therapy resistance and relate it to the parallel occurrence of different mechanisms: (1) preexisting epigenetic profiles of subclones associated with survival advantages, (2) converging phenotypic adaptation of genetically distinct subclones, and (3) subclone-specific interactions of myeloma and bone marrow microenvironment cells. Our study showcases how an integrative multiomics analysis can be applied to track and characterize distinct multidrug-resistant subclones over time for the identification of molecular targets against them.

Oral supplementation of choline attenuates the development of alcohol-related liver disease (ALD).

BACKGROUND: Chronic alcohol intake is associated with alterations of choline metabolism in various tissues. Here, we assessed if an oral choline supplementation attenuated the development of alcohol-related liver disease (ALD) in mice. METHODS: Female C57BL/6 J mice (n = 8/group) were either pair-fed a liquid control diet, or a Lieber DeCarli liquid diet (5% ethanol) ± 2.7 g choline/kg diet for 29 days. Liver damage, markers of intestinal permeability and intestinal microbiota composition were determined. Moreover, the effects of choline on ethanol-induced intestinal permeability were assessed in an ex vivo model. RESULTS: ALD development as determined by liver histology and assessing markers of inflammation (e.g., nitric oxide, interleukin 6 and 4-hydroxynonenal protein adducts) was attenuated by the supplementation of choline. Intestinal permeability in small intestine being significantly higher in ethanol-fed mice was at the level of controls in ethanol-fed mice receiving choline. In contrast, no effects of the choline supplementation were found on intestinal microbiota composition. Choline also significantly attenuated the ethanol-induced intestinal barrier dysfunction in small intestinal tissue ex vivo, an effect almost entirely abolished by the choline oxidase inhibitor dimbunol. CONCLUSION: Our results suggest that an oral choline supplementation attenuates the development of ALD in mice and is related to a protection from intestinal barrier dysfunction.

A weekly 4-methylpyrazole treatment attenuates the development of non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in male mice: Role of JNK.

BACKGROUND: 4-methylpyrazole (4MP, fomepizole) is a competitive inhibitor of alcohol dehydrogenase (ADH) preventing the metabolism of ethylene glycol and methanol, respectively, into their toxic metabolites. 4MP seems also to possess a potential in the treatment of intoxication from other substance, for example, acetaminophen, and to modulate JNK-dependent signalling. Here, we determined if a treatment with 4MP once weekly affects the development of diet-induced non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in C57BL/6 mice. METHODS: Male C57BL/6 mice (6-8 weeks old, n = 7-8/group) were pair-fed either a liquid control diet (C) or a liquid sucrose-, fat- and cholesterol-rich diet (SFC) for 8 weeks while being concomitantly treated with 4MP (50 mg/kg bw i.p.) or vehicle once a week. Liver damage, inflammatory markers and glucose tolerance were assessed. Moreover, in endotoxin-challenged J774A.1 cells pretreated with 4MP, pro-inflammatory markers were assessed. RESULTS: The concomitant treatment of SFC-fed mice with 4MP attenuated the increase in JNK phosphorylation and pro-inflammatory markers like IFNγ, IL-6 and 3-nitrotyrosine protein adducts in liver tissue found in vehicle-treated SFC-fed mice, while not affecting impairments of glucose tolerance or the increase in portal endotoxin levels. Moreover, a pretreatment of endotoxin-stimulated J774A.1 cells with 4MP significantly attenuated the increases in JNK phosphorylation and pro-inflammatory mediators like IL-6 and Mcp1. CONCLUSIONS: Taken together, our results suggest that a treatment with 4MP once weekly attenuates the activation of JNK and dampens the development of non-obese MASLD in mice.

Fructose: a modulator of intestinal barrier function and hepatic health?

PURPOSE: Consumption of fructose has repeatedly been discussed to be a key factor in the development of health disturbances such as hypertension, diabetes type 2, and non-alcoholic fatty liver disease. Despite intense research efforts, the question if and how high dietary fructose intake interferes with human health has not yet been fully answered. RESULTS: Studies suggest that besides its insulin-independent metabolism dietary fructose may also impact intestinal homeostasis and barrier function. Indeed, it has been suggested by the results of human and animal as well as in vitro studies that fructose enriched diets may alter intestinal microbiota composition. Furthermore, studies have also shown that both acute and chronic intake of fructose may lead to an increased formation of nitric oxide and a loss of tight junction proteins in small intestinal tissue. These alterations have been related to an increased translocation of pathogen-associated molecular patterns (PAMPs) like bacterial endotoxin and an induction of dependent signaling cascades in the liver but also other tissues. CONCLUSION: In the present narrative review, results of studies assessing the effects of fructose on intestinal barrier function and their impact on the development of health disturbances with a particular focus on the liver are summarized and discussed.

Alterations of nitric oxide homeostasis as trigger of intestinal barrier dysfunction in non-alcoholic fatty liver disease.

Changes in intestinal nitric oxide metabolism are discussed to contribute for the development of intestinal barrier dysfunction in non-alcoholic fatty liver disease (NAFLD). To induce steatosis, female C57BL/6J mice were pair-fed with a liquid control diet (C) or a fat-, fructose- and cholesterol-rich diet (FFC) for 8 weeks. Mice received the diets ± 2.49 g L-arginine/kg bw/day for additional 5 weeks. Furthermore, mice fed C or FFC ± L-arginine/kg bw/day for 8 weeks were concomitantly treated with the arginase inhibitor Nω -hydroxy-nor-L-arginine (nor-NOHA, 0.01 g/kg bw). Liver damage, intestinal barrier function, nitric oxide levels and arginase activity in small intestine were assessed. Also, arginase activity was measured in serum from 13 patients with steatosis (NAFL) and 14 controls. The development of steatosis with beginning inflammation was associated with impaired intestinal barrier function, increased nitric oxide levels and a loss of arginase activity in small intestine in mice. L-arginine supplementation abolished the latter along with an improvement of intestinal barrier dysfunction; nor-NOHA attenuated these effects. In patients with NAFL, arginase activity in serum was significantly lower than in healthy controls. Our data suggest that increased formation of nitric oxide and a loss of intestinal arginase activity is critical in NAFLD-associated intestinal barrier dysfunction.

Oral intake of xanthohumol attenuates lipoteichoic acid-induced inflammatory response in human PBMCs.

PURPOSE: The aim of the study was to determine if xanthohumol, a prenylated chalcone found in Hop (Humulus lupulus), has anti-inflammatory effects in healthy humans if applied in low doses achievable through dietary intake. METHODS: In a placebo-controlled single-blinded cross-over design study, 14 healthy young men and women either consumed a beverage containing 0.125 mg xanthohumol or a placebo. Peripheral blood mononuclear cells (PBMCs) were isolated before and 1 h after the intake of the beverages. Subsequently, PBMCs were stimulated with or without lipoteichoic acid (LTA) for 24 and 48 h. Concentrations of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and soluble cluster of differentiation (sCD14) protein were determined in cell culture supernatant. Furthermore, hTLR2 transfected HEK293 cells were stimulated with LTA in the presence or absence of xanthohumol and sCD14. RESULTS: The stimulation of PBMCs with LTA for 24 and 48 h resulted in a significant induction of IL-1ß, IL-6, and sCD14 protein release in PBMCs of both, fasted subjects and subjects after the ingestion of the placebo. In contrast, after ingesting xanthohumol, LTA-dependent induction of IL-1ß, IL-6, and sCD14 protein release from PBMCs was not significantly higher than in unstimulated cells after 48 h. In hTLR2 transfected HEK293 cells xanthohumol significantly suppressed the LTA-dependent activation of cells, an effect attenuated when cells were co-incubated with sCD14. CONCLUSION: The results of our study suggest that an ingestion of low doses of xanthohumol can suppress the LTA-dependent stimulation of PBMCs through mechanisms involving the interaction of CD14 with TLR2. Study registered at ClinicalTrials.gov (NCT04847193, 22.03.2022).

A Xanthohumol-Rich Hop Extract Diminishes Endotoxin-Induced Activation of TLR4 Signaling in Human Peripheral Blood Mononuclear Cells: A Study in Healthy Women.

Infections with Gram-negative bacteria are still among the leading causes of infection-related deaths. Several studies suggest that the chalcone xanthohumol (XN) found in hop (Humulus lupulus) possesses anti-inflammatory effects. In a single-blinded, placebo controlled randomized cross-over design study we assessed if the oral intake of a single low dose of 0.125 mg of a XN derived through a XN-rich hop extract (75% XN) affects lipopolysaccharide (LPS)-induced immune responses in peripheral blood mononuclear cells (PBMCs) ex vivo in normal weight healthy women (n = 9) (clinicaltrials.gov: NCT04847193) and determined associated molecular mechanisms. LPS-stimulation of PBMCs isolated from participants 1 h after the intake of the placebo for 2 h resulted in a significant induction of pro-inflammatory cytokine release which was significantly attenuated when participants had consumed XN. The XN-dependent attenuation of proinflammatory cytokine release was less pronounced 6 h after the LPS stimulation while the release of sCD14 was significantly reduced at this timepoint. The LPS-dependent activation of hTLR4 transfected HEK293 cells was significantly and dose-dependently suppressed by the XN-rich hop extract which was attenuated when cells were co-challenged with sCD14. Taken together, our results suggest even a one-time intake of low doses of XN consumed in a XN-rich hop extract can suppress LPS-dependent stimulation of PBMCs and that this is related to the interaction of the hop compound with the CD14/TLR4 signaling cascade.

Microbiota profiling in aging-associated inflammation and liver degeneration.

BACKGROUND: The number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called 'inflammaging', and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood. OBJECTIVE: The aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration. METHODS: Blood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed. RESULTS: Pairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon's diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice. CONCLUSION: Despite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age.

Aging-related liver degeneration is associated with increased bacterial endotoxin and lipopolysaccharide binding protein levels.

Aging is a risk factor in the development of many diseases, including liver-related diseases. The two aims of the present study were 1) to determine how aging affects liver health in mice in the absence of any interventions and 2) if degenerations observed in relation to blood endotoxin levels are critical in aging-associated liver degeneration. Endotoxin levels and markers of liver damage, mitochondrial dysfunction, insulin resistance, and apoptosis as well as the Toll-like receptor 4 (Tlr-4) signaling cascade were studied in liver tissue and blood, respectively, of 3- and 24-mo-old male C57BL/6J mice. In a second set of experiments, 3- to 4-mo-old and 14-mo-old female lipopolysaccharide-binding protein (LBP)-/- mice and littermates fed standard chow, markers of liver damage, insulin resistance, and mitochondrial dysfunction were assessed. Plasma activity of aspartate aminotransferase and histological signs of hepatic inflammation and fibrosis were significantly higher in old C57BL/6J mice than in young animals. The number of neutrophils, CD8α-positive cells, and mRNA expression of markers of apoptosis were also significantly higher in livers of old C57BL/6J mice compared with young animals, being also associated with a significant induction of hepatic Tlr-4 and LBP expression as well as higher endotoxin levels in peripheral blood. Compared with age-matched littermates, LBP-/- mice display less signs of senescence in liver. Taken together, our data suggest that, despite being fed standard chow, old mice developed liver inflammation and beginning fibrosis and that bacterial endotoxin may play a critical role herein.NEW & NOTEWORTHY Old age in mice is associated with marked signs of liver degeneration, hepatic inflammation, and fibrosis. Aging-associated liver degeneration is associated with elevated bacterial endotoxin levels and an induction of lipopolysaccharide-binding protein (LBP) and Toll-like receptor 4-dependent signaling cascades in liver tissue. Furthermore, in old aged LBP-/- mice, markers of senescence seem to be lessened, supporting the hypothesis that bacterial endotoxin levels might be critical in aging-associated decline of liver.

Moderate consumption of fermented alcoholic beverages diminishes diet-induced non-alcoholic fatty liver disease through mechanisms involving hepatic adiponectin signaling in mice.

PURPOSE: Results of some epidemiological studies suggest that moderate alcohol consumption may be associated with a decreased risk to develop NAFLD. Here, the effect of the consumption of moderate beer and diluted ethanol, respectively, on the development of NAFLD were assessed. METHODS: Female C57BL/6J mice were fed a control diet (C-D) or a diet rich in fructose, fat and cholesterol (FFC) enriched isocalorically and isoalcoholically with beer (FFC + B) or plain ethanol (FFC + E) (2.5 g ethanol/kg body weight/day) for 7 weeks. Liver damage was assessed by histology using NAFLD activity score. Markers of inflammation, insulin resistance and adiponectin signaling were measured at mRNA and protein levels. Using J774A.1 cells as a model of Kupffer cells, the effect of alcoholic beverages on adiponectin receptor 1 (Adipor1) was assessed. RESULTS: Hepatic triglyceride concentration, neutrophil granulocytes, iNOS protein concentrations and early signs of insulin resistance found in FFC-fed mice were significantly attenuated in FFC+ B-fed mice (P < 0.05 for all). These findings were associated with a super-induction of Adipor1 mRNA expression (+ ~ 18-fold compared to all other groups) and a decrease of markers of lipid peroxidation in liver tissue of FFC + B-fed mice when compared to FFC-fed animals. Similar differences were not found between FFC- and FFC+ E-fed mice. Expression of Adipor1 was also super-induced (7.5-fold) in J774A.1 cells treated with beer (equivalent to 2 mmol/L ethanol). CONCLUSIONS: These data suggest that moderate intake of fermented alcoholic beverages such as beer at least partially attenuates NAFLD development through mechanisms associated with hepatic AdipoR1 expression.

Oral Supplementation of Glutamine Attenuates the Progression of Nonalcoholic Steatohepatitis in C57BL/6J Mice.

Background: Universally accepted therapeutic strategies for the treatment of nonalcoholic steatohepatitis (NASH) are still lacking. Studies suggest a preventive effect of oral Gln supplementation on the development of NASH; however, whether Gln also has therapeutic potential for pre-existing NASH has not yet been clarified.Objective: The aim of the present study was to determine whether Gln prevents the progression of diet-induced NASH in mice.Methods: For 8 wk, female C57BL/6J mice (6-8 wk old) were pair-fed a liquid Western-style diet [WSD, 25% of energy from fat, 50% wt:wt fructose, 0.16% wt:wt cholesterol] or control diet (C diet) to induce liver damage. From week 8 to 13, they were pair-fed the C diet or WSD alone or supplemented with l-Gln to provide 2.1 g/kg body weight (C diet + Gln or WSD + Gln). Energy intake was adjusted to the group with the lowest energy intake. Indexes of liver damage and inflammation, intestinal barrier function, and toll-like receptor 4 (Tlr4) signaling in the liver were determined.Results: The liver histology scores significantly increased from 8 to 13 wk (+31%) in WSD-fed mice and were significantly higher than in controls (P ≤ 0.05 for both time comparisons), whereas scores did not differ between C diet-fed and WSD + Gln-fed mice after 13 wk of feeding. The occludin protein concentrations in the small intestinal tissue were similarly reduced in both WSD-fed groups when compared with controls [WSD compared with C diet (-53%) and C diet + Gln (-42%), P ≤ 0.05; WSD + Gln compared with C diet + Gln (-34%), P ≤ 0.05] after 13 wk, whereas the expression of myeloid differentiation primary response gene 88 mRNA and concentration of inducible nitric oxide synthase and 4-hydroxynonenal protein adducts were significantly higher only in livers of WSD-fed mice (P ≤ 0.05 for the WSD group compared with all other groups; WSD + Gln group compared with the C diet groups: NS).Conclusion: Taken together, our data suggest that oral Gln supplementation protects mice from the progression of pre-existing, WSD-induced NASH.

Stigmatization of psychiatrists and general practitioners: results of an international survey.

The stigma of mental illness affects psychiatry as a medical profession and psychiatrists. The present study aimed to compare the extent and correlation patterns of perceived stigma in psychiatrists and general practitioners. An international multicenter survey was conducted in psychiatrists and general practitioners from twelve countries. Responses were received from N = 1,893 psychiatrists and N = 1,238 general practitioners. Aspects of stigma assessed in the questionnaire included perceived stigma, self-stigma (stereotype agreement), attitudes toward the other profession, and experiences of discrimination. Psychiatrists reported significantly higher perceived stigma and discrimination experiences than general practitioners. Separate multiple regression analyses showed different predictor patterns of perceived stigma in the two groups. Hence, in the psychiatrists group, perceived stigma correlated best with discrimination experiences and self-stigma, while in the general practitioners group it correlated best with self-stigma. About 17% of the psychiatrists perceive stigma as a serious problem, with a higher rate in younger respondents. Against this background, psychiatry as a medical profession should set a high priority on improving the training of young graduates. Despite the number of existing antistigma interventions targeting mental health professionals and medical students, further measures to improve the image of psychiatry and psychiatrists are warranted, in particular improving the training of young graduates with respect to raising awareness of own stigmatizing attitudes and to develop a better profession-related self-assertiveness.

Oral Supplementation of Phosphatidylcholine Attenuates the Onset of a Diet-Induced Metabolic Dysfunction-Associated Steatohepatitis in Female C57BL/6J Mice.

BACKGROUND & AIMS: Changes in phosphatidylcholine levels in the liver have been associated with the development of metabolic dysfunction-associated steatotic liver disease. Here, the effects of supplementing phosphatidylcholine on the development of early signs of metabolic dysfunction-associated steatohepatitis were assessed. METHODS: Male and female C57BL/6J mice were fed a liquid control or a fructose-, fat-, and/or cholesterol-rich diet for 7 or 8 weeks. The diets of female mice were fortified ± phosphatidylcholine (12.5 mg/g diet). In liver tissue and portal blood, indices of liver damage, inflammation, and bacterial endotoxemia were measured. J774A.1 cells and human monocytes preincubated with phosphatidylcholine (0.38 mmol/L) were challenged with lipopolysaccharide (50-100 ng/mL) ± the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (10 µmol/L) or ± a liver receptor homolog 1 (LRH-1) antagonist 1-(3'-[1-(2-[4-morpholinyl]ethyl)-1H-pyrazol-3-yl]-3-biphenylyl)ethanon (1-10 µmol/L). RESULTS: In fructose-, fat-, and/or cholesterol-rich diet-fed mice the development of fatty liver and the beginning of inflammation were associated with significantly lower hepatic phosphatidylcholine levels when compared with controls. Supplementing phosphatidylcholine significantly attenuated the development of fatty liver and inflammation, being associated with protection against the induction of PPARγ2, and activation of nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor α whereas Lrh1 expression was unchanged. The protective effects of phosphatidylcholine on the lipopolysaccharide-induced activation of J774A.1 cells and human monocytes were attenuated significantly by the PPARγ activator pioglitazone and the LRH-1 antagonist. CONCLUSIONS: Our data suggest that phosphatidylcholine levels in the liver are lower in early metabolic dysfunction-associated steatohepatitis in mice and that supplementation of phosphatidylcholine can diminish the development of metabolic dysfunction-associated steatotic liver disease through mechanisms involving LRH-1/PPARγ2/ nuclear factor κ-light-chain enhancer of activated B-cell signaling.

MASLD is related to impaired alcohol dehydrogenase (ADH) activity and elevated blood ethanol levels: Role of TNFα and JNK.

Elevated fasting ethanol levels in peripheral blood frequently found in metabolic dysfunction-associated steatohepatitis (MASLD) patients even in the absence of alcohol consumption are discussed to contribute to disease development. To test the hypothesis that besides an enhanced gastrointestinal synthesis a diminished alcohol elimination through alcohol dehydrogenase (ADH) may also be critical herein, we determined fasting ethanol levels and ADH activity in livers and blood of MASLD patients and in wild-type ± anti-TNFα antibody (infliximab) treated and TNFα-/- mice fed a MASLD-inducing diet. Blood ethanol levels were significantly higher in patients and wild-type mice with MASLD while relative ADH activity in blood and liver tissue was significantly lower compared to controls. Both alterations were significantly attenuated in MASLD diet-fed TNFα-/- mice and wild-type mice treated with infliximab. Moreover, alcohol elimination was significantly impaired in mice with MASLD. In in vitro models, TNFα but not IL-1ß or IL-6 significantly decreased ADH activity. Our data suggest that elevated ethanol levels in MASLD patients are related to TNFα-dependent impairments of ADH activity.

Antibiotics attenuate diet-induced nonalcoholic fatty liver disease without altering intestinal barrier dysfunction.

To date the role of the alterations of intestinal microbiota in the development of intestinal barrier dysfunction in settings of nonalcoholic fatty liver disease (NAFLD) has not been fully understood. Here, we assessed the effect of antibiotics on development of NAFLD and their impact on intestinal barrier dysfunction. Male C57BL/6J mice were either pair-fed a liquid control diet (C) or fat- and fructose-rich diet (FFr) +/- antibiotics (AB, ampicillin/vancomycin/metronidazole/gentamycin) for 7 weeks. Fasting blood glucose was determined and markers of liver damage, inflammation, intestinal barrier function, and microbiota composition were assessed. The development of hepatic steatosis with early signs of inflammation found in FFr-fed mice was significantly abolished in FFr+AB-fed mice. Also, while prevalence of bacteria in feces was not detectable and TLR4 ligand levels in portal plasma were at the level of controls in FFr+AB-fed mice, impairments of intestinal barrier function like an increased permeation of xylose and iNOS protein levels persisted to a similar extent in both FFr-fed groups irrespective of AB use. Exposure of everted small intestinal tissue sacs of naïve mice to fructose resulted in a significant increase in tissue permeability and loss of tight junction proteins, being not affected by the presence of AB, whereas the concomitant treatment of tissue sacs with the NOS inhibitor aminoguanidine attenuated these alterations. Taken together, our data suggest that intestinal barrier dysfunction in diet-induced NAFLD in mice may not be predominantly dependent on changes in intestinal microbiota but rather that fructose-induced alterations of intestinal NO-homeostasis might be critically involved.

Pre-Clinical Assessment of SAR442257, a CD38/CD3xCD28 Trispecific T Cell Engager in Treatment of Relapsed/Refractory Multiple Myeloma.

Current treatment strategies for multiple myeloma (MM) are highly effective, but most patients develop relapsed/refractory disease (RRMM). The anti-CD38/CD3xCD28 trispecific antibody SAR442257 targets CD38 and CD28 on MM cells and co-stimulates CD3 and CD28 on T cells (TCs). We evaluated different key aspects such as MM cells and T cells avidity interaction, tumor killing, and biomarkers for drug potency in three distinct cohorts of RRMM patients. We found that a significantly higher proportion of RRMM patients (86%) exhibited aberrant co-expression of CD28 compared to newly diagnosed MM (NDMM) patients (19%). Furthermore, SAR442257 mediated significantly higher TC activation, resulting in enhanced MM killing compared to bispecific functional knockout controls for all relapse cohorts (Pearson's r = 0.7). Finally, patients refractory to anti-CD38 therapy had higher levels of TGF-ß (up to 20-fold) compared to other cohorts. This can limit the activity of SAR442257. Vactoserib, a TGF-ß inhibitor, was able to mitigate this effect and restore sensitivity to SAR442257 in these experiments. In conclusion, SAR442257 has high potential for enhancing TC cytotoxicity by co-targeting CD38 and CD28 on MM and CD3/CD28 on T cells.

The circadian clock is functional in eosinophils and mast cells.

Allergic diseases are frequently exacerbated between midnight and early morning, suggesting a role for the biological clock. Mast cells (MC) and eosinophils are the main effector cells of allergic diseases and some MC-specific or eosinophil-specific markers, such as tryptase or eosinophil cationic protein, exhibit circadian variation. Here, we analysed whether the circadian clock is functional in mouse and human eosinophils and MC. Mouse jejunal MC and polymorphonuclear cells from peripheral blood (PMNC) were isolated around the circadian cycle. Human eosinophils were purified from peripheral blood of non-allergic and allergic subjects. Human MC were purified from intestinal tissue. We found a rhythmic expression of the clock genes mPer1, mPer2, mClock and mBmal1 and eosinophil-specific genes mEcp, mEpo and mMbp in murine PMNC. We also found circadian variations for hPer1, hPer2, hBmal1, hClock, hEdn and hEcp mRNA and eosinophil cationic protein (ECP) in human eosinophils of both healthy and allergic people. Clock genes mPer1, mPer2, mClock and mBmal1 and MC-specific genes mMcpt-5, mMcpt-7, mc-kit and mFcεRI α-chain and protein levels of mMCPT5 and mc-Kit showed robust oscillation in mouse jejunum. Human intestinal MC expressed hPer1, hPer2 and hBmal1 as well as hTryptase and hFcεRI α-chain, in a circadian manner. We found that pre-stored histamine and de novo synthesized cysteinyl leukotrienes, were released in a circadian manner by MC following IgE-mediated activation. In summary, the biological clock controls MC and eosinophils leading to circadian expression and release of their mediators and, hence it might be involved in the pathophysiology of allergy.

Acute Intake of Sucrose but Not of the Intense Sweetener Sucralose Is Associated with Post-Prandial Endotoxemia in Healthy Young Adults-A Randomized Controlled Trial.

Sugar-rich diets, but also the use of intense sweeteners, may alter intestinal barrier function. Here, we assessed the effect of sucrose and sucralose on post-prandial endotoxemia in a randomized placebo-controlled single-blinded crossover-designed study. Following a 2-day standardization of their diet, healthy men and women received a beverage containing either sucrose, sucralose (iso-sweet) or an isocaloric combination of sucralose + maltodextrin. Plasma endotoxin levels were measured after consumption of the respective beverages. Moreover, the effect of sucrose and sucralose on intestinal permeability was assessed in Caco-2 cells and ex vivo in an everted gut sac model. The nutritional standardization recommended by nutrition societies was associated with a significant decrease in plasma endotoxin levels. The intake of the sucrose-sweetened beverage resulted in a significant increase in plasma endotoxin levels while being unchanged after the intake of sucralose-sweetened beverages. In Caco-2 cells, the challenge with sucrose but not with sucralose significantly increased the permeation of the bacterial endotoxin across the cell monolayer. Xylose permeation in small intestinal everted tissue sacs was significantly higher upon the challenge with sucrose while remaining unchanged in sucralose-challenged sacs. Our data suggest that an acute intake of physiologically relevant amounts of sucrose but not of sucralose can result in post-prandial endotoxemia.

TNFα is a key trigger of inflammation in diet-induced non-obese MASLD in mice.

Tumor necrosis factor alpha (TNFα) is thought to be a critical factor in the development of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we determined the effects of a treatment with the anti-TNFα antibody infliximab and a genetic deletion of TNFα, respectively, in the development of non-obese diet-induced early metabolic dysfunction-associated steatohepatitis (MASH) in mice. The treatment with infliximab improved markers of liver damage in mice with pre-existing early MASH. In TNFα-/- mice, the development of early signs of MASH and insulin resistance was significantly attenuated compared to wild-type animals. While mRNA expression of proinflammatory cytokines like interleukin 1ß (Il1b) and interleukin 6 (Il6) were significantly lower in livers of MASH-diet-fed TNFα-/- mice compared to wild-type mice with early MASH, markers of intestinal barrier function were similarly impaired in both MASH-diet-fed groups compared to controls. Our data suggest that TNFα is a key regulator of hepatic inflammation and insulin resistance associated with the development of early non-obese MASH.

Supplementing L-Citrulline Can Extend Lifespan in C. elegans and Attenuate the Development of Aging-Related Impairments of Glucose Tolerance and Intestinal Barrier in Mice.

L-Citrulline (L-Cit) is discussed to possess a protective effect on intestinal barrier dysfunction but also to diminish aging-associated degenerative processes. Here, the effects of L-Cit on lifespan were assessed in C. elegans, while the effects of L-Cit on aging-associated decline were determined in C57BL/6J mice. For lifespan analysis, C. elegans were treated with ±5 mM L-Cit. Twelve-month-old male C57BL/6J mice (n = 8-10/group) fed a standard chow diet received drinking water ± 2.5 g/kg/d L-Cit or 5 g/kg/d hydrolyzed soy protein (Iso-N-control) for 16 or 32 weeks. Additionally, 4-month-old C57BL/6J mice were treated accordingly for 8 weeks. Markers of senescence, glucose tolerance, intestinal barrier function, and intestinal microbiota composition were analyzed in mice. L-Cit treatment significantly extended the lifespan of C. elegans. The significant increase in markers of senescence and signs of impaired glucose tolerance found in 16- and 20-month-old control mice was attenuated in L-Cit-fed mice, which was associated with protection from intestinal barrier dysfunction and a decrease in NO2- levels in the small intestine, while no marked differences in intestinal microbiota composition were found when comparing age-matched groups. Our results suggest that pharmacological doses of L-Cit may have beneficial effects on lifespan in C. elegans and aging-associated decline in mice.