An Enterobacteriaceae bloom in aging animals is restrained by the gut microbiome.

The gut microbiome plays important roles in host function and health. Core microbiomes have been described for different species, and imbalances in their composition, known as dysbiosis, are associated with pathology. Changes in the gut microbiome and dysbiosis are common in aging, possibly due to multi-tissue deterioration, which includes metabolic shifts, dysregulated immunity, and disrupted epithelial barriers. However, the characteristics of these changes, as reported in different studies, are varied and sometimes conflicting. Using clonal populations of Caenorhabditis elegans to highlight trends shared among individuals, we employed 16s rRNA gene sequencing, CFU counts and fluorescent imaging, identifying an Enterobacteriaceae bloom as a common denominator in aging animals. Experiments using Enterobacter hormaechei, a representative commensal, suggested that the Enterobacteriaceae bloom was facilitated by a decline in Sma/BMP immune signaling in aging animals and demonstrated its potential for exacerbating infection susceptibility. However, such detrimental effects were context-dependent, mitigated by competition with commensal communities, highlighting the latter as determinants of healthy versus unhealthy aging, depending on their ability to restrain opportunistic pathobionts.

Microbiome remodeling through bacterial competition and host behavior enables rapid adaptation to environmental toxins.

Human activity is altering the environment in a rapid pace, challenging the adaptive capacities of genetic variation within animal populations. Animals also harbor extensive gut microbiomes, which play diverse roles in host health and fitness and may help expanding host capabilities. The unprecedented scale of human usage of xenobiotics and contamination with environmental toxins describes one challenge against which bacteria with their immense biochemical diversity would be useful, by increasing detoxification capacities. To explore the potential of bacteria-assisted rapid adaptation, we used Caenorhabditis elegans worms harboring a defined microbiome, and neomycin as a model toxin, harmful for the worm host and neutralized to different extents by some microbiome members. Worms raised in the presence of neomycin showed delayed development and decreased survival but were protected when colonized by neomycin-resistant members of the microbiome. Two distinct mechanisms facilitated this protection: gut enrichment driven by altered bacterial competition for the strain best capable of modifying neomycin; and host avoidance behavior, which depended on the conserved JNK homolog KGB-1, enabling preference and acquisition of neomycin-protective bacteria. We further tested the consequences of adaptation, considering that enrichment for protective strains may represent dysbiosis. We found that neomycin-adapted gut microbiomes caused increased susceptibility to infection as well as an increase in gut lipid storage, suggesting metabolic remodeling. Our proof-of-concept experiments support the feasibility of bacteria-assisted host adaptation and suggest that it may be prevalent. The results also highlight trade-offs between toxin adaptation and other traits of fitness.

An Enterobacteriaceae bloom in aging animals is restrained by the gut microbiome.

The gut microbiome plays important roles in host function and health. Core microbiomes have been described for different species, and imbalances in their composition, known as dysbiosis, are associated with pathology. Changes in the gut microbiome and dysbiosis are common in aging, possibly due to multi-tissue deterioration, which includes metabolic shifts, dysregulated immunity, and disrupted epithelial barriers. However, the characteristics of these changes, as reported in different studies, are varied and sometimes conflicting. Using clonal populations of C. elegans to highlight trends shared among individuals, and employing NextGen sequencing, CFU counts and fluorescent imaging to characterize age-dependent changes in worms raised in different microbial environments, we identified an Enterobacteriaceae bloom as a common denominator in aging animals. Experiments using Enterobacter hormachei, a representative commensal, suggested that the Enterobacteriaceae bloom was facilitated by a decline in Sma/BMP immune signaling in aging animals and demonstrated its detrimental potential for increasing susceptibility to infection. However, such detrimental effects were context-dependent, mitigated by competition with commensal communities, highlighting the latter as determinants of healthy versus unhealthy aging, depending on their ability to restrain opportunistic pathobionts.

Real-world experience with belantamab mafodotin therapy for relapsed/refractory multiple myeloma: A multicentre retrospective study.

Belantamab mafodotin, an immuno-conjugate targeting B-cell maturation antigen, showed single-agent activity in phase 1 and 2 studies, and was recently approved for heavily pretreated relapsed/refractory multiple myeloma (RRMM) patients. Real-world data and long-term follow-up are scarce. We conducted a multisite retrospective study aimed to assess safety and efficacy of belantamab mafodotin monotherapy administered via the GSK expanded access compassionate care programme. One-hundred and six RRMM patients were treated with belantamab mafodotin between July 2019 and March 2021. The median age was 69.4 years. Patients were heavily pretreated with a median of six (range 2-11) prior therapy lines. Major adverse effects included ocular toxicity (keratopathy 68.4%, grade ≥3: 40.5%; blurred vision 36.8%, grade ≥3: 6.3%), thrombocytopenia (27.4%, grade ≥3: 17.9%) and infections (11.3%, grade ≥3: 7.5%). Median follow-up time was 11.9 [95% confidence interval (CI) 10.0-13.8] months. Overall response rate was 45.5%. Median progression-free survival was 4.7 (95% CI 3.5-5.9) months in the entire cohort and 8.8 (95% CI 6.6-10.9) months among responders. Median overall survival was 14.5 (95% CI 9.5-19.6) months, and not reached for responders. To conclude, in a real-world setting, belantamab mafodotin monotherapy showed efficacy comparable with the prospective clinical trials, with a tolerable toxicity profile.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab growth in microbially diverse and natural-like environments. Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Host Preference of Beneficial Commensals in a Microbially-Diverse Environment.

Gut bacteria are often described by the neutral term commensals. However, the more we learn about their interactions with hosts, the more apparent it becomes that gut commensals often contribute positively to host physiology and fitness. Whether hosts can prefer beneficial bacteria, and how they do so, is not clear. This is of particular interest in the case of the bacterivore C. elegans, which depends on bacteria as food source, but also as gut colonizers that contribute to its physiology, from development to immunity. It is further unclear to what extent worms living in their microbially-diverse habitats can sense and distinguish between beneficial bacteria, food, and pathogens. Focusing on Enterobacteriaceae and members of closely related families, we isolated gut bacteria from worms raised in compost microcosms, as well as bacteria from the respective environments and evaluated their contributions to host development. Most isolates, from worms or from the surrounding environment, promoted faster development compared to the non-colonizing E. coli food strain. Pantoea strains further showed differential contributions of gut isolates versus an environmental isolate. Characterizing bacterial ability to hinder pathogenic colonization with Pseudomonas aeruginosa, supported the trend of Pantoea gut commensals being beneficial, in contrast to the environmental strain. Interestingly, worms were attracted to the beneficial Pantoea strains, preferring them over non-beneficial bacteria, including the environmental Pantoea strain. While our understanding of the mechanisms underlying these host-microbe interactions are still rudimentary, the results suggest that hosts can sense and prefer beneficial commensals.

The Impact of Induction Regimes on Immune Responses in Patients with Multiple Myeloma.

Current standard frontline therapy for newly diagnosed patients with multiple myeloma (NDMM) involves induction therapy, autologous stem cell transplantation (ASCT), and maintenance therapy. Major efforts are underway to understand the biological and the clinical impacts of each stage of the treatment protocols on overall survival statistics. The most routinely used drugs in the pre-ASCT "induction" regime have different mechanisms of action and are employed either as monotherapies or in various combinations. Aside from their direct effects on cancer cell mortality, these drugs are also known to have varying effects on immune cell functionality. The question remains as to how induction therapy impacts post-ASCT immune reconstitution and anti-tumor immune responses. This review provides an update on the known immune effects of melphalan, dexamethasone, lenalidomide, and bortezomib commonly used in the induction phase of MM therapy. By analyzing the actions of each individual drug on the immune system, we suggest it might be possible to leverage their effects to rationally devise more effective induction regimes. Given the genetic heterogeneity between myeloma patients, it may also be possible to identify subgroups of patients for whom particular induction drug combinations would be more appropriate.

Eltrombopag for enhancement of platelet engraftment in patients undergoing allogeneic cord blood transplantation.

Platelet recovery after allogeneic umbilical cord blood (UCB) transplantation is delayed compared to other graft sources. We conducted a multicenter phase 2a study to explore whether eltrombopag, a thrombopoietin-receptor agonist, would enhance platelet recovery after UCB transplantation. Between 02/2013 and 07/2016, 12 (10 adults, 2 children) individuals (median age 50; range 6-74 years) with hematological malignancies in complete remission were enrolled. Eltrombopag was given for a median of 76 (range 15-175) days and was safe even at doses of 300 mg/day. Median time to neutrophil engraftment was 23 (range 16-40) days. Median time to platelets >20,000/µl and >50,000/µl was 55 (range 25-199) and 66 (range 31-230) days, respectively. A historical cohort comparison did not reveal an advantage for eltrombopag. In conclusion, in the present study eltrombopag seems safe. Based on our limited data, it seems unlikely that eltrombopag could enhance platelet engraftment after UCB transplantation.

miR-71 mediates age-dependent opposing contributions of the stress-activated kinase KGB-1 in Caenorhabditis elegans.

Studying the evolutionary processes that shaped aging offers a path for understanding the causes of aging. The antagonistic pleiotropy theory for the evolution of aging proposes that the inverse correlation between age and natural selection strength allows positive selection of gene variants with early-life beneficial contributions to fitness despite detrimental late-life consequences. However, mechanistic understanding of how this principle manifests in aging is still lacking. We previously identified antagonistic pleiotropy in the function of the Caenorhabditis elegans JNK homolog KGB-1, which provided stress protection in developing larvae, but sensitized adults to stress and shortened their lifespan. To a large extent, KGB-1's contributions depended on age-dependent and opposing regulation of the stress-protective transcription factor DAF-16, but the underlying mechanisms remained unknown. Here, we describe a role for the microRNA miR-71 in mediating effects of KGB-1 on DAF-16 and downstream phenotypes. Fluorescent imaging along with genetic and survival analyses revealed age-dependent regulation of mir-71 expression by KGB-1-upregulation in larvae, but downregulation in adults-and showed that mir-71 was required both for late-life effects of KGB-1 (infection sensitivity and shortened lifespan), as well as for early life resistance to cadmium. While mir-71 disruption did not compromise development under protein-folding stress (known to depend on KGB-1), disruption of the argonaute gene alg-1, a central component of the microRNA machinery, did. These results suggest that microRNAs play a role in mediating age-dependent antagonistic contributions of KGB-1 to survival, with mir-71 playing a central role and additional microRNAs potentially contributing redundantly.

Interactions with a Complex Microbiota Mediate a Trade-Off between the Host Development Rate and Heat Stress Resistance.

Animals and plants host diverse communities of microorganisms, and these microbiotas have been shown to influence host life history traits. Much has been said about the benefits that host-associated microbiotas bestow on the host. However, life history traits often demonstrate tradeoffs among one another. Raising Caenorhabditis elegans nematodes in compost microcosms emulating their natural environment, we examined how complex microbiotas affect host life history traits. We show that soil microbes usually increase the host development rate but decrease host resistance to heat stress, suggesting that interactions with complex microbiotas may mediate a tradeoff between host development and stress resistance. What element in these interactions is responsible for these effects is yet unknown, but experiments with live versus dead bacteria suggest that such effects may depend on bacterially provided signals.

CeMbio - The Caenorhabditis elegans Microbiome Resource.

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g., human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans.

TGFß/BMP immune signaling affects abundance and function of C. elegans gut commensals.

The gut microbiota contributes to host health and fitness, and imbalances in its composition are associated with pathology. However, what shapes microbiota composition is not clear, in particular the role of genetic factors. Previous work in Caenorhabditis elegans defined a characteristic worm gut microbiota significantly influenced by host genetics. The current work explores the role of central regulators of host immunity and stress resistance, employing qPCR and CFU counts to measure abundance of core microbiota taxa in mutants raised on synthetic communities of previously-isolated worm gut commensals. This revealed a bloom, specifically of Enterobacter species, in immune-compromised TGFß/BMP mutants. Imaging of fluorescently labeled Enterobacter showed that TGFß/BMP-exerted control operated primarily in the anterior gut and depended on multi-tissue contributions. Enterobacter commensals are common in the worm gut, contributing to infection resistance. However, disruption of TGFß/BMP signaling turned a normally beneficial Enterobacter commensal to pathogenic. These results demonstrate specificity in gene-microbe interactions underlying gut microbial homeostasis and highlight the pathogenic potential of their disruption.

Augmented myeloablative conditioning with thiotepa in acute myeloid leukemia - improved outcomes with similar toxicity.

Myeloablative doses of busulfan (Bu) with fludarabine (Flu) have reduced toxicity, however, limited by an increased relapse rate. We aimed to improve outcome of Flu-Bu regimen by augmentation with thiotepa (TT) (10 mg/kg). Eighty-nine patients with AML, 44 patients conditioned with Flu-Bu (group 1), and 45 patients augmented with TT (Flu-Bu-TT, group 2), were retrospectively analyzed. Primary objectives were toxicity and outcomes. Major toxicities were comparable: mucositis (p = 1.0), sepsis (p = .7), severe venocclusive disease of liver (VOD) (p = 1.0), and non-relapse mortality (NRM) (22 vs. 22%, p = .7). Five-year disease-free survival was significantly better in group 2 compared to group 1 (62 vs. 38%, p = .02). Five-year overall survival (OS) showed trend toward benefit in group 2 (62 vs. 42%, p = .06). Lower relapse rate in group 2 (14 vs. 46%, p = .005) contributed to better outcomes. Augmented regimen has better disease-free survival (DFS) (mainly due to reduced relapse rate) and similar toxicities as compared to Flu-Bu. Key points  Assessing the addition of TT to myeloablative conditioning (Flu, Bu) in patients undergoing allogeneic stem cell transplant for acute myeloid leukemia with regard to relapse rate, disease-free survival and toxicity.  Addition of thiotepa improves disease-free survival and shows trend toward benefit in overall survival, by reducing relapses without additional toxicity.

Integration of Stress Signaling in Caenorhabditis elegans Through Cell-Nonautonomous Contributions of the JNK Homolog KGB-1.

Dealing with physiological stress is a necessity for all organisms, and the pathways charged with this task are highly conserved in Metazoa . Accumulating evidence highlights cell-nonautonomous activation as an important mode of integrating stress responses at the organism level. Work in Caenorhabditis elegans highlighted the importance of such regulation for the unfolded protein response (UPR) and for gene expression downstream of the longevity-associated transcription factor DAF-16 Here we describe a role for the JNK homolog KGB-1 in cell-nonautonomous regulation of these two response modules. KGB-1 protects developing larvae from heavy metals and from protein folding stress (which we found to be independent of canonical UPR pathways), but sensitizes adults to the same stress, further shortening life span under normal conditions. This switch is associated with age-dependent antagonistic regulation of DAF-16 Using transgenic tissue-specific KGB-1 expression or tissue-specific KGB-1 activation we examined the contributions of KGB-1 to gene regulation, stress resistance, and life span. While cell-autonomous contributions were observed, particularly in the epidermis, cell-nonautonomous contributions of neuronal KGB-1 (and also in muscle) were effective in driving intestinal gene induction, age-dependent regulation of intestinal DAF-16, and stress resistance, and did not require KGB-1 expression in the target tissue. Additional genetic analyses revealed requirement for UNC-13 in mediating neuronal contributions, indicating involvement of neurotransmission. Our results expand the role of KGB-1 in stress responses from providing local cellular protection to integrating stress responses at the level of the whole organism.

A Bortezomib-Based Protocol Induces a High Rate of Complete Remission with Minor Toxicity in Adult Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia.

The treatment of relapsed/refractory acute lymphoblastic leukemia (RR-ALL) presents a true clinical challenge. In 2012, a protocol combining bortezomib, dexamethasone, asparaginase, doxorubicin, and vincristine administered to children with RR-ALL was published with encouraging results. Over the past 5 years, we have implemented this protocol in the adult RR-ALL population (> 18 years) and addressed its feasibility in terms of remission rate and toxicity. Here, we present the results of our experience in 9 patients, all of whom received multiple previous chemotherapy protocols, two of them relapsing after an allogeneic bone marrow transplantation. All of the five B-ALL patients, and two of the four T-ALL achieved complete remission. Of the seven patients achieving complete remission, two patients were referred for allogeneic bone marrow transplantation, two patients were subsequently given blinatumomab, and one patient subsequently received donor lymphocyte infusion followed by blinatumomab. Thus, five out of nine patients treated (55%) were able to proceed to best available therapy in a complete remission. We observed minimal adverse effects, mainly hematological toxicity. We conclude that the bortezomib-based protocol should be evaluated as an effective and well-tolerated treatment option for adult patients either unfit for or failing standard salvage chemotherapy, as a bridge to immunotherapy or allogeneic bone marrow transplantation.

Fludarabine-based reduced toxicity yet myeloablative conditioning is effective and safe particularly in patients with high-risk thalassemia undergoing allogeneic transplantation.

INTRODUCTION: Thalassemia major (TM) is an inherited disorder caused by ineffective erythropoiesis. At the present time, allogeneic stem cell transplantation (allo-SCT) is a curative option. Conventional busulfan and cyclophosphamide based myeloablative conditioning regimens are limited by increased toxicity, especially in high-risk patients. Replacement of cyclophosphamide with fludarabine has reduced toxicity and nonrelapse mortality (NRM), thus improving outcomes. We analyzed long-term data of our fludarabine-based myeloablative, reduced toxicity protocol, specifically in high-risk patients. METHODS: We retrospectively analyzed a cohort of 47 consecutive patients with TM undergoing allo-SCT from matched donors, using the fludarabine-based regimen (reduced toxicity regimen). The median age of the cohort was 10 years. Thirty-eight patients (80%) were in the high-risk and nine patients (20%) were in the low-risk category. The primary aim of this analysis was thalassemia-free survival (TFS). RESULTS: The rejection rate was 11% within high-risk patients with NRM of 2%. With a median follow-up period of 7 years (1-15 years), the 10-year TFS in the entire cohort was 87%, and the overall survival (OS) was 97%. The 10-year TFS and OS among the low-risk and high-risk groups were 90% versus 84%, respectively (P = 0.45) and 100% versus 96%, respectively (P = 0.5), and both subsets of patients did equally well. CONCLUSION: In conclusion, replacement of high-dose cyclophosphamide with fludarabine is well tolerated with minimal regimen-related toxicity and acceptable rejection rates, especially in high-risk patients.

Thiotepa, Etoposide, Cyclophosphamide, Cytarabine, and Melphalan (TECAM) Conditioning Regimen for Autologous Stem Cell Transplantation in Lymphoma.

BACKGROUND: High-dose chemotherapy and autologous stem cell transplantation (ASCT) is the current standard of care for relapsed non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Conditioning regimens with high-dose carmustine have been associated with idiopathic pneumonitis syndrome. We, therefore, created a modified alternative TECAM conditioning regimen, consisting of etoposide, thiotepa, cytarabine, cyclophosphamide, and melphalan. PATIENTS AND METHODS: We retrospectively analyzed our cohort of 212 NHL and HL patients, who had undergone ASCT with the TECAM conditioning regimen from 2000 to 2013. Although toxicity and engraftment were analyzed for all 212 patients, the survival analysis was performed for the 2 largest groups of patients, those with diffuse large B-cell lymphoma (DLBCL) and those with HL (n = 127) to minimize heterogeneity. RESULTS: The 3-year overall survival among the DLBCL and HL patients was 0.618 (95% confidence interval [CI], 0.490-0.722) and 0.828 (95% CI, 0.701-0.904), respectively. Stage IV disease at transplantation was a statistically significant poor prognostic factor. Higher Eastern Cooperative Oncology Group performance status and progressive disease at transplantation were found to be borderline significant. No idiopathic pneumonitis syndrome cases were reported in our cohort. Six patients died of treatment-related toxicity during the first 100 days. The 3-year progression-free survival was 0.5 (95% CI, 0.37-0.61) for HL patients and 0.49 (95% CI, 0.36-0.60) for DLBCL patients. CONCLUSION: Our results are encouraging and justify evaluation of TECAM versus BEAM (carmustine, etoposide, cytarabine, melphalan) in a prospective multicenter study in a large homogenous patient population.

Host-microbiota interactions in Caenorhabditis elegans and their significance.

As a useful genetic model, C. elegans can facilitate investigation of the genetic underpinnings of host-microbiota interactions. However, decades of feeding it with Escherichia coli left a gap in our understanding of its interactions with microbes, hindering such use. This is changing, with recent studies characterizing the gut microbiota of worms in their natural habitats, comparing them to those in their environment, and evaluating the significance of gut and environmental commensals. This work defined a shared core gut microbiota significantly influenced by host genetics, and unraveled bacterial contributions to life history traits. Establishing C. elegans as a new model of host-microbiota interactions will benefit from existing knowledge about bacterial modulation of worm physiology, and could draw mechanistic insights from characterized interactions between parasitic nematodes and their symbionts.