DietaAnti-Inflamatoria or DAIN: A Crohn's disease management strategy tailored for Puerto Ricans.

Diet has been increasingly shown to be of therapeutic benefit for patients with inflammatory bowel diseases (IBD), especially Crohn's disease (CD). Yet dietary guidelines are nonexistent. Moreover, diets tailored to Puerto Ricans with IBD living on the island, have not been developed and tested. The rising prevalence of IBD in Puerto Rico warrants exploring the use of diet as part of the treatment strategies for these patients [1]. Here, we describe the study design of "Dieta Anti-Inflamatoria" or DAIN, a parallel two-arm randomized pilot trial aiming at testing the efficacy of IBD-Anti-inflammatory diet (IBD-AID) adapted for adults with CD living in Puerto Rico (clinical trial registration number: NCT05627128). We tailored the IBD-AID to the local cuisine preferences and food availability by creating and adapting recipes consistent with the IBD-AID principles [2,3]. In focus groups with a Community Research Advisory Panel and one-on-one consultations with implementation experts, we identified several aspects of the intervention to adapt before the implementation. The objectives of the stakeholder/expert-informed adaptation were to improve feasibility and compliance while developing the culturally tailored dietary intervention. DAIN was designed for adults living in Puerto Rico with CD and geared to be affordable, appropriate, and acceptable for patients with mild-to-moderate CD. The significance of this work is the validation of culturally appropriate nutritional guidelines to help manage CD symptoms. DAIN provides a blueprint for a comprehensive nutritional program that can be adapted to regional preferences and local food availability allowing wider implementation of diet as an adjunct treatment in diverse clinical settings.

Antibiotics Modulate Intestinal Regeneration.

The increased antibiotics usage in biomedical and agricultural settings has been well documented. Antibiotics have now been shown to exert effects outside their purposive use, including effects on physiological and developmental processes. We explored the effect of various antibiotics on intestinal regeneration in the sea cucumber Holothuria glaberrima. For this, holothurians were eviscerated and left to regenerate for 10 days in seawater with different penicillin/streptomycin-based cocktails (100 µg/mL PS) including: 100 µg/mL kanamycin (KPS), 5 µg/mL vancomycin (VPS), and 4 µg/mL (E4PS) or 20 µg/mL (E20PS) erythromycin. Immunohistological and histochemical analyses were performed to analyze regenerative processes, including rudiment size, extracellular matrix (ECM) remodeling, cell proliferation, and muscle dedifferentiation. A reduction in muscle dedifferentiation was observed in all antibiotic-treated animals. ECM remodeling was decreased by VPS, E4PS, and E20PS treatments. In addition, organisms subjected to E20PS displayed a significant reduction in the size of their regenerating rudiments while VPS exposure altered cell proliferation. MTT assays were used to discard the possibility that the antibiotics directly affect holothurian metabolic activity while bacterial cultures were used to test antibiotic effects on holothurian enteric microbiota. Our results demonstrate a negative effect on intestinal regeneration and strongly suggest that these effects are due to alterations in the microbial community.

The Role of the Microbiota in Regeneration-Associated Processes.

The microbiota, the set of microorganisms associated with a particular environment or host, has acquired a prominent role in the study of many physiological and developmental processes. Among these, is the relationship between the microbiota and regenerative processes in various organisms. Here we introduce the concept of the microbiota and its involvement in regeneration-related cellular events. We then review the role of the microbiota in regenerative models that extend from the repair of tissue layers to the regeneration of complete organs or animals. We highlight the role of the microbiota in the digestive tract, since it accounts for a significant percentage of an animal microbiota, and at the same time provides an outstanding system to study microbiota effects on regeneration. Lastly, while this review serves to highlight echinoderms, primarily holothuroids, as models for regeneration studies, it also provides multiple examples of microbiota-related interactions in other processes in different organisms.

Insights into intestinal regeneration signaling mechanisms.

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/ß-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro, using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of ß-catenin activity was determined using an antibody against phosphorylated ß-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated ß-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined.

The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 during inflammation. However, the roles of TLT-1 in hemostasis and inflammation are not well understood. We are developing the mouse model of TLT-1 to mechanistically test clinical associations of TLT-1 in health and disease. To facilitate our studies, monoclonal murine TLT-1 (mTLT-1) antibodies were produced by the immunization of a rabbit using the negatively charged region of the mTLT-1 extracellular domain 122PPVPGPREGEEAEDEK139. In the present study, we demonstrate that two selected clones, 4.6 and 4.8, are suitable for the detection of mTLT-1 by western blot, immunoprecipitation, immunofluorescent staining, flow cytometry and inhibit platelet aggregation in aggregometry assays. In addition, we found that the topical administration of clone 4.8 delayed the wound healing process in an experimental burn model. These results suggest that TLT-1 plays an important role in wound healing and because both clones specifically detect mTLT-1, they are suitable to further develop TLT-1 based models of inflammation and hemostasis in vivo.