Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice.

Bone fractures are among the most prevalent musculoskeletal injuries, and pain management is an essential part of fracture treatment. Fractures heal through an early inflammatory phase, followed by repair and remodeling. Nonsteroidal anti-inflammatory drugs (NSAIDs) are not recommended for fracture pain control as they potently inhibit the inflammatory phase and, thus, impair the healing. Opioids do not provide a better alternative for several reasons, including abuse potential. Accordingly, there is an unmet clinical need for analgesics that effectively ameliorate postfracture pain without impeding the healing. Here, we investigated the analgesic efficacy of two nonpsychotropic cannabinoids, cannabidiol (CBD) and cannabigerol (CBG), in a mouse model for tibial fracture. Mice with fractured tibiae exhibited increased sensitivity to mechanical, cold, and hot stimuli. Both CBD and CBG normalized pain sensitivity to all tested stimuli, and their analgesic effects were comparable to those of the NSAIDs. Interestingly, CBD and CBG promoted bone healing via multiple mechanisms during the early and late phases. During the early inflammatory phase, both cannabinoids increased the abundance of periosteal bone progenitors in the healing hematoma and promoted the osteogenic commitment of these progenitors. During the later phases of healing, CBD and CBG accelerated the fibrocartilaginous callus mineralization and enhanced the viability and proliferation of bone and bone-marrow cells. These effects culminated in higher bone volume fraction, higher bone mineral density, and improved mechanical quality of the newly formed bone. Together, our data suggest CBD and CBG as therapeutic agents that can replace NSAIDs in managing postfracture pain as both cannabinoids exert potent analgesic effects and, at the same time, promote bone healing. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing.

Maturation of the 3' end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3' untranslated regions (UTRs). The 3'UTR contains motifs that regulate mRNA metabolism; thus, changing the 3'UTR length via APA can significantly affect gene expression. Endochondral ossification is a central process in bone healing, but the impact of APA on gene expression during this process is unknown. Here, we report the widespread occurrence of APA, which impacts multiple pathways that are known to participate in bone healing. Importantly, the progression of endochondral ossification involves global 3'UTR shortening, which is coupled with an increased abundance of shortened transcripts relative to other transcripts; these results highlight the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of transcripts that undergo APA in the fracture callus revealed an intricate regulatory network in which APA enhances the expression of the collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA exerts this effect by shortening the 3'UTRs of the Col1a1 and Col1a2 mRNAs, thus removing the binding sites of miR-29a-3p, which would otherwise strongly promote the degradation of both transcripts. Taken together, our study is the first to characterize the crucial roles of APA in regulating the 3'UTR landscape and modulating gene expression during fracture healing.

Transurethral Resection of the Bladder Tumour as a Treatment Method in Children with Transitional Cell Carcinoma of the Bladder--Analysis of Our Material and Literature Review.

BACKGROUND: Bladder cancer occurs mainly in adults. In children, younger than 10 years in particular, it is very rare. OBJECTIVES: The aim of the study is to retrospectively evaluate the efficacy of transurethral resection of the bladder tumour (TUR-BT) of transitional cell carcinoma (TCC) of the bladder in children. MATERIAL AND METHODS: Transurethral resection of the bladder tumour was performed in 7 boys aged 4 to 17 years (median 12.1 years). In all cases laboratory tests, ultrasound, and cystoscopic tumour biopsy were carried out prior to the resection. Doxorubicin was additionally instilled intravesically as one dose in two patients. The Foley catheter was left in the bladder for 1 to 4 days (median 1.85 days). The follow-up period ranged from 10 months to 10 years (median 4 years). RESULTS: Papillary urothelial neoplasm of low malignant potential (PUNLMP) was diagnosed in 5 patients and urothelial papilloma in 2. Local recurrence was observed in one case two years after the resection. In all other cases complete remission was achieved. CONCLUSIONS: Transitional cell carcinoma of the bladder in children is usually benign and endoscopic treatment (TUR-BT) seems to be the treatment of choice. To determine a follow-up schedule a more substantial group of children with bladder cancer should be analysed.

Erk/MAP kinase signaling pathway and neuroendocrine differentiation of non-small-cell lung cancer.

INTRODUCTION: Almost all small-cell lung cancers (SCLC) and carcinoid tumors express neuroendocrine differentiation (NED), and 10% to 20% of non-small-cell lung cancers (NSCLC) are associated with NED. Although distinct clinical features and histology of SCLC and carcinoid tumors are well recognized, the clinical significance and the molecular basis of NED in NSCLC remain unclear. METHODS: To explore the potential molecular pathway involved in NED of NSCLC and its clinical relevance, we conducted investigations using an NSCLC cell line (NCI-H157) as a NED induction model, and explored the potential intracellular signal transduction pathways involved in NED of NSCLC. We confirmed our findings using activators versus inhibitors to these signal transduction pathways in vitro. We also performed immunohistochemical stains of phospho-Erk1/2 of lung cancer specimens known to have NED and explored its clinical relevance. RESULTS: We discovered that NED of NSCLC was associated with the activation of Erk1/2-mitogen-activated protein kinases (MAPK) signal transduction pathway, and the inhibition of the Akt signal transduction pathway. Using specific activator (Pb) and inhibitors (siRNA-Erk1/2 and U0126) to the Erk1/2-MAP-kinase pathway, as well as the inhibitor (LY294002) to the Akt pathway, we found that Erk1/2-MAP-kinase activation was essential for NED of NCI-H157 cells. Staining of Erk1/2-MAP-kinase pathway revealed a high rate of positivity in NSCLC tumors with NED when compared with other neuroendocrine lung tumors. CONCLUSIONS: To our knowledge, our findings are the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in the association with NED. Further investigation of the Erk/MAPK signal transduction pathway of NSCLC may yield discoveries in identifying specific molecular targets for the treatment of NSCLC with NED.

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis.

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.

Response kinetics of radiation-induced micronucleated reticulocytes in human bone marrow culture.

The frequency of micronucleated reticulocytes (MN-RETs) in the bone marrow or peripheral blood is a sensitive indicator of cytogenetic damage. While the kinetics of MN-RET induction in rodent models following irradiation has been investigated and reported, information about MN-RET induction of human bone marrow after radiation exposure is sparse. In this report, we describe a human long-term bone marrow culture (LTBMC), established in three-dimensional (3D) bioreactors, which sustains long-term erythropoiesis. Using this system, we measured the kinetics of human bone marrow red blood cell (RBC) and reticulocyte (RET) production, as well as the kinetics of human MN-RET induction following radiation exposure up to 6Gy. Human bone marrow established in the 3D bioreactor demonstrated an average percentage of RBCs among total viable cells peaking at 21% on day 21. The average percentage of RETs among total viable cells reached a maximum of 11% on day 14, and remained above 5% by day 28, suggesting that terminal erythroid differentiation was still active. Time- and dose-dependent induction of MN-RET by gamma radiation was observed in the human 3D LTBMC, with peak values occurring at approximately 3 days following 1Gy irradiation. A trend towards delayed peak to 3-5 days post-radiation was observed with radiation doses ≥2Gy. Our data reveal valuable information on the kinetics of radiation-induced MN-RET of human bone marrow cultured in the 3D bioreactor, a synthetic bioculture system, and suggest that this model may serve as a promising tool for studying MN-RET formation in human bone marrow, thereby providing opportunities to study bone marrow genotoxicity testing, mitigating agent effects, and other conditions that are not ordinarily feasible to experimental manipulation in vivo.

Validating high-throughput micronucleus analysis of peripheral reticulocytes for radiation biodosimetry: benchmark against dicentric and CBMN assays in a mouse model.

Automation of radiation biodosimetry is one of the top priority tasks considered by the Office of Science and Technology Policy and the Homeland Security Council in preparation for the nation's readiness for a possible radionuclear terrorist attack. The Center for Biophysical Assessment and Risk Management Following Irradiation, a consortium of researchers and institutions centered at the University of Rochester, has been investigating automated scoring of radiation-induced micronucleus formation in reticulocytes for high-throughput radiation biodosimetry. The collaborative project is based on a commercially-available product by Litron Laboratories in Rochester, New York. The study was designed to validate the flow-cytometry based analysis of micronucleated reticulocyte expression for radiation biodosimetry by benchmarking against the standard lymphocyte-based biodosimetry methods in a mouse model. C57B1/6 mice and C3H mice were exposed to Cs total-body radiation from 0-3 Gy. Blood samples were subsequently analyzed for CD71+ micronucleated reticulocyte and reticulocyte frequencies by flow cytometry. Results showed a linear dose-response of MN-RET up to 1 Gy for C57B1/6 and 2 Gy for C3H mice. On the other hand, robust and good dose-response curves were obtained with lymphocyte-based dicentric assay and cytokinesis-block micronucleus assay up to 3 Gy. High-throughput, automated analyses of micronucleated reticulocytes is a sensitive and reproducible method for detecting recent radiation exposure. In mice, the dose range of detection is useful up to 1 Gy (C57Bl/6) and 2 Gy (C3H) but not reliable beyond these dose limits. The utilization of this automated analysis for human radiation biodosimetry is currently under investigation.

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: effect of age.

The effect of age on the formation of radiation-induced micronucleated reticulocytes (MN-RETs) and reticulocytes (RETs) was investigated by exposing female C57BL/6J mice to graded doses of gamma rays from a (137)Cs source. Age at time of irradiation was 6, 16, or 32 weeks, and doses ranged from 0.5 to 3 Gy. A flow cytometric technique based on anti-CD71 labeling was used to measure RET and MN-RET frequencies in blood specimens collected 43 h post-irradiation. Mean RET frequencies declined in a dose-dependent manner for each age group. There was only one significant difference among the ages, that is, %RETs were not significantly reduced in the oldest animals at 0.5 Gy, whereas this dose did have a significant effect on the other age groups. MN-RET data were more complex. Age was observed to influence the baseline frequency of MN-RET, with the oldest mice exhibiting a significantly higher mean value. Each group's %MN-RETs values increased up to 1 Gy, but past this dose the frequencies plateaued or decreased. Age was observed to influence micronucleus frequency, with older mice exhibiting higher mean MN-RET values, especially at the high doses where the response was saturated (2-3 Gy). We hypothesize that these dissimilar responses can largely be explained by an age-related down-regulation of apoptosis whereby younger animals eliminate damaged bone marrow erythroid precursors with a greater efficiency compared with aged mice.

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: dose-response and time-course profiles measured by flow cytometry.

A flow cytometric, anti-CD71-based method was used to measure peripheral blood reticulocyte and micronucleated reticulocyte frequencies in response to (137)Cs total body irradiation (TBI). In three independent experiments, groups of five female C57BL/6N mice were irradiated at graded doses up to 3 Gy, and peripheral blood specimens were collected at 43 h post-irradiation. Whereas the frequency of reticulocytes declined over the range of doses studied, micronucleated reticulocyte incidence was observed to increase in a dose-dependent manner up to 1 Gy. At doses greater than approximately 1 Gy, micronucleated reticulocyte frequencies declined with increasing exposure. These responses were highly reproducible, with significant effects on reticulocyte and micronucleated reticulocyte frequencies observed for the lowest dose studied (0.125 Gy). A time-course experiment was performed to test whether radiation-induced cell cycle delay may explain saturation of the micronucleated reticulocyte endpoint at doses >1 Gy. For this experiment, groups of four female C57BL/6N mice were exposed to 1, 1.5, or 2 Gy TBI, and blood collection occurred at 12h intervals from 43 to 115 h post-exposure. Reduced reticulocyte frequencies were observed for each dose studied, and the recovery of reticulocytes was increasingly delayed with higher radiation doses. Maximal micronucleated reticulocyte frequencies were observed at 43 or 55 h, with progressively lower values at later time points. At no time did micronucleated reticulocyte frequencies induced by 1.5 or 2 Gy significantly exceed that observed for 1 Gy at 43 h. These time-course data suggest that radiation-induced cell cycle delay cannot account for the micronucleated reticulocyte downturn phenomenon observed at doses greater than 1 Gy. An alternate hypothesis is discussed whereby apoptotic elimination of severely damaged bone marrow erythroid precursors plays a dominant role in saturating the radiation-induced micronucleated reticulocyte response observed for C57BL/6N mice.

[Selected aspects of the epidemiological analysis of celiac disease and diabetes mellitus type 1 coexistance in children and youth]. / Wybrane aspekty analizy epidemiologicznej wspólwystepowania celiakii i cukrzycy typu 1 u dzieci i mlodziezy.

UNLABELLED: Modern serological tests and changed clinical appearance are the causes of more frequent occurrence of celiac disease (CD) in general population, also in risk groups for instance in patients suffering from diabetes mellitus type 1 (DM t. 1). The aim of the study was to estimate the frequency of CD in patients with DM t. 1 and attempt at evaluation of some factors making to incidence. MATERIAL AND METHODS: It was examined 446 patients (titre of anty-endomysial antibodies (anty EMA), endoscopic biopsy in these with titre > 1OIF). The frequency of CD elevated 5.16%, it was sex and duration of DM t. 1 independent. Children with confirmed CD were younger and earlier suffered from DM t. 1 in comparison with these negative anty EMA. RESULTS: Higher frequency of CD in younger children, who suffered from DM t. 1 to the completion of the fourth year was shown. It wasn't stated the relationship between duration of DM t. 1 and frequency of CD. CONCLUSIONS: This study indicates the necessity to perform serological studies of CD in diabetic patients. Annual screening tests of CD in first fourth years of DM t. 1 in children before the completion of the fourth year of life seem to be justified.

[Morgagni-Adams-Stokes after adenosine injection in a patient with WPW syndrome--a case report]. / Zespól Morgagni-Adams-Stockesa po wstrzyknieciu adenozyny u pacjentki z zespolem WPW.

A case of a 16-year-old girl with left sided accessory pathway is presented. Following adenosine-induced termination of atrio-ventricular reentrant tachycardia the patient developed polymorphic ventricular tachycardia followed by preexcited atrial fibrillation with very rapid ventricular response and syncope. Arrhythmia was terminated by amiodarone infusion. Potential complications after adenosine injection are discussed.

Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes.

Interleukin-6 (IL-6) is one of several pro-inflammatory cytokines implicated in insulin resistance during infection, cachexia, and obesity. We recently demonstrated that IL-6 inhibits insulin signaling in hepatocytes (Senn, J. J., Klover, P. J., Nowak, I. A., and Mooney, R. A. (2002) Diabetes 51, 3391-3399). Members of the suppressors of cytokine signaling (SOCS) family associate with the insulin receptor (IR), and their ectopic expression inhibits IR signaling. Since several SOCS proteins are induced by IL-6, a working hypothesis is that IL-6-dependent insulin resistance is mediated, at least in part, by induction of SOCS protein(s) in insulin target cells. To examine the involvement of SOCS protein(s) in IL-6-dependent inhibition of insulin receptor signaling, HepG2 cells were treated with IL-6 (20 ng/ml) for periods from 1 min to 8 h. IL-6 induced SOCS-3 transcript at 30 min with a maximum effect at 1 h. SOCS-3 protein levels were also markedly elevated at 1 h. Transcript and protein levels returned to near basal levels by 2 h. SOCS-3 induction by IL-6 paralleled IL-6-dependent inhibition of IR signal transduction. Ectopically expressed SOCS-3 associated with the IR and suppressed insulin-dependent receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase, and activation of Akt. SOCS-3 was also a direct inhibitor of insulin receptor autophosphorylation in vitro. In mice exposed to IL-6 for 60-90 min, hepatic SOCS-3 expression was increased. This was associated with inhibition of hepatic insulin-dependent receptor autophosphorylation and IRS-1 tyrosine phosphorylation. These data suggest that induction of SOCS-3 in liver may be an important mechanism of IL-6-mediated insulin resistance.

Interleukin-6 induces cellular insulin resistance in hepatocytes.

Interleukin (IL)-6 is one of several proinflammatory cytokines that have been associated with insulin resistance and type 2 diabetes. A two- to threefold elevation of circulating IL-6 has been observed in these conditions. Nonetheless, little evidence supports a direct role for IL-6 in mediating insulin resistance. Here, we present data that IL-6 can inhibit insulin receptor (IR) signal transduction and insulin action in both primary mouse hepatocytes and the human hepatocarcinoma cell line, HepG2. This inhibition depends on duration of IL-6 exposure, with a maximum effect at 1-1.5 h of pretreatment with IL-6 in both HepG2 cells and primary hepatocytes. The IL-6 effect is characterized by a decreased tyrosine phosphorylation of IR substrate (IRS)-1 and decreased association of the p85 subunit of phosphatidylinositol 3-kinase with IRS-1 in response to physiologic insulin levels. In addition, insulin-dependent activation of Akt, important in mediating insulin's downstream metabolic actions, is markedly inhibited by IL-6 treatment. Finally, a 1.5-h preincubation of primary hepatocytes with IL-6 inhibits insulin-induced glycogen synthesis by 75%. These data suggest that IL-6 plays a direct role in insulin resistance at the cellular level in both primary hepatocytes and HepG2 cell lines and may contribute to insulin resistance and type 2 diabetes.