Corticosteroid-associated osteonecrosis of the femoral head in an 87-year-old patient following treatment for COVID-19-associated pneumonia: A case report.

Coronavirus disease 2019 is known to cause severe acute respiratory syndrome, and serious cases need to be treated with corticosteroids. Herein, we report an 87-year-old woman who developed bilateral osteonecrosis of the femoral head after corticosteroid treatment for coronavirus disease 2019-related pneumonia. Sixteen months after treatment, she developed right hip pain without any evidence of trauma. A diagnosis of bilateral osteonecrosis of the femoral head was made based on sclerotic bands on plain radiographs and low-signal bands on T1-weighted magnetic resonance images. The patient underwent right total hip arthroplasty 4 months after symptom onset. Histological examination of the resected femoral head revealed pathological evidence of osteonecrosis. The postoperative course was good, and the patient can now walk unassisted. To the best of our knowledge, this is the first report of histologically proven osteonecrosis after corticosteroid therapy for coronavirus disease 2019-related disease.

Incidence and clinical outcome of lateral femoral cutaneous nerve injury after periacetabular osteotomy.

Aims: Lateral femoral cutaneous nerve (LFCN) injury is a complication after periacetabular osteo-tomy (PAO) using an anterior approach, which might adversely affect the outcome. However, no prospective study has assessed the incidence and severity of this injury and its effect on the clinical outcomes over a period of time for longer than one year after PAO. The aim of this study was to assess the incidence and severity of the symptoms of LFCN injury for ≥ three years after PAO and report its effect on clinical outcomes. Methods: A total of 40 hips in 40 consecutive patients who underwent PAO between May 2016 and July 2018 were included in the study, as further follow-up of the same patients from a previous study. We prospectively evaluated the incidence, severity, and area of symptoms following LFCN injury. We also recorded the clinical scores at one year and ≥ three years postoperatively using the 36-Item Short Form Health Survey (SF-36) and Japanese Orthopaedic Association Hip Disease Evaluation Questionnaire (JHEQ) scores. Results: A total of 20 patients (50%) had symptoms of a LFCN injury at one year after PAO. At ≥ three years postoperatively, the symptoms had completely resolved in seven of these patients and 13 (33%) had persistent symptoms. The severity and area of symptoms did not significantly differ between one and ≥ three years postoperatively. The JHEQ showed significant differences in the patient satisfaction and mental scores between those with and those without sypmtoms of LFCN injury at ≥ three years postoperatively, while there was no significant difference in the mean SF-36 scores. Conclusion: The incidence of LFCN injury after PAO using an anterior approach is high. The outcome of PAO, ≥ three years postoperatively, is poorer in patients with persistent symptoms from a perioperative LFCN injury, in that patient satisfaction and mental health scores are adversely affected.

Life Course Epidemiology of Hip Osteoarthritis in Japan: A Multicenter, Cross-Sectional Study.

BACKGROUND: The incidence of developmental dysplasia of the hip (DDH) in Japanese newborns has reduced drastically following a primary prevention campaign initiated around 1972 to 1973; this perinatal education campaign promoted maintaining the hips of newborns in the naturally flexed-leg position. The purpose of the present study was to describe the life course epidemiology of hip osteoarthritis (OA) in adolescent and adult patients and to assess its association with exposure to the primary prevention campaign for DDH. METHODS: We included new patients with hip OA diagnosed from January 1, 2022, to December 31, 2022, at 12 core hospitals (8 special-function hospitals and 4 regional medical care support hospitals). The trend in the percentage of hips with a history of DDH treatment in childhood was estimated with use of a centered moving average using the birth year of the patient. We compared the prevalence of severe subluxation (Crowe type II, III, or IV) between patients with secondary hip OA due to hip dysplasia who were born in or before 1972 and those who were born in or after 1973. RESULTS: Overall, 1,095 patients (1,381 hips) were included. The mean age at the time of the survey was 63.5 years (range, 15 to 95 years). A total of 795 patients (1,019 hips; 73.8% of hips) were diagnosed with secondary OA due to hip dysplasia. Approximately 13% to 15% of hips among patients born from 1963 to 1972 had a history of DDH treatment in childhood; however, the percentage decreased among patients born in or after 1973. The prevalence of severe subluxation (Crowe type II, III, or IV) among patients born in or after 1973 was 2.4%, which was significantly less than that among patients born in or before 1972 (11.1%; odds ratio, 0.20; p < 0.001). CONCLUSIONS: As of 2022, secondary hip OA due to hip dysplasia is still responsible for most new cases of adolescent and adult hip OA seen in core hospitals in Japan. However, the perinatal education campaign initiated 50 years ago, which utilized a population approach and advocated for maintaining the hips of newborns in the naturally flexed-leg position, may have improved the environmental factors of DDH, as indicated by the apparently reduced need for treatment of DDH in childhood and the associated severe subluxation. This may result in a reduced need for challenging hip surgery later in life. LEVEL OF EVIDENCE: Prognostic Level III . See Instructions for Authors for a complete description of levels of evidence.

The role of torsional stress in the development of subchondral insufficiency fracture of the femoral head: A finite element model analysis.

BACKGROUND: Subchondral insufficiency fracture of the femoral head generally occurs without evidence of trauma or with a history of minor trauma. Insufficient bone quality is considered one cause; however, the detailed mechanism of fracture development at the subchondral area (SA) is not understood. The aim of this study was to clarify the directions of force that cause subchondral fracture using finite element model analysis. METHODS: Two types of finite element models were generated from the CT data of femurs obtained from three individuals without osteoporosis (normal models) and another three with osteoporosis (osteoporosis models). Three directions of force, including compressive, shearing, and torsional, were applied to the femoral head. The distribution of von Mises stress (Mises stress) was evaluated at the SA, principal compressive trabeculae (PC), and principal tensile trabeculae. RESULTS: Under compressive force, the mean Mises stress value was greatest at the PC in both the normal and osteoporosis models. Under shearing force, the mean Mises stress value tended to be greatest at the SA in the normal model and at the PC in the osteoporosis model. Under torsional force, the mean Mises stress value was greatest at the SA in both types of models. CONCLUSIONS: The torsional force showed the greatest Mises stress at the SA in both the normal and osteoporosis models, suggesting the importance of torsion as a possible force responsible for subchondral insufficiency fracture development.

Side-to-side variability in the femoral neck anteversion angle: A study of the Japanese population with osteonecrosis of the femoral head.

BACKGROUND: Side-to-side variability in the femoral neck anteversion angle (FA) reportedly varies from 0.0° to 17.3°. To investigate the side-to-side variability in the FA and the relationship between the FA and the morphology of the acetabulum in the Japanese population, we performed a three-dimensional computed tomography (CT)-based study involving patients with osteonecrosis of the femoral head (ONFH). METHODS: CT data were obtained from 170 nondysplastic hips of 85 patients with ONFH. The FA and acetabular coverage parameters, including the acetabular anteversion angle, acetabular inclination angle, and acetabular sector angle in the anterior, superior, and posterior directions, were measured using three-dimensional CT. The distribution of the side-to-side variability in the FA was evaluated separately for each of the five degrees. RESULTS: The mean side-to-side variability in the FA was 6.7° ± 5.3° (range, 0.2°-26.2°). The distribution of the side-to-side variability in the FA was 0.0°-5.0° in 41 patients (48.2%), 5.1°-10.0° in 25 patients (29.4%), 10.1°-15.0° in 13 patients (15.3%), 15.1°-20.0° in 4 patients (4.7%), and >20.1° in 2 patients (2.4%). There was a weak negative correlation between the FA and anterior acetabular sector angle (r = -0.282, P < 0.001) and a very weak positive correlation between the FA and acetabular anteversion angle (r = 0.181, P < 0.018). CONCLUSIONS: The mean side-to-side variability in the FA was 6.7° ± 5.3° (range, 0.2°-26.2°) in Japanese nondysplastic hips, and about 20% of the patients had a side-to-side variability of >10°.

Assessment of the Hip Flexor Muscles Shortly after Curved Periacetabular Osteotomy using Magnetic Resonance Imaging.

BACKGROUND: Curved periacetabular osteotomy requires detachment and retraction of the hip flexors. In this study, we evaluated hip flexor muscle status by magnetic resonance imaging (MRI) shortly after curved periacetabular osteotomy. METHODS: We retrospectively evaluated 60 hips of 56 patients by MRI 1 week and 3 months after curved periacetabular osteotomy performed from August 2017 to December 2019. We classified the condition of the flexors as follows: Grade 0, normal; Grade I, strain/edema; Grade II, partial tear; and grade III, complete tear. RESULTS: At 1 week after surgery, the iliacus muscle was classified as grades I and II in 12.0 and 88.0% of hips; psoas as grades 0, I and II in 22.0, 72.0, and 6.0%; sartorius muscle as grades 0, I and II in 6.0, 62.0, and 32.0%; and rectus femoris muscle as grades 0 and I in 86.0 and 14.0%, respectively. At 3 months, 82.0, 88.0, and 96.0% of psoas, sartorius, and rectus femoris muscles, respectively, had improved to grade 0, whereas the iliacus was grades I and II in 94.0 and 6.0%, respectively. These changes in the iliacus muscle at 3 months were not significantly associated with patient characteristics, radiographic data, or clinical scores. CONCLUSIONS: All the iliacus, 78% of psoas, 94% of sartorius, and 14% of rectus femoris muscles appeared abnormal on MRI 1 week after curved periacetabular osteotomy. However, at 3 months, only 18% of psoas, 12% of sartorius, and 4% of rectus femoris muscles appeared abnormal, whereas all iliacus muscles still appeared abnormal. These abnormalities did not significantly affect clinical scores.

A case of total hip arthroplasty for nonunion after femoral trochanteric fracture surgery with complete breakage of only the compression screw of the InterTAN nail.

Intramedullary nails are an effective treatment for common femoral trochanteric fractures. However, one of their complications is implant breakage due to poor reduction and nonunion after surgery. We herein report a case of a 54-year-old man who underwent total hip arthroplasty for nonunion after internal fixation of a femoral trochanteric fracture. The femoral trochanteric fracture was treated by internal fixation using the Trigen InterTAN nail. The patient developed symptoms of hip pain 6 months after internal fixation. Nine months after internal fixation, hip radiographs and computed tomography scans showed breakage of only the compression screw. During total hip arthroplasty, we were unable to remove the lag screw and compression screw before the femoral head dislocation because no gap was present between the two screws. Thus, we removed these screws with the femoral head after dislocation of the femoral head. The removed nail was partially damaged at the lag screw hole. This change was retrospectively observed on the preoperative computed tomography scan. Three months after total hip arthroplasty, the patient was able to walk unaided and the hip pain had resolved. If only the compression screw is completely broken after internal fixation with the Trigen InterTAN nail, both the lag screw and compression screw will be difficult to remove with preservation of the femoral head. We effectively managed such a case by not only revision internal fixation but also total hip arthroplasty.

Going beyond Binary: Rapid Identification of Protein-Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach.

Kinetic target-guided synthesis (KTGS) is a powerful screening approach that enables identification of small molecule modulators for biomolecules. While many KTGS variants have emerged, a majority of the examples suffer from limited throughput and a poor signal/noise ratio, hampering reliable hit detection. Herein, we present our optimized multifragment KTGS screening strategy that tackles these limitations. This approach utilizes selected reaction monitoring liquid chromatography tandem mass spectrometry for hit detection, enabling the incubation of 190 fragment combinations per screening well. Consequentially, our fragment library was expanded from 81 possible combinations to 1710, representing the largest KTGS screening library assembled to date. The expanded library was screened against Mcl-1, leading to the discovery of 24 inhibitors. This work unveils the true potential of KTGS with respect to the rapid and reliable identification of hits, further highlighting its utility as a complement to the existing repertoire of screening methods used in drug discovery.

Clinical Outcomes for Total Hip Arthroplasty with and without Previous Curved Periacetabular Osteotomy.

There are currently no reports on the clinical outcomes after total hip arthroplasty (THA) with previous curved periacetabular osteotomy (CPO), although the outcomes after THA with non-CPO types of periacetabular osteotomy have been reported. This study aimed to clarify the differences in clinical outcomes and radiographic features after THA with or without previous CPO. We performed a retrospective case-control with individual matching study. The participants were 10 patients with 11 hips that underwent cementless THA between October 1998 and October 2018 with previous CPO (osteotomy group). For the control group, we matched age, sex, and follow-up period, and included 32 patients with 33 hips that underwent cementless THA without previous CPO at a 1:3 ratio. The Harris Hip Score (HHS), cup size, position, and alignment, global offset (GO), operative time, perioperative blood loss, frequency of osteophyte removal, and major complications were compared between the two groups. The osteotomy group had no cases with revision surgery and dislocation. No significant differences were found between the two groups as follows: mean HHS, 94.9 points in the osteotomy group versus 92.7 points in the control group at the final follow-up; mean GO, 70.1 mm in the osteotomy group versus 71.4 mm in the control group; cup size, position, and alignment after THA; operative time; and perioperative blood loss. The frequency of osteophyte removal was higher in the osteotomy group. The take-home messages were that the clinical outcomes, including HHS, and radiographic features, including GO, after THA were equivalent in the two groups.

Mechanical Influence of Pubic Nonunion on the Stress Distribution After Curved Periacetabular Osteotomy: Patient-Specific Three-Dimensional Finite Element Analysis.

BACKGROUND: Pubic nonunion after curved periacetabular osteotomy (CPO) reportedly occurs in 1%-17% of patients and causes pubic pain in 21%. Furthermore, pubic nonunion is associated with a risk of ischial ramus stress fracture, but the mechanical influence of pubic nonunion has not been fully clarified. METHODS: Patient-specific finite element (FE) analysis was performed using Mechanical Finder software. Three FE models (pre-CPO, union, and nonunion models) were constructed from preoperative and postoperative computed tomographic data. The contact area (mm2) and contact pressure (MPa) in the hip joint as well as the equivalent stress (MPa) at the ischial ramus were evaluated among the 3 FE models. RESULTS: Patient-specific FE models were generated using 18 consecutive hips treated with CPO. The mean contact pressure in the hip joint was not significantly different between the union and nonunion models (0.50 ± 0.10 vs 0.50 ± 0.09 MPa, P = .88). However, the mean equivalent stress at the ischial ramus in the nonunion models was 1.7 times higher than that in the union models (1.13 ± 0.77 vs 0.64 ± 0.45 MPa, P < .01). CONCLUSION: FE analysis revealed that pubic nonunion did not affect the mechanical distribution in the hip joint itself but increased the mean equivalent stress at the ischial ramus. This finding suggests the importance of achieving pubic union after CPO to avoid the risk of ischial ramus stress fracture.

Acid ceramidase promotes drug resistance in acute myeloid leukemia through NF-κB-dependent P-glycoprotein upregulation.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

SKI-178: A Multitargeted Inhibitor of Sphingosine Kinase and Microtubule Dynamics Demonstrating Therapeutic Efficacy in Acute Myeloid Leukemia Models.

AIM: To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178. METHODS: Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, in vitro and in vivo assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML). RESULTS: Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2. We also present evidence that, in addition to its actions as a Sphingosine Kinase Inhibitor, SKI-178 functions as a microtubule network disrupting agent both in vitro and in intact cells. Interestingly, we separately demonstrate that simultaneous SphK inhibition and microtubule disruption synergistically induces apoptosis in AML cell lines. Furthermore, we demonstrate that SKI-178 is well tolerated in normal healthy mice. Most importantly, we demonstrate that SKI-178 has therapeutic efficacy in several mouse models of AML. CONCLUSION: SKI-178 is a multi-targeted agent that functions both as an inhibitor of the SphKs as well as a disruptor of the microtubule network. SKI-178 induced apoptosis arises from a synergistic interaction of these two activities. SKI-178 is safe and effective in mouse models of AML, supporting its further development as a multi-targeted anti-cancer therapeutic agent.

A carcinogenicity study of diphenylarsinic acid in F344 rats in drinking water for 104 weeks.

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical used as a chemical warfare agent, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. We previously demonstrated that DPAA promotes diethylnitrosamine-induced liver carcinogenesis in a medium-term rat liver bioassay. The purpose of the present study was to evaluate the potential carcinogenicity of DPAA, including investigation of whether the bile duct hyperplasia in the liver that was observed in a previous 52 week rat chronic study develops into a tumor, when administered to rats in their drinking water for 104 weeks. DPAA was administered to groups 1-4 at concentrations of 0, 5, 10, and 20 ppm in their drinking water for 104 weeks. A significant decrease in survival rate was found for females in the 20 ppm DPAA group. Body weights of males in the 20 ppm and females in the 10 and 20 ppm DPAA groups were significantly decreased compared to the controls. Overall histopathological evaluation of neoplasms in all tissues showed no significant increase of tumor incidence in any organ or tissue of the 5, 10, or 20 ppm DPAA-treated male or female F344 rats. In conclusion, the present study demonstrated that DPAA is not a complete carcinogen in male or female F344 rats.

Chemopreventive Action by Ethanol-extracted Brazilian Green Propolis on Post-initiation Phase of Inflammation-associated Rat Colon Tumorigenesis.

BACKGROUND/AIM: Propolis has since long been utilized in numerous folk medicines with a variety of medicinal properties. In this study, the effects of ethanol-extracted (EEP) and water-extracted (WEP) Brazilian green propolis on the post-initiation phase of inflammation-associated rat colon tumorigenesis were directly compared. MATERIALS AND METHODS: Male F344 rats at 6 weeks of age were subcutaneously injected with 1,2-dimethylhydrazine (DMH) at 40 mg/kg body weight twice during the first week, followed by 1% dextran sodium sulfate (DSS) in drinking water for one week. After a 1-week no-treatment period, animals were administered either basal Oriental MF powdered diet, or 1% EEP or 1% WEP in the basal diet until week 32. RESULTS: Post-initiation treatment with EEP significantly reduced the multiplicity of colorectal carcinomas compared to the control (0.40±0.13/rat vs. 2.29±0.84/rat, respectively, p<0.05), and EEP also reduced the tumor volume. Immunohistochemically, expression of inflammation-associated proteins inducible nitric oxide synthase, tumor necrotic factor alpha, nuclear factor kappa B and glutathione peroxidase-2 were significantly diminished in colorectal tumors from EEP-treated rats. CONCLUSION: Suppression of inflammation and oxidative stress, which had been triggered by DMH and promoted by DSS, was a primary mechanism by which EEP suppressed carcinogenesis.

A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks.

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to rats in their drinking water for 52 weeks. DPAA was administered to groups 1-4 at concentrations of 0, 5, 10, and 20ppm in their drinking water for 52 weeks. There were no significant differences in the final body weights between the control groups and the treatment groups in male or female rats. In serum biochemistry, in females 20ppm DPAA significantly increased alkaline phosphatase and γ-glitamyl transferase compared to controls, and 10 and 20ppm DPAA significantly increased total cholesterol compared to controls. Absolute and relative liver weights were significantly increased in females treated with 20ppm DPAA compared to the control group. Dilation of the common bile duct outside the papilla of Vater and stenosis of the papilla of Vater was observed in all male and female rats administered 20ppm DPAA. The incidence of intrahepatic bile duct hyperplasia was significantly increased in male and female rats treated with 20ppm DPAA compared to the control groups. These results suggest that DPAA is toxic to the bile duct epithelium in rats. The no-observed adverse effect levels of DPAA were estimated to be 10ppm (0.48mg/kg b.w./day) for males and 5ppm (0.35mg/kg b.w./day) for females under the conditions of this study.

Examination of in vivo mutagenicity of sodium arsenite and dimethylarsinic acid in gpt delta rats.

Arsenic is a well-known human bladder and liver carcinogen, but its exact mechanism of carcinogenicity is not fully understood. Dimethylarsinic acid (DMAV) is a major urinary metabolite of sodium arsenite (iAsIII) and induces urinary bladder cancers in rats. DMAV and iAsIII are negative in in vitro mutagenicity tests. However, their in vivo mutagenicities have not been determined. The purpose of present study is to evaluate the in vivo mutagenicities of DMAV and iAsIII in rat urinary bladder epithelium and liver using gpt delta F344 rats. Ten-week old male gpt delta F344 rats were randomized into 3 groups and administered 0, 92mg/L DMAV, or 87mg/L iAsIII (each 50mg/L As) for 13weeks in the drinking water. In the mutation assay, point mutations are detected in the gpt gene by 6-thioguanine selection (gpt assay) and deletion mutations are identified in the red/gam genes by Spi- selection (Spi- assay). Results of the gpt and Spi- assays showed that DMAV and iAsIII had no effects on the mutant frequencies or mutation spectrum in urinary bladder epithelium or liver. These findings indicate that DMAV and iAsIII are not mutagenic in urinary bladder epithelium or liver in rats.

Acid ceramidase is upregulated in AML and represents a novel therapeutic target.

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.

Ethanol-Extracted Brazilian Propolis Exerts Protective Effects on Tumorigenesis in Wistar Hannover Rats.

The present study was conducted over a course of 104 weeks to estimate the carcinogenicity of ethanol-extracted Brazilian green propolis (EEP). Groups of 50 male and 50 female Wistar Hannover rats, 6-week-old at commencement were exposed to EEP at doses of 0, 0.5 or 2.5% in the diet. Survival rates of 0.5% and 2.5% EEP-treated male and female rats, respectively, were significantly higher than those of respective control groups. Overall histopathological evaluation of neoplasms in rat tissues after 2 years showed no significant increase of tumors or preneoplastic lesions in any organ of animals administered EEP. Significantly lower incidences of pituitary tumors in 0.5% EEP male and 2.5% EEP female groups, malignant lymphoma/leukemia in both 2.5% EEP-treated males and females and total thyroid tumors in 0.5% EEP male group were found. Administration of EEP caused significant decreases of lymphoid hyperplasia of the thymus and lymph nodes in 2.5% EEP-treated rats, tubular cell hyperplasia of kidneys in all EEP groups, and cortical hyperplasia of adrenals in EEP-treated females. In the blood, significant reduction of neutrophils in all EEP-treated males and band neutrophils in 2.5% EEP-treated females was found indicating lower levels of inflammation. Total cholesterol and triglicerides levels were significantly lower in the blood of 2.5% EEP-treated female rats. In conclusion, under the conditions of the 2-year feeding experiment, EEP was not carcinogenic, did not induce significant histopathological changes in any organ, and further exerted anti-inflammatory and antitumorigenic effects resulting in increase of survival of Wistar Hannover rats.

Role of deltaNp63(pos)CD44v(pos) cells in the development of N-nitroso-tris-chloroethylurea-induced peripheral-type mouse lung squamous cell carcinomas.

The role of cells expressing stem cell markers deltaNp63 and CD44v has not yet been elucidated in peripheral-type lung squamous cell carcinoma (pLSCC) carcinogenesis. Female A/J mice were painted topically with N-nitroso-tris-chloroethylurea (NTCU) for induction of pLSCC, and the histopathological and molecular characteristics of NTCU-induced lung lesions were examined. Histopathologically, we found atypical bronchiolar hyperplasia, squamous metaplasia, squamous dysplasia, and pLSCCs in the treated mice. Furthermore, we identified deltaNp63(pos)CD44v(pos)CK5/6(pos)CC10(pos) clara cells as key constituents of early precancerous atypical bronchiolar hyperplasia. In addition, deltaNp63(pos)CD44v(pos) cells existed throughout the atypical bronchiolar hyperplasias, squamous metaplasias, squamous dysplasias, and pLSCCs. Overall, our findings suggest that NTCU induces pLSCC through an atypical bronchiolar hyperplasia-metaplasia-dysplasia-SCC sequence in mouse lung bronchioles. Notably, Ki67-positive deltaNp63(pos)CD44v(pos) cancer cells, cancer cells overexpressing phosphorylated epidermal growth factor receptor and signal transducer and activator of transcription 3, and tumor-associated macrophages were all present in far greater numbers in the peripheral area of the pLSCCs compared with the central area. These findings suggest that deltaNp63(pos)CD44v(pos) clara cells in mouse lung bronchioles might be the origin of the NTCU-induced pLSCCs. Our findings also suggest that tumor-associated macrophages may contribute to creating a tumor microenvironment in the peripheral area of pLSCCs that allows deltaNp63(pos)CD44v(pos) cancer cell expansion through activation of epidermal growth factor receptor signaling, and that exerts an immunosuppressive effect through activation of signal transducer and activator of transcription 3 signaling.

Ethanol-extracted propolis enhances BBN-initiated urinary bladder carcinogenesis via non-mutagenic mechanisms in rats.

Ethanol-extracted propolis (EEP) is used for medical, dietetic and cosmetic purposes. In this study, the effects of EEP on urinary bladder carcinogenesis, its underlying mechanism and in vivo genotoxicity were investigated. In experiment 1, rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 2 or 4 weeks followed by dietary administration of 0.125, 0.25, 0.5 or 1% EEP for 4 or 32 weeks, respectively. At week 6, the mRNA levels of top2a, cyclin D1 and survivin were significantly elevated in the 0.5 and 1% EEP groups. At week 36, the incidence and multiplicity of urothelial carcinomas and total tumors were markedly elevated in all EEP groups. In experiment 2, rats were fed basal diet or the 1% EEP diet for 13 weeks without carcinogen initiation. Increases in urinary precipitate, cell proliferation and incidence of simple hyperplasia were observed in the 1% EEP group. In experiment 3, dietary administration of 2.5% EEP to gpt delta rats for 13 weeks did not induce any obvious mutagenicity in the urinary bladder urothelium. Taken together, EEP enhanced BBN-initiated rat urinary bladder carcinogenesis in a non-genotoxic manner through increasing formation of urinary precipitate, enhancing cell proliferation and inhibiting apoptosis during the early stages of carcinogenesis.