Developmental mutant mouse models for external genitalia formation

Development of external genitalia and perineum is the subject of developmental biology as well as toxicology and teratology researches. Cloaca forms in the lower (caudal) end of endoderm. Such endodermal epithelia and surrounding mesenchyme interact with various signals to form the external genitalia. External genitalia (the anlage termed as genital tubercle: GT) formation shows prominent sexually dimorphic morphogenesis in late embryonic stages, which is an unexplored developmental research field because of many reasons. External genitalia develop adjacent to the cloaca which develops urethra and corporal bodies. Developmental regulators including growth factor signals are necessary for epithelia‐mesenchyme interaction (EMI) in posterior embryos including the cloaca and urethra in the genitalia. In the case of male type urethra, formation of tubular urethra proceeds from the lower (ventral) side of external genitalia as a masculinization process in contrast to the case of female urethra. Mechanisms for its development are not elucidated yet due to the lack of suitable mutant mouse models. Because of the recent progresses of Cre (recombinase)‐mediated conditional target gene modification analyses, many developmental regulatory genes become increasingly analyzed. Conditional gene knockout mouse approaches and tissue lineage approaches are expected to offer vital information for such sexually dimorphic developmental processes. This review aims to offer recent updates on the progresses of these emerging developmental processes for the research field of congenital anomalies.     

Serial profiling of circulating tumor DNA for optimization of anti‐VEGF chemotherapy in metastatic colorectal cancer patients

Understanding the molecular changes in tumors in response to anti‐VEGF chemotherapy is crucial for optimization of the treatment strategy for metastatic colorectal cancer. We prospectively investigated changes in the amount and constitution of circulating tumor DNA (ctDNA) in serial peripheral blood samples during chemotherapy. Sixty‐one plasma samples taken at different time points (baseline, remission, and post‐progression) and pre‐treatment tumor samples were collected from 21 patients who received bevacizumab‐containing first‐line chemotherapy. Extracted DNA was sequenced by next‐generation sequencing using a panel of 90 oncogenes. Candidate ctDNAs in plasma were validated using mutational data from matching tumors. ctDNAs encoding one to six trunk mutations were found in all 21 cases, and the mutant allele frequency (MAF) was distributed over a wide range (1‐89%). Significant decreases in the MAF at remission and increases in the MAF after progression were observed (p < 0.001). Reduction in the MAF to below 2% in the remission period was strongly associated with better survival (16.6 vs. 32.5 months, p < 0.001). In two cases, mutations (in CREBBP and FBXW7 genes) were newly detected in ctDNA at a low frequency of around 1% in the post‐progression period. The use of ctDNA allows elucidation of the tumor clonal repertoire and tumor evolution during anti‐VEGF chemotherapy. Changes in ctDNA levels could be useful as predictive biomarkers for survival. Mutations newly detected in ctDNA in the late treatment period might reveal the rise of a minor tumor clone that may show resistance to anti‐VEGF therapy.     

Idiopathic Neonatal Hepatitis Presenting as Neonatal Hepatic Siderosis and Steatosis

Idiopathic neonatal hepatitis (INH) is aheterogenous disease of undetermined cause. We report aretrospective histologic reevaluation of INH. Sixtypatients with INH were reviewed along with 32 biliary atresia (BA) patients. Histologic findings,iron and fat deposits, giant cell transformation, portalfibrosis, and bile duct proliferation weresemiquantitatively graded from 0 to 4+. Significanthistologic findings were defined as 2+. Frequencies ofpatients with significant histologic findings in the INHgroup were compared with those of the BA group. Amongthe patients with significant histologic findings, those in the INH group had significantly lessiron deposits (P < 0.01), portal fibrosis (P <0.01), and bile duct proliferation (P < 0.01) thanthose of the BA group. A combination of significanthepatic macrovesicular steatosis and siderosis was observed in 10 INHpatients but not in any BA patient (10/60 vs 0/32, P< 0.05). Without extensive treatment, the 10 INHpatients all recovered, and hepatic abnormalitiesnormalized by the age of 12 months. In conclusion, thepresent study showed that the recognition of hepaticsiderosis is helpful to distinguish BA from INH and thatin a subset of INH patients hepatic macrovesicular steatosis and siderosis occurs.     

Changes in the rewarding effects induced by tramadol and its active metabolite M1 after sciatic nerve injury in mice

INTRODUCTION: The present study was designed to investigate the rewarding effects induced by tramadol and its active metabolite O-desmethyltramadol (M1) under a neuropathic pain-like state. RESULTS: In opioid receptor binding and G protein activation, we confirmed that M1, but not tramadol, showed mu-opioid receptor (MOR) agonistic activity. Furthermore, we found that the subcutaneous (s.c.) injection of tramadol and M1 each produced a significant place preference in mice, and these effects were significantly suppressed by pretreatment with the MOR antagonist beta-funaltrexamine. The dopamine level in the mouse nucleus accumbens was significantly increased by s.c. injection of either tramadol or M1. Mice with sciatic nerve ligation exhibited a marked decrease in the latency of paw withdrawal in response to a thermal stimulus only on the ipsilateral side. Under these neuropathic pain-like conditions, the rewarding effect induced by s.c. injection of either tramadol or M1 was dramatically inhibited after sciatic nerve ligation. Furthermore, the M1-induced G protein activation in the lower midbrain area was suppressed after sciatic nerve ligation. DISCUSSION: Our present data support the notion that the rewarding effect induced by tramadol is mediated mainly through metabolism to its active metabolite M1 via MOR. Furthermore, the suppression of the M1-induced G protein activation in the lower midbrain area caused by sciatic nerve ligation may be responsible for inhibiting the rewarding effects induced by s.c. injection of tramadol and M1 under a neuropathic pain-like state.     

Mycobacterial receptor,Clec4d (CLECSF8, MCL), is coregulated with Mincle and upregulated on mouse myeloid cells following microbial challenge

The C‐type lectin receptor (CTLR), Clec4d (MCL, CLECSF8), is a member of the Dectin‐2 cluster of CTLRs, which also includes the related receptors Mincle and Dectin‐2. Like Mincle, Clec4d recognizes mycobacterial cord factor, trehalose dimycolate, and we recently demonstrated its key role in anti‐mycobacterial immunity in mouse and man. Here, we characterized receptor expression in naïve mice, under inflammatory conditions, and during Mycobacterium bovis BCG infection using newly generated monoclonal antibodies. In naïve mice, Clec4d was predominantly expressed on myeloid cells within the peritoneal cavity, blood, and bone marrow. Unexpectedly, basal expression of Clec4d was very low on leukocytes in the lung. However, receptor expression was significantly upregulated on pulmonary myeloid cells during M. bovis BCG infection. Moreover, Clec4d expression could be strongly induced in vitro and in vivo by various microbial stimuli, including TLR agonists, but not exogenous cytokines. Notably, we show that Clec4d requires association with the signaling adaptor FcRγ and Mincle, but not Dectin‐2, for surface expression. In addition, we provide evidence that Clec4d and Mincle, but not Dectin‐2, are interdependently coregulated during inflammation and infection. These data show that Clec4d is an inducible myeloid‐expressed CTLR in mice, whose expression is tightly linked to that of Mincle.