Calcineurin inhibitors exacerbate coronary arteritis via the MyD88 signalling pathway in a murine model of Kawasaki disease.

Calcineurin inhibitors (CNIs) have been used off-label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in wild-type, severe combined immunodeficiency (SCID), caspase-associated recruitment domain 9 (CARD9)-/- and myeloid differentiation primary response gene 88 (MyD88)-/- mice. We also performed in-vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1-mediated coronary arteritis in a dose-dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9-/- mice but not in MyD88-/- mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88-dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.

Mid-trimester residual cervical length and the risk of preterm birth in pregnancies after abdominal radical trachelectomy: a retrospective analysis.

OBJECTIVE: To investigate the association between mid-trimester residual cervical length (CL) and the risk of preterm birth in pregnancies after abdominal radical trachelectomy (RT). DESIGN: Retrospective cohort study. SETTING: University hospital. POPULATION: A total of 33 deliveries after 22 weeks' gestation in 30 women who underwent abdominal RT including prophylactic cervical cerclage and perinatal care between January 2002 and May 2016. METHODS: The association between mid-trimester residual CL (the distance between the cerclage and the external cervical os) and gestational age at delivery was investigated. Receiver-operating characteristics (ROC) curve analysis was performed to estimate the optimal cut-off values of the mid-trimester residual CL for the prediction of preterm birth. MAIN OUTCOME MEASURES: Preterm birth before 34 weeks' gestation. RESULTS: Mid-trimester residual CL showed a significant correlation with gestational age at delivery (r = 0.36, P < 0.05). There was a significant difference in residual CL between women who did and those who did not give birth before 34 weeks (P < 0.05). Mid-trimester residual CL < 13 mm was a good predictor of birth before 34 weeks, with a sensitivity of 67%, specificity of 75%, positive predictive value of 55% and negative predictive value of 86% (area under ROC curve, 0.75). CONCLUSIONS: Mid-trimester residual CL is significantly correlated with gestational age at delivery. Residual CL assessment could be used to reassure physicians and women that there is only a small chance of preterm birth in pregnancies after abdominal RT. TWEETABLE ABSTRACT: Mid-trimester residual cervical length is a good predictor of preterm birth after radical trachelectomy.

Kawasaki disease: a matter of innate immunity.

Kawasaki disease (KD) is an acute systemic vasculitis of childhood that does not have a known cause or aetiology. The epidemiological features (existence of epidemics, community outbreaks and seasonality), unique age distribution and clinical symptoms and signs of KD suggest that the disease is caused by one or more infectious environmental triggers. However, KD is not transmitted person-to-person and does not occur in clusters within households, schools or nurseries. KD is a self-limited illness that is not associated with the production of autoantibodies or the deposition of immune complexes, and it rarely recurs. Regarding the underlying pathophysiology of KD, innate immune activity (the inflammasome) is believed to play a role in the development of KD vasculitis, based on the results of studies with animal models and the clinical and laboratory findings of KD patients. Animal studies have demonstrated that innate immune pathogen-associated molecular patterns (PAMPs) can cause vasculitis independently of acquired immunity and have provided valuable insights regarding the underlying mechanisms of this phenomenon. To validate this concept, we recently searched for KD-specific PAMPs and identified such molecules with high specificity and sensitivity. These molecules have structures similar to those of microbe-associated molecular patterns (MAMPs), as shown by liquid chromatography-tandem mass spectrometry. We propose herein that KD is an innate immune disorder resulting from the exposure of a genetically predisposed individual to microbe-derived innate immune stimulants and that it is not a typical infectious disease.

Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor.

BACKGROUND: Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated. METHODS: The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ. RESULTS: In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells. CONCLUSIONS: NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.

Allele-specific PCR for a cost-effective & time-efficient diagnostic screening of spinal muscular atrophy.

BACKGROUND & OBJECTIVES: Genetic diagnosis of spinal muscular atrophy (SMA) is complicated by the presence of SMN2 gene as majority of SMA patients show absence or deletion of SMN1 gene. PCR may amplify both the genes non selectively in presence of high amount of DNA. We evaluated whether allele-specific PCR for diagnostic screening of SMA is reliable in the presence of high amount of genomic DNA, which is commonly used when performing diagnostic screening using restriction enzymes. METHODS: A total of 126 blood DNA samples were tested in amounts ranging 80-200 ng, referred for the genetic diagnosis of SMA using both conventional PCR-RFLP and allele-specific PCR. RESULTS: The results from both methods showed agreement. Further, allele-specific PCR was found to be a time-efficient and cost-effective method. INTERPRETATION & CONCLUSIONS: Our study demonstrated the accuracy of our allele-specific PCR and the results were comparable compatible with that of PCR-RFLP, indicating its practical application in SMA diagnostic screening.

Infectivity of HBV DNA positive donations identified in look-back studies in Hyogo-Prefecture, Japan.

AIMS/OBJECTIVES: To clarify transfusion incidence of hepatitis B virus (HBV) infected blood negative for mini pool-nucleic acid amplification testing (MP-NAT). BACKGROUND: Japanese Red Cross (JRC) blood centres screen donated blood to avoid contamination with HBV. However, a low copy number of HBV may be overlooked. METHODS/MATERIALS: In Hyogo-Prefecture, JRC blood centres screened 787 695 donations for HBV from April 2005 to March 2009. Of these, 685 844 were donations from the repeat donors. To detect the donors with HBV, serological tests, MP-NAT and/or individual donation (ID)-NAT were performed. To detect the recipients with transfusion-transmitted HBV infection (TTHBI), serological analysis and/or ID-NAT were performed. RESULTS: In this study, 265 of the 685 844 repeat donations were serologically and/or MP-NAT positive for HBV. Their repository samples from the previous donation were examined in a look-back study; 13 of the 265 repository samples proved ID-NAT positive. Twelve recipients were transfused with HBV-infected blood components derived from 10 of the 13 HBV-infected donors. Only 1 of the 12 recipients was identified as TTHBI case. Seven of the 12 recipients escaped from our follow-up study and 4 recipients were negative for HBV during the observation period. CONCLUSION: On the basis of the look-back study among the repeat donors in Hyogo-Prefecture, Japan, donations with HBV-infected blood negative for MP-NAT occurred with a frequency of 13 in 685 844 donations (∼1/53 000 donations). However, more than half of the recipients transfused with HBV-infected blood negative for MP-NAT could not be followed up. It is necessary to establish a more cautious follow-up system.

Percutaneous transhepatic biliary drainage catheter tract recurrence in cholangiocarcinoma.

BACKGROUND: The aim of the study was to clarify the incidence, risk factors and treatment of percutaneous transhepatic biliary drainage (PTBD) catheter tract recurrence in patients with resected cholangiocarcinoma. METHODS: The medical records of 445 patients with perihilar and distal cholangiocarcinoma who underwent resection following PTBD were reviewed retrospectively. RESULTS: PTBD catheter tract recurrence was detected in 23 patients (5.2 per cent). The mean(s.d.) interval between surgery and onset of the recurrence was 14.4(13.8) months. On multivariable analysis, duration of PTBD (60 days or more), multiple PTBD catheters and macroscopic papillary tumour type were identified as independent risk factors. In four patients with synchronous metastasis, the PTBD sinus tract was resected simultaneously, at the time of initial surgery. Of 19 patients with metachronous metastasis, 15 underwent surgical resection of the metastasis. Survival of the 23 patients with PTBD catheter tract recurrence was poorer than that of the 422 patients without recurrence (median 22.8 versus 27.3 months; P = 0.095). Even after surgical resection of PTBD catheter tract recurrence, survival was poor. CONCLUSION: PTBD catheter tract recurrence is not unusual. The prognosis for these patients is generally poor, even after resection. To prevent this troublesome complication, endoscopic biliary drainage is first recommended when drainage is indicated.

Value of indocyanine green clearance of the future liver remnant in predicting outcome after resection for biliary cancer.

BACKGROUND: It is difficult to predict hepatic functional reserve accurately before major hepatectomy. The aim of this study was to analyse the usefulness of the future liver remnant plasma clearance rate of indocyanine green (ICGK-F, calculated as plasma clearance rate of indocyanine green (ICGK) x proportion of the future liver remnant) in predicting death after major hepatectomy. METHODS: Data on ICGK and ICGK-F were collected prospectively and analysed retrospectively for 274 patients who underwent right hepatectomy, right trisectionectomy or left trisectionectomy for biliary cancer between 1991 and 2008. The mortality rate and incidence of postoperative complications were analysed. Patients were separated into two groups according to year of operation (85 patients operated on between 1991 and 2000; 189 from 2001 to 2008). RESULTS: In multiple logistic regression analyses, an ICGK-F less than 0.05 had the strongest impact on the incidence of postoperative mortality (odds ratio 8.06; P < 0.001). The postoperative mortality rate was significantly lower in the later period (P < 0.001). In patients with an ICGK-F value between 0.040 and 0.049, the mortality rate in the early period was 30 per cent, whereas it was only 8 per cent in the later period. CONCLUSION: An ICGK-F of 0.05 is a useful cut-off value for predicting mortality and morbidity. With careful perioperative patient management in an experienced institution, this cut-off value can be lowered further.

Comparison of stress distribution in partially and completely edentulous mandibles around splinted and non-splinted implant prostheses: A finite element study.

BACKGROUND: In some treatments using multiple dental implants, the implants are inserted in the bone with splinted or non-splinted implant prostheses. There are some reports about the influence of the splinted and non-splinted implants on stress distribution in the bone using the finite element method (FEM), and there is a controversy in the literature regarding whether the splinted or non-splinted implants prostheses reduce the stress generated on the implant-surrounding bone more efficiently. Additionally, the simple shape of the jaw bones with limited bone area was used for FEM analysis in many studies at the expense of accurate analysis. OBJECTIVE: The aim of this study was to evaluate the difference in stress distribution in the bone between the splinted and non-splinted implants, and between completely and partially edentulous mandibles. METHODS: The implants were inserted in the first premolar, second premolar, and first molar regions of the partial and complete mandibles, and the splinted and non-splinted crowns were attached to the implants. Vertical load (100 N) or oblique load (70 N, 30° from its long axis towards the lingual) was applied on the first premolar. RESULTS: When vertical load was applied to the partially edentulous mandible model, the stress was concentrated intensively on the cortical bone around the first premolar regardless of whether splinted or non-splinted implants were used. On the other hand, the vertical load applied to the completely edentulous mandible model caused the stress to be concentrated intensively on the cortical bone around the first premolar with non-splinted implants. With respect to the oblique load, the stress was concentrated intensively on the cortical bone around the first premolar only with the non-splinted implants, in both the partial and complete mandibles. CONCLUSION: This study shows the different stress distributions of the cortical bone around the implants between the partial and complete mandible. This indicates that the complete mandible should be used for the analysis of bone stress distribution around the implants using FEM.

The concept of perihilar cholangiocarcinoma is valid.

BACKGROUND: The term perihilar cholangiocarcinoma has been used for all tumours involving or requiring resection of the hepatic confluence. However, it does not distinguish between intrahepatic and extrahepatic hilar tumours, and has no clinicopathological basis. This retrospective study examined whether the concept of perihilar cholangiocarcinoma is valid clinically. METHODS: Some 250 patients with perihilar cholangiocarcinoma were divided into extrahepatic (EHC, 167 patients) and intrahepatic (IHC, 83) groups based on tumour location. Clinicopathological data were compared between these groups. RESULTS: Liver, portal vein, venous and lymphatic invasion, and nodal metastasis were more common in IHCs than EHCs, whereas histological grade and incidence of perineural invasion were similar. IHCs were more advanced at the time of surgery; stage III or IV disease was found in 37.7 per cent of EHCs and 59 per cent of IHCs. Survival was marginally better for patients with EHCs than for those with IHCs (29.3 versus 20 per cent at 5 years; P = 0.057), but survival rates were similar for each tumour stage in the American Joint Committee on Cancer classification. CONCLUSION: Combining EHC and IHC under the term perihilar cholangiocarcinoma is valid, as these tumours have comparable biological behaviour, with similar clinical management depending on stage and invasion.

Non-steroidal anti-inflammatory drug-induced small intestinal damage is Toll-like receptor 4 dependent.

BACKGROUND: Enterobacteria and cytokines both play roles in the pathophysiology of NSAID-induced enteropathy. Toll-like receptor (TLR) 4 recognises lipopolysaccharide (LPS), resulting in activation of an inflammatory cascade via the accessory protein MyD88. AIMS: To investigate role of TLR4 in inflammatory responses in indomethacin-induced enteropathy. METHODS: Indomethacin was administered p.o. to non-fasting rats and mice to induce small intestinal damage. The extent of such damage was evaluated by measuring the injured area stained dark blue with Evans blue. Rats were given antibiotics (ampicillin, aztreonam or vancomycin) p.o., or intraperitoneal LPS (a TLR4 ligand) or neutralising antibodies against neutrophils, tumour necrosis factor (TNF)-alpha, or monocyte chemotactic protein (MCP)-1. Furthermore, the intestinal ulcerogenicity of indomethacin was examined in TLR4-mutant, TLR4(-/-), and MyD88(-/-) mice. RESULTS: Indomethacin induced small intestinal damage with an increase in expression of TNF-alpha and MCP-1 in both rats and mice. Antibodies against neutrophils, TNF-alpha and MCP-1 inhibited the damage by 83%, 67% and 63%, respectively, in rats. Ampicillin and aztreonam also inhibited this damage, and decreased the number of Gram-negative bacteria in the small intestinal contents of the rat. However, vancomycin, which exhibited no activity against Gram-negative bacteria, had no preventive effect against this damage. Administration of LPS 1 h after indomethacin aggravated the damage, whereas LPS pretreatment inhibited it with reduction of expression of TLR4 and cytokines. In TLR4-mutant mice, the damage and cytokine expression were markedly inhibited. TLR4(-/-) and MyD88(-/-) mice were also resistant to the damage. CONCLUSIONS: Indomethacin may injure the small intestine through a TLR4/MyD88-dependent pathway.

Appropriate management of high-flow priapism based on color Doppler ultrasonography findings in pediatric patients: four case reports and a review of the literature.

INTRODUCTION: High-flow priapism in children is a very rare condition, and there is no clear consensus on its management. High-flow priapism is associated with increased cavernosal blood flow and broadly divided into two groups based on the presence or absence of arteriocavernous fistula in the corpora cavernosa. OBJECTIVE: This study aimed to determine the appropriate management of high-flow priapism based on the existence of arteriocavernous fistula using penile color Doppler ultrasonography (CDU) findings in the pediatric population. STUDY DESIGN: The cases of four boys aged between 6 and 11 years with high-flow priapism treated between 2009 and 2017 are reported. Two boys had prior perineal trauma, one boy had blunt penile glans trauma, and one had no obvious cause for the condition. All boys initially underwent penile CDU and were treated conservatively or via selective arterial embolization depending upon the presence or absence of an arteriocavernous fistula. RESULTS: Penile CDU revealed an arteriocavernous fistula inside the corpus cavernosum penis in two of four boys and increased blood flow inside the corpus spongiosum in the remaining boys. The former two boys underwent selective arterial embolization and one boy underwent repeated embolization because of remaining arteriocavernous fistula feeding from the contralateral cavernosal artery, whereas the boys with no arteriocavernous fistula on CDU were managed conservatively. All boys were successfully treated within 1 month, and they had normal morning erection and no evidence of recurrent priapism at the follow-up. DISCUSSION: Unlike low-flow priapism, high-flow priapism is not a medical emergency. Therefore, conservative therapy is an appropriate initial treatment, although selective arterial embolization can be effective for high-flow priapism with arteriocavernous fistula, with a success rate of 97% and no reported complications to date. Penile CDU is an imaging technique that can detect focal areas of turbulent flow with sensitivity close to 100%. This study has several limitations including a small number of cases, limited follow-up duration, and possibility of spontaneous arteriocavernous fistula closure in cases treated by arterial embolization. CONCLUSION: Penile CDU could be a reliable tool to diagnose high-flow priapism and detect the presence or absence of arteriocavernous fistula. Although conservative therapy remains the first choice, selective arterial embolization may be an early treatment option when CDU reveals an arteriocavernous fistula.

E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.

The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of beta-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing 52-bp deletion was skipped during splicing, although the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y. Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 87:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is lost in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover, an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA in a dose- and time-dependent manner. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy.

Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 bp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site.

Insertion of a 5' truncated L1 element into the 3' end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy.

We report here the second evidence of retrotransposition of L1, which was found inserted into the dystrophin gene of a patient, causing Duchenne muscular dystrophy (DMD). When the PCR was used to amplify a region of the dystrophin gene encompassing exon 44 from genomic DNA of two Japanese brothers with DMD, it was found to be approximately 600 bp larger than expected. Both the normal and the abnormally large products were amplified from the DNA of their mother. However, the maternal grandparents did not have the abnormal allele, and the mutation must therefore have occurred in the mother. Analysis of nucleotide sequence of the amplified product from a patient disclosed that the insertion was present zero to two bases upstream from the 3' end of exon 44 and that two to four bases of the exon sequence were deleted from the insertion site. The insertion sequence was found to be composed of 606-608 bp and to be almost identical to the inverse complement of 3' portion of the L1 retrotransposon consensus sequence. The dystrophin gene transcript from peripheral lymphocytes of one of the patients was analyzed by using reverse transcription/semi-nested PCR. The size of the amplified product encompassing exon 42 to 46 was smaller than expected. Sequencing of the amplified product disclosed that the sequence of exon 43 was directly joined to that of exon 45. Exon 44 of the transcript was thus shown to be skipped during splicing. This novel mutation of the dystrophin gene has important implications regarding retrotransposition of an active L1 element and provides a new insight into the origins of mutations in the dystrophin gene.

Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter.

The dystrophin gene, which is mutated in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously unidentified alternative promoter. The case study presented is that of a patient with Duchenne muscular dystrophy who had a deletion extending from the 5' end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5' part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5' end of the new transcript indicated that the 5' end of exon 3 was extended by 9 codons, only the last (most 3') of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5' of the previously identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

Outcome following repeat liver resection for colorectal liver metastases.

AIM: Our aim was to determine independent predictors of survival after second liver resection and to confirm whether the type of first resection influences survival after repeat resection. METHODS: Fifty-four patients who underwent a second liver resection for colorectal liver metastases were analyzed. To find independent predictors of survival, possible prognostic factors regarding the primary tumor, and the first and second resections were used in the Cox regression analysis. RESULTS: There were three postoperative deaths within 90 days of surgery. The 3- and 5-year overall survival rates were 53% and 46%, respectively. The size of the tumor (>50mm) (p=0.005), serum carcinoembryonic antigen level (>30microg/L) (p=0.002), and the presence of a positive surgical margin at the second resection (p=0.006) were independent predictors of poor survival following the second resection. The type of first resection was not associated with survival but was associated with the ability to achieve a histological negative surgical margin at the second liver resection (p=0.01). CONCLUSION: Three independent predictors of survival were identified. Major initial liver resection was associated with a reduced ability to achieve surgical clearance at the second resection. For colorectal liver metastases, major resection should only be performed if a negative margin cannot be achieved by minor resection.

Involvement of prostaglandin E receptor EP3 subtype and prostacyclin IP receptor in decreased acid response in damaged stomach.

We investigated the roles of cyclooxygenase (COX) isozymes and prostaglandin E (PGE) receptor EP1 and EP3 subtypes or prostacyclin IP receptors in the decrease in acid secretion in the damaged mouse stomach. Male C57/BL6 mice, both wild type and animals lacking EP1, EP3, or IP receptors, were used after 18 h of fasting. Under urethane anesthesia, the stomach was mounted on an ex-vivo chamber and perfused with saline, and acid secretion as well as transmucosal potential difference (PD) was measured before and after exposure to 20 mM taurocholate Na (TC) for 20 min. Indomethacin, SC-560 or rofecoxib was given i.d. 30 min before TC. Mucosal exposure to TC in wild-type mice caused a reduction in PD, followed by decrease in acid secretion. Indomethacin attenuated the decrease in acid secretion after exposure to TC in wild-type mice, an effect mimicked by SC-560 but not rofecoxib, yet none of these drugs affected the decrease in PD. An altered acid response after exposure to TC was similarly observed in EP1 (-/-) mice but mitigated in mice lacking either EP3 or IP receptors, although a decrease in PD was observed in all groups. Furthermore, the decreased acid response was also attenuated by prior administration of the EP3- but not EP1- antagonist. Mucosal levels of PGE(2) and 6-keto PGF(1a) increased after exposure to TC in all groups of mice. In conclusion, the decrease in acid secretion in the damaged stomach is mediated by endogenous PGs derived from COX-1, through PGE(2)/EP3 receptors and prostacyclin/IP receptors.