Atherosclerotic cardiovascular disease is the main cause of morbidity and mortality in chronic kidney disease patients. There is a raft of evidence showing that in the general population dyslipidaemia is associated with an increased risk of cardiovascular events, as well as with a greater prevalence of chronic kidney disease. Consequently, the use of statins in the general population with dyslipidaemia is not controversial. Nevertheless, the benefits of statins in patients with chronic kidney disease are more elusive. The authors review the possible effects of statins on the progression of renal disease and cardiovascular events in chronic kidney disease patients. Atherosclerotic cardiovascular disease is the main cause of morbidity and mortality in chronic kidney disease patients , 2, withIt is still uncertain whether dyslipidaemia itself causes kidney disease progression or whether kidney impairment and proteinuria are responsible for both renal disease progression and dyslipidaemia . EvidencThe severity of the hypercholesterolemia correlates with proteinuria. In rats with kidney disease caused by unilateral nephrectomy, such diet augmented the glomerular lesions in the remaining kidney . Renal bSamuelsson et al., in a small study with 73 adult nondiabetic patients with primary chronic renal disease, showed that lipoprotein abnormalities can contribute to the progression of kidney failure in CKD patients and demonstrated an association between progression of renal disease and hyperlipidaemia . The PhyThe Helsinki Heart Study documented an association between dyslipidaemia and progressive kidney disease in 2702 middle-aged dyslipidaemic men. Renal function deteriorated by a mean of 3% over 5 years, and although hypertension accelerated this change, the decline was faster by 20% in men with an LDL : HDL ratio >4.4 than in men with a ratio <3.2. After multiple regression analyses, the only measures having a significant effect on the observed phenomenon were an increased LDL : HDL ratio (negative outcome) and an increased HDL-C level (protective outcome) .The lipid profile is influenced by the severity of renal dysfunction and the presence of proteinuria. Patients with CKD have, in general, lower levels of HDL, LDL, and total cholesterol and higher levels of triglycerides. There is a clustering of low HDL and elevated Lp(a) and TG-rich ApoB containing VLDL and IDL . PatientIn the last decades the medical literature confirmed that oxidative stress and inflammation are major players in the pathogenesis of atherosclerosis , and morIf dyslipidaemia promotes renal injury, then reducing dyslipidaemia should slow or prevent the progression of CKD. Experimental models show that statins decrease the severity of glomerular damage and preserve renal function. For example, New Zealand rabbits fed a diet rich in cholesterol became hypercholesterolaemic, with evidence of endothelial dysfunction in renal segmental arteries as well as glomerular hypertrophy and diffuse glomerulosclerosis. In this model, atorvastatin attenuated the increase in plasma cholesterol and prevented renal artery endothelial dysfunction, glomerular hypertrophy, and most of the glomerulosclerosis . Data onThe presence of proteinuria is an indicator of kidney disease with an increased probability of progressive kidney failure and is associated with faster loss of GFR compared with little or no proteinuria . In a meA meta-analysis by Douglas et al. evaluated 15 studies involving a total of 1,384 patients and examined the proportional reduction in proteinuria with the use of statins. It was shown that statins reduced albuminuria and proteinuria in 13 of the 15 studies. The reduction of excretion was greater among studies with greater baseline albuminuria or proteinuria. More specifically, 440 patients with albuminuria ≥30 mg/day showed a 48% reduction of albuminuria relative to placebo . Another meta-analysis of six randomised placebo control trials including 311 patients showed that compared to placebo, statins reduced proteinuria significantly (−0.73 g/24 h) . A diffeP = 0.0056) [In PLANET I, atorvastatin (80 mg) significantly reduced proteinuria by about 15%, whereas rosuvastatin (10 or 40 mg) had no significant effect on proteinuria. In PLANET II, atorvastatin (80 mg) reduced proteinuria by 23.8% ( 0.0056) . Discrep 0.0056) , 20, 24.The National Lipid Association Statin Safety Assessment Task Force reported that “proteinuria is at least possible with all statins at some concentration, but is more likely to be seen with statins that are potent inhibitors of HMG-CoA reductase.” The report concludes that statin-induced proteinuria is not associated with either renal impairment or renal failure , 25. AccP < 0.0001) [Two trials—the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) study and the Aggressive Lipid-Lowering Initiation Abates New Cardiac Events (ALLIANCE) study—evaluated atorvastatin therapy versus usual care in patients with coronary artery disease (CAD). In both studies, creatinine clearance declined by an average of 4.4% during 4 years in patients assigned to usual care. In contrast, patients assigned to atorvastatin therapy had an 11.6% increase in creatinine clearance in the GREACE study. In the atorvastatin group in the ALLIANCE study, although creatinine clearance did not improve, no decline was seen. The difference between treatments was highly significant in both studies ( 0.0001) , 27.Post Hoc analysis of the Cholesterol and Recurrent Events (CARE) trial, a secondary prevention trial of pravastatin versus placebo, showed a significant difference in the rate of decline with pravastatin  in those with severe CKD at baseline (estimated GFR <40 mL/min/1.73 m2) [A 1.73 m2) . The afo1.73 m2) . 2, and 237 patients with a mean eGFR of 74.9 mL/min/1.73 m2 at baseline, respectively. Recent results showed that patients on atorvastatin lost about 1 to 2 mL/min per 1.73 m2 over 52 weeks, those on rosuvastatin 10 mg/day lost about 4 mL/min per 1.73 m2 and those on rosuvastatin 40 mg/day lost close to 8 mL/min per 1.73 m2 [The Study of Heart and Renal Protection (SHARP), a prospective, randomised, controlled study, assessed outcomes in approximately 6300 CKD patients given combination simvastatin/ezetimibe therapy. Concerning the secondary endpoint of progression to ESRD in SHARP, no difference was seen between groups. In fact, one third of the patients in both arms progressed to dialysis or transplantation . Concern 1.73 m2 . In nondiabetic patients (PLANET II), the effects of the treatments on kidney function were slightly less pronounced. There was a significant decline in eGFR with rosuvastatin 40 mg/day but not in the other two treatment groups –32. AtorThe available data and the evidence are inconclusive to answer the question if statins slow the kidney disease progression. It is premature to recommend statin therapy for renal protection alone. Additional prospective, randomised trials are also needed to determine whether statins are truly renoprotective. Addressing safety, the use of high doses of statins in CKD patients has largely been demonstrated to be safe and well tolerated , 23.Statins improve the lipid profile in renal patients and exert several pleiotropic effects without major adverse consequences. However, respecting cardiovascular outcomes is critical to the timing of the initiation of the therapy. In the SHARP trial the use of simvastatin plus ezetimibe versus placebo reduced major atherosclerotic events  only in the group of patients not on dialysis (risk reduction of 20.2%). In dialysis patients the difference did not reach statistical significance . This observation corroborates the result of the Prospective Pravastatin Pooling Project that included three large trials: CARE, WOSCOPS, and LIPID studies . PravastOn the other hand, it is known from several randomised controlled trials  , 35, 36 The apparent lack of benefit of using statins in patients in dialysis can have quite a few explanations: the existence of other pathogenic pathways contributing to cardiovascular disease, the high mortality of dialysis patients due to sudden death and cardiomyopathy, situations not preventable by statins, or just because it is too late to interfere with the natural history of atherosclerosis. Moreover, inflammation can play an important role in the dialysis population. In the JUPITER study, the administration of rosuvastatin in patients with mild dyslipidaemia but with high CRP levels reduced the number of cardiovascular events . This finding was not unexpected, since statins have an anti-inflammatory effect. This fact can also explain the negative results in the 4D and AURORA trials, because in these studies the CRP levels remained high despite the use of atorvastatin or rosuvastatin, respectively .Nevertheless, in a recent post hoc analysis of the 4D trial, it was shown that atorvastatin decreased the risk of fatal and nonfatal cardiac events in dialysis diabetic patients with high levels of LDL cholesterol levels .Statins may have antiproteinuric effects, although the clinical significance of these benefits remains uncertain. The available data and the evidence are inconclusive to answer the question if these agents slow the kidney disease progression. It is premature to recommend statin therapy for renal protection alone.Regarding prevention of cardiovascular disease, there is a strong clinical evidence of benefit in using statins in early stages of CKD, but there is no clear advantage to its use in dialysis patients, a particular population with a huge cardiovascular risk. Nevertheless these drugs have several pleiotropic effects, and remembering the musical “The Fiddler on the Roof” and the song “If I Were a Rich Man…” maybe we can also use statins in selected dialysis patients; namely, in those with higher LDL cholesterol levels, their use may be advantageous.


Human kallikrein gene 6 (KLK6) is a member of the human kallikrein gene family . Human kallikrein-related peptidase 6 (hK6) is a trypsin-like serine protease encoded by the KLK6, has been reported to be highly expressed in several cancers including gastric cancer. In this study, we investigated the the correlation of hK6 expression with clinicopathological characteristics, tumor recurrence and prognosis in advanced gastric carcinoma after curative resection.We retrospectively analyzed the clinical data of 129 cases advanced gastric cancer after curative gastrectomy. The expression of hK6 in advanced gastric cancer tissues compared to adjacent noncancerous tissues were examined, and the relationship between hK6 expression and clinicopathological characteristics was evaluated. In additional, these patients were followed up to investigate the relationship between hK6 expression and the survival time.The positive rate of hK6 expression was significantly higher in advanced gastric cancer tissue, than that in adjacent noncancerous and gastric ulcer tissues . There was a close relationship between hK6 expression and TNM stage (P = 0.005), vascular invasion (P = 0.037) and perineural invasion (P = 0.035). Furthermore, patients with hK6 positive showed significantly higher recurrence and poorer prognosis than those with hK6 negative. Multivariate analysis showed that hK6 expression was a significant independent factor for tumor recurrence and overall survival.hK6 is overexpressed in advanced gastric cancer tissues. Its clinical utility may be used as an unfavorable indicator in predicting tumor recurrence and prognosis for advanced gastric cancer after operation. This study also suggests that hK6 might be a potential therapeutic target for gastric cancer.http://www.diagnosticpathology.diagnomx.eu/vs/8558403578787206The virtual slide(s) for this article can be found here:  Gastric cancer is one the most common malignancies and the second leading cause of death among all cancers in the clinical , and surThe KLKs consist of 15 homologous genes which encoding secreted serine proteases, localized in tandem on chromosome 19q13.4. The KLKs have similar genomic organizations, and show significant homology at both the nucleotide and protein level. The same gene of the family is expressed in different tissues, and multiple genes are commonly expressed in the same tissue .KLK6 is a member of the KLKs, which encodes for human kallikrein-related peptidase 6 (hK6). It is a serine protease made up of 223 amino acids with trypsin-like activity. hK6 has been cloned independently by three groups, using a differential display technique from primary and metastatic breast cancer cell lines. Anisowicz et al.  cloned tTo our best knowledge, few studies have been investigated concerning the clinical significance of hK6 expression in advanced gastric cancer with recurrence and prognosis. In this study, we evaluated the expression of hK6 in the surgical specimens of advanced gastric cancer tissues, paired adjacent noncancerous tissues and gastric ulcer tissues using immunohistochemistry HRP, and analyzed their correlations with clinicopathological characteristics and patients survival to clarify the significance of hK6 in advanced gastric cancer after curative surgery.A total of 129 samples were obtained from patients from January 2007 to November 2011 who underwent curative surgery for advanced gastric cancer in HuiZhou Municipal Central Hospital of GuangDong. Resection samples were confirmed to be primary advanced gastric cancer by clinical pathology, none of the patients received any chemotherapy, radiotherapy and other adjuvant therapy prior to operation. 52 blocks from adjacent noncancerous gastric tissues (at least 5 cm away from the cancer margin) were obtained from the patients. In addition, 36 specimens obtained from patients who underwent surgery and were confirmed to be gastric ulcer used as controls in the same period., clinicopathological characteristics such as gender and age, tumor location, size, differentiation, T stage, lymph node metastasis, TNM stage and whether recurrence were retrospectively reviewed. The mean age was 60 years (range: 28 ~ 80 years)with 56 women and 73 men. All the patients were staged based on the TNM classification of the American Joint Committee on Cancer  [,s Ethics Committee of HuiZhou Municipal Central Hospital. All the specimens were fixed in 10% neutralized maldehyde, embedded in paraffin, cut into 3-μm thick sections, and mounted on glass slides for immunohistochemistry analysis.PatientsImmunohistochemistry was performed using the Horseradish Peroxidase two-step method, which avoids the interference of endogenous biotin, thus reducing the nonspecific false-positive background staining. Immunohistochemistry was carried out in eight steps according to the instructions: 1, The paraffin sections were deparaffinized in xylene, rehydrated through gradient ethanol. 2, Antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) with a pressure cooker. 3, Block the endogenous peroxidase with 3% hydrogen peroxide. 4, Then incubated with the purified rabbit polyclonal antibody, antigen retrieval and dilution were determined by preliminary experiments. 5, Detection of hK6 immunocomplex was incubated with the PV9000 kits . 6, Stained with diaminobenzidine substrate chromogen solution  until the optimal staining was achieved. 7, The sections were counterstained with hematoxylin, differentiated, returned blue. 8, Finally, dehydrated through gradient alcohol, cleared in xylene, neutral gum mounted. Then observed under the optical microscope.All slides were assessed separately by two senior pathologists with no prior knowledge of clinicopathological parameters, the final score for their average. For evaluation of hK6 immunohistochemical staining, the percentage of positive cells was evaluated quantitatively 10 fields within the tumor were selected randomly, expression in 1000 cancer cells (100 cells per field) was evaluated using a high-power magnifcation (400×) and an average was evaluated quantitatively and scored as: 0 for the percentage of positive cells ≤ 10%, 1 for staining of 11 ~ 25%, 2 for staining of 26 ~ 50%, and 3 for staining of 51 ~ 75%, 4 for staining of > 75% of the cells examined , a totalAll these patients received fluorouracil based adjuvant chemotherapy after surgery. As a regular follow-up, all the patients were checked every 3 months during the first 2 years and every 6 months during the third to the fifth year, once a year thereafter. Medical work-up consisted of history and physical examination, hematology and biochemical tests including serum CEA, CA19-9 and CA72-4, and imaging including chest radiography, barium meal, abdominal ultrasonography (US), computed tomography (CT), and endoscopy according to the clinical situation. The follow-up time had the day of surgery as a starting point, the time of tumor recurrence and death were recorded, these two points were evaluated for prognostic analysis. If the follow-ups were incomplete, patients or their families were contacted by telephone, the closing date for follow-up was May 2012, the median follow-up period after surgery was 25 months (range 4–64 months).2) and Fisher’s exact test. For survival analysis, two end points were examined:recurrence-free survival (RFS) and overall survival (OS) respectively. RFS is defined as the time interval between the date of surgery and the date of first detected recurrence or metastasis, and OS is defined as the time interval between the date of surgery and the date of death or the date of last follow-up for those who were alive at the end of the study. The Kaplan–Meier model was used to examine survival between the patients positive and negative hK6 expression. The significance of any difference in the survival curves was determined with the log-rank test. Variables achieving a significance level in the univariate analysis were subsequently introduced in a forward LR proportional hazard analysis (Cox’s model)to identify those variables independently associated with survival, the p value of 0.05 (or less)was considered as statistically significant.Statistical analysis was carried out using the SPSS 13.0, the relationships between hK6 expression and patient clinicopathological characteristics were analyzed with the Chi-square  patients had died, A total of 61 patients died because of tumor progression and 7 due to other causes. In all, there were 69 (53.5%) patients developed tumor recurrence, 31 of whom developed as loco-regional relapse, 19 as distant metastases , 14 as peritoneal recurrence and 5 as remnant; All but eight patients with tumor recurrence died from it.Kaplan–Meier survival curves indicated patients with positive hK6 expression were more likely to have both a shorter RFS .Studies found that hK6 was secreted to the extracellular matrix and involved in the degradation of matrix proteins including fibronectin, laminin, vitronectin, collagen and induced E-cadherin ectodomain shedding, reduced the cell-cell adhesion ,12 and lTo date, it has been shown that KLK6 is upregulated in such gastrointestinal malignancies as pancreatic and colon cancers . Ogawa eOgawa et al.  reportedA plenty of evidence has demonstrated that several members of the KLKs are potential cancer biomarkers . The mosStudies have suggested that high KLK6 expression was closely associated with shorter overall and recurrence-free survival for ovarian cancer  and coloWith the development of molecular and proteomics in gastric cancer, many proteins have been the potential markers for early diagnosis, prognosis and therapeutic targets of gastric carcinoma, among them c-Met , PKP2 anFew previous studies investigated the clinicopathologic significance of hK6 expression in advanced gastric cancer tissues. Thus, in this study, we examined the correlation of hK6 expression with tumor recurrence and prognosis in advanced gastric cancer. The findings indicate that the expression of hK6 was significantly associated with TNM stage, vascular invasion, perineural invasion and poor prognosis for patients with advanced gastric cancer. Moreover, we found that patients with positive hK6 expression were more likely to have recurrence, and lower RFS and OS rates compared with those with negative hK6 expression. Multivariate analysis proved that hK6 expression was a significant independent prognostic factor of RFS and OS in patients with advanced gastric cancer. Our findings support that hK6 might be used as a potential biomarker and therapeutic target for gastric cancer. Furthermore, positive expression of hK6 in advanced gastric cancer tissues may provide valuable information to understand the biological behavior of gastric cancer.In conclusion, our study showed that hK6 expression in advanced gastric cancer significantly correlated with TNM stage, vascular invasion and perineural invasion as well as RFS and OS rates. This is a rare report to evaluate hK6 expression and its correlation with tumor recurrence and poor prognosis in advanced gastric cancer. Further studies will be needed to explore the possibilities whether hK6 might be used as a valuable biomarker in predicting tumor recurrence and a therapeutic target for gastric cancer by using serum samples from patients. And we also will examine the KLK6 at the mRNA level from the tissues samples next step.The authors declared that they have no competing interest.XL and XY carried out pathological examination, HX and JL analyzied the data, YH drafted the manuscript. All authors read and approved the final manuscript.


An ideal animal model for the study of a human disease is one which utilizes a route of infection that mimics the natural transmission of the pathogen; the ability to obtain disease with an infectious dose equivalent to that causing disease in humans; as well having a disease course, morbidity and mortality similar to that seen with human disease. Additionally, the animal model should have a mode(s) of transmission that mimics human cases. The development of small animal models for the study of monkeypox virus (MPXV) has been quite extensive for the relatively short period of time this pathogen has been known, although only a few of these models have been used to study anti-poxvirus agents. We will review those MPXV small animal models that have been developed thus far for the study of therapeutic agents. Additionally, the animal model should have a mode(s) of transmission that mimics human cases. Factors which subsequently allow more detailed inferences about disease pathogenesis include the availability of reagents to evaluate host innate and adaptive immune responses to the pathogen, and histopathological changes in the host which result from infection or the host response to infection. These findings can then be compared to what is known of human disease. The utility of a small animal model of human disease for study of therapeutic efficacy is augmented when large numbers of animals are available for use in appropriately, well-powered studies. Even if all aspects of an animal model of disease are not completely faithful to what is known of human disease, important information regarding therapeutic efficacy can be gleaned from their use in “pre-clinical” studies.The published literature on clinical manifestations of systemic human orthopoxvirus disease is derived from historic literature descriptions of human smallpox and more recent descriptions of human monkeypox disease. The clinical-descriptive literature on human monkeypox is expected to grow in the next five years, as data acquisition and analysis from an ongoing study in the Democratic Republic of Congo is finalized. Currently available literature is largely derived from WHO-sponsored surveillance efforts in West Africa and the Congo Basin in the 1980s, after the first recognition of human disease in these areas, and subsequent analyses of public health response data and human research studies following the introduction of West African clade virus into the U.S. in 2003. Human monkeypox, as described through the active surveillance and case ascertainment studies sponsored by WHO in the 1980s, was depicted as resembling discrete ordinary smallpox. In natural human infection, exposure leading to infection is believed to occur via a respiratory route, with subsequent progressive viremias/lymphemia, ultimately leading to seeding of the skin to generate a generalized rash. Percutaneous exposure, also leading to generalized rash formation, has also been described for both viral infections. The disease pathogenesis has been conjectured and modeled largely from animal studies; initial models were using ectromelia infection of mice; some kinetic observations of virus shedding and viremia have been made in human studies of smallpox and monkeypox. The time course of disease is generally thought to include an asymptomatic phase of 10–12 days, during which time the virus initially enters the host, replicates, seeds reticuloendothelial organs, replicates, then spreads via the bloodstream (inducing a febrile response) which is the first symptomatic hallmark of disease. The fever is usually described as occurring 10–12 days post initial exposure/infection. The range has been 7–17 days. Fever is accompanied by other symptoms, including headache, backache, myalgias, and or abdominal pain. Two to three days following the fever, rash develops—initially presenting as a macular, then papular, then vesicular and pustular eruption. Scabbing then begins. Each stage of rash lasts 1–2 days. Approximately 2–3 weeks post initial symptoms, scabs begin to separate from the skin. Death and disease severity have had some correlation with rash burden in epidemiologic studies of hospitalized smallpox patients. Severe outcomes are more frequent in unvaccinated, younger age groups; death occurs within the first week of illness in cases with hemorrhagic manifestations, and during the second or third week of illness in “ordinary” cases. In the human monkeypox cases studied in Zaire/DRC between 1981–1986, of the 33 deaths among 338 patients, all occurred in unvaccinated children less than eight years of age. Death occurred during the first week of illness in 21%, the second week in 52%, and the third week of illness in the remaining 27% [The development of small animal models for the study of monkeypox virus (MPXV) has been quite extensive for the relatively short period of time this pathogen has been known. Initial animal models were designed to address natural history in potential or surrogate reservoir host species, as well as studies of disease in primates. Routes of exposure were designed to evaluate disease if respiratory or percutaneous exposures occurred, or in some cases to simply address whether virus would replicate in the animal model system. Factors that influence the outcome of a challenge study include the age of the animal at time of infection, inoculation route used, and the viral dosage given. Additionally, the strain of MPXV (currently delineated as belonging to Congo Basin clade or West African clade) used in the study may influence the disease severity.2.7) [Guinea pigs and golden hamsters were found to be relatively resistant to MPXV (West African clade Copenhagen strain) infection by multiple routes. Guinea pigs were challenged via an intracardial, intranasal (IN), oral or foot pad (FP) inoculation with no observable symptoms of disease except for edema at the FP inoculation site. Golden hamsters were also resistant to MPXV infection via several routes of infection with no observable signs of disease, even with large dosages of virus (1.5–5.9 × 107) .Rabbits have also been considered as a possible animal model for the study of MPXV –3; susce1 to 103 plaque forming units (pfu) of West African MPXV were not susceptible to infection with intravenous, IN, or cutaneous routes of infection. However, newborn white rats (1–3 days old) developed adynamia leading to death in 5–8 days when challenged with MPXV intranasally [5 pfu via an intravenous route of infection, 100% mortality was seen 4–5 days post infection (p.i.). The infection was characterized by difficulty breathing, cough, sneezing, cyanosis, rhinitis, purulent conjunctivitis, and progressive emaciation. An IN MPXV challenge in cotton rats caused 50% mortality with a clinical picture such as that seen with the intravenous route [Several rat species including white rats, cotton rats and multimammate rats have been challenged with MPXV. Adult white rats inoculated with 10anasally . Cotton us route ,7. Multiet al. challenged adult common squirrels (Sciurus vulgaris) with 106 pfu of MPXV Z-249 (Congo Basin clade) via IN, oral or scarification routes of infection [et al. challenged six African squirrel species  with Congo Basin MPXV via an IN infection (105 or 106 pfu/0.1 mL) [Marennikova Spermophilus tridecemlineatus) are very susceptible to MPXV infection. Tesh et al. challenged adult ground squirrels with the West African MPXV clade either IP or IN with 105.1 pfu [50 values were similar for the two strains using the ground squirrel MPXV model (0.35 pfu for Congo Basin and 0.46 pfu for West African MPXV).Ground squirrels  via an IN or scarification route of infection [50 for the prairie dog MPXV model is approximately a hundred-times lower for the Congo Basin clade compared to the West African clade  [Prairie dogs have also been looked at as a possible MPXV animal model. Xiao ectively . Hutson nfection . Animalsctively) , utiliziet al. challenged African dormice, Graphiuris kelleni, with 1.4 × 104 pfu of a Congo Basin clade of MPXV via FP route and observed 92% mortality [50 as 12 pfu. Animals became symptomatic at day 3 (conjunctivitis and dehydration), and animals that succumbed to disease had a mean time to death of 7.9 ± 1 to 12.3 ± 5 days (depending on dose). Morbidity for those animals that succumbed to disease included decreased activity, hunched posture, unkempt hair coat, dehydration, and conjunctivitis; lesions did not develop on any animals. Weight loss was highest with 2,000 pfu (the highest dosage given), but also was seen in animals given 200 or 20 pfu. Weight loss was not observed in the lowest dosage groups (2 and 0.2 pfu). Disease pathogenesis was described as localized inflammation, viral replication and hemorrhage in the nasal mucosa, followed by dissemination around day 3 with subsequent necrosis of liver, spleen, lung and gastrointestinal tract tissues. When the West African strain of MPXV was used to challenge dormice by an IN infection, similar days until death and mortality rates were seen as the Congo Basin MPXV challenged animals.Schultz ortality . The auti.e., wild strains or laboratory strains). In early studies [et al. compared the two clades of virus (105 pfu) in immunocompetent BALB/c and C57BL/6 laboratory mouse strains via an IN or FP route of infection [et al. compared both clades of virus by an IP inoculation in either BALB/c or severe combined immune deficient (SCID) BALB/c mice [et al. utilized a laboratory mouse strain lacking STAT1 (C57BL/6stat-/-), an important protein involved in Type I and Type II IFN signaling [et al. screened 38 inbred mouse strains and identified three that are highly susceptible to MPXV [5 or 106 pfu Congo Basin MPXV) lost up to 28% of the starting body weight and 100% died between days 5–8. Animals challenged with 104 pfu all died between days 8–10. Animals challenged with 103 pfu had an even longer delay in weight loss and death and 40% of the animals recovered. No animals perished in the 102 pfu challenge group. The calculated LD50 for the Congo Basin clade in the CAST/EiJ mice given an IN challenge was 680 pfu. Animals were found to be even more sensitive with an IP Congo Basin MPXV infection and the calculated LD50 was 14 pfu. Challenging mice with 105 or 106 pfu of a West African MPXV strain resulted in rapid weight loss and 100% mortality by day 8 p.i. Lower dosages of West African MPXV resulted in less weight loss and lower amounts of death than what was observed for the Congo Basin MPXV; the calculated LD50 was 7,600 pfu, more than a log higher than for the Congo Basin clade.As is the case for many pathogens, mice have been utilized numerous times for the study of MPXV. Results have varied greatly depending on the type of mice used . Herein we will summarize those studies, efficacy data, and discuss the advantages, and limitations, of the animal models used.et al. utilized ground squirrels to test the efficacy of ST-246 against a MPXV challenge [50) via a subcutaneous route of inoculation. Squirrels (8–9 per group) were divided into five treatment groups; drug was given either at 0 hours of infection, 24 hours, 48 hours, 72 hours or 96 hours p.i. 100 mg/kg of drug was given once a day for 14 days. Two animals in each group were sacrificed at day 7 to measure objective morbidity; the remainder of the animals were used to calculate survival rates. Animals in the placebo group, that were not given ST-246, showed signs of illness beginning on day 4 and all died between days 6–9. Signs of disease included lethargy, anorexia, nosebleeds, and terminal respiratory distress. At day 7, a sampling of placebo-treated animals exhibited significant leukocytosis, transaminitis, and coagulopathy; almost 105 pfu/mL of infectious monkeypox was found in blood; at this time, between 107 and 108 pfu /mL of infectious MPXV was observed in 10% organ homogenates of liver, spleen and lung. Animals treated on days 0, 24, 48 or 72 hours, before symptomatic disease onset, all survived infection and showed no signs of disease. At day 7, in a sampling of animals treated at hour 0, 24, 48 or 72 p.i., no virus was found in the liver, spleen, lung, or blood; although some abnormal values were apparently recorded, no clear trends in leukocytosis, transaminitis or coagulopathy were noted with delay in treatment onset. In animals initiating treatment at 96 hours p.i., concurrent with symptomatic disease onset, 67% of animals survived infection. 2/4 survivors showed signs of disease. In those animals that succumbed to infection, ST-246 prolonged the time to death; the mean time to death was day 7 for animals receiving placebo and day 13 for those receiving ST-246 in the 96 hour p.i. treatment group. The sampling of animals at day 7, initiating ST-246 at 96 hour p.i., demonstrated lower levels of viremia (∼3 log decrease) and ∼5 logs less virus in liver, spleen and lungs than that seen on the placebo treated animals at day 7. Although some evidence of transaminitis was present, leukocytosis and coagulopathy were not observed in this treatment group. Pathologic examination of tissues in general showed greater tissue necrosis in animals treated at later times p.i. This study was able to demonstrate a survival benefit in animals treated prior to, or at the onset of disease symptoms, in a disease model that has a time course attenuated with respect to what is seen in human disease.Sbrana hallenge . The autet al. infected African dormice with a lethal challenge of Congo Basin clade virus MPXV-ZAI-79 via an IN route of infection to evaluate the efficacy of Cidofovir as post exposure prophylaxis [3, or 5 × 103 pfu of MPXV, animals were intraperitoneally administered 100 mg/kg cidofovir . Aggregate data from all challenges showed animals treated with cidofovir had a mortality rate of 19% (7/36), whereas vehicle treated animals all (41/41) succumbed to disease. Treatment initiation at later times p.i. was not evaluated; effects on viral load or histopathologic changes were not reported.Schultz phylaxis . Four hoet al. utilized a laboratory mouse strain lacking STAT1 (C57BL/6stat-/-), which has been found to be sensitive to a range of viruses including SARS, murine norovirus 1, respiratory viruses, dengue virus and MPXV [As inbred mice have historically shown little disease symptomatology or pathogenesis post monkeypox infection, Stabenow and MPXV ,22–25. Tet al. tested the efficacy of ST246 in a prairie dog MPXV model [5, equal to 65 × LD50 for the prairie dog model. Animals were divided into three treatment groups; prophylactic (day 0), post exposure (day 3) and therapeutic (varying day based on rash onset), and a control vehicle treated group. ST246 was formulated at 30 mg/mL and administered daily, by oral gavage, for 14 days. Animals initiating treatment at day 0 or 3 were protected from death and apparent signs of illness. Animals treated at rash onset had symptoms similar to the placebo control group; however symptoms were less severe in the treated animals. Although all animals treated at rash onset survived infection, animals lost 10–24% of body weight and did develop generalized rash  and shown survival benefit. Routes of infection have used methods that attempt to simulate potential human routes of infection and resultant human illness courses. Given the uncertainties of what a human infectious or lethal monkeypox dose is, it is difficult to extrapolate the potential “best fit” of any of these models for human disease. The clinical time course of disease in the prairie dog model, however, has a temporal relationship that is close to what has been described with human systemic orthopoxvirus (variola or monkeypox) disease. However, a limitation of the prairie dog and some of the other described animal systems, with the exception of the mouse model, is a paucity of immune reagents. There are a handful of antiviral compounds which show promise in these small animal models using monkeypox virus as the challenge. Additional studies evaluating treatment benefit when used in later stages of disease, their effect on elicitation of a protective immune response, evaluation of antiviral resistance, and their effect on viral shedding will improve our understanding of how they may be used in treatment of human disease, or in response to epidemic disease.Animal models permit an advance beyond what can be gleaned from tissue culture evaluation of an antiviral effect. The evaluation of an antiviral, in the context of a host with a functioning immune system, enables better understanding of therapeutics’ potential efficacy. The evaluation of an antiviral in the context of an impaired immune system enables better understanding of therapeutic use in a particular immunosuppressed population. Pathogen host range, especially if not a simple issue of receptor utilization, can confound the ability to interpret, and extrapolate to the human, some of the nuances of the host pathogen interaction and prediction of potential human therapeutic benefit. Of the small animal models used to evaluate antiviral efficacy, all have used stringent virus challenges (all greater than 10 × LD


Bactrocera dorsalis (Diptera: Tephritidae) is mainly distributed in tropical and subtropical Asia and in the Pacific region. Despite its economic importance, very few studies have addressed the question of the wide genetic structure and potential source area of this species. This pilot study attempts to infer the native region of this pest and its colonization pathways in Asia. Combining mitochondrial and microsatellite markers, we evaluated the level of genetic diversity, genetic structure, and the gene flow among fly populations collected across Southeast Asia and China. A complex and significant genetic structure corresponding to the geographic pattern was found with both types of molecular markers. However, the genetic structure found was rather weak in both cases, and no pattern of isolation by distance was identified. Multiple long-distance dispersal events and miscellaneous host selection by this species may explain the results. These complex patterns may have been influenced by human-mediated transportation of the pest from one area to another and the complex topography of the study region. For both mitochondrial and microsatellite data, no signs of bottleneck or founder events could be identified. Nonetheless, maximal genetic diversity was observed in Myanmar, Vietnam and Guangdong (China) and asymmetric migration patterns were found. These results provide indirect evidence that the tropical regions of Southeast Asia and southern coast of China may be considered as the native range of the species and the population expansion is northward. Yunnan (China) is a contact zone that has been colonized from different sources. Regions along the southern coast of Vietnam and China probably served to colonize mainly the southern region of China. Southern coastal regions of China may also have colonized central parts of China and of central Yunnan. Determining the source area and understanding the colonization routes of invasive pests are key issues when developing management strategies Bactrocera dorsalis (Hendel), the oriental fruit fly, is one of the most notorious species in the family Tephritidae. This species belongs to the B. dorsalis species complex K-selected demographic strategy B. dorsalis would be adapted for growth and establishment in near optimal environmental conditions, taking advantage of the most productive niches B. dorsalis has been regarded as a typical invasive species causing high economic losses, and many research programs have been already carried out in the fields of ecology and biology B. dorsalis in these regions is complex, and its interpretation was not straightforward. Some populations at the western limit of the range  or in which the fly was recently introduced  were somewhat differentiated, while high gene flow was suggested among Southeast Asian countries, namely Laos, Thailand and Cambodia. Aketarawong et al. B. dorsalis, and that genetic structure is weak, even at this geographical scale. The authors The question of identifying the source of origin and understanding the colonization pathway for this species has received less attention. Recently, two studies focused on this issue. One paper was based on microsatellites only B. dorsalis over a large geographical scale in China and Southeast Asia. We also aimed to identify the native region of this pest species and its colonization pathways in Asia.The present study is based on an extensive geographical sampling of the oriental fruit fly, as it includes most regions studied by Akeratawong et al. Bactrocera oleae (Gmelin), Bactrocera cucurbitae (Coquillett), or Ceratitis capitata (Wiedemann) represent cases of successful invasive species, which have caused tremendous economic losses in many countries Bactrocera invadens, a recently described species of Tephritidae, has expanded throughout Africa within only two years after its first discovery and is currently causing extensive agricultural losses Phytophagous flies of the Tephritidae family, also called “true fruit flies”, are among the most important pests of fruits and vegetables. One-third of the species in this large family (more than 4000 known species) attack soft fruits, including many commercially and economically important species B. dorsalis typically occurs in tropical areas of South, East, and Southeast Asia  as well as in some Pacific islands  B. dorsalis was first reported in 1934 in Hainan province B. dorsalis infestations were reported in southwestern and southeastern parts of China B. dorsalis is now distributed mainly in provinces located south of the Yangtze River in mainland China No specific permits were required in our studies of this widespread agriculture pest. We confirm that the study locations were not privately owned or protected. This work did not involve endangered or protected species.B. dorsalis adults were collected from 21 localities in China and Southeast Asian countries. In China, we sampled 9 provinces, namely Yunnan, Guizhou, Guangxi, Fujian, Guangdong, Hainan, Jiangxi, Hubei and Hunan. From Southeast Asia we sampled in Myanmar, Vietnam, Laos, Thailand and Cambodia. Details about sampling sites and sampling sizes are given in A 601 bp fragment of the mitochondrial cytochrome oxidase gene (COI) was amplified and sequenced using the protocols described in Shi et al. Seven microsatellite loci were used in this study. Technical details are given in Dai et al. Numbers and distribution of haplotypes, numbers of unique haplotypes, polymorphic sites, within-population mean number of pairwise differences between pairs of haplotypes (π) and nucleotide diversity na); number of private alleles (np); frequency of private alleles (AP); observed heterozygosity (HO) and expected heterozygosity (HE). Gene diversity (HS) and allelic richness (RS) were calculated using FSTAT 2.9.3.2 For each population, the following genetic diversity estimates were calculated as averages over all microsatellite loci with GENEPOP 4.0 ST estimates for both types of markers. They were calculated using ARLEQUIN for mitochondrial sequences, and estimated using the ENA (Excluding Null Alleles) correction described in Chapuis & Estoup The degree of genetic differentiation between pairs of populations was measured by pairwise FST values) were compared with the matrix of geographic distances by means of a simple Mantel test To detect isolation by distance, matrices of pairwise estimates of genetic differentiation (FDs) calculated with microsatellite data Genealogical relationships between mitochondrial haplotypes were reconstructed using TCS 1.21 CT values should reach a maximum or a plateau. Second, the configurations with one or more single-population groups should be excluded because this indicates that the group structure is disappearing Spatial analysis of molecular variance (SAMOVA) was performed with both types of markers separately using SAMOVA 1.0 For microsatellite markers, the Bayesian approach implemented in STRUCTURE 2.3.3 AMOVAs were run on the mitochondrial data set as well as on the microsatellite data to test the effect of the host plants on the genetic structure of the fly. Flies were thus grouped depending on the host they were sampled from . Such analyses were performed using ARLEQUIN.D and Fu's SF for the 8 identified SAMOVA groups. We then tested whether these indices significantly deviated from 0. All demographic analyses were performed using ARLEQUIN.To study the demographic history of the groups of populations identified previously, we studied mismatch distributions The program BOTTLENECK 1.2.02 The coalescent-based strategy implemented in MIGRATE 3.2.17 The GENECLASS 2.0 software B. dorsalis from 21 populations. We merged these sequences with previously published sequences  from five populations in Yunnan province  and three Southeast Asian populations  B. dorsalis flies from 29 localities. The total alignment was 519 base pairs (bp) long. There were 85 polymorphic sites and 73 haplotypes were observed , number of haplotypes and number of unique haplotypes were calculated to measure genetic diversity within population see . Among tB. dorsalis flies sampled across 21 populations and genotyped at 7 microsatellite loci. Number of alleles per locus ranged from 12 to 27. After the sequential Bonferroni correction Our data were based on 420 ST estimates were used to measure the genetic structure of the 29 B. dorsalis populations using mitochondrial sequences, and 21 populations genotyped with microsatellites. ST values that ranged from 0.01 (YK/YY) to 0.4 (YL/YQ). Concerning microsatellite data, ST values after ENA correction, that ranged from 0.01 (TB/TC and FX/VP) to 0.22 (HC/YQ). Most values were significant.Pairwise FST were not significant , indicating the absence of IbD.The Mantel tests performed with both kinds of markers showed that the correlations between geographic distances and pairwise FAn unrooted NJ tree  was consThe NJ tree of populations constructed with the microsatellite data  also showed five monophyletic clades . Three pCT value was highest for K = 8 , as shown by the likelihood curve  and subjected to AMOVA. The results based either on mitochondrial or on microsatellite data showed that no significant differences were found among groups (see To test the effect of host plant species on the genetic structure of oups see . Most moD and Fu's Fs) were performed on mitochondrial data for the whole data set and for each of the 8 groups identified by SAMOVA. Significant negative values were found in the whole dataset  and three of the SAMOVA groups, namely Mt-C, Mt-G and Mt-F  was found for TC (P = 0.023) and GJ (P = 0.012). On the contrary, a significant heterozygote deficit  was detected in YY (P = 0.039), YK (P = 0.023), HA (P = 0.027), HC (P = 0.034) and JA (P = 0.019), suggesting a recent population expansion for these populations. However, under the more relaxed hypothesis of a certain proportion of mutations encompassing more than a single repeat unit (TPM), the results suggested that no populations had faced a bottleneck. The population expansion signal was confirmed in JA (P = 0.039) and HC (P = 0.043).We tested the hypothesis of a recent bottleneck or a recent population expansion for each of the 21 genotyped We also tested the hypothesis of a recent bottleneck or expansion for each of the 7 microsatellite SAMOVA groups. Under the SMM model, population groups Msat-A (P = 0.016) and Msat-E (P = 0.016) showed signs of recent significant bottlenecks, while groups Msat-C (P = 0.0195), Msat-F (P = 0.039) and Msat-G (P = 0.008) experienced a recent population expansion. When using the TPM model, no bottleneck was found for any of the 7 groups, but groups Msat-C (P = 0.039), Msat-E (P = 0.039), Msat-F (P = 0.039) still showed signs of recent expansion.GENECLASS 2.0 was used to estimate the proportion of immigrants (m) into each population see . The diaB. dorsalis in Asia to unravel the patterns of genetic differentiation of an important insect pest over a large area of its geographical distribution, including its potential invasive range. Our results go well beyond previous studies In this study, we obtained data using both mitochondrial and nuclear DNA markers of an extensive sampling of Mitochondrial and microsatellite data both revealed a weak but significant genetic structure that corresponds to a geographic pattern see , 3. The B. dorsalis was already discussed in previous studies Our data allowed for the identification of some patterns that did not appear previously, probably because of the limited sampling studied in previous papers. Some of the identified groups  correspond either to geographically distant sites, sometimes at the limit of the fly's range  and range expansion (see below). Even though we did not find any significant genetic structure due to host groups .(TIF)Click here for additional data file.Figure S2CT and LnP(D) for SAMOVA groups and STRUCTURE clusters.Values of F A: FCT values from K = 2 to 10 based on mitochondrial SAMOVA results. B: FCT values from K = 2 to 10 based on microsatellite SAMOVA results. C: Log-likelihood probability LnP(D) of the number of inferred clusters (K) as a function of K using STRUCTURE, for K = 2 to 13, with 10 independent runs for each K.(TIFF)Click here for additional data file.Table S1ST values based on mitochondrial sequences  and microsatellite loci .Pairwise F Some of the values were not significantly different from zero (ns)(XLS)Click here for additional data file.Table S2Effective immigration rate between populations pairs estimated from mitochondrial data using MIGRATE. Instances of asymmetrical gene flow are indicated in bold. The source population is indicated in columns, the target population in row.(XLS)Click here for additional data file.


In patients undergoing primary percutaneous coronary intervention (PPCI) ticagrelor is superior to clopidogrel in reducing cardiovascular events. This study sought to evaluate the effect of clopidogrel pretreatment on the pharmacodynamics of ticagrelor in patients undergoing PPCI.We measured platelet reactivity using the VerifyNow P2Y12 assay at baseline, 1, 2, 4, 6, 12, 24, and 48 hours following ticagrelor bolus in patients previously loaded with clopidogrel (C+T) and in thienopyridine-naive patients (T) referred to our centre for PPCI.In total, 52 consecutive eligible patients with ST-elevation myocardial infarction (STEMI) were enrolled (27 C+T and 25 T). Baseline characteristics and mean baseline platelet reactivity units (PRUs) were similar between the groups. The primary endpoint, the proportion of patients achieving a PRU<208 at 2 hours, was more frequently achieved in the C+T group compared to T treatment . Notably, C+T therapy resulted in fewer patients with high platelet reactivity at 1 hour (56.0% vs. 14.8%), 4 hours (100.0% vs. 61.5%) and 6 hours . Furthermore, C+T therapy was associated with lower PRU values from 2 to 48 hours.In patients referred for PPCI, ticagrelor bolus following clopidogrel resulted in more rapid and profound platelet inhibition, demonstrating a positive pharmacodynamic interaction. Further study is needed to determine if this pharmacodynamic effect translates into reduced clinical events. Patients undergoing primary percutaneous coronary intervention (PPCI) for ST-elevation myocardial infarction (STEMI) are routinely treated with a P2Y12 inhibitor in addition to aspirin. Recently, ticagrelor was shown to be superior to clopidogrel in acute coronary syndrome (ACS) patients in the PLATelet inhibition and patient Outcomes (PLATO) trial Rapid inhibition of platelet aggregation is paramount in patients undergoing PCI as delayed onset of drug activity or high on treatment platelet reactivity (HPR) is associated with increased risk of cardiovascular events, including ST In the PLATO trial, over 40% of patients received pre-randomization clopidogrel at doses ranging from 75–600 mg The University of Ottawa Heart Institute regional STEMI program serves a population of approximately 1.3 million residents in eastern Ontario, receiving patients from 17 referral hospitals as well as direct transport by paramedics from the field All patients undergoing PPCI at our center receive a ticagrelor 180 mg loading dose irrespective of previous administration of clopidogrel by a referring center. During the enrollement period of this study, referral centers bolused patients with 600 mg of clopidogrel prior to transfer. Thus, we elected to perform an open label non-randomized prospective study to understand the effect of clopidogrel co-therapy in patients receiving bolus ticagrelor for PPCI. The groups consisted of patients who received ticagrelor 180 mg bolus alone (T) or patients who had received a bolus of clopidogrel 600 mg, at the discretion of the point of medical contact physician, and were then reloaded with ticagrelor prior to PPCI (C+T). All patients subsequently received ticagrelor 90 mg twice daily. Patients were consented on arrival to the PCI center. Adjuvant pharmacotherapy included aspirin 160 mg to chew followed by 81 mg daily, an unfractionated heparin bolus of 60 units/kg to a maximum of 4,000 units followed by bivalirudin administered at the time of PCI as a bolus of 0.75 mg/kg followed by an infusion of 1.75 mg/kg/hr during the procedure and at 0.25 mg/kg/hr afterward for a total of 2 hours.Inhibition of platelet reactivity was assessed by the VerifyNow P2Y12 assay and reported as platelet reactivity units (PRU). Blood samples were collected in 2.7 mL citrate (3.2%) tubes by venipuncture and analyzed as per manufacturer’s instructions. Baseline blood samples were defined as time point zero and were drawn just prior to ticagrelor bolus, which was administered during transfer to the catheterization laboratory. Samples were subsequently assessed at 1, 2, 4, 6, 12, 24, and 48 hours. The primary outcome was the proportion of patients achieving PRU<208 at 2 hours. Secondary outcomes included comparison of absolute PRU values assessed as a continuous variable in addition to the proportion of patients achieving a PRU<208 at the remaining time points. Baseline demographic data was collected prospectively on all patients as well as assessment of hemoglobin concentration, platelet count, and mean platelet volume at baseline and at 48 hours. Serial creatinine kinase (CK) serum levels were collected and infarct size estimated by peak CK values All continuous variables were described as mean (± standard deviation) or median (and interquartile range) as appropriate and categorical variables as number (%). Categorical variables were compared by Fisher exact test or Chi-square test and continuous variables by student t-test or Mann-Whitney rank sum test, as appropriate. Correlation between PRU values and MI size was assessed using a Pearson correlation coefficient. To calculate sample size, we estimated that 20% of patients in the T arm would achieve PRUs<208 at 2 hours compared to 60% of patients in the C+T arm This study was reviewed and approved by the University of Ottawa Heart Institute human research ethics board and written informed consent was obtained. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.From June 2012 to November 2012, 328 patients were indexed in our regional STEMI program of which 52 eligible patients were enrolled and underwent baseline and serial PRU testing with the VerifyNow P2Y12 assay Figure 1The groups were well balanced in terms of baseline characteristics, including age, sex, body mass index, renal function, atherosclerotic risk factors, and cardiovascular history Table 1.The primary outcome, the proportion of patients achieving target PRU<208, was significantly higher in the C+T cohort compared to T alone (76.0 vs 44.4%, p = 0.026,  & When analyzed as a continuous variable, C+T therapy was associated with a marked reduction in absolute PRU values. As expected, baseline PRUs and hematologic laboratory values were similar between the groups  & Table 3Our report is the first to study the effect of bolusing ticagrelor after clopidogrel administration on the pharmacodynamics of platelet inhibition in patients undergoing PPCI. Compared to ticagrelor alone, we observed that concurrent clopidogrel and ticagrelor was associated with a reduced risk of HPR with a greater number of patients achieving target PRUs at all time points analyzed within the first 6 hours. Moreover, the administration of a clopidogrel loading dose prior to ticagrelor resulted in a significant reduction in the absolute PRUs persisting to 48 hours, suggesting a positive pharmacodynamic interaction between the drugs. Finally, though not directly compared in our study, our findings suggest that the onset of ticagrelor action is significantly delayed in patients with STEMI compared to healthy controls – a finding consistent with a recent report comparing ticagrelor and prasugrel in PPCI The most important finding in our study is the pharmacodynamic impact of clopidogrel administration prior to ticagrelor – an interaction previously suggested in other studies describing cross-over from clopidogrel to ticagrelor therapy. In the RESPOND study, clopidogrel-responsive patients crossing over from clopidogrel to ticagrelor demonstrated an additional 20% platelet inhibition compared to initiation of ticagrelor alone and prasugrel in patients presenting with STEMI, with 12 of 27 patients exhibiting HPR at 2 hours. Our results replicate these findings, with 12 of 27 clopidogrel-naïve patients exhibiting HPR. In contrast, 76% of patients pre-treated with clopidogrel achieved a PRU<208 by 2 hours with no HPR observed in any patient by 4 hours. Indeed, the pharmacodynamic profile achieved with clopidogrel plus ticagrelor combination therapy more closely approximated that seen in stable coronary artery disease patients in the RESPOND and ONSET/OFFSET studies While recent studies have drawn into question the measurement of platelet function as a therapeutic target, there is considerable evidence to suggest that HPR is associated with adverse events in patients undergoing PCI Our study is certainly not without limitations, the most important of which is its non-randomized, observational design, which resulted in an imbalance in the number of inter-hospital transfers between the groups. However, the cohorts were otherwise similar with respect to baseline variables thereby rendering the large differences observed in PRUs unlikely to be the result of confounding variables. Secondly, we set out to assess the potential impact of co-therapy on the pharmacodynamic profile of ticagrelor in patients with STEMI. Accordingly, the current study was underpowered to assess the impact of more rapid platelet inhibition on clinical outcomes such as ST, re-infarction, and death. We instead elected to use a clinically validated PRU cut-off, which has previously been demonstrated to be associated with adverse clinical events, to define ‘therapeutic platelet inhibition’. The VerifyNow P2Y12 assay was selected as a modality for measuring platelet function as it has been shown to be superior to other assays in predicting clinical events In patients referred for PPCI, reloading with ticagrelor following clopidogrel resulted in more rapid and profound platelet inhibition, demonstrating a positive pharmacodynamic interaction and resulting in fewer patients with HPR at 2 hours. This interaction has important implications for both clinical and pharmacodynamic studies. The impact of this interaction on cardiovascular events remains to be established.


Closterovirus within the family Closteroviridae, is the causal agent of citrus tristeza disease. Previous studies revealed that the negative selection, RNA recombination and gene flow were the most important forces that drove CTV evolution. However, the CTV codon usage was not studied and thus its role in CTV evolution remains unknown.Citrus tristeza virus (CTV), a member of the genus A detailed comparative analysis of CTV codon usage pattern was done in this study. Results of the study show that although in general CTV does not have a high degree of codon usage bias, the codon usage of CTV has a high level of resemblance to its host codon usage. In addition, our data indicate that the codon usage resemblance is only observed for the woody plant-infecting closteroviruses but not the closteroviruses infecting the herbaceous host plants, suggesting the existence of different virus-host interactions between the herbaceous plant-infecting and woody plant-infecting closteroviruses.Based on the results, we suggest that in addition to RNA recombination, negative selection and gene flow, host plant codon usage selection can also affect CTV evolution. Protein synthesis takes place when genetic codes stored in the genome is translated at ribosomes in a three-nucleotide manner from the 5' to the 3' end. Each three-nucleotides represents a unique genetic codon for an amino acid or as a translation stop codon. There are 64 codons for the 20 standard amino acids and three stop codons, resulting in more than one codon for most of the 20 amino acids. Codons encode the same amino acid are known as synonymous codons. The synonymous codons are not used in the same frequency in different genes or organisms, indicating the existence of biases in codon usage . Bias inViruses are obligate intracellular parasites which dependent on host cells for their genome replication and protein synthesis. It was reported that viral codon usage bias is determined by both virus itself and its host. Similar to other organisms, both mutation pressure and translation selection play a key role in shaping viral codon usage bias -10. OtheClosterovirus in the family of Closteroviridae[ORF1aORF1bp33p6p65p61p27 (encodes the minor coat protein), p25 (encodes the major coat protein), p18p13p20, and p23. The 12 ORFs are finally translated into at least 19 different proteins [ORF1a and ORF1b are translated directly from the gRNA and encode proteins that are required for CTV replication. The ORFs, present on 3′-coterminal subgenomic RNAs, encode proteins that are necessary for CTV replication (e.g. p65 and p61), virion assembly  [Citrus tristeza virus (CTV), the causal agent of citrus tristeza disease, is a notorious plant RNA virus. CTV causes tremendous economic losses to the citrus industries worldwide . CTV is roviridae. Genome proteins . ORF1a aand p25) , symptomand p25) , and supand p25) . Functioi.e. yellowing canopies, declining and stunting of trees, and stem pitting) on different indicator citrus plants, indicating the existence of a highly diversified genetic population of CTV in nature [Isolates of CTV can cause different disease symptoms  of the 20 selected CTV isolates was determined to generate an overall view of the codon usage patterns. Table C values of the 20 selected CTV isolates varied from 51.9 to 54.8, with an average value of 53.0 ± 0.6641. This fining suggests that CTV does not possess an excessive overall codon usage bias and the variation of codon usage bias among CTV isolates is small.The effective number of codons , it is clear that the U3 value is the highest, ranging from 35.9 to 37.1% with an average at 36.5 ± 0.36. Thus the major codons used by CTV are U-ended. Further comparison of the U, C, G and A contents with the U3 C3, G3, and A3 contents indicated that the U and C contents were significantly enriched at the synonymous sites, whereas the G and A were significantly decreased at these synonymous sites . To generate a visual display of the main features of codon usage pattern as reported previously by Wright [C-plot, a plot showing NCvs. (G + C)3. In this NC-plot , the codons with the highest RSCU values are all ended with U. For amino acids that have two synonymous codons and are ended with U or C , only Tyr displayed a weak preference to codons ended with C (UAC). The RSCU values for amino acids that have two synonymous codons and are ended with A or G  are similar, indicating that a similar codon usage frequency  values for the 20 selected CTV isolates with a total number of 123,535 synonymous codons (Table C. sinensis from the Codon Usage Database (http://www.kazusa.or.jp/codon/). Interestingly, our analysis shows that most of the C. sinensis preferred codons are also U-ended   were clustered in the group3  for BYV, Daucus carrot (carrot) for CYLV, and Vitis vinifera (grapevine) for GRSLaV and GRSLaV-2. Results shown in Figure P < 0.01) is observed between grapevine and its two viruses (GRSLaV and GRSLaV-2) but not between beet and BYV or carrot and CYLV. This finding shows that codon usage adaptation to a host is not a common phenomenon of closteroviruses. It occurs only in some closteroviruses.The high degree of CTV codon usage adaptation to its host suggests that the adaptation may be a common phenomenon between closteroviruses and their hosts. To confirm this hypothesis, the full length genome sequences of beet yellows virus , carrot yellow leaf virus , grapevine rootstock stem lesion associated virus  and grapevine leafroll-associated virus 2  were downloaded from the GenBank. The empirical codon frequency of each virus was calculated and compared with that of its host plant: C = 53.0, Table C-plot cannot be simply explained by the mutational bias as suggested by Wright previously [In this study, a detailed comparative analysis was done to determine CTV codon usage bias. Our results show that in general CTV does not have a high degree of codon usage bias  both CTV and citrus have significantly higher content of U at the synonymous codon sites; ii) most of the preferred codons in CTV and citrus are the same; iii) a high correlation exists in codon frequencies between CTV and citrus. This result is understandable when consider the specific relationship between CTV and its host. CTV is restricted to citrus and it is generally accepted that the virus co-evolved with the host species . WhereasBahir et al. suggested previously that adaptation of codon usage varied among different viral genes and the highest degree of adaptation was observed for genes that expressed to high levels in cells, such as the viral CP . In thisFlaviviridae, and bacteria-infecting and human viruses [Closterovirus since the codon usage patterns of different closteroviruses are highly resemblance to each other (data not shown). However, our results show that the high degree of codon resemblance is only observed between the woody plant-infecting closteroviruses and their woody hosts, but not the herbaceous plants-infecting closteroviruses and their herbaceous hosts . To establish a sequence data set for CTV, isolates share less than 98% sequence identity were downloaded and the final data set consists of 20 CTV isolates , which were tabulated based on all available sequences in the international DNA sequence databases [The codon usage pattern of atabases .Phylogenetic tree was constructed using the Neighbor-joining (NJ) method described in the MEGA 5.0 software . The nuc3, A3, U3, G3 and C3, was performed using the CodonW version 1.4.2 . The nucleotide contents at the first and second codon positions were calculated as described by Wang et al. previously [Analysis of compositional properties of all CTV ORFs, including (G + C), (G + C)eviously .C) has been used as a measurement for synonymous codon usage bias in genes and is considered to be independent of the gene length and amino acid composition [C value ranging from 20 to 61 is often used to determine the degree of codon usage bias in a gene [C value at or below 35 is considered to have a strong codon usage bias, whereas a gene with a NC value of 61 indicates that all available codons are used equally [C values were calculated using the CodonW version 1.4.2.Effective number of codons .CTV, Citrus tristeza virus; ORF, Open reading frame; NC, Effective number of codon; RSCU, Relative synonymous codon usage; COA, Correspondence analysis.The authors declare no competing interests.XC involved in data calculation, results analysis and manuscript preparation; XW involved in data collection, results analysis and manuscript revision; HW and YS involved in data analysis and manuscript preparation; YQ and LL involved in data visualization; All authors have read and approved the final submission of the manuscript.


SCNN1A gene. Renal PHA type 1 responds well to salt supplementation, whereas management of patients with systemic PHA type 1 proves more difficult as they are likely to get frequent episodes of electrolyte imbalance requiring urgent correction.Type 1 pseudohypoaldosteronism (PHA) is a rare heterogeneous group of disorders characterised by resistance to aldosterone action. There is resultant salt wasting in the neonatal period, with hyperkalaemia and metabolic acidosis. Only after results confirm isolated resistance to aldosterone can the diagnosis of type 1 PHA be confidently made. Type 1 PHA can be further classified into i) renal type 1 ) and ii) multiple target organ defect/systemic type 1 ). The aim of this case series was to characterise the mode of presentation, management and short-term clinical outcomes of patients with PHA type 1. Case notes of newly diagnosed infants presenting with PHA type 1 were reviewed over a 5-year time period. Seven patients were diagnosed with PHA type 1. Initial presentation ranged from 4 to 28 days of age. Six had weight loss as a presenting feature. All subjects had hyperkalaemia, hyponatraemia, with elevated renin and aldosterone levels. Five patients have renal PHA type 1 and two patients have systemic PHA type, of whom one has had genetic testing to confirm the AR gene mutation on the Patients with type 1 PHA are likely to present in the neonatal period with hyponatraemia, hyperkalaemia and metabolic acidosis and can be diagnosed by the significantly elevated plasma renin activity and aldosterone levels.The differential diagnosis of type 1 PHA includes adrenal disorders such as adrenal hypoplasia and congenital adrenal hyperplasia; thus, adrenal function including cortisol levels, 17-hydroxyprogesterone and a urinary steroid profile are required. Secondary (transient) causes of PHA may be due to urinary tract infections or renal anomalies; thus, urine culture and renal ultrasound scan are required respectively.A differentiation between renal and systemic PHA type 1 may be made based on sodium requirements, ease of management of electrolyte imbalance, sweat test results and genetic testing.Management of renal PHA type 1 is with sodium supplementation, and requirements often decrease with age.Systemic PHA type 1 requires aggressive and intensive fluid and electrolyte management. Securing an enteral feeding route and i.v. access are essential to facilitate ongoing therapy.In this area of the UK, the incidence of AD PHA and AR PHA was calculated to be 1:66 000 and 1:166 000 respectively. Type 1 pseudohypoaldosteronism (PHA) is a heterogeneous syndrome characterised by salt wasting resulting from target organ unresponsiveness to mineralocorticoids. As the principle effect of aldosterone is to promote potassium and hydrogen secretion, the condition typically presents in the neonatal period with hyperkalaemia, metabolic acidosis and an elevated plasma aldosterone concentration. Type 1 PHA can be further classified into two main clinical entities: renal PHA and systemic type/multiple target organ defects.Renal type 1 PHA has an autosomal dominant (AD) pattern of inheritance with heterogeneous mutations on the gene coding for the mineralocorticoid receptor The systemic form of type 1 PHA is an autosomal recessive (AR) disorder caused by mutations on the epithelial sodium channel (ENaC) Owing to the rarity of the condition and transient nature of renal PHA type 1, the published literature has focussed mainly on case reports or case series The aim of this study was to characterise the mode of presentation, management and short-term clinical outcomes of patients with PHA type 1 and for the first time calculate a clinical incidence for both renal PHA type 1 and systemic PHA type 1.Patients with PHA were selected by searching the Supra-regional Assay (SAS) laboratory database for all elevated aldosterone concentrations (>5000 pmol/l) between January 2006 and December 2010. This was further corroborated by contacting all regional paediatric endocrinologists and nephrologists. The assays were performed in the SAS Service Steroid Centre, one of three national units performing aldosterone and renin assays for England. Aldosterone is analysed with an in-house RIA. Assays have been continuously monitored by external quality assurance schemes.We retrospectively analysed the case notes of all children with confirmed PHA type 1. Secondary or transient forms of PHA (or type 3 PHA), which are associated with renal tract anomalies or urine tract infections, were excluded. The children were from Leeds Children's Hospital, Bradford Teaching Hospital and Sheffield Children's Hospital, UK. The initial presentation, management, biochemical evaluation and outcomes are documented.In Cases 1–5, the diagnosis of renal PHA type 1 was made on the biochemical results and relatively mild disease severity.This male infant was born at term and presented at 3 weeks of life with vomiting and weight loss. Initial blood samples revealed hyponatraemia with a Na of 119 mmol/l. He was clinically stable and was initially managed for gastro-oesophageal reflux disease. When the plasma sodium failed to normalise after 6 days, a synacthen (1 μg) test was carried out, revealing a normal peak cortisol level of 1152 nmol/l. He was subsequently started on sodium supplements, at a dose of 3–5 mmol/kg per day, and fludrocortisone treatment was initiated. After receipt of his elevated aldosterone and normal 17-hydroxyprogesterone (17-OHP) levels, the fludrocortisone treatment was stopped and sodium supplementation was increased. His subsequent clinical course has been uneventful.This patient presented at 4 weeks of age with poor feeding, poor weight gain, hair loss and dry skin. He was found to be hyponatraemic and hyperkalaemic, with a metabolic acidosis on capillary blood gas sampling. Initial management was with i.v. fluids of 0.45% saline with 5% dextrose. He was started on oral sodium supplements, with a view to commencing mineralocorticoids if he became clinically unwell. After investigation, results indicated a diagnosis of PHA, his dose of sodium supplements was increased and low potassium formula milk was commenced.This female infant presented on day 8 of life with significant weight loss. Biochemical investigations revealed hyperkalaemia and hyponatraemia. Initial management involved sodium supplementation, hydrocortisone, fludrocortisone and calcium resonium. After the diagnosis of PHA was made, the hydrocortisone and fludrocortisone were stopped, and calcium resonium was gradually decreased. Subsequently, her management has been with sodium supplements alone.This male infant was born prematurely at 33 weeks gestation to a mother with diabetes. There was antenatal polyhydramnios, and at birth, the baby was macrosomic and hypoglycaemic. These findings were attributed to maternal diabetes. At day 4 of life, he was noted to have hypertension with macroscopic haematuria, which was thought to be secondary to prior umbilical artery catheterisation. The haematuria rapidly resolved and no renal abnormalities were identified. At day 10, the patient had profound hyponatraemia and hyperkalaemia, with a metabolic acidosis. A cortisol level at this time was 1070 nmol/l.Initial management involved treatment of hyperkalaemia with salbutamol nebulisers and calcium resonium, administration of hydrocortisone and fludrocortisone, low potassium feeds and sodium supplementation. After establishing the diagnosis of PHA, the glucocorticoid and mineralocorticoid treatment was stopped and management has been with weaning doses of sodium supplementation.This normal term female infant presented at day 23 of age with profuse vomiting and failure to thrive, with an initial plasma sodium of 118 mmol/l and potassium of 7.3 mmol/l. She was managed on i.v. fluids, anti-reflux medications and an elemental milk formula. Plasma aldosterone and renin levels were elevated at 48 000 pmol/l and 16 nmol/l per h respectively. Subsequently, a diagnosis of AD PHA was made and sodium supplements were started. These were eventually weaned and discontinued and she was discharged from follow-up by 15 months of age.Infants 6 and 7 have been diagnosed with systemic PHA type 1 due to the severity and longevity of electrolyte disturbance, positive sweat test results and systemic features of the condition. Patient 7 has had genetic testing which confirmed systemic PHA.This female infant presented at day 5 with jaundice and weight loss, after an uncomplicated pregnancy to consanguineous parents of Pakistani origin. She was admitted for establishment of feeds and developed progressively worsening hyponatraemia and hyperkalaemia by day 10. At this stage, her hyperkalaemia was managed with salbutamol nebulisers, and she was given low potassium feeds, i.v. hydrocortisone, sodium chloride supplements (3 mmol/kg per day) and sodium bicarbonate (1 mmol/kg per day). After 4 days of treatment, her electrolytes normalised, but 4 days later, hyperkalaemia redeveloped. After the diagnosis of PHA was confirmed, sodium supplements were increased, and mineralocorticoids were stopped. She has had recurrent episodes of weight loss, hyperkalaemia and i.v. fluid requirement necessitating an inpatient stay for many months. At 2 months of age, she required paediatric intensive care admission to manage an episode of severe hyperkalaemia.A trial of treatment with sodium resonium was unsuccessful, and at the age of 2 years, she was started on indomethacin. This has helped to stabilise her urine output, reducing the need for i.v. fluids, and has allowed her to spend longer periods of time at home.At times, she has required high doses of sodium chloride (up to 10 mmol/kg per day) and sodium bicarbonate (13 mmol/kg per day), without which she would have acute decompensation resulting in weight loss, irritability and hyperkalaemia. Significant reflux with gastrointestinal dysmotility and recurrent vomiting has resulted in poor oromotor skills. She had a gastrostomy inserted at 5 months of age, followed by fundoplication, but due to persistent retching, a jejunostomy was required at 6 months of age. She has had one episode of peritonitis at the age of 2 years, requiring maximal sodium supplementation in parenteral nutrition and i.v. 0.9% saline to maintain sodium concentrations.This male infant was born at full term to consanguineous Pakistani parents. He initially suffered from respiratory distress syndrome but was able to be discharged home after 2 days. At 10 days of age, he presented to hospital with a history of weight loss and drowsiness. Initial results revealed hyponatraemia, hyperkalaemia and a metabolic acidosis, which was managed as in Case 6. After 4 days of treatment, his electrolytes normalised, and hydrocortisone and fludrocortisone were stopped after a diagnosis of PHA was made. He was subsequently managed with sodium bicarbonate, sodium chloride supplementation and low potassium formula feeds.in situ for emergency management. He has suffered from recurrent lower respiratory tract infections, and a sweat test was positive. He was therefore commenced on prophylactic flucloxacillin, with subsequent clinical improvement. He has had recurrent tonsillitis for which he receives prophylactic phenoxymethylpenicillin prior to a tonsillectomy.He has had recurrent episodes of hyperkalaemia, requiring hospital admissions. The severity of electrolyte imbalance has resulted in episodes of cardiac arrest requiring resuscitation, and he has a central line Owing to difficulties feeding, nasogastric feeds were required prior to gastrostomy tube insertion. He has had recurrent episodes of vomiting and aspiration of feeds, eventually resulting in a fundoplication.A summary of the cases with the initial biochemistry and treatment is presented in Cases 1–5 presented with significant hyponatraemia and hyperkalaemia. The diagnosis of PHA type 1 was made after receipt of their significantly elevated aldosterone levels (ranging from 35 700 to 83 390 pmol/l) and increased plasma renin activity (ranging from 16 to >250 nmol/l per h).Differential diagnoses based on the initial electrolytes includes hypoaldosteronism, secondary PHA, adrenal hypoplasia, congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, or specific hormonal blocks within the adrenal biosynthetic pathway. Some of these enzyme deficiencies may be associated with indeterminate genitalia and consequently a range of biochemical abnormalities; neither of which were apparent in our infants. A detailed discussion of these is beyond the scope of this report.All five cases of renal type 1 PHA had normal 17-OHP levels, renal ultrasound scans, sterile urine cultures, and a urinary steroid profile, thus excluding 21-hydroxylase deficiency or disorders of the adrenal biosynthetic pathway. Owing to the ease of management with good long-term outcomes, it was not possible to justify genetic testing for the children with renal type PHA.SCNN1A gene.Cases 6 and 7 had hyponatraemia and hyperkalaemia; however, in Case 6, this only became evident by day 10. Both patients had elevated plasma aldosterone levels and plasma renin activity , with noPatients 1–5 have been successfully treated with sodium chloride supplementation alone. The dose of this has varied, with initial requirements between 1.5 and 4 mmol/kg per day. Sodium supplementation has reduced with time with one patient not requiring any supplementation by a year of age.Patients 6 and 7 have required varying sodium supplementation with sodium chloride and sodium bicarbonate, and Patient 6 continues to have an intermittent requirement for i.v. 0.9% sodium chloride. Both patients have low potassium feeds, yet hyperkalaemia has remained an ongoing problem.Ion exchange resins were utilised for the treatment of hyperkalaemia. Calcium resonium binds to potassium in the gastrointestinal tracts, thus limiting its absorption, whereas sodium resonium is another cation exchange resin that swaps sodium ions for potassium ions. Its use should result in a simultaneous correction of both hyponatraemia and hyperkalaemia. However, Patient 6 did not respond to a trial of sodium resonium unlike the reported cases with AR PHA Owing to recurrent bacterial infections resulting in clinical decompensation and electrolyte imbalance, Patient 7 also received prophylactic antibiotics with a reduction in infection frequency.Both patients with systemic PHA type 1 have had significant difficulties with enteral feeding, with severe gastro-oesophageal reflux requiring early fundoplication with gastrostomy or jejunostomy.Our follow-up for Patients 1–5 ranges from 14 to 62 months. They are all thriving and remain on low doses of sodium supplements, apart from one case in which treatment has been discontinued.in situ. She continues to have regular episodes of hyperkalaemia and acute decompensation, often precipitated by intercurrent infections requiring hospital admission and management with i.v. fluids. Despite her positive sweat test, she has not had frequent or severe respiratory tract infections.At the age of 3 years, Patient 6 has had longer periods of time at home and she is currently on treatment with sodium bicarbonate, sodium chloride, indomethacin, gaviscon and ranitidine. She remains on full jejunostomy feeds with low potassium formula milk. Long-term i.v. access remains Patient 7 is now 42 months of age, and over the last year, the number of admissions into hospital with severe hyperkalaemia has significantly decreased. He continues to have recurrent central line infections requiring regular assessments as an inpatient. He receives weekly blood testing at home and remains gastrostomy fed.PHA type 1 is a condition that may present with mild or profound salt wasting with hyperkalaemia.The initial biochemical picture is similar to adrenal hypoplasia, CAH or secondary PHA and investigations should include plasma renin activity, aldosterone, 17-OHP, cortisol levels, renal ultrasound scan, urine cultures and a urine steroid profile.Sodium and potassium homeostasis are regulated by the effect of aldosterone on epithelial cells in the collecting duct of the nephron AD PHA type 1 is more common, and indeed five of the infants had this diagnosis. In most cases, patients presented in the early neonatal period with weight loss and biochemical analysis showed hyperkalaemia and hyponatraemia, with a variable metabolic acidosis. A negative sweat test would further differentiate these patients from AR PHA1. Treatment in AD PHA type 1 is with sodium supplementation, which generally becomes unnecessary by 1–3 years of age The literature describes a wide clinical spectrum, with some patients apparently unaffected, others with elevated renin activity and aldosterone concentrations but normal electrolytes, and others with clinically significant renal salt loss SCNN1A gene. Homozygous mutations in the SCNN1A gene in patients with AR PHA type 1 has been previously described SCNN1B gene SCNN1G gene Our patients with AR PHA type 1 had a much more difficult clinical course, requiring intensive support for stabilisation of electrolyte imbalances and feeding. Infant 7 has a confirmed gene mutation on the Both patients have had positive sweat tests, helping to confirm the diagnosis of systemic PHA type 1. Unless clinicians are aware of this association, a positive sweat test may result in an incorrect diagnosis of cystic fibrosis, as both disorders cause excessive salt loss from sweat glands.Unlike previous reports Case 7 had neonatal respiratory distress syndrome at birth, which is the first time this has been described in a term infant with AR PHA type 1. He has subsequently had recurrent lower respiratory tract infections. In AR PHA type 1, patients typically demonstrate a chronic pulmonary syndrome, characterised by recurrent coughing and wheezing without identifiable bacterial airway infection Management of AR PHA type 1 is with extensive salt supplementation, and vigilance for the systemic features of the disease, such as the respiratory complications. Despite close monitoring of these patients, the speed and severity of the electrolyte imbalance in Patient 7 led to a number of cardiac arrests requiring resuscitation, and Patient 6 has required intensive care admission due to severe hyperkalaemia. Gastrostomy/jejunostomy feeding and secure venous access have been crucial in the management of these two patients, and this is something we would recommend obtaining early after a diagnosis of AR PHA has been secured. AR PHA type 1 requires lifelong salt supplementation, but both of our patients have shown some improvement in clinical severity over time.Other phenotypic features of patients with AR PHA type 1 can occur as a consequence of ENaC expression in other tissues. These include cholelithiasis It is important to differentiate patients with systemic PHA type 1 from those with renal PHA type 1 as these patients are more likely to severely decompensate even with apparently trivial symptoms. It should be noted from our results that normalisation of PRA and aldosterone on treatment cannot be used as an indication for or against the genetic subtype of PHA. A positive sweat test may help endorse a diagnosis of AR PHA and genetic testing can be confirmatory.www.statistics.gov.uk/statbase, accessed 09/07/2011), the birth rate in the Yorkshire and Humber area was found to be 66 400 per year. Thus, the calculated incidence of PHA type 1 was 1:47 000, with the incidence of AD PHA and AR PHA calculated at 1:66 000 and 1:166 000 respectively. It is possible that some infants may not survive the initial salt-wasting episode or may be misclassified as adrenal hypoplasia, CAH or sudden neonatal death. Similarly, AD PHA type 1 may be underdiagnosed as it typically runs a much milder course and is easily treated with sodium supplementation.There were found to be seven cases of confirmed PHA type 1 over a 5-year period. Using 2009 data from the office of national statistics (Patient consent has been obtained for the two patients with AR PHA type 1, as these patients may be identified from the report.J H Barth and H P Field gave inputs for the biochemical analysis and were involved in writing the report. Physicians: N P Wright, T Makaya, S Frazer, E Finlay, K Tyerman, G Savill, N S Alvi and T Mushtaq are responsible for patients.


Early (around two weeks after onset) and late (one month after onset) outcomes  and efficacy (changes of NIHSS scores) of HBOT were evaluated. The baseline clinical characteristics were similar in both groups. Both early and late outcomes of the HBOT group showed significant difference (P ≤ 0.001). In the control group, there was only significant difference in early outcome (P = 0.004). For early efficacy, there was no difference when comparing changes of NIHSS scores between the two groups (P = 0.140) but there was statistically significant difference when comparing changes of NIHSS scores at one month (P ≤ 0.001). The HBOT used in this study may be effective for patients with acute ischemic stroke and is a safe and harmless adjunctive treatment.The role of hyperbaric oxygen therapy (HBOT) in the treatment of acute ischemic stroke is controversial. This prospective study assessed the efficacy and safety of HBOT as adjuvant treatment on 46 acute ischemic stroke in patients who did not receive thrombolytic therapy. The HBOT group ( In most developed countries, cerebrovascular disease is always ranked in the top ten causes of death. In Taiwan, about 70% of hospitalized acute stroke patients have ischemic stroke. Although the mortality rate of acute ischemic stroke is less than that of hemorrhagic stroke, it still results in patient disabilities and complications that often lead to significant costs to individuals, families, and society.Traditional treatment for acute ischemic stroke includes thrombolytic therapy by injecting t-PA within three hours after onset of symptoms, and antiplatelet and/or anticoagulant agents administered within the first 48 hours. Clinically, the narrow time window of thrombolytic therapy and coexisting contraindications limit the use of t-PA , 2. ThusHyperbaric oxygen therapy (HBOT) is valuable in treating acute CO poisoning, air or gas embolism , 4 and iHowever, the viewpoint and shortcomings of this trial were challenged by others who suggested that further studies were needed to determine the feasibility of lower pressures, greater numbers of dives with better clinical outcomes, and suitable stroke subtype. In 2006, Bennett et al. included three randomized controlled trials (106 participants) to assess the benefits and safety of adjunctive HBOT in the treatment of acute ischemic stroke. Their systematic review did not demonstrate that HBOT could improve clinical outcomes. However, due to the lack of guidelines for study design, the use of HBOT in stroke patients could not be justified . Moreover, a previous report showed a 56-year-old Chinese patient suffering from acute ischemic stroke on the left corona radiata leading to right hemiparesis and dysarthria, had great improvement after repetitive (10 dives) HBOT with lower pressure (2.0 ATA) and short duration (60 min) .This study prospectively assessed the efficacy and feasibility of applying 10 repetitions of HBOT at 2.0 ATA for one hour in patients with mild acute ischemic stroke within 3–5 days after stroke onset to reinvestigate the role of HBOT in treating acute ischemic stroke.This prospective, open-label study was conducted in a local teaching hospital and compared the efficiency of receiving HBOT with traditional therapy for treatment of acute ischemic stroke. This hospital's Institutional Review Board approved the study and all participants provided informed consent.Adult (aged >18 years) patients were included if they had a diagnosis of acute ischemic stroke within 48 hours after onset, according to imaging findings by brain computed tomography (CT) without evidence of hemorrhage, upon admission to the hospital. No patient received thrombolytic therapy. Upon admission, the vital signs, personal history , laboratory data including CBC/DC, electrolytes, and lipid profiles, and past medical history, including type 2 diabetes mellitus, hypertension, or cardiovascular disease were reviewed. 2 retention, pneumothorax, or seizure disorder). The severity of disease upon admission was scored using the National Institutes of Health Stroke Scale (NIHSS). “Mild” disease was defined as score of 0–14, “moderate” as 15–28, and “severe” as 29–42. Patients with moderate or severe disease were not eligible, as well as those with contraindications or risk factors for HBOT  early efficacy, or changes in NIHSS scores at the time before HBOT and after 10 sessions of HBOT in HBOT group were compared to changes of scores between the early days and 14 days after stroke onset in the control group, and (2) late efficacy, where changes of NIHSS scores between pretreatment and one month posttreatment in the HBOT group were compared with changes of scores between the early days and one month after stroke onset in the control group.t-test . To compare paired data, the Shapiro-Wilk test was used for the HBOT group and the Shapiro-Wilk test and Wilcoxon Signed Rank test were used in the control group to test the data of early and long-term clinical responses. Variables in both groups  were compared by Equal Variance Test for early efficacy and by the Mann-Whitney Rank Sum Test for late efficacy. For all analysis, P < 0.05 was considered statistically significant.Data analysis was performed using the SPSS Statistics version 19 software and the figures were constructed by Sigmaplot version 12 software. Baseline categorical variables were analyzed by Fisher's exact test, whereas numerical variables were analyzed by Student's t-test showed no statistically significant difference between the two groups in each item  to all the patients in the HBOT group. Treatments lasted 60 minutes for 10 sessions, once a day, for two weeks. Treatment outcome was evaluated by the NIHSS score immediately after HBOT and at one month after onset of stroke attack. The schedule of testing by the NIHSS score in the control group was the same. t-test was used to compare the NIHSS scores before HBOT (mean ± SE 9.313 ± 3.459) with either the 2 weeks post-HBOT scores (6.188 ± 3.920) or the scores at one month after the stroke (4.438 ± 3.245). The NIHSS scores were significantly decreased immediately after HBOT and at one month later compared to the pre-HBOT data (P < 0.001)  .t-test (P = 0.004). However, the scores between early and late outcomes were not significantly different (P = 0.054)  .P = 0.647) and there was no statistically significant difference on early efficacy between the groups (P = 0.140). However, comparison of late efficacy showed a statistically significant difference between the groups (P = 0.024)  . The present study demonstrates better clinical outcome evaluated by NIHSS in acute stroke patients with adjunctive HBOT therapy than those without, which is different from results of previous clinical studies. Compared to several successful animal studies in acute ischemic stroke, human studies contain many factors that may influence outcomes, such as age, severity of stroke, social circumstances, prior disability, and comorbidities . MoreoveThird, the protocol of HBOT was using lower pressure (2.0 ATA) and shorter duration (60 mins) to avoid possible oxidative stress caused by high pressure , 12, 18.P = 0.024), although outcome in the early days did not show significant difference (P = 0.140). All patients in the HBOT group showed improvement except for one patient who presented with worse outcome after 10 repetitions of HBOT (NIHSS score from 7 to 9), but no more disease progression after one month (NIHSS score from 9 to 9). Although the early efficacy of HBOT did not reach the statistic significance, it did reveal the tendency of better outcome compared to the control group. There are possible explanations. One, the mechanisms of HBOT for ischemic stroke  repair and generate new blood vessels to the parts of the brain that have been injured, inhabited by apoptosis, and improve metabolism after ischemia [The present study suggests that HBOT may be effective for acute ischemic stroke patients, and the efficacy of therapy is more obvious later in the disease course. A comparison of the two groups at one month revealed that the HBOT group had better outcome than the control group (ischemia . All of ischemia . Three, This study has several limitations. First, although the case number is small, increasing study number may show the more significant results. Second, outcome evaluation is merely with the NIHSS score; however, it may not completely represent functioning status . Even thThe present study of HBOT with 10 repetitions at 2 ATA 100% oxygen for 60 minutes appears to be effective for patients of mild acute ischemic stroke. It also appears as a safe and harmless adjunctive treatment. However, because of the limitations described above, the effects of HBOT for acute ischemic stroke cannot be completely revealed and require more large-scale trials with carefully planned investigation. The protocol here set up a pilot study for future efforts about adjuvant stroke treatment by HBOT.