For example, some viruses interfere with the major histocompatibility complex class I presentation of viral antigens, whereas others modulate lymphocyte and macrophage functions, including cytokine production.12-16 In our previous study, we detected an increasing number of mutations in the HCV genome isolated from JFH-1 patient

serum–infected Ibrutinib chimpanzees. Thus, we reasoned that these detected mutations might have imparted some advantage to this virus for long-time survival. To examine this hypothesis, we compared the phenotypes of JFH-1 variant strains emerged at early and late stages of infection in JFH-1 patient serum–infected and JFH-1cc–infected chimpanzees and found that the JFH-1/S2 strain isolated from the patient serum–infected chimpanzee at a later time point of infection replicated slowly, produced more infectious viruses, and displayed reduced susceptibility to cytokine-induced apoptosis. The JFH-1 variant strain JFH-1/C, which contains seven nonsynonymous mutations identified in the JFH-1cc–infected chimpanzee at week 7, showed comparatively slower replication kinetics and slightly enhanced infectious virus production in cell culture. The intracellular

specific infectivity of this https://www.selleckchem.com/products/cx-4945-silmitasertib.html strain in Huh7-25 cells was 3.9 times higher than that of JFH-1/wt (Table 1). These characteristics might have imparted some advantage to this strain for establishing productive infection in the chimpanzee. The other JFH-1 variant strains, JFH-1/S1 and JFH-1/S2, contain 6 and 17 nonsynonymous mutations identified in the JFH-1 patient serum–infected chimpanzee at weeks 2 and 23 postinfection, respectively. Replication kinetics and infectious virus production of the JFH-1/S1 strain were comparable to that of JFH-1/wt in cultured cells (Fig. 1, Table 1). In contrast, the JFH-1/S2 strain showed lower replication efficiency. Although the intracellular HCV RNA level of this strain in Huh7-25 cells was lower than that of JFH-1/wt and JFH-1/S1, and almost the same as that of JFH-1/C (Table 1), intracellular specific infectivity was 18.0 and 12.9 times

BCKDHA higher than that of JFH-1/wt and JFH-1/S1, respectively, suggesting a significant increase in the assembly of infectious virus particles (P < 0.005, Table 1). The enhanced capacity of this strain to assemble infectious virus particles resulted in a higher extracellular infectivity titer that contributed to the rapid spread of virus to surrounding cells. Flow cytometry analyses of cells transfected with JFH-1/wt and variant strains revealed that the percentage of the HCV NS5A-positive population in JFH-1/S2–transfected cells was higher, but the mean fluorescence intensity of the anti-NS5A signal was lower than that in JFH-1/wt–transfected cells, thus confirming higher spread and lower replication of this strain. Taken together, both JFH-1/C and JFH-1/S2 exhibited a tendency toward decreased replication and increased infectious virus production.

2). All of the predesignated hepatocyte-specific genes were more highly expressed in parenchymal

segments compared with portal tracts irrespective of fibrosis stage, confirming that LCM of hepatic parenchyma predominantly yielded hepatocytes: albumin expression in the parenchyma was ≈5-fold greater than in the portal tract (FDR < 1.67 × 10−6), whereas the expression of a representative hepatic microsomal enzyme, CYP2C9, in the parenchyma was ≈14 times the expression in portal tracts (FDR < 2 × 10−13). Due to this higher expression of hepatocyte specific genes in the parenchyma, we assume that the extracted parenchymal section consisted mainly of hepatocytes and hence refer to them as hepatocytes for the rest of the study. On comparison between hepatocytes in PC and NF groups, 74 genes were found to be differentially expressed. (Fig. 3A; Supporting Table 1): 73 genes were down-regulated and only MK 1775 one gene was up-regulated in hepatocytes from PC tissues. Using gene

ontogeny (GO) analysis, oxidative-reductive processes were found to be the most down-regulated processes in PC tissues, with 8/73 (P > 0.05, due to small n) belonging to this category. Other genes with decreased expression were associated with metabolic processes, such as steroid and alcohol metabolism genes and genes involved in small molecule and drug metabolism. Hepatic parenchyma from PC tissues had only one up-regulated gene in contrast to ubiquitin D (UBD), which has been described

earlier.17 Notably, HCV RNA was detected in 51/54 hepatocyte segments; hence, differences JAK inhibitor in gene expression between captured hepatocytes from PC and NF liver tissue were more likely to reflect fibrosis rather than HCV replication. For validation, genes were randomly selected at different positions in the rank list so as not to bias the validation toward outlier genes and tested Tobramycin by qPCR using gene-specific primers. Representative captured material was tested from each subject, showing close agreement between microarray and qPCR results (Fig. 3B). Importantly, albumin was not differentially expressed between PC and NF hepatocytes, indicating that hepatic synthetic function was preserved in PC hepatocytes. BCHE had the most suppressed expression in PC tissues, showing 5-fold lower expression by microarray (FDR = 2 × 10−4), and a log2 (FC) of 13.51 by qPCR (Fig. 4A), and was still significant after exclusion of the PC tissue with Ishak Stage 5. BCHE protein is synthesized in the liver and widely distributed in the body, including plasma, brain, and lung. Notably, BCHE is the predominant enzyme that metabolizes cocaine and plays a role in heroin metabolism.18-22 SBA, a surrogate of the highly polymorphic protein, was measured in an expanded sample of chronic HCV-infected participants to confirm and validate that gene expression differences were manifest at the level of protein expression.

All 69 patients had no previous treatment with TACE or hepatic arterial infusion. No serious adverse events were observed in either group. The response rates, including complete response (CR) and partial response (PR), of the EPIR group and the MPT group were 85.7% and 81.5%, respectively, with a time to treatment failure of 5.1 and 7.5 months, respectively. Excluding whole liver TACE cases, time to buy Tamoxifen treatment failure was 5.4 months for the EPIR group and 10.1 months for the MPT group. In TACE naïve cases, there was no significant difference in local control between EPIR and MPT. “
“The stress-activated mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal kinase (JNK), and

p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal-regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1-deficient (ASK1−/−) www.selleckchem.com/products/BKM-120.html and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1−/− mice as in WT mice. Although JNK and

p38 activation were attenuated in ASK1−/− HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types Verteporfin cell line of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl-2 family member, were suppressed in the ASK1−/− HCCs. ASK1−/− mice showed remarkable resistance to Fas-induced hepatocyte apoptosis in vivo, probably because of attenuated JNK-mediated BimEL phosphorylation and

mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1−/− mouse liver using an adenoviral vector restored Fas-induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha-induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage-induced p21 up-regulation through a p38 pathway. Conclusion: ASK1 is involved in death receptor-mediated apoptosis and DNA-damage response by way of stress-activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress- activated MAPK signaling in hepatocarcinogenesis. (HEPATOLOGY 2011;) Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality; thus, understanding the molecular carcinogenic mechanism is an important issue.1 Several molecular pathways have been reported to play important roles in hepatocarcinogenesis.

“
“Objective.— To investigate bilateral widespread pressure pain hyperalgesia selleck kinase inhibitor in deep tissues over symptomatic (trigemino-cervical) and nonsymptomatic (distant pain-free) regions in patients with cluster headache (CH). Background.— Central sensitization is claimed to play a relevant role in CH. No study has previously searched for widespread pressure hyperalgesia in deep tissues over both symptomatic (trigemino-cervical) and nonsymptomatic (distant pain-free) regions in patients with CH. Methods.— Sixteen men (mean age: 43 ± 11 years) with CH in a remission phase and 16 matched controls were recruited. Pressure pain thresholds (PPTs) were bilaterally measured over the supra-orbital

(V1), infra-orbital (V2), mental (V3), median (C5), radial (C6), and ulnar (C7) nerves, C5-C6 zygapophyseal joint, mastoid process, and tibialis anterior muscle by an assessor blinded to the subjects’ condition. Results.— The results showed Dinaciclib that PPT levels were significantly decreased bilaterally in patients with CH as compared with healthy controls (all sites, P < .001). A greater degree of sensitization over the mastoid process (P < .001) and a lower degree of sensitization over the tibialis anterior muscle (P < .01) was found. Conclusions.— Our findings revealed bilateral widespread pressure pain hypersensitivity in patients with CH confirming the presence

of central sensitization mechanisms in this headache condition. “
“Migraine is generally recognized as a complex condition, which is often challenging to treat. Patients are often open to novel approaches to understanding why this pain occurs and how to prevent future attacks. Ayurvedic medicine, which is a 5000-year-old healing system, offers additional understanding on this disease by categorizing patients into a unique dosha (mind–body) type. Specific herbals, dietary modifications, and lifestyle changes have been utilized for thousands of years to create balance in

the system to improve chronic conditions. Evaluating migraine patients utilizing the Ayurvedic model allows patients and practitioners a further layer of understanding and offers additional treatment options for the patient. Ayurveda is recognized as one of the oldest healing systems Cobimetinib datasheet known to mankind. Ayurveda is a Sanskrit word which literally means “the science” of life. This is the knowledge of health and disease predisposition based on understanding oneself as an individual in an environment that is constantly influencing us to shift and change from our baseline state of balance. Our current Western, allopathic, view of medicine is to base treatment on presenting symptoms. Medications and injectables are often used to treat the symptoms, allowing relief of the symptoms at hand. Preventive medications are given to decrease the severity of disease and give relief of acute symptomatology. The Eastern, specifically Ayurvedic, approach offers a layer on the allopathic model.

[24] This transposon-based model provides exceptional genetic

flexibility and a short induction time. However, due to the simultaneous development of multiple tumor foci, the mosaic model cannot be used for investigations of novel therapies in the adjuvant setting, where potentially curative R0-resection is mandatory. We investigated in this study an approach to induce an autochthonously grown and resectable tumor by local transfection of the liver parenchyma using electroporation techniques. To this end, we used transposase-mediated oncogenic KRas-G12V-insertion combined with Cre-mediated p53-knockout, which resulted in locally restricted formation of an intrahepatic tumor. Histopathologic analyses and coimmunostainings

demonstrated Kinase Inhibitor Library purchase development of ICC characterized by expression of the biliary marker CK19 within the tumor cells. CK19 expression learn more was clearly connected to cellular insertion of the oncogenic KRas-G12V transposon. Additionally, the desmoplastic stroma surrounding the characteristic glandular tumor structures was visualized by vimentin staining of CAFs. Primary tumor growth analysis and survival showed that the oncogenic effect of the KRas-G12V mutant was only exerted in combination with p53-knockout. Within the investigated time, KRas-G12V insertion or p53-knockout alone did not result in any tumor formation. Since we anticipated the predominant transduction of hepatocytes but not cholangiocytes, the exclusive development of liver tumors with characteristic histologic features of ICC was remarkable. By lineage-tracing experiments we indeed identified hepatocytes as primary target cells of electroporation-mediated gene transfer but not cholangiocytes, which have been expected as a source of ICC. However, the originating cell type of ICC is currently a matter of debate since an in vitro study has provided initial evidence for transdifferentiation of mature hepatocytes into bile duct

cells.[39] Our results are also consistent with recent reports demonstrating that hepatocytes STK38 can be a source for ICC. Fan et al.[12] observed that cooperation of activated Notch and Akt-signaling lead to ICC formation by lineage conversion of hepatocytes. Further evidence for the Notch-driven conversion of hepatocytes was confirmed with an elaborate transgenic mouse model of ICC by Sekiya and Suzuki.[13] In contrast, we investigated the role of the most abundant genetic alterations of ICC and demonstrated that genetic engineering of adult hepatocytes in vivo by oncogenic KRas-activation and p53-inactivation also resulted in ICC formation. Our results suggest the existence of Notch-independent mechanisms enabling hepatobiliary transdifferentiation.

Ascher, John P. Roberts “
“Studies using

surrogate NVP-BGJ398 purchase estimates show high prevalence of insulin resistance in hepatitis C infection. This study prospectively evaluated the correlation between surrogate and directly measured estimates of insulin resistance and the impact of obesity and ethnicity on this relationship. Eighty-six nondiabetic, noncirrhotic patients with hepatitis C virus (age = 48 ± 7 years, 74% male, 44% white, 22% African American, 26% Latino, 70% genotype 1) were categorized into normal-weight (body mass index [BMI] < 25, n = 30), overweight (BMI = 25-29.9, n = 38), and obese (BMI ≥ 30, n = 18). Insulin-mediated glucose uptake was measured by steady-state plasma glucose (SSPG) concentration during a 240-minute insulin suppression test. Surrogate estimates included: fasting glucose and insulin, glucose/insulin, homeostasis model assessment (HOMA-IR), quantitative insulin sensitivity check index (QUICKI), insulin (I-AUC) and glucose (G-AUC) area under the curve during oral glucose tolerance test, and the Belfiore and Stumvoll indexes. All surrogate estimates correlated with SSPG, but the magnitude of correlation varied (r = 0.30-0.64). The correlation check details coefficients were highest in the obese. I-AUC had the highest correlation

among all ethnic and weight groups (r = 0.57-0.77). HOMA-IR accounted for only 15% of variability in SSPG in the normal weight group. The common HOMA-IR cutoff of ≤3 to define insulin resistance had high misclassification rates especially in the overweight group independent of ethnicity. HOMA-IR > 4 had the lowest misclassification rate (75% sensitivity, 88% specificity). Repeat HOMA-IR measurements had higher within-person variation in the obese (standard deviation = 0.77 higher than normal-weight, 95% confidence interval = 0.25-1.30,

P = 0.005). Conclusion: Because of limitations of surrogate estimates, caution should be used in interpreting data evaluating insulin resistance especially in nonobese, nondiabetic patients with HCV. HEPATOLOGY 2010 Epidemiologic studies support an association between chronic hepatitis C virus (HCV) infection and type 2 diabetes mellitus.1-3 The mechanism by which HCV may induce diabetes is thought to be related to insulin resistance and potential defects in insulin signaling pathways.4, 5 Guanylate cyclase 2C Studies to date have shown a higher prevalence of insulin resistance in HCV infection compared to hepatitis B virus infection and other causes of liver disease.6 Insulin resistance and diabetes in the setting of HCV infection is of great importance due to its association with increased rates of fibrosis and faster progression of liver disease7, 8 as well as potentially lower response to antiviral HCV therapy.9-11 To date, all human studies except for one that evaluated insulin resistance in the setting of HCV have used surrogate estimates of insulin resistance rather than direct measurements of insulin-mediated glucose uptake (IMGU).

dipsaci populations obtained in our study shared a 99–100% identity with Roxadustat datasheet each other as well as with other D. dipsaci populations deposited previously in databases. The

only population (S) isolated from V. faba spp. minor was identified as D. gigas. Comparison of the nucleotide sequence of this population revealed a 99% identity with other D. gigas populations described so far. The populations D8, 1 and 2, of D. destructor, compared with other populations of this species present in GenBank, showed an identity level between 68.5 and 99.8%. American and Chinese populations described as haplotype C (Subbotin et al. 2011) showed the highest identity level (99.0–99.8%) with the D8, 1 and 2, of D. destructor populations. Polish populations of D. destructor analysed share only a 93% identity with the Polish population (Stu3), described previously (Marek et al. 2010). The phylogenetic analysis for D. dipsaci revealed a phylogenetic tree which was similar to that obtained by Subbotin et al. (2005). Two separate clades for diploid races and polyploidy

races were indicated (Fig. 1). It is important to note that when looking at the tree topology, populations isolated from the same host are rarely grouped together. This can be observed (e.g. for D. dipsaci populations isolated from Allium cepa or Cichorium spp.), and it cannot be explained PF-02341066 research buy by host or geographic origin. In the case of D. dipsaci, there are more than 30 distinguished host races that are supposed to be at different stages of speciation; however, some authors indicate that there is ambiguity about how they Cyclin-dependent kinase 3 should be defined (Sturhan and Brzeski 1991). Phylogenetic analysis

for D. destructor populations was performed for populations isolated from S. tuberosum, I. batatas and Astragalus mongholicus. For D. destructor, length variability of the ITS1 fragment was found, and eight haplotypes were separated (Subbotin et al. 2011). The haplotype A isolated from sweet potato from China is the most distinct and formed a separate clade. The previously reported population (Stu3) from Poland was assigned as haplotype G (Marek et al. 2010; Subbotin et al. 2011). The populations described in this study (D8, 1, 2), however, grouped together with haplotype C populations on a phylogenetic tree (Fig. 2). This indicates that in Poland, there are at least two haplotypes present. It is worth noting that most populations isolated from the sweet potato cluster separately from those isolated from potato. But at the same cluster with the presently described D. destructor populations from Poland grouping members of haplotype C, there is also a Chinese population from I. batatas (Fig. 2). Phylogenetic analysis was carried out with D. gigas found on V. faba minor seeds in Poland. In spite of a very high identity level with other populations reported so far from Europe and Northern Africa, D. gigas from Poland grouped separately from all of them.

08 g/mL in iodixanol gradient (corresponding to 1.18 g/mL in sucrose gradient) and were infectious as indicated by passage to naïve HepaRG cells; (3) HCV E1E2 and core protein accumulation in the cytoplasm of infected cells 1 month p.i.; (4) complete reduction of HCV RNA and infectious virus in HepaRG culture supernatants (97% at 3 weeks

Adriamycin supplier p.i.) by E1E2-specific mAb D32.107-9 at low concentration (0.5 μg/mL) even when the infection was performed in the presence of NHS; (5) ability of infected-HepaRG cells to produce high titers of HCV RNA (4 to 5log10) as complete virus particles in culture media after freezing/thawing and subculture(s) followed by induction of the differentiation process; (6) production of apoE/apoB-associated HCV virions by the HepaRG cells similar to authentic patient-derived HCV particles11, 14; (7) observation of typical positive-strand RNA click here virus-induced membrane rearrangements18 and detection of HCV E1E2 antigen in association with vesicular structures in ER and at a submembranous localization in HCVsp-RG cells. Very recently, a cell-culture-based system was established using PHHs inoculated with HCVcc.16 Even if freshly isolated PHHs are currently the in vitro “gold standard” of human liver

cells, the HepaRG human hepatic cell line is now increasingly used as a surrogate for PHHs in pharmaceutical research and development for metabolism studies.17

Here, our results cAMP demonstrate that HepaRG cells can be infected with serum-derived HCV of genotype 3 and persistently produce infectious enveloped HCV particles with biophysical and immunological properties similar to circulating7, 11 and infected liver-derived10 HCV. The major contributions of our study were to use a genuine HCV isolate from patients distinct from the JFH-1 or Jc1 virus of genotype 2a together with the HepaRG cell line, which possesses key features of authentic hepatocytes. Of course, the current Huh-7-derived HCVcc system remains the “gold standard,” and it would have been optimal to successfully infect HepaRG cells with HCVcc. Unfortunately, only a weak transient replication was obtained in our laboratory when we tried to inoculate differentiated HepaRG cells with a highly infectious JFH-1 inoculum (Durantel et al., unpubl. data). This could be due to the production of type-I interferons in the culture medium,18 which likely should inhibit HCV replication and spreading. This could also explain why the HepaRG cells are only susceptible to HCVsp infection when they exhibit dedifferentiated, depolarized epithelial phenotype associated with an immature innate immunity, resistance to apoptosis, and cellular growth.

There is no consensus on imaging the head and neck for vascular anomalies in the absence of symptoms, as surgical intervention is unlikely under those circumstances. However, repair of a coarctation of the abdominal aorta or renal artery stenosis

should be considered prior to LT.[161] Comorbidities resulting from multiorgan selleck involvement have a significant impact on the outcome of LT, with structural cardiac disease being the most important contributor to mortality.[152, 162] Increased intraoperative fluid requirements place a significant burden on cardiac function and pulmonary blood flow. Patients with AGS often have established right ventricular hypertrophy which, coupled with increased pulmonary vascular resistance associated with pulmonary see more artery stenosis, may increase the risk of diminished cardiac output and poor graft perfusion. Echocardiogram alone may be insufficient to assess the descending aorta and peripheral pulmonary artery branches.[151] Utilization of a dynamic stress test with dobutamine during cardiac catheterization can identify those patients who can successfully increase their cardiac output by over 40%, the necessary cardiac response for successful LT.[163] Posttransplant survival rates vary between

82.9% and 87% at 1 year, 78.4% and 86% at 5 years,[164] and 80.9% at 10 years.[165] Long-term survival rates between AGS and all other pediatric liver transplant recipients were reported to be similar in a single-center experience.[165] However, a review of the UNOS database revealed 5-year graft and patient survival

was worse for AGS compared BA patients, 61.5% versus 70% (P = 0.02) and 78.4% versus 84% (P = 0.01), respectively.[164] Risk factors for poor outcome among AGS patients included neurological and cardiac complications. Renal disease associated with AGS will require a renal-sparing immunosuppressive www.selleck.co.jp/products/obeticholic-acid.html regimen to minimize the risk of renal dysfunction following LT.[166] 36. Patients with AGS should be carefully assessed for evidence of extrahepatic manifestations of this multisystem disorder; decisions regarding liver transplant should be individualized to include potential nontransplant treatment options for nonlife-threatening complications such as intractable pruritus and deforming xanthoma with biliary diversion or ileal exclusion. (1-B). 37. Realistic expectations related to growth potential following LT should be made clear to the family. (1-B) 38. Careful assessment of cardiac and renal function should occur during LT evaluation in all liver transplant candidates. (2-B) 39. Pretransplant vascular imaging of the intra-abdominal vasculature should be performed (2-B); vascular imaging of the head and neck may be considered. (2-C) Wilson’s disease (WD) is a chronic liver condition with a myriad of presentations. WD may be clinically indistinguishable from autoimmune hepatitis, nonalcoholic fatty liver disease, or cryptogenic cirrhosis.

The methodological quality was defined as the control of bias in the treatment comparison. The assessment was based on published reports and information provided by the authors of included trials. Based on previous evidence, the randomization methods were classified as the primary measure of bias control.21, 22 The randomization methods were evaluated by the allocation sequence generation (classified as adequate if based on a table of random numbers, computer-generated random numbers, or similar) and allocation concealment (classified as adequate if based on central randomization, identically Vemurafenib appearing coded drug containers, serially numbered opaque sealed

envelopes, or similar). We also extracted blinding (whether the trial was described as double-blind or single-blind, the method of blinding; whether patients, investigators, outcome assessors or other persons involved in the trial were blinded; and whether the adequacy of blinding was assessed),23 the risk of attrition bias (numbers and reasons for dropouts and withdrawals and whether all patients

were accounted for in the report and analysis of the trial), whether the primary outcome measure was defined and reported, whether a crossover design was used, whether sample size calculations were performed, Maraviroc and whether the preset sample size was achieved. For trials terminated prematurely, we registered whether this was based on predefined criteria. The analyses were performed using RevMan version 5.0.5 (Nordic Cochrane Centre, Copenhagen, Denmark). Meta-analyses were performed using random effects models due to expected clinical heterogeneity. Results are presented as the relative risk (RR) for binary and weighted mean differences for continuous outcomes, both with 95% confidence intervals (CIs).

I2 values were calculated as measures of the degree of intertrial heterogeneity. Data on all patients randomized were extracted to allow intention-to-treat analyses. For patients with missing data, carry-forward of the last observed response was used. Only data from the first period of crossover trials were included. For the primary outcome measure, we performed subgroup analyses of trials stratified by the treatment regimen, the type of HRS, and methodological quality. Based on differences in the duration of follow-up in individual Calpain trials, we performed a post hoc analysis to evaluate the relationship between the treatment effect on mortality and the duration of follow-up. Based on discrepancies between the number of patients who survived and the number of patients with reversal of HRS, we performed a post hoc analysis that combined these two outcome measures. We originally planned to perform regression analyses to detect the risk of bias, including publication bias.24 However, we did not perform these analyses, because the power to detect bias was insufficient due to the small number of trials included.