Transit time ultrasound perivascular flow probe technology is superior to MR imaging on hepatic blood flow measurement in a porcine model

Background: The hepatic hemodynamics is an essential parameter in surgical planning as well as in various disease processes. The transit time ultrasound(TTUS) perivascular flow probe technology is widely used in clinical practice to evaluate the hepatic inflow, yet invasive. The phase-contrast-MRI(PC-MRI) is not invasive and potentially applicable in assessing the hepatic blood flow. In the present study, we compared the hepatic inflow rates using the PC-MRI and the TTUS probe, and evaluated their predictive value of post-hepatectomy adverse events. Methods: Eighteen large white pigs were anaesthetized for PC-MRI and approximately 75% hepatic resection was performed under a unified protocol. The blood flow was measured in the hepatic artery(Qha), the portal vein(Qpv), and the aorta above the celiac trunk(Qca) using PC-MRI, and was compared to the TTUS probe. The Bland-Altman method was conducted and a partial least squares regression(PLS) model was implemented. Results: The mean Qpv measured in PC-MRI was 0.55 ± 0.12 L/min, and in the TTUS probe was 0.74 ± 0.17 L/min. Qca was 1.40 ± 0.47 L/min in the PC-MRI and 2.00 ± 0.60 L/min in the TTUS probe. Qha was 0.17 ± 0.10 L/min in the PC-MRI, and 0.13 ± 0.06 L/min in the TTUS probe. The Bland-Altman method revealed that the estimated bias of Qca in the PC-MRI was 32%(95% CI:-49% to 15%); Qha 17%(95% CI:-15% to 51%); and Qpv 40%(95% CI:-62% to 18%). The TTUS probe had a higher weight in predicting adverse outcomes after 75% resection compared to the PC-MRI( β= 0.35 and 0.43 vs β = 0.22 and 0.07, for tissue changes and premature death, respectively). Conclusions: There is a tendency of the PC-MRI to underestimate the flow measured by the TTUS probes. The TTUS probe measures are more predictive of relevant post-hepatectomy outcomes.     

Anastomotic salvage after rectal cancer resection using the Turnbull–Cutait delayed anastomosis

Background

Turnbull–Cutait abdominoperineal pull-through followed by delayed coloanal anastomosis (DCA) was first described in 1961. Studies have described its use for challenging colorectal conditions. We reviewed our experience with Turnbull–Cutait DCA as a salvage procedure for complex failure of colorectal anastomosis.

Methods

We performed a retrospective cohort study from October 2010 to September 2011, with analysis of postoperative morbidity and mortality.

Results

Seven DCAs were performed for anastomotic complications (3 chronic leaks, 2 rectovaginal fistulas, 1 colovesical fistula, 1 colonic ischemia) following surgery for rectal cancer. Six patients had a diverting ileostomy constructed as part of previous treatment for anastomotic complications before the salvage procedure. No anastomotic leaks were observed. All procedures but 1 were completed successfully. One patient who underwent DCA subsequently required an abdominoperineal resection and a permanent colostomy for postoperative extensive colonic ischemia. No 30-day mortality occurred.

Conclusion

Salvage Turnbull–Cutait DCA appears to be a safe procedure and could be offered to patients with complex anastomotic complications. This procedure could be added to the surgeon’s armamentarium as an alternative to the creation of a permanent stoma.     

Early lesions of follicular lymphoma: a genetic perspective

The pathogenesis of follicular lymphoma is a multi-hit process progressing over many years through the accumulation of numerous genetic alterations. Besides the hallmark t(14;18), it is still unclear which other oncogenic hits contribute to the early steps of transformation and in which precursor stages these occur. To address this issue, we performed high-resolution comparative genomic hybridization microarrays on laser-capture micro-dissected cases of follicular lymphoma in situ (n=4), partial involvement by follicular lymphoma (n=4), and duodenal follicular lymphoma (n=4), assumed to represent, potentially, the earliest stages in the evolution of follicular lymphoma. Cases of reactive follicular hyperplasia (n=2), uninvolved areas from follicular lymphoma in situ lymph nodes, follicular lymphoma grade 1–2 (n=5) and follicular lymphoma grade 3A (n=5) were used as controls. Surprisingly, alterations involving several relevant (onco)genes were found in all entities, but at significantly lower proportions than in overt follicular lymphoma. While the number of alterations clearly assigns all these entities as precursors, the pattern of partial involvement by follicular lymphoma alterations was quantitatively and qualitatively closer to that of follicular lymphoma, indicating significant selective pressure in line with its faster rate of progression. Among the most notable alterations, we observed and validated deletions of 1p36 and gains of the 7p and 12q chromosomes and related oncogenes, which include some of the most recurrent oncogenic alterations in overt follicular lymphoma (TNFRSF14, EZH2, MLL2). By further delineating distinctive and hierarchical molecular and genetic features of early follicular lymphoma entities, our analysis underlines the importance of applying appropriate criteria for the differential diagnosis. It also provides a first set of candidates likely to be involved in the cascade of hits that pave the path of the various progression phases to follicular lymphoma development.     

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.With a mortality rate exceeding 1.5 million deaths annually, Mycobacterium tuberculosis remains one of the world''s most important pathogens (1). M. tuberculosis succeeds as a pathogen because of productive infection of the endosomal network of phagocytes. Its residence within the phagosome protects it from immune responses during its decades long infection cycle. However, intracellular survival depends on active inhibition of pH-dependent killing mechanisms, which occurs for M. tuberculosis but not species with low disease-causing potential (2). Intracellular survival is also enhanced by an unusually hydrophobic and multilayered protective cell envelope. Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (3), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass and LipidDB databases (4, 5).To broadly compare the lipid profiles of virulent and avirulent mycobacteria, we took advantage of a recently validated metabolomics platform (4). This high performance liquid chromatography–mass spectrometry (HPLC-MS) system uses methods of extraction, chromatography, and databases that are specialized for mycobacteria. After extraction of total bacterial lipids into organic solvents, HPLC-MS enables massively parallel detection of thousands of ions corresponding to diverse lipids that range from apolar polyketides to polar phosphoglycolipids. Software-based (XCMS) ion finding algorithms report reproducibly detected ions as molecular features. Each feature is a 3D data point with linked mass, retention time, and intensity values from one detected molecule or isotope. All features with equivalent mass and retention time from two bacterial lipid extracts are aligned, allowing pairwise comparisons of MS signal intensity to enumerate molecules that are overproduced in one strain with a false-positive rate below 1% (4).This comparative lipidomics system allowed an unbiased, organism-wide analysis of lipids from M. tuberculosis and the attenuated vaccine strain, Mycobacterium bovis Bacillus Calmette–Guérin (BCG). BCG was chosen because of its worldwide use as a vaccine and its genetic similarity to M. tuberculosis (6). We reasoned that any features that are specifically detected in M. tuberculosis might be clinically useful as markers to distinguish tuberculosis-causing bacteria from vaccines. Furthermore, given the differing potential for productive infection by the two strains, any M. tuberculosis-specific compounds would be candidate virulence factors. Comparative genomics of M. tuberculosis and BCG successfully identified “regions of deletion” (RD) that encode genes that were subsequently proven to promote productive M. tuberculosis infection (7), including the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 (ESX-1) (8, 9). We reasoned that a metabolite-based screen might identify new virulence factors because not all functions of RD genes are known. Also, biologically important metabolites could emerge from complex biosynthetic pathways that cannot be predicted from single-gene analysis.Comparison of M. tuberculosis and BCG lipid profiles revealed more than 1,000 differences, among which we identified a previously unknown M. tuberculosis-specific diterpene-linked adenosine and showed that it is produced by the enzyme Rv3378c. Previously, Rv3378c was thought to generate free tuberculosinol and isotuberculosinol (10–12). This discovery revises the enzymatic function of Rv3378c, which acts as a virulence factor to inhibit phagolysosome fusion (13). Whereas current models of prenyl transferase function emphasize iterative lengthening of prenyl pyrophosphates using one binding pocket, the crystal structure of Rv3378c identifies two pockets in the catalytic site, establishing a mechanism for heterologous prenyl transfer to nonprenyl metabolites.     

Accuracy of Physician Reporting in Routine Public Health Surveillance for Hepatitis C Virus Infection

Objective

From January 2007 to December 2008, the Montréal Public Health Department sent postal questionnaires to physicians and conducted patient interviews for all those newly diagnosed with hepatitis C virus (HCV) infection. We evaluated physician responses to risk factor questions for non-acute HCV cases.

Methods

We compared physician and patient responses with each of nine risk factor questions, determined the sensitivity and specificity of physician responses compared with patient responses, and evaluated agreement using Gwet''s agreement coefficient (AC₁). We ranked risk factors and compared the distributions by principal exposure category according to physician reporting vs. patient interview using the Chi-square test.

Results

The completeness of physicians'' responses (yes, no, or unknown) varied by risk factor question from 90.8% to 96.7%. For risk factors present among more than 5% of cases, sensitivity of physician responses ranged from 26.9% to 87.7% and specificity ranged from 93.0% to 98.6%. The AC₁ coefficients for agreement between physician and patient responses to lifetime risk factors considered most important in HCV acquisition were 0.80 for injection drug use, 0.95 for blood transfusion before 1990, and 0.86 for birth in a country with high HCV prevalence. Risk distributions by principal exposure category according to physician reporting vs. patient interview were not statistically different (χ²[4] = 2.17, p=0.704).

Conclusion

Postal questionnaires completed by physicians appear valid for determining the principal exposure category among non-acute HCV cases. Physician reporting can be a useful and low-cost component of routine HCV surveillance.Hepatitis C virus (HCV) infection is an important health problem affecting all countries, with an estimated global prevalence of 2.35%, representing 160 million chronically infected individuals.¹ Initial infection, most commonly by parenteral exposure to infected blood, is usually asymptomatic. Three out of four infections become chronic and may not be diagnosed until years later, when the patient develops abnormal liver function or presents with complications such as cirrhosis, liver failure, or hepatocellular carcinoma. A modeling study of the incidence and prevalence of HCV infection and sequelae in Canada estimated that about 242,500 individuals, or 0.78% of the population, were living with HCV infection at the end of 2007.² According to the modeling results, 11% of prevalent cases had been infected through blood transfusion, 36% were past injection drug users (IDUs), 22% were current IDUs, and 31% had been infected through other routes.Risk factors for HCV infection have changed over time, both in North America and globally.^3–5 Before 1990, when the first serological test for HCV became available for donor screening, HCV-contaminated blood was a well-recognized cause of post-transfusion hepatitis. Since the introduction of nucleic acid testing for HCV in 1999, the per-unit risk of HCV contamination of blood or blood products in Canada has fallen to approximately one in three million.^6,7 Among people born outside of Canada, the likelihood of HCV infection prior to immigration depends on age, risk behaviors, and the extent of unscreened blood donation or inadequate sterilization of reusable medical devices in the country of origin.^4,8,9Sharing drug injection equipment is the predominant risk factor for new HCV infections in Canada,^10,11 and the importance of screening current IDUs for HCV is understood. In contrast, people who injected drugs decades ago may not be questioned about past risk behaviors, and immigrants who acquired infection through other routes may also remain undiagnosed until complications arise. Because of the late emergence of sequelae, it is expected that an increasing number of people will require treatment for HCV-related liver disease in the coming years.² A recent cost-effectiveness simulation study in the United States found that one-time HCV screening among adults born between 1945 and 1965 could potentially prevent 82,000 HCV-related deaths compared with the status quo.¹² Characterizing the demographic and risk profiles of people infected in the past but diagnosed today is, therefore, integral to evaluating HCV screening policies and practices and planning appropriate treatment services for different populations.

Surveillance context

HCV infection became a notifiable disease in the province of Québec in 2002, requiring diagnostic laboratories to report all positive HCV test results and physicians to report each newly detected case to the regional public health department. In practice, laboratories report regularly and physicians report rarely. Given the high number of HCV case reports at the time (about 1,000 annually),¹³ the Montréal Public Health Department (MPHD) opted in 2003 to implement a surveillance system whereby physicians who had ordered the HCV diagnostic test were asked to complete a short postal questionnaire.In 2005, the MPHD began participating in the Enhanced Hepatitis Strain Surveillance System (EHSSS) funded by the Public Health Agency of Canada.¹⁰ Under the EHSSS protocol, systematic efforts were made to contact all newly diagnosed HCV cases to conduct a telephone interview using a standardized questionnaire and, when required for the purpose of case classification, to obtain information from the clinic chart.This study was conducted to assess the usefulness of physician reporting in determining the principle exposure category for non-acute cases in the context of routine HCV surveillance. The study objective was to evaluate physician responses to risk factor questions on a postal questionnaire when used for non-acute HCV cases. We compared physician and patient responses to the same risk factor question, determined the accuracy of physician responses to the responses obtained through patient interview, and evaluated agreement between the two. After ranking risk factors and assigning cases to a principal exposure category, we compared the risk distributions obtained by each surveillance method.     

Tumor necrosis factor receptor family costimulation increases regulatory T-cell activation and function via NF-κB

Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3⁺ regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.