Faecal haemoglobin concentration thresholds for reassurance and urgent investigation for colorectal cancer based on a faecal immunochemical test in symptomatic patients in primary care.

BACKGROUND: Faecal haemoglobin concentration (f-Hb), estimated using a faecal immunochemical test, can be safely implemented in primary care to assess risk of colorectal cancer (CRC). Clinical outcomes of patients presenting with symptoms of lower gastrointestinal disease were examined using an extensive range of f-Hb thresholds to decide on reassurance or referral for further investigation. METHODS: All patients who attended primary care and submitted a single faecal specimen faecal immunochemical test in the first year of the routine service had f-Hb estimated using HM-JACKarc: f-Hb thresholds from <2 to ≥ 400 µg Hb/g faeces (µg/g) were examined. RESULTS: Low f-Hb thresholds of <2, <7, <10 and <20 µg/g gave respective CRC risks of 0.1, 0.3, 0.3 and 0.4%, numbers needed to scope for one CRC of 871, 335, 300 and 249, and 'false negative' rates of 2.9, 11.4, 13.3 and 17.1%. With thresholds of <2, <7, <10 and <20 µg/g, 48.6, 74.6, 78.1 and 83.2% respectively of symptomatic patients could be managed without further investigation. With reassurance thresholds of <2 µg/g, <7 µg/g and <10 µg/g, the thresholds for referral for urgent investigation would be >400 µg/g, ≥200 µg/g and ≥100 µg/g. However, patients with a f-Hb concentration of <10 or <20 µg/g with iron deficiency anaemia, or with severe or persistent symptoms, should not be denied further investigation. CONCLUSIONS: In primary care, f-Hb, in conjunction with clinical assessment, can safely and objectively determine individual risk of CRC and decide on simple reassurance or urgent, or routine referral.

A prospective single centre pilot evaluation of a serum calprotectin assay in unselected GI patients.

BACKGROUND: Whilst C-reactive protein (CRP) is an established serum marker of inflammation, its use in gastroenterology has been limited by its poor sensitivity and specificity for GI disease. Faecal calprotectin (FC) has been adopted into mainstream GI practice as a sensitive but non-specific marker of intestinal inflammation. However, stool samples collection for FC can be challenging and the possibility of utilising a sensitive and specific serum biomarker of intestinal inflammation in luminal gastroenterology is an attractive prospect. This work investigates the performance of serum calprotectin (SC) compared to current biomarkers, FC and CRP, in an unselected cohort of patients attending our GI unit. METHODS: Patients attending in and outpatients within an adult GI service who submitted a stool sample for FC analysis were identified. A total of 109 who had a serum sample obtained within one day of stool sample collection had the serum analysed for CRP and SC and the correlation between these biomarkers was investigated. RESULTS: The intraclass correlation coefficient (ICC) between SC, FC and CRP was 0.10, 95% CI -0.09-0.28 and 0.18, 95% CI -0.01-0.35, respectively. The ICC between FC and CRP was 0.18, 95% CI -0.01-0.35. CONCLUSIONS: Our data reveals that there is no significant correlation between SC and FC, nor between SC and CRP in a large unselected cohort of GI patients. Therefore, as a serum biomarker for intestinal inflammation, SC is unlikely to be of clinical utility and the search for an appropriate serum GI biomarker continues.

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells.

The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution.

The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae.

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.

Insulin-regulated trafficking of GLUT4 requires ubiquitination.

A major consequence of insulin binding its receptor on fat and muscle cells is translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the cell surface where it serves to clear glucose from the bloodstream. Sorting of GLUT4 into its insulin-sensitive store requires the GGA [Golgi-localized, γ-ear-containing, ADP ribosylation factor (ARF)-binding] adaptor proteins, but the signal on GLUT4 to direct this sorting step is unknown. Here, we have identified a role for ubiquitination of GLUT4 in this process. We demonstrate that GLUT4 is ubiquitinated in 3T3-L1 adipocytes, and that a ubiquitin-resistant version fails to translocate to the cell surface of these cells in response to insulin. Our data support a model in which ubiquitination acts as a signal for the trafficking of GLUT4 from the endosomal/trans-Golgi network (TGN) system into its intracellular storage compartment, from where it is mobilized to the cell surface in response to insulin.