Biophotonic tools for probing extracellular matrix mechanics.

The complex, hierarchical and heterogeneous biomechanics of the extracellular matrix (ECM) are central to the health of multicellular organisms. Characterising the distribution, dynamics and above all else origins of ECM biomechanics are challenges that have captivated researchers for decades. Recently, a suite of biophotonics techniques have emerged as powerful new tools to investigate ECM biomechanics. In this mini-review, we discuss how the non-destructive, sub-micron resolution imaging capabilities of Raman spectroscopy and nonlinear microscopy are being used to interrogate the biomechanics of thick, living tissues. These high speed, label-free techniques are implemented during mechanical testing, providing unprecedented insight into the compositional and structural response of the ECM to changes in the mechanical environment.

Micro-mechanical damage of needle puncture on bovine annulus fibrosus fibrils studied using polarization-resolved Second Harmonic Generation(P-SHG) microscopy.

Needle injection has been widely used in spinal therapeutic or diagnostic processes, such as discography. The use of needles has been suspected in causing mild disc degeneration which can lead to long-term back pain. However, the localised microscopic damage caused by needles has not been well studied. The local progressive damage on a microscopic level caused by needle punctures on the surface of bovine annulus fibrosus was investigated. Four different sizes of needle were used for the puncture and twenty-nine bovine intervertebral discs were studied. Polarization-resolved second harmonic generation and fluorescent microscopy were used to study the local microscopic structural changes in collagen and cell nuclei due to needle damage. Repeated 70 cyclic loadings at ±5% of axial strain were applied after the needle puncture in order to assess progressive damage caused by the needle. Puncture damage on annulus fibrosus were observed either collagen fibre bundles being pushed aside, being cut through or combination of both with part being lift or pushed in. The progressive damage was found less relevant to the needle size and more progressive damage was only observed using the larger needle. Two distinct populations of collagen, in which one was relatively more organised than the other population, were observed especially after the puncture from skewed distribution of polarization-SHG analysis. Cell shape was found rounder near the puncture site where collagen fibres were damaged.

Can faecal calprotectin predict relapse in inflammatory bowel disease: a mini review.

Crohn's disease and ulcerative colitis are chronic inflammatory disorders affecting the gastrointestinal tract. Faecal calprotectin is a protein complex of the S-100 family of calcium-binding proteins present in inflammatory cells that can be measured in stool samples, which act as a biomarker for bowel inflammation. Elevated faecal calprotectin has been shown to reflect the presence of ongoing mucosal inflammation, which improves with mucosal healing. The aim of this review was to evaluate the available evidence on the ability of faecal calprotectin to predict a relapse in inflammatory bowel disease. Multiple retrospective studies have shown that patients who relapse have significantly higher levels of calprotectin in their stool compared with non-relapsers, especially in ulcerative colitis. Elevated faecal calprotectin postoperatively in Crohn's disease was also shown to be indicative of a relapse. However, the association of a raised faecal calprotectin and relapse is not universal and may be explained by the different patterns of mucosal inflammatory activity that exist. In conclusion, we put forward our hypothesis that changes such as a rise in faecal calprotectin levels may be more predictive of a relapse than absolute values.

The micromechanics of the superficial zone of articular cartilage.

OBJECTIVE: To investigate the relationships between the unique mechanical and structural properties of the superficial zone of articular cartilage on the microscopic scale. DESIGN: Fresh unstained equine metacarpophalangeal cartilage samples were mounted on tensile and compressive loading rigs on the stage of a multiphoton microscope. Sequential image stacks were acquired under incremental loads together with simultaneous measurements of the applied stress and strain. Second harmonic generation was used to visualise the collagen fibre network, while two photon fluorescence was used to visualise elastin fibres and cells. The changes visualised by each modality were tracked between successive loads. RESULTS: The deformation of the cartilage matrix was heterogeneous on the microscopic length scale. This was evident from local strain maps, which showed shearing between different regions of collagen under tensile strain, corrugations in the articular surface at higher tensile strains and a non-uniform distribution of compressive strain in the axial direction. Chondrocytes elongated and rotated under tensile strain and were compressed in the axial direction under compressive load. The magnitude of deformation varied between cells, indicating differences in either load transmission through the matrix or the mechanical properties of individual cells. Under tensile loading the reorganisation of the elastin network differed from a homogeneous elastic response, indicating that it forms a functional structure. CONCLUSIONS: This study highlights the complexity of superficial zone mechanics and demonstrates that the response of the collagen matrix, elastin fibres and chondrocytes are all heterogeneous on the microscopic scale.

Micromechanical response of articular cartilage to tensile load measured using nonlinear microscopy.

Articular cartilage (AC) is a highly anisotropic biomaterial, and its complex mechanical properties have been a topic of intense investigation for over 60 years. Recent advances in the field of nonlinear optics allow the individual constituents of AC to be imaged in living tissue without the need for exogenous contrast agents. Combining mechanical testing with nonlinear microscopy provides a wealth of information about microscopic responses to load. This work investigates the inhomogeneous distribution of strain in loaded AC by tracking the movement and morphological changes of individual chondrocytes using point pattern matching and Bayesian modeling. This information can be used to inform models of mechanotransduction and pathogenesis, and is readily extendable to various other connective tissues.

Measurement of faecal calprotectin and lactoferrin in inflammatory bowel disease.

Crohn's disease and ulcerative colitis are chronic relapsing gastrointestinal conditions characterised by an influx of inflammatory cells to the affected gut mucosa. The mainstay of diagnosing and re-evaluating these conditions in clinical practice and research is by invasive serological, radiological, endoscopic and histological assessment. In clinical trials, disease activity is often evaluated using a combination of the above tests plus clinical indices such as the Crohn's Disease Activity Index and Ulcerative Colitis Activity Index. These tools rely on subjective assessment of symptoms and so, often, do not correlate with mucosal inflammation or mucosal healing, which may be the preferred therapeutic end point for long-term inflammatory bowel disease (IBD) management. The faecal biomarkers calprotectin and lactoferrin are neutrophil derived proteins that are stable in faeces and can be detected by quantitative ELISA in small stool samples. Concentrations of both are raised in patients with gastrointestinal mucosal inflammation. They provide a unique, inexpensive, non-invasive method of testing for active inflammatory disease. They can be used to screen for IBD and as a surrogate marker of mucosal healing they are useful in monitoring the response to therapeutic intervention or surgery. They may also predict the clinical course of the disease. This clinical review aims to discuss the current evidence, limitations and potential future uses of these biomarkers in IBD.

Anti-tissue transglutaminase antibodies in the follow-up of adult coeliac disease.

BACKGROUND: The detection of auto antibodies directed against tissue transglutaminase (anti-tTG antibodies) has a well-established role in the diagnosis of coeliac disease, but the value of these antibodies in long-term follow-up is controversial. AIMS: To determine if serial anti-tTG antibody measurements could confirm adherence to a gluten-free diet (GFD) and identify patients at risk of disease complications. METHODS: In a 54-month cohort follow-up study, 182 adult patients were assessed. Data recorded included self-assessment of GFD adherence; anti-tTG antibody concentration and serum ferritin, vitamin B12 and folate. Where available, bone mineral density (BMD) and duodenal histology data were retrieved. RESULTS: Persistently elevated anti-tTG antibody levels were significantly associated with abnormal duodenal histology (P < 0.001), low ferritin (P < 0.01) and poor adherence to the GFD (P < 0.001). The specificity was >85% while the sensitivity was 39-60%. Anti-tTG antibody concentrations fell rapidly following successful initiation of a GFD, and maintenance of normalization identified those who continued to be adherent to the diet. CONCLUSIONS: This study supports a strategy of using anti-tTG antibody concentrations to monitor newly diagnosed and established patients with coeliac disease, and to target dietetic intervention to reduce the risk of complication.

Faecal calprotectin or lactoferrin can identify postoperative recurrence in Crohn's disease.

BACKGROUND: Identifying Crohn's disease recurrence in symptomatic patients after ileocaecal resection is difficult. The aim of this study was to evaluate faecal concentrations of granulocyte degradation products in this setting. METHODS: A postoperative cohort of 13 patients was followed prospectively for 1 year with regular faecal calprotectin (FC) and lactoferrin (FL) measurements. A second postoperative cohort (median 24 months after resection) of 104 patients provided a single stool sample. Faecal measurements were compared with symptom diaries, the Harvey Bradshaw Index, endoscopic examination, C-reactive protein and platelet measurement. RESULTS: In the uncomplicated course, both markers normalized within 2 months. Both FC and FL correlated significantly with Harvey Bradshaw Index (P < 0.001). Twenty-eight patients with severely clinically active disease had high mean(s.e.) levels of FC (661.1(119.1) microg/g) and FL (116.6(32.2) microg/g); and 43 with clinically inactive disease had low levels of FC (70.2(27.1) microg/g) and FL (5.9(2.4) microg/g). In patients with mild to moderately clinically active disease, FC and FL identified individuals with and without recurrent inflammatory disease. Faecal markers were more accurate at predicting clinical disease activity than C-reactive protein, platelet count or endoscopic appearance. CONCLUSION: FC and FL are non-invasive tests that can help to identify disease recurrence in symptomatic postoperative patients.

A randomized, double-blind, placebo-controlled trial of lenalidomide in the treatment of moderately severe active Crohn's disease.

BACKGROUND: Therapy targeted at tumour necrosis factor-alpha has an established role in Crohn's disease. Lenalidomide, an analogue of thalidomide, is an oral immunomodulatory agent with powerful antitumour necrosis factor-alpha properties. It is licensed for myeloma and myelodysplastic syndrome. Based upon reports of thalidomide efficacy, lenalidomide was evaluated in Crohn's disease. AIM: To evaluate the efficacy and safety of lenalidomide in subjects with moderately severe active Crohn's disease. METHODS: In a multicentre, double-blind, placebo-controlled parallel group study 89 subjects were randomized to lenalidomide 25 mg daily, 5 mg daily or placebo. Subjects were treated for 12 weeks. The primary end point was a 70-point reduction in Crohn's Disease Activity Index. RESULTS: The overall clinical response rate was not significantly different between the three groups: lenalidomide 25 mg 26%, lenalidomide 5 mg 48% and placebo 39%. Lenalidomide was generally well tolerated with only one serious adverse event, a deep vein thrombosis, being attributed to treatment. CONCLUSION: Lenalidomide, an oral agent with antitumour necrosis factor-alpha properties, was not effective in active Crohn's disease in contrast to reports of benefit from thalidomide. The reasons for this lack of efficacy are speculative, other physiological activities may offset its action on inflammatory cytokines, or its antitumour necrosis factor-alpha action without apoptosis may be insufficient for activity in Crohn's disease.

Influence of IL-6, COL1A1, and VDR gene polymorphisms on bone mineral density in Crohn's disease.

BACKGROUND: Osteoporosis is an important cause of morbidity in patients with Crohn's disease. The pathogenesis of reduced bone mineral density (BMD) is multifactorial. A range of genetic factors have been implicated in other populations of patients with osteoporosis. AIM: To investigate the influence of interleukin 6 (IL-6), collagen type 1alpha1 (COL1A1), and vitamin D receptor gene (VDR) single nucleotide polymorphisms (SNP) on BMD in patients with Crohn's disease. PATIENTS: A cohort of 245 well characterised patients with Crohn's disease were recruited from the inflammatory bowel disease register at the Freeman Hospital and Royal Victoria Infirmary, Newcastle upon Tyne, and the Queen Elizabeth Hospital, Gateshead, UK. METHODS: Patients were genotyped for IL-6 C-174G SNP, COL1A1 Sp1 binding site G T SNP, VDR Taq1, and Fok1 SNPs, and CARD15 R702W, G908R, and L1007fs SNPs. BMD was measured at the lumbar spine (LSP) and hip using dual energy x ray absorptiometry. RESULTS: A total of 158 female and 87 male patients, aged 24-70 years (mean 44), were recruited. There were no significant differences in the distribution of the tested SNPs when analysed for age, body mass index, pre/post-menopausal status, smoking, or steroid use. Two hundred and thirteen patients were genotyped for the IL-6 SNP. LSP and total hip BMD was significantly lower in patients with the GG genotype (48%) than the CC genotype (15%) (p = 0.041, p = 0.014). One hundred and eighty patients were genotyped for the COL1A1 SNP. There was no significant difference in BMD at LSP. Hip BMD was significantly lower in heterozygous patients compared with homozygous wild-types (p = 0.034). There were no significant differences in BMD between genotypes for the two VDR SNPs or the CARD15 genotypes examined. CONCLUSION: IL-6 and COL1A1 gene polymorphisms influence BMD in patients with Crohn's disease but the particular VDR gene polymorphisms studied do not have a major effect.

Review article: smoking cessation as primary therapy to modify the course of Crohn's disease.

This article aims to offer an updated review of the effects of smoking on inflammatory bowel disease, and provide a review of the methods of achieving smoking cessation. A systematic review of Embase and Medline databases was conducted. Smoking causes opposing effects on ulcerative colitis and Crohn's disease. The odds ratio of developing ulcerative colitis for smokers compared with lifetime non-smokers is 0.41. Conversely, smokers with Crohn's disease have a more aggressive disease requiring more therapeutic intervention. Smoking cessation is associated with a 65% reduction in the risk of a relapse as compared with continued smokers, a similar magnitude to that obtained with immunosuppressive therapy. Although difficult to achieve smoking cessation can best be encouraged by accessing appropriate counselling services, nicotine replacement therapy and bupropion. Using a combination of these treatments there is an improved chance of success of up to 20% compared with an unassisted quit attempt. Smoking cessation unequivocally improves the course of Crohn's disease and should be a primary therapeutic aim in smokers with Crohn's disease.

Combined percutaneous and endoscopic procedures for bile duct obstruction: simultaneous and delayed techniques compared.

BACKGROUND/AIMS: In patients with obstructive jaundice, when the endoscopic approach fails to achieve biliary drainage, percutaneous cannulation and combined endoscopic/percutaneous endoprosthesis insertion can be performed simultaneously or in stages. This study compared these two approaches. METHODOLOGY: Over a three-year period 41 patients were studied. All had obstructive jaundice for which endoscopic drainage had failed. In 22 patients (group 1) percutaneous transhepatic drainage was followed a few days later by combined endoscopic and percutaneous procedure. In 19 patients (group 2) the percutaneous transhepatic drainage and combined drainage were performed at the same session. In the multiple stage group the mean interval between the first endoscopic retrograde cholangiopancreatography and final combined procedure was 9 days (SD 5.2). The groups were similar for sex, underlying pathology and reasons for failure of endoscopic approach. Group 1 patients were older 73 vs. 65 years (p < 0.05). RESULTS: Patients in group 2 had a more rapid recovery and discharge home: mean 6 days, compared to mean 18 days from the initial procedure for group 1 (p < 0.001). Five patients died of their disease without leaving hospital (4 in group 1, 1 in group 2). In each group drainage failed in 1 patient. Complications were more common in group 1: 73% vs. 37% (p < 0.05). Pancreatitis (3 vs. 2) and septicemia (4 in group 1, 3 in group 2) were similar but group 1 had complications from the external drain: cholangitis and pyrexia in 4 patients, 3 bile leaks, and 1 catheter displacement. CONCLUSIONS: When endoscopic drainage alone fails, a combined percutaneous/endoscopic procedure should only be performed if it can be carried out simultaneously.

Management of irritable bowel syndrome.

The diagnosis of irritable bowel syndrome (IBS) is made on clinical grounds with appropriate limited investigations to exclude organic disease. IBS is common and may have a significant impact on a patient's quality of life. Psychological symptoms are common. IBS may benefit from pharmacological and non-pharmacological management. Specific measures should be directed towards the dominant symptoms of constipation or diarrhoea. Several new drugs are currently under evaluation and may prove valuable for subgroups of patients with IBS. Successful management requires a combination of reassurance and explanation about the natural history of the condition.

A double-blind comparison of balsalazide, 6.75 g daily, and sulfasalazine, 3 g daily, in patients with newly diagnosed or relapsed active ulcerative colitis.

BACKGROUND: Sulfasalazine is well established in the treatment of active ulcerative colitis. Intolerance to sulfasalazine, however, is a common problem. Balsalazide has been designed to deliver 5-aminosalicylic acid to the colon without the poor tolerability of sulfasalazine. AIM: To compare the safety and efficacy of balsalazide, 6.75 g daily, with sulfasalazine, 3 g daily, in the treatment of active ulcerative colitis of all grades of severity. METHODS: Balsalazide and sulfasalazine were compared in a multicentre, double-blind, parallel group study over 12 weeks. Patients were stratified for disease severity and topical and/or oral steroids were co-administered where clinically necessary. RESULTS: Fifty-seven patients were randomized: 28 to receive balsalazide and 29 to receive sulfasalazine. Significantly fewer patients withdrew from the balsalazide group due to adverse events (2/28 vs. 9/29, P=0.041). These data confirm that balsalazide is better tolerated than sulfasalazine. In patients able to tolerate the treatment, similar improvements were recorded in clinical, sigmoidoscopic and histological assessments in both treatment groups. CONCLUSIONS: This study confirms the better tolerability of balsalazide compared to sulfasalazine, and supports the use of balsalazide in ulcerative colitis of all grades of severity.

A double-blind comparison of balsalazide, 6.75 g, and sulfasalazine, 3 g, as sole therapy in the management of ulcerative colitis.

BACKGROUND: Sulfasalazine is accepted therapy for active ulcerative colitis, but side-effects and intolerance are common. Balsalazide is an azo-bonded pro-drug which also releases 5-aminosalicylic acid into the colon, but uses an inert carrier molecule. AIM: To compare the safety and efficacy of sul- fasalazine, 3 g, with balsalazide, 6.75 g, in the initial daily treatment of mild to moderate ulcerative colitis. METHODS: A randomized, multicentre, double-blind, parallel group study was performed, with a treatment duration of 8 weeks. Patients on previous maintenance treatment were excluded. The trial medication was the sole treatment for the colitis. Efficacy was assessed by patient diaries, symptom assessment, sigmoidoscopic appearance and histology. RESULTS: Fifty patients were recruited: 26 allocated to the balsalazide group and 24 to the sulfasalazine group. More patients withdrew due to adverse events in the sulfasalazine group (nine patients vs. one patient in the balsalazide group, P=0.004). Improvement occurred in both groups, with a tendency to a faster response with balsalazide. Of the patients taking balsalazide, 61% achieved clinical and sigmoidoscopic remission. CONCLUSIONS: Balsalazide, 6.75 g, is effective as the sole treatment for patients with mild to moderately active ulcerative colitis, with significantly fewer withdrawals due to side-effects than in a similar group of patients taking sulfasalazine, 3 g.

Role of polymorphisms in the interleukin-10 gene in determining disease susceptibility and phenotype in inflamatory bowel disease.

Interleukin-10 (IL-10) has a key role in regulating mucosal inflammation. The role of functional polymorphisms at positions -627 and -1117 in the IL-10 gene as candidate susceptibility loci in inflammatory bowel disease and their importance in determining disease extent were evaluated in 159 patients with ulcerative colitis (83 left-sided; 76 extensive), 90 patients with Crohn's disease (22 small bowel; 29 large bowel; 39 both), and 227 controls. Genotyping was performed either by PCR-RFLP assays (-627 site) or SSCP analysis (-1117 site). An excess of -627A allele was observed in patients with left-sided colitis (52%) compared with controls (33%; P = 0.004) suggesting that IL-10 may influence the extent of the disease. These results were not replicated in a newly recruited group (N = 100) of patients with UC. We conclude that polymorphisms at -627 and -1117 sites in the IL-10 gene do not contribute to the susceptibility to IBD or determining the extent of the disease in our population.