A novel approach to measuring skin elasticity in systemic sclerosis: results from a pilot study.

OBJECTIVES: Systemic sclerosis (SSc) is characterized by progressive fibrosis of various organs, and causes hard, tethered, and inelastic skin. The modified Rodnan score is used to quantify skin involvement, but this method is subjective and user dependent. The aim of this study was to test the ability of a new skin torsion device to measure skin elasticity in patients with SSc. METHODS: The study included 16 female SSc patients and 58 healthy controls. Skin elasticity was assessed on the forearms and backs of the hands using a new hand-held device that gently rotates the skin for 15 s to a maximum of 40 deg, and measures the speed of rotation and the angle of rotation at 15 s. Total and localized modified Rodnan scores were also documented. RESULTS: Measurements produced by the skin torsion device had good intra-subject reproducibility, particularly in the control group. The SSc patients had significantly lower skin elasticity than an age-matched subgroup of control subjects, as determined by the median speed of rotation of the device in the hands (1.91 vs. 2.60 deg/s, p < 0.0001) and forearms (1.84 vs. 2.46 deg/s, p < 0.0001), and the rotation at 15 s in the hands (28.6 vs. 39.0 deg, p < 0.0001) and forearms (27.6 vs. 36.9 deg, p < 0.0001). The presence of SSc disease was the only independent predictor of skin elasticity. CONCLUSIONS: This pilot study has shown the potential value of a new skin torsion device to assess skin involvement in patients with SSc.

The inhibitory effects of local anaesthetics on the vascular flare responses to bradykinin and substance P in human skin.

Bradykinin and substance P are involved in inflammation and act through Gq-protein-coupled receptors. Local anaesthetics inhibit the signalling of these receptors and have potent anti-inflammatory actions. The aim of this study was to investigate the effects of local anaesthetics on the cutaneous flare responses to bradykinin and substance P. Skin blood flow responses to intradermal injections of bradykinin and substance P were assessed in the absence and presence of anaesthetic and analgesic concentrations of lidocaine, levobupivacaine and ropivacaine. All local anaesthetics significantly attenuated the vascular responses to bradykinin (p = 0.001) and substance P (p < 0.001). There were no differences in this effect between the different agents, but anaesthetic concentrations had a greater attenuating effect than analgesic concentrations on the substance P response (p < 0.001). Local anaesthetics may therefore be useful in the suppression of inflammation and the prevention of postoperative hyperalgesia.

Improvement in systemic endothelial condition following amputation in patients with critical limb ischemia.

AIM: Most patients with critical limb ischemia (CLI) have co-existing coronary heart disease, which is the main cause of their increased mortality. Peripheral ischemic tissue produces circulating toxic molecules, which may worsen endothelial function systemically and contribute to the general atherosclerotic process within the body. We looked at whether markers of endothelial function improve after amputation of the ischemic limb, when this potential source of toxins has been removed. METHODS: We measured blood levels of vascular endothelial growth factor (VEGF), homocysteine, endothelin-1, vascular cell adhesion molecule-1, E-selectin, thrombomodulin and von Willebrand factor (vWF) in 40 patients with CLI. We also assessed peripheral microvascular function in forearm skin by measuring responses to iontophoresis of acetylcholine and sodium nitroprusside. The measurements were repeated 6 months after amputation. RESULTS: We found abnormally high levels of endothelial products in the patients, and 6 months later VEGF and vWF had both reduced significantly from previous values (by 70% and 40%, respectively; P<0.01 in both cases). CONCLUSION: Improvements in these two markers after amputation are consistent with the hypothesis that peripheral ischemic tissue has a systemic effect on the vascular endothelium and may contribute to the progression of coronary heart disease in patients with CLI.

Effects of poor glucose handling on arterial stiffness and left ventricular mass in normal children.

AIM: Cardiovascular risk factors can be present in children and young adults. We previously found abnormal microvascular function in children who had glucose intolerance and insulin resistance. The aim of the present study was to investigate whether they also have abnormalities in left ventricular mass (LVM) and arterial stiffness. METHODS: We measured heart dimensions and LVM using echocardiography, and arterial stiffness using pulse wave analysis in 23 children with good glucose handling (postfeeding glucose: 3.9 to 5 mmol/L) and 21 with poor glucose handling (7.7 to 11.4 mmol/L). RESULTS: The time to pulse reflection was slightly shorter in the poorer glucose handlers (mean+/-SD: 143+/-10 vs 153+/-20 ms, P=0.04), suggestive of increased arterial stiffness. Also in this group, there were significant relationships between intraventricular septal thickness, blood pressure and body mass index, but not in the normal glucose handlers. CONCLUSIONS: We have found that normal children who are in the lowest quintile of glucose tolerance in comparison with their peers are exhibiting the first signs of arterial stiffening. In addition, we have seen the beginnings of a relationship between blood pressure, body mass index and left ventricular enlargement in this group. While these changes may not yet be clinically significant, their emergence might be further evidence of early predisposition to cardiovascular disease.

An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases.

Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases.

Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1-1: microconformational changes induced by S-hexyl glutathione.

The glutathione S-transferase (GST) isoenzyme A1-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within alpha-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

A novel isoform of human membrane cofactor protein (CD46) mRNA generated by intron retention.

The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine-threonine-rich region (ST-A), together with an antisense exon 12 primer, RT-PCR with primers for other exons both 5' and 3' of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5' and 3' to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.

Case 4: a patient with symptomatic bradycardia.

Both the clinical and electrocardiographic presentations of sick sinus syndrome are highly variable. As illustrated by this month's case of Interactive Grand Rounds, the initial challenge to the clinician is to establish the correct diagnosis in the patient who has symptomatic bradyarrhythmias.

Skin blood flow changes in response to intradermal injection of bupivacaine and levobupivacaine, assessed by laser Doppler imaging.

BACKGROUND AND OBJECTIVES: The vascular effects of local anesthetics are important determinants of their therapeutic activity. Drugs that vasoconstrict have the potential clinical advantages of limited systemic uptake and prolonged duration of effect. The aim of this study was to assess quantitatively the cutaneous vasoactivity of racemic bupivacaine and one of its enantiomers, levobupivacaine. METHODS: Four concentrations of each drug (0.1 mL each of 0.125%, 0.25%, 0.5%, and 0.75%), as well as normal saline, were injected intradermally into randomly assigned sites on the forearms of 10 volunteers. We measured skin blood perfusion using laser Doppler imaging before injection and at 2.5, 10, 20, 40, 60, and 90 minutes thereafter. RESULTS: Both drugs produced a rapid, dose-dependent increase in skin perfusion (P <.001). Saline also caused an increase in perfusion, although less sustained. By 40 minutes, most responses had returned to baseline levels. However, after this time, perfusion continued to decrease, below baseline, for both bupivacaine and levobupivacaine. The exception to this was 0.75% bupivacaine, the response to which was significantly higher than the same concentration of levobupivacaine over this later period (P <.05). CONCLUSIONS: Bupivacaine and levobupivacaine both have a biphasic effect on skin microvessels. The vasoconstriction observed after 40 minutes may occur when the quantity of drug remaining at the administration site has decreased to a lower level. The continued vasodilatation caused by bupivacaine is more difficult to interpret. The results suggest that these local anesthetics cause vasodilatation at high doses and vasoconstriction at lower, subclinical doses. This hypothesis and the clinical relevance of these effects warrant further investigation.

Comparison of macro- and micro-lightguide spectrophotometric measurements of microvascular haemoglobin oxygenation in the tuberculin reaction in normal human skin.

The changes in haemoglobin oxygenation (SO2) occurring in the tuberculin reaction in human skin were measured using macro- and micro-lightguide spectrophotometry and the results compared. A significant difference was found between the measurements from the respective instruments, demonstrating that the micro-lightguide technique measures only in the most superficial capillaries. Laser Doppler flux (LDF) and transcutaneous oxygen (tcpO2) measurements were also obtained concurrently. At the height of the reaction, heating did not significantly change SO2 or LDF, showing that the vessels in the skin were maximally vasodilated. Although SO2 was increased in the reaction, tcpO2 decreased. This suggests that the infiltrating cells may present a diffusion barrier to oxygen between the capillaries and the tissue cells. This study has shown that micro-lightguide spectrophotometry gives a local picture of intracapillary oxygen supply, which is useful in elucidating the pathophysiological changes occurring during chronic inflammation.

Vascular endothelial growth factor in patients with critical limb ischemia before and after amputation.

BACKGROUND: It is known that levels of vascular endothelial growth factor (VEGF) in biological fluids increase with inflammation and vascular proliferation. Theraputic angiogenesis by injection of VEGF or genes encoding for it may be a promising strategy for treatment of critical limb ischemia. However growth factors are also implicated in the development of vascular disease by smooth muscle cell proliferation. METHODS: We have previously shown VEGF levels to be increased in juvenile diabetic subjects with no clinical evidence of vascular disease. We have measured serum VEGF using an enzyme linked immunosorbent assay and cellular VEGF expression using flow cytometry in patients with critical limb ischemia prior to and 6 months postamputation to determine whether removal of the ischemic limb leads to changes in systemic endothelial cell function. RESULTS: Baseline VEGF levels were significantly increased in the patient group compared to controls with levels returning to control levels at 6 months postsurgery. Monocyte and neutrophil VEGF expression was significantly reduced in the patient group. Platelet expression of VEGF was also reduced but this failed to reach statistical significance. CONCLUSIONS: The results suggest that it may be useful to determine the balance between VEGF production and cellular receptor expression prior to treatment.

Amputation level assessment using lightguide spectrophotometry.

The aim of this experimental study was to investigate whether lightguide spectrophotometry in the visible wavelength range in skin could be used to predict stump healing viability in patients with critical lower limb ischaemia. Remission spectra recorded at two sites (medial and lateral) on the line of a proposed trans-tibial amputation (TTA) and at 10mm intervals along the leg were analysed to give haemoglobin oxygenation (SO2). Degree of tissue hypoxia (DTH) along the leg was defined as the percentage of values along the leg less than 10% SO2. DTH and mean SO2 values were compared with skin blood flow values ((I125) 4-Iodoantipyrine clearance technique) and clinical outcome of trans-tibial amputation, (TTA) or trans-femoral amputation (TFA), in 41 patients. SO2 histograms were also measured in 12 normal subjects for comparison. The results of the study allowed the establishment of criteria for the accurate prediction of flap healing potential. Successful TTAs all displayed a minimum mean SO2 at the medial and lateral measurement sites of 30%, together with a maximum degree of tissue hypoxia of 15% along the limb. The combination of these criteria gave a sensitivity and selectivity of 1.0 for prediction of a successful outcome of TTA.

Role of endothelium-derived hyperpolarising factor in acetylcholine-mediated vasodilatation in skin.

AIM: Acetylcholine (ACh) is an endothelium-dependent vasodilator used to investigate endothelial function in the microcirculation. The mediators of its vasodilatory effects are not clear, but endothelium-derived hyperpolarising factor (EDHF) is thought to contribute, and appears to have particular importance in smaller peripheral vessels. The aim of this study was to investigate the role of EDHF in ACh-mediated vasodilator responses in human forearm skin. METHODS: Laser Doppler imaging was used to measure forearm skin blood flow responses to iontophoretic administration of ACh in 7 healthy men. ACh in a 10-mg/mL solution was administered in accumulating doses using increasing delivery currents of 10, 15, 20, 50 and 100 µA. The measurements were repeated on subsequent visits when the effects of EDHF were blocked using intra-arterial sulphaphenazole at 2 mg/min (a cytochrome P-450 inhibitor), nitric oxide (NO) was blocked using intra-arterial administration of the NO synthetase inhibitor l-NG-monomethyl arginine (l-NMMA) at 4 µmol/min, and prostanoids were blocked with oral aspirin 1 g. RESULTS: The microvascular response to ACh was significantly attenuated by sulphaphenazole alone (P=0.018), l-NMMA alone (P<0.001) and the combination of sulphaphenazole plus l-NMMA (P<0.001), and aspirin had no additional effect. CONCLUSION: EDHF is a significant contributor to the vasodilatory effects of ACh in the human dermal microcirculation. Information about abnormalities in specific pathways of endothelial function in patient groups may help in the targeting of appropriate drug therapies.

Detecting diabetes and impaired glucose tolerance in patients with atherosclerotic peripheral arterial disease.

AIM: In patients with peripheral arterial disease (PAD), diabetes mellitus is associated with increased mortality rates. The aim of this study was to estimate the prevalence of undiagnosed diabetes in PAD patients, and to assess whether a glucose tolerance test is more sensitive than a simple fasting glucose measurement for diagnosis in this group. METHODS: A standard glucose tolerance test and fasting glucose measurements were performed in 53 patients with PAD, who were then categorised into diagnostic groups according to each test result. RESULTS: Using the glucose tolerance test results, 11.5% of patients were diagnosed with diabetes mellitus and 28.8% had either impaired fasting glucose or impaired glucose tolerance. Using fasting glucose levels only, 7.7% received a diagnosis of diabetes mellitus and 17.3% had impaired fasting glucose. The glucose tolerance data and the fasting glucose data were in agreement in 82.7% of cases, but the glucose tolerance test identified an additional 3.8% of cases with diabetes and an additional 13.5% of cases with impaired glucose tolerance. CONCLUSION: Undiagnosed diabetes and impaired glucose homeostasis are common in patients with PAD. Routine screening using a simple glucose tolerance test should be considered in the clinical assessment of this group.