Effects of reduced dietary protein and supplemental rumen-protected essential amino acids on the nitrogen efficiency of dairy cows.

When fed to meet the metabolizable protein requirements of the National Research Council, dairy cows consume an excess of N, resulting in approximately 75% of dietary N being lost to the environment as urine and feces. Reductions in environmental N release could be attained through an improvement in N efficiency. The objective of this study was to determine if the predicted reduction in milk yield associated with feeding a low-protein diet to lactating dairy cows could be avoided by dietary supplementation with 1 or more ruminally protected (RP) AA. Fourteen multiparous and 10 primiparous Holstein cows, and 24 multiparous Holstein × Jersey crossbred cows were used in a Youden square design consisting of 8 treatments and 3 periods. The 8 dietary treatments were (1) a standard diet containing 17% crude protein [CP; positive control (PC)], (2) a 15% CP diet [negative control (NC)], (3) NC plus RP Met (+M), (4) NC plus RP Lys (+K), (5) NC plus RP Leu (+L), (6) NC plus RP Met and Lys (+MK), (7) NC plus RP Met and Leu (+ML), and (8) NC plus RP Met, Lys, and Leu (+MKL). Dry matter intake was not affected by treatment. Crude protein intake was lower for NC and RP AA treatments compared with the PC treatment. No detrimental effect was detected of the low-CP diet alone or in combination with AA supplementation on milk and fat yield. However, milk protein yield decreased for NC and +MKL diets, and lactose yield decreased for the +MKL compared with the PC diet. Milk urea N concentrations were lower for all diets, suggesting that greater N efficiency was achieved by feeding the low-protein diet. Minimal effects of treatments on arterial plasma essential AA concentrations were detected, with only Ile and Val being significantly lower in the NC than in the PC diet. Phosphorylation ratios of signaling proteins known to regulate mRNA translation were not affected by treatments. This study highlights the limitations of requirement models aggregated at the protein level and the use of fixed postabsorptive efficiency to calculate milk protein requirements. Milk protein synthesis regulation by signaling pathways in vivo is still poorly understood.

Plasma IL-6 levels correlate with clinical and ultrasound measures of arthritis in patients with systemic lupus erythematosus.

OBJECTIVE: The objective of this paper is to elucidate the role of specific cytokines in lupus (SLE) arthritis. METHODS: Fifty SLE and 40 RA patients had an ultrasound (US) scan of their hand as per standardized protocols. US scores were expressed per joint and as a total 'US activity' score, (sum of power Doppler (PD) and grey-scale synovial hypertrophy scores in all joints) and a total erosion score. SLE disease activity was assessed (BILAG and SELENA-SLEDAI). Plasma levels of IL-6, TNF-alpha and BLyS were measured using sandwich ELISA kits (Quantikine kits, R & D). RESULTS: On the basis of the US results SLE patients were divided into three groups: erosive arthritis (n = 20), non-erosive arthritis (n = 18) and those with a normal US scan (n = 12). Across the SLE groups plasma IL-6 levels correlated with CRP (p < 0.001), hand deformity scores (p = 0.005), BILAG musculoskeletal score (p = 0.009), wrist PD score (p = 0.01), the presence of tenosynovitis (p = 0.008) and total US activity score (p < 0.001) (which remained constant when corrected for total BILAG score). Neither TNF-alpha nor BLyS levels correlated with US or clinical measures of lupus arthritis; however, TNF-alpha correlated with total BILAG score (p < 0.001). CONCLUSION: This is the first study to examine levels of specific cytokines in a cohort of SLE patients stratified in terms of joint disease by US, where the most significant finding is that IL-6 levels correlated both with clinical and US measures of arthritis disease activity.

Colour Doppler ultrasound of the ocular circulation in patients with systemic lupus erythematosus identifies altered microcirculatory haemodynamics.

We assessed whether quantitative analysis of Doppler flow velocity waveforms is able to identify subclinical microvascular abnormalities in SLE and whether eigenvector analysis can detect changes not detectable using the resistive index (RI). Fifty-four SLE patients with no conventional cardiovascular risk factors, major organ involvement or retinopathy were compared to 32 controls. Flow velocity waveforms were obtained from the ophthalmic artery (OA), central retinal artery (CRA) and common carotid artery (CA). The waveforms were analysed using eigenvector decomposition and compared between groups at each arterial site. The RI was also determined. The RI was comparable between groups. In the OA and CRA, there were significant differences in the lower frequency sinusoidal components (P < 0.05 for each component). No differences were apparent in the CA between groups. Eigenvector analysis of Doppler flow waveforms, recorded in proximity of the terminal vascular bed, identified altered ocular microvascular haemodynamics in SLE. Altered waveform structure could not be identified by changes in RI, the traditional measure of downstream vascular resistance. This analytical approach to waveform analysis is more sensitive in detecting preclinical microvascular abnormalities in SLE. It may hold potential as a useful tool for assessing disease activity, response to treatment, and predicting future vascular complications.

Enhancement of undergraduate rheumatology teaching through the use of musculoskeletal ultrasound.

OBJECTIVES: Musculoskeletal ultrasound (MSUS) is increasingly being used by rheumatologists in routine clinical practice to aid with diagnosis and therapy monitoring in the rheumatic conditions. Undergraduate teaching in anatomy is often regarded as problematic and various approaches are in use in UK medical schools. To our knowledge no study to date describes the use of MSUS to facilitate understanding of pathology in the musculoskeletal system at undergraduate level. Accordingly, we wished to explore the usefulness of a short practical ultrasound course for medical undergraduates in learning basic and pathological features of the musculoskeletal system. METHODS: As part of the third-year undergraduate medical musculoskeletal attachment at Queens University Belfast, a 12-week student-selected component (SSC) entitled 'Diagnostic Musculoskeletal Ultrasound in Rheumatology' was offered. The course was a combination of lectures, hand-outs, practical demonstration and supervised hands-on scanning with three methods of summative assessment employed (MCQ paper, practical examination and general attitude). RESULTS: The course involved approximately 30 h work for the two tutors. Seven students completed the course with an overall mean score of 85.3% attained in the assessment. The feedback on the course from the students was extremely positive. CONCLUSION: Musculoskeletal ultrasound could be utilized to enhance the delivery of undergraduate teaching in rheumatology.

A randomised interventional trial of omega-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus.

OBJECTIVE: To determine the clinical effect of dietary supplementation with low-dose omega-3-polyunsaturated fatty acids on disease activity and endothelial function in patients with systemic lupus erythematosus. METHODS: A 24-week randomised double-blind placebo-controlled parallel trial of the effect of 3 g of omega-3-polyunsaturated fatty acids on 60 patients with systemic lupus erythematosus was performed. Serial measurements of disease activity using the revised Systemic Lupus Activity Measure (SLAM-R) and British Isles Lupus Assessment Group index of disease activity for systemic lupus erythematosus (BILAG), endothelial function using flow-mediated dilation (FMD) of the brachial artery, oxidative stress using platelet 8-isoprostanes and analysis of platelet membrane fatty acids were taken at baseline, 12 and 24 weeks. RESULTS: In the fish oil group there was a significant improvement at 24 weeks in SLAM-R (from 9.4 (SD 3.0) to 6.3 (2.5), p<0.001); in BILAG (from 13.6 (6.0) to 6.7 (3.8), p<0.001); in FMD (from 3.0% (-0.5 to 8.2) to 8.9% (1.3 to 16.9), p<0.001) and in platelet 8-isoprostanes (from 177 pg/mg protein (23-387) to 90 pg/mg protein (32-182), p = 0.007). CONCLUSIONS: Low-dose dietary supplementation with omega-3 fish oils in systemic lupus erythematosus not only has a therapeutic effect on disease activity but also improves endothelial function and reduces oxidative stress and may therefore confer cardiovascular benefits.

Effect of ethanol/trisodium citrate lock on the mechanical properties of carbothane hemodialysis catheters.

AIMS: The objective of this study was to investigate the effect of three locking solutions on the mechanical properties of carbothane hemodialysis catheters. METHODS: Catheters were exposed in vitro to one of three locking solutions (heparin 5000 U/ml; 4% trisodium citrate (TSC) or 30% ethanol/4% TSC). Each solution was locked in six catheters and bathed at 37 degrees C for 9 weeks. Changes in the mechanical properties namely, force at break, elongation at break and elastic modulus of the catheters were determined by tensile testing. RESULTS: The ethanol/TSC lock has an effect on the properties of carbothane hemodialysis catheters. The force at break was significantly lower in the ethanol/TSC group compared to the heparin and TSC groups (113.26 N, 191.97 N and 229.72 N, respectively, p < 0.01). Similarly, elongation at break was lower in the ethanol/TSC group, compared to the heparin and TSC groups (stretched 21.97, 38.29, and 42.42 times original length respectively, p < 0.01). The elastic modulus was not significantly different. CONCLUSIONS: The effect of the ethanol/TSC lock on the catheters is unlikely to prohibit clinical use. After 9 weeks of exposure to the solution, the catheter segments could still be stretched to 22 times their length and withstand 11.5 kg (113 N) of force. Clinically produced forces during dialysis are many times smaller than the force required to break the catheters examined in this study. Therefore, the ethanol/TSC lock shows promise as a new catheter locking solution for the treatment of catheter-related infections. Further clinical studies are required.

Distinctive effects of G-CSF, GM-CSF and TNFalpha on neutrophil apoptosis in systemic lupus erythematosus.

OBJECTIVE: To investigate the influence of culture with G-CSF GM-CSF and TNFalpha on neutrophil apoptosis, comparing neutrophils from SLE patients with rheumatoid arthritis (RA) patients and healthy control subjects. METHODS: Neutrophils were isolated from SLE (n= 10), RA (n= 10) and healthy control subjects (n= 10), and cultured with two different concentrations of G-CSF, GM-CSF and TNFalpha. Proportion of apoptotic neutrophils at T=0, T=2hrs and T=24hrs was measured using FITC-labelled annexinV and flow cytometry. RESULTS: Significantly more neutrophils were apoptotic at T=0 in the SLE subjects than in the other groups (median, range--Control 3.5% (0.3-7.9) SLE 9.5% (2.9-29.1) RA 3.0% (0.4-23.0) p<0.05). Following culture for 24 hours with 1ng/ml G-CSF the proportion of apoptotic neutrophils from SLE subjects was significantly increased (median, range = 51.6% (27.0-84.0) without G-CSF v 66.8% (31.8-89.2) with G-CSF p<0.05). This was not observed with RA or control subjects, in whom the trend was towards inhibition of apoptosis. Similar trends were seen with GM-CSF There was significant induction of apoptosis in SLE neutrophils after 2 hr culture with 1ng/ml TNFalpha (median, range = 2.3% (0.1-8.0) without TNFalpha v 5.2% (1.0-22.4) with TNFalpha). No significant change was seen in the other groups. There was an inverse correlation between total neutrophil count and the degree of induction of apoptosis by G-CSF and GM-CSF, determined at a range of time-points and cytokine concentrations CONCLUSIONS: Neutrophils from SLE patients display resistance to the apoptosis-inhibiting effects of G-CSF and possibly GM-CSF, and appear more susceptible to the apoptosis-inducing action of TNFalpha, the greatest resistance being observed in the more neutropenic patients.

Ethanol/trisodium citrate for hemodialysis catheter lock.

AIMS: The objective of this study was to confirm the compatibility of ethanol and 4% trisodium citrate (TSC) for potential use as a catheter locking solution. METHODS: Increasing concentrations of ethanol were combined with 4% TSC in glass test tubes and stored at 37 degrees C over 72 hours. Each tube was visually inspected to determine the highest compatible concentration. To confirm visual compatibility, HPLC analysis was used to compare the concentration of TSC in control solutions (n = 6) to solutions containing both TSC and the highest concentration of ethanol that was visually compatible (n = 6). Compatibility in carbothane hemodialysis catheters was then confirmed in vitro. RESULTS: Results of the compatibility tests indicated that 30% ethanol was the maximum concentration visually compatible with 4% TSC. Ethanol concentrations of 35% or above form a crystalline precipitate in the glass test tubes within 72 hours. HPLC analysis showed no difference in the concentration of TSC in the control solutions compared to the TSC/ethanol solutions when incubated in glass test tubes. A slight, but statistically significant increase in the TSC concentration (1.27%; p < 0.0001) was observed when the ethanol/TSC solution was incubated in carbothane hemodialysis catheters. This slight increase may be due to ethanol absorption into the catheter polymer. Further studies are underway to determine if an ethanol/TSC lock affects the mechanical properties of these carbothane hemodialysis catheters. CONCLUSIONS: We conclude that 30% ethanol is compatible with 4% trisodium citrate in carbothane hemodialysis catheters in vitro. Until the lock's affect on carbothane hemodialysis catheters is known, it cannot yet be recommended for clinical use.

Can diagnostic triage by general practitioners or rheumatology nurses improve the positive predictive value of referrals to early arthritis clinics?

OBJECTIVES: To determine whether diagnostic triage by general practitioners (GPs) or rheumatology nurses (RNs) can improve the positive predictive value of referrals to early arthritis clinics (EACs). METHODS: Four GPs and two RNs were trained in the assessment of early inflammatory arthritis (IA) by four visits to an EAC supervised by hospital rheumatologists. Patients referred to one of three EACs were recruited for study and assessed independently by a GP, an RN and one of six rheumatologists. Each assessor was asked to record their clinical findings and whether they considered the patient to have IA. Each was then asked to judge the appropriateness of the referral according to predetermined guidelines. The rheumatologists had been shown previously to have a satisfactory level of agreement in the assessment of IA. RESULTS: Ninety-six patients were approached and all consented to take part in the study. In 49 cases (51%), the rheumatologist judged that the patient had IA and that the referral was appropriate. The assessments of GPs and RNs were compared with those of the rheumatologists. Levels of agreement were measured using the kappa value, where 1.0 represents total unanimity. The kappa value was 0.77 for the GPs when compared with the rheumatologists and 0.79 for the RNs. Significant stiffness in the morning or after rest and objective joint swelling were the most important clinical features enabling the GPs and RNs to discriminate between IA and non-IA conditions. CONCLUSION: Diagnostic triage by GPs or RNs improved the positive predictive value of referrals to an EAC with a degree of accuracy approaching that of a group of experienced rheumatologists.

Interleukin-10 receptor expression in systemic lupus erythematosus and rheumatoid arthritis.

OBJECTIVE: We aimed to determine the expression of the interleukin-10 receptor (IL10R) on circulating leukocytes in SLE and rheumatoid arthritis, and correlate this with plasma IL-10 levels and disease activity. METHODS: Peripheral blood was sampled from 20 SLE patients, 14 rheumatoid arthritis patients, and 14 healthy controls. IL-10R expression was determined by immunofluorescence labelling and flow cytometric analysis. Plasma IL-10 levels were measured by ELISA. RESULTS: IL-10R was highly expressed on monocytes, and to a lesser degree on neutrophils in all 3 patient groups. Only a small percentage of lymphocytes expressed IL-10R in all three groups. There was no significant difference in IL-10R expression on the surface of monocytes, neutrophils or lymphocytes in any of the 3 groups. IL-10R expression did not correlate with plasma IL-10 levels or disease activity. CONCLUSION: This study has shown no difference in surface IL-10R expression between SLE, rheumatoid arthritis and normal subjects. Deficient or excessive circulating leukocyte surface IL-10R expression therefore does not seem to play a role in the pathogenesis of SLE or rheumatoid arthritis. Functional IL-10R studies would be of interest.

Neutrophils from systemic lupus erythematosus patients demonstrate increased nuclear DNA damage.

OBJECTIVE: (i) To determine the levels of nuclear DNA damage in freshly isolated and cultured neutrophils from SLE patients (SLE), rheumatoid arthritis patients (RA) and healthy individuals and relate these to the percentage of apoptotic neutrophils. (ii) To assess rates of repair of neutrophil oxidative DNA damage. METHODS: The comet assay was used to quantify nuclear DNA damage in neutrophils from SLE patients (n = 20), control subjects (n = 15) and RA patients (n = 15). Levels of DNA damage were related to apoptosis as assessed by annexin V binding and morphology. Rates of repair of neutrophil oxidative DNA damage was measured by incorporating formamidopyrimidine-DNA glycosylase (FPG) into the comet assay. RESULTS: Nuclear DNA damage in freshly isolated and cultured (20 h) neutrophils was significantly greater in SLE patients (median = 12.5%, 27.3%; respectively) compared with RA patients (median = 9.4%, p = 0.002; 19.3%, p = 0.002; respectively) and control subjects (median = 8.2%, p = 0.003; 18.7%, p = 0.01, respectively). Significantly higher levels of circulating apoptotic neutrophils were demonstrated in SLE patients compared to RA and control subjects. Similar findings were observed following 20 h cultured neutrophil preparations. However, no significant direct correlation between neutrophil apoptosis and DNA damage was observed. Neutrophils from 3 of 5 SLE patients demonstrated an impaired ability to repair oxidatively modified DNA. CONCLUSION: Neutrophils from SLE patients display increased DNA damage and, additionally, may demonstrate defective repair of oxidative DNA damage. These features, in addition to increased rates of neutrophil apoptosis, may act as contributing factors to autoantigen excess and immune activation.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody.

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity.

BACKGROUND: Increased numbers of apoptotic neutrophils, and impaired monocyte/macrophage clearance of apoptotic cells, have been demonstrated in systemic lupus erythematosus (SLE). CD44 is implicated in the clearance of apoptotic neutrophils. OBJECTIVE: To determine the expression of CD44 on peripheral blood monocytes and neutrophils in SLE, and examine the relations with disease activity and numbers of circulating apoptotic neutrophils. METHODS: Peripheral blood was sampled from 31 patients with SLE, 19 healthy normal subjects, and 19 patients with rheumatoid arthritis (RA). Monocyte and neutrophil density of surface CD44 expression was determined by immunofluorescence labelling and flow cytometry, and results expressed as mean channel fluorescence (MCF) values. Neutrophil apoptosis was measured by morphology in 15 patients with SLE, nine with RA, and six normal subjects. RESULTS: Monocyte CD44 expression was significantly lower in SLE (median MCF 4.71) than in healthy normal subjects (median MCF 5.61) and controls with RA (median MCF 5.39). Neutrophil CD44 expression was also significantly lower in SLE (median MCF 1.95) than in healthy normal subjects (median MCF 2.37) and controls with RA (median MCF 2.60). Monocyte, but not neutrophil, CD44 expression correlated negatively with the percentage of apoptotic neutrophils. There was no significant correlation of monocyte or neutrophil CD44 expression in SLE with disease activity or damage. CONCLUSIONS: Monocyte and neutrophil CD44 expression is reduced in SLE, and this may contribute to the impaired recognition and clearance of apoptotic neutrophils by monocyte derived macrophages.