Novel use of immunohistochemistry for phenotypic evaluation of circulating neoplastic lymphocyte populations.

CASE HISTORY: A 5-year-old male neutered Poodle cross presented with a 2-week history of non-specific gastrointestinal signs including vomiting, inappetence and lethargy (Case 1). A 14-year-old male neutered Staffordshire Bull Terrier presented with a 6-week history of progressive inappetence and lethargy (Case 2). CLINICAL FINDINGS: On presentation, Case 1 was dehydrated and had repeatable cranial abdominal pain. Mild hypoproteinemia with hypoalbuminemia, and electrolyte disturbances were found on biochemistry profile. Abdominal ultrasonography showed moderate diffuse small intestinal wall irregularity and moderate local lymphadenopathy. Haematology was repeated 2 days after initial presentation and showed a marked leucocytosis associated with an elevated circulating neoplastic cell population. On presentation, Case 2 was dehydrated, had palpable hepatomegaly and a mild generalised lymphadenopathy. A low number of large, atypical lymphocytes were found on haematology. Sonographically there was hepatomegaly with diffuse parenchymal changes and severe mesenteric lymphadenopathy. LABORATORY FINDINGS: Aspirates from the abdominal lymph nodes (Cases 1 and 2) and liver (Case 2), and blood smears revealed atypical neoplastic lymphoid populations, predominantly comprising large lymphocytes. Immunocytochemistry failed to determine the lymphoid phenotype in Case 1 but supported a T cell phenotype in Case 2. Immunohistochemistry of formalin-fixed paraffin-embedded (FF-PE) blood clots was able to identify a T cell phenotype in both cases. PCR for antigen receptor rearrangement results from both cases were consistent with an expanded T cell population. DIAGNOSIS: In both cases, immunohistochemistry on FF-PE blood clots revealed a circulating T cell lymphocyte population, consistent with T cell lymphoproliferative neoplasia. CLINICAL RELEVANCE: Immunohistochemistry on FF-PE blood clots offers clinicians a reliable, inexpensive and minimally invasive method of phenotyping neoplastic cells in circulation. Compared to other available methods, prolonged sample stability allowing for transport and retrospective examination is a distinct advantage. This technique should be considered a useful adjunct to the currently available methods for the phenotypic evaluation of lymphoid leukaemia in dogs.

Effectiveness of enzyme replacement therapy in adults with late-onset Pompe disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapy (ERT) for adults with late-onset Pompe disease. DESIGN: A longitudinal cohort study including prospective and retrospective clinical outcome data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment. Untreated patients contributed natural history data. PARTICIPANTS: Consenting adults (N = 62) with a diagnosis of late-onset Pompe disease who attended a specialist treatment centre in England. This cohort represented 83 % of all patients in the UK with a confirmed diagnosis of this rare condition. At study entry, all but three patients were receiving ERT (range of treatment duration, 0 to 3.1 years). OUTCOME MEASURES: Percent predicted forced vital capacity (%FVC); ventilation dependency; mobility; 6 min walk test (6MWT); muscle strength and body mass index (BMI). RESULTS: An association was found between time on ERT and significant increases in the distance walked in the 6MWT (p < 0.001) and muscle strength scores (p < 0.001). Improvements in both these measures were seen over the first 2 years of treatment with ERT. No statistically significant relationship was found between time on ERT and respiratory function or in BMI. CONCLUSIONS: These data provide some further evidence of the effectiveness of ERT in adults with late-onset Pompe disease. SYNOPSIS: The results of this longitudinal cohort study of 62 adults with late-onset Pompe disease, provide further evidence on the effectiveness of ERT in this rare condition.

Long-term effectiveness of enzyme replacement therapy in Fabry disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapy (ERT) for adults and children with Fabry disease. DESIGN: Cohort study including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment and untreated patients contributed natural history data. PARTICIPANTS: Consenting adults (N = 289) and children (N = 22) with a confirmed diagnosis of Fabry disease attending a specialist Lysosomal Storage Disorder treatment centre in England. At recruitment 211 adults and seven children were on ERT (range of treatment duration, 0 to 9.7 and 0 to 4.2 years respectively). OUTCOME MEASURES: Clinical outcomes chosen to reflect disease progression included left ventricular mass index (LVMI); proteinuria; estimated glomerular filtration rate (eGFR); pain; hearing and transient ischaemic attacks (TIA)/stroke. RESULTS: We found evidence of a statistically significant association between time on ERT and a small linear decrease in LVMI (p = 0.01); a reduction in the risk of proteinuria after adjusting for angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (p < 0.001) and a small increase in eGFR in men and women without pre-treatment proteinuria (p = 0.01 and p < 0.001 respectively). The same analyses in children provided no statistically significant results. No associations between time on ERT and pain, risk of needing a hearing aid, or risk of stroke or TIAs, were found. CONCLUSIONS: These data provide some further evidence on the long-term effectiveness of ERT in adults with Fabry disease, but evidence of effectiveness could not be demonstrated in children.

Long-term effectiveness of enzyme replacement therapy in children with Gaucher disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapies (ERT) for children with Gaucher disease (GD). DESIGN: A longitudinal cohort study including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Children on treatment contributed data before and during treatment. Children not on treatment contributed natural history data. PARTICIPANTS: Consenting children (N = 25, aged 1.1 to 15.6 years) with a diagnosis of GD (14 with GD1 and 11 with GD3) who attended a specialist treatment centre in England. At recruitment, 24 patients were receiving ERT (mean treatment duration, 5.57 years; range 0-13.7 years). OUTCOME MEASURES: Clinical outcomes chosen to reflect disease progression, included platelet count; haemoglobin and absence/presence of bone pain. RESULTS: Duration of ERT was associated with statistically significant improvements in platelet count (p < 0.001), haemoglobin (p < 0.001), and reported bone pain (p = 0.02). The magnitude of effect on haematological parameters was greater in children with GD3 than in those with GD1. CONCLUSIONS: These data provide further evidence of the long-term effectiveness of ERT in children with GD.

The NCS-LSD cohort study: a description of the methods and analyses used to assess the long-term effectiveness of enzyme replacement therapy and substrate reduction therapy in patients with lysosomal storage disorders.

Lysosomal storage disorders (LSDs) comprise more than 50 extremely rare, inherited metabolic diseases resulting from a deficiency of specific lysosomal enzymes required for normal macromolecular metabolism. The National Collaborative Study for Lysosomal Storage Disorders (NCS-LSD), was a longitudinal cohort study which collected prospective and retrospective clinical data, and patient-reported data from adults and children with a confirmed diagnosis of Gaucher disease, Fabry disease, mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), Pompe disease and Niemann Pick disease type C (NPC) in the UK. The study aimed to determine the natural history of these conditions and estimate the effectiveness and cost of therapies. Clinical outcomes were chosen to reflect disease progression. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment while untreated patients contributed natural history data. A total of 711 adults and children were recruited to this study from the seven LSD treatment centres in England. Data was collected from 2008 to 2011. This paper describes the methods used to collect and analyse clinical data for this study. The clinical findings are reported separately in a series of condition-specific articles in this issue.

Long-term effectiveness of enzyme replacement therapy in adults with Gaucher disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapies (ERT) for adults with Gaucher disease (GD). DESIGN: A longitudinal, multi-centre cohort study, including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment. Untreated patients contributed natural history data. PARTICIPANTS: Consenting adults (N = 150, aged 16 to 83 years) with a diagnosis of GD who attended a specialist treatment centre in England. At recruitment, 131 patients were receiving ERT (mean treatment duration, 10.8 years; range 0-18 years). OUTCOME MEASURES: Clinical outcomes chosen to reflect disease progression, included platelet count; haemoglobin; absence/presence of bone pain; spleen and liver volumes and AST levels. RESULTS: One hundred and fifty adults were recruited. Duration of ERT was associated with statistically significant improvements in platelet count (p < 0.001), haemoglobin (p < 0.001), liver and spleen volumes (p < 0.001) and AST levels (p = 0.02). CONCLUSIONS: These data provide further evidence of the long-term effectiveness of ERT in adults with GD.

Selective serotonin reuptake inhibitors for premenstrual syndrome.

BACKGROUND: Severe premenstrual syndrome affects between 3-5% of women of reproductive age. Such severe PMS is classified under the Diagnostic and Statistical Manual of Mental Disorders as premenstrual dysphoric disorder, PMDD. Selective serotonin reuptake inhibitors (SSRIs) are increasingly being used as a front-line therapy for premenstrual syndrome (PMS). A systematic review was undertaken on the efficacy of SSRIs in the management of severe PMS/PMDD, to assess the evidence for this treatment option. OBJECTIVES: The objective of this review was to evaluate the effectiveness of SSRIs in reducing premenstrual syndrome symptoms in women diagnosed with severe premenstrual syndrome. SEARCH STRATEGY: Electronic searches for relevant randomised controlled trials of the Cochrane Menstrual Disorders and Subfertility Group specialised register of controlled trials, Cochrane Controlled Trials Register, MEDLINE, EMBASE and PsychLit were undertaken. References were searched interactively to identify missed trials. Where insufficient data were presented original authors were contacted for further details. SELECTION CRITERIA: All trials were considered in which women with a prospective diagnosis of PMS/ PMDD were randomised to receive SSRIs or placebo in a double blind trial for the treatment of premenstrual syndrome. DATA COLLECTION AND ANALYSIS: 31 randomised controlled trials were identified which reported the use of SSRIs in the management of PMS. 16 trials were excluded, 15 trials were included in the systematic review, and ten trials were included in the main analyses. The reviewers extracted the data independently and standardised mean differences for continuous outcomes were estimated from the data. MAIN RESULTS: The primary analysis of reduction in overall symptomatology included data on 844 women with premenstrual syndrome. SSRIs were found to be highly effective in treating premenstrual symptoms. Secondary analysis showed that they were as effective in treating physical as well as behavioural symptoms. There was no significant difference between trials funded by pharmaceutical companies and those independently funded. Withdrawals due to side effects were 2.5 times more likely to occur in the treatment group, particularly at higher doses. REVIEWER'S CONCLUSIONS: There is now very good evidence to support the use of selective serotonin reuptake inhibitors in the management of severe premenstrual syndrome.

Determination of total menstrual blood loss.

OBJECTIVE: To develop and validate a simple method of measuring total menstrual blood loss using a pictorial representation of blood loss, the menstrual pictogram. DESIGN: A prospective evaluation of total menstrual blood loss measurement by the menstrual pictogram compared to the alkaline hematin technique. SETTING: Academic menorrhagia research clinic. PATIENT(S): One hundred twenty-one women; 62 women complaining of heavy menstrual blood loss, 59 women who considered their menstrual blood loss to be normal. INTERVENTION(S): Participants were asked to complete the menstrual pictogram through the period and collect their feminine hygiene products for an alkaline hematin assessment. MAIN OUTCOME MEASURE(S): Percentage agreement between blood loss measured by the gold standard alkaline hematin method and the menstrual pictogram. Extraneous blood loss was measured using a semiquantitative pictorial method. RESULT(S): The menstrual pictogram had a high level of agreement for blood collected on feminine hygiene products compared with the alkaline hematin method. Some women also lose a significantly large amount of extraneous blood, which is not proportional to the alkaline hematin blood loss assessment. CONCLUSION(S): The menstrual pictogram provides a simple means of measuring menstrual blood loss. It is no longer appropriate to ignore extraneous blood loss, particularly as there is no correlation between extraneous blood loss and that measured on feminine hygiene products.

Efficacy of selective serotonin-reuptake inhibitors in premenstrual syndrome: a systematic review.

BACKGROUND: Selective serotonin-reuptake inhibitors (SSRIs) are increasingly being used as first-line therapy for severe premenstrual syndrome (PMS). We undertook a meta-analysis on the efficacy of SSRIs in this disorder. METHODS: We searched medical and scientific databases, approached pharmaceutical companies, and reviewed citations of relevant articles to identify 29 studies of the use of SSRIs in PMS. 14 were excluded (no placebo group, preliminary report of included trial, or low quality). 15 randomised placebo-controlled trials were included. Information on study design, participants, drugs used and dosing regimens, outcome measures, side-effects, and sources of funding was extracted. Standardised mean differences between treatment and placebo groups were calculated to obtain an overall estimate of efficacy. The primary outcome measure was a reduction in overall PMS symptoms. FINDINGS: The primary analysis included data on 904 women (570 assigned active treatment and 435 assigned placebo, including 101 in crossover trials). The overall standardised mean difference was -1.066 (95% CI -1.381 to -0.750), which corresponds to an odds ratio of 6.91 (3.90 to 12.2) in favour of SSRIs. SSRIs were effective in treating physical and behavioural symptoms. There was no significant difference in symptom reduction between continuous and intermittent dosing or between trials funded by pharmaceutical companies and those independently funded. Withdrawal due to side-effects was 2.5 times more likely in the active-treatment group than in the placebo group. INTERPRETATION: SSRIs are an effective first-line therapy for severe PMS. The safety of these drugs has been demonstrated in trials of affective disorder, and the side-effects at low doses are generally acceptable.

Pancreatic pseudocyst causing extrahepatic biliary obstruction in a dog.

A 3-year-old Rhodesian Ridgeback was examined because of recurrent pancreatitis of 2 months duration. The dog had signs of abdominal pain and jaundice. Blood biochemical findings were consistent with extrahepatic bile duct obstruction, but on abdominal ultrasonography no cause of obstruction was identified. At surgery a pancreatic pseudocyst was found in the body of the pancreas. Cystoduodenostomy, cystic omentalization and biliary diversion resulted in excellent long-term recovery.

Efficacy of vitamin B-6 in the treatment of premenstrual syndrome: systematic review.

OBJECTIVE: To evaluate the efficacy of vitamin B-6 in the treatment of premenstrual syndrome. DESIGN: Systematic review of published and unpublished randomised placebo controlled trials of the effectiveness of vitamin B-6 in the management of premenstrual syndrome. SUBJECTS: Nine published trials representing 940 patients with premenstrual syndrome. MAIN OUTCOME MEASURES: Proportion of women whose overall premenstrual symptoms showed an improvement over placebo. A secondary analysis was performed on the proportion of women whose premenstrual depressive symptoms showed an improvement over placebo. RESULTS: Odds ratio relative to placebo for an improvement in overall premenstrual symptoms was 2.32 (95% confidence interval 1.95 to 2.54). Odds ratio relative to placebo for an improvement in depressive symptoms was 1.69 (1.39 to 2.06) from four trials representing 541 patients. CONCLUSION: Conclusions are limited by the low quality of most of the trials included. Results suggest that doses of vitamin B-6 up to 100 mg/day are likely to be of benefit in treating premenstrual symptoms and premenstrual depression.

The effect of prostanoid precursors and inhibitors on platelet angiotensin II binding.

Pregnancy-induced hypertension is characterised by an imbalance of arachidonic acid metabolites: Prostacyclin (PGI2) is vasodilatory and a potent inhibitor of platelet reactivity. Thromboxane (TXA2) induces vasoconstriction and platelet aggregation. Previous intervention studies have been aimed at increasing vasodilatation and decreasing platelet aggregation using low dose aspirin or dietary manipulation of prostaglandins. The aim of this study was to investigate the value of combining low dose aspirin with dietary fatty acid supplementation and its effects on platelet angiotensin II binding in non-pregnant women. Sixty non-pregnant, healthy female volunteers were recruited and randomly allocated to one of six treatment regimens which included aspirin taken alone and in combination with fish oil or evening primrose oil. A control group took no treatment. Platelet AII binding was determined before and after treatment for 1 month. There was no change in platelet angiotensin II binding after 1 month in the control group or in those who received evening primrose oil or fish oil alone. A significant decrease in binding was found in those who took aspirin in combination with fish oil (P = 0.03). An increase in binding was seen in those who took aspirin only, although this was not statistically significant (P = 0.14). A decrease was found in those who took aspirin in combination with evening primrose oil but again this was not statistically significant (P = 0.07). This study found that the combined effect of low-dose aspirin and fish oil causes a significant decrease in platelet angiotensin II binding not caused by either compound taken alone. Given that angiotensin II exerts its effect in part by direct interaction with vascular AII receptors, (platelets being used as 'models' of vascular myocytes), and that pre-eclampsia is associated with major pathophysiological changes in prostanoid metabolism, these pilot data provide a basis for further investigation.

Ranolazine increases active pyruvate dehydrogenase in perfused normoxic rat hearts: evidence for an indirect mechanism.

Ranolazine has shown anti-anginal efficacy in humans and cardiac anti-ischaemic activity in models, but without affecting haemodynamics or baseline contraction. In isolated normoxic rat hearts, Langendorff-perfused for 30 min with 11 mM glucose, 3% albumin, and 0.4 mM or 0.8 mM palmitate, 20 microM ranolazine significantly increased active, dephosphorylated, pyruvate dehydrogenase (PDHa), but not with no palmitate or 1.2 mM palmitate. Dichloroactetate (DCA, 1 mM), a PDHa kinase inhibitor, significantly increased PDHa in hearts perfused with 0, 0.4 or 0.8 mM but not 1.2 mM palmitate. PDHa was significantly increased with 1.2 mM palmitate by DCA plus ranolazine, and additive effects were also seen at 0.8 mM palmitate. Activation of PDH by ranolazine and promotion of glucose oxidation offers a plausible means by which the drug may be anti-ischaemic nonhaemodynamically. Extensive studies with extracted enzymes and isolated rat heart mitochondria failed to demonstrate any effects of ranolazine on PDH kinase or phosphatase, or on PDH catalytic activity, whereas effects of other known effectors (such as DCA) were readily demonstrable, suggesting that ranolazine activates PDH indirectly. Further analyses of the hearts revealed that ranolazine reduced acetyl CoA content under all conditions where fatty acid was present, and +/- DCA which itself had little effect. In the absence of fatty acid, ranolazine and/or DCA raised acetyl CoA. In perfusions where octanoate (+/- albumin) replaced palmitate, ranolazine still decreased acetyl CoA, but not when acetate replaced palmitate. In octanoate-perfused hearts, the contents of the C4, C6 and C8 CoA esters were all increased by ranolazine. This is consistent with ranolazine causing an inhibition of fatty acid beta-oxidation leading to decreased acetyl CoA and activation of PDH.

On the roles of long-chain acyl carnitine accumulation and impaired glucose utilization in ischaemic contracture development and tissue damage in the guinea-pig heart.

It has been proposed that the presence of increasing concentrations of fatty acids may accelerate the development of ischaemic contracture and cardiac damage, and that this may be due to long-chain acyl carnitine accumulation and/or impairment of glucose utilization. In isolated guinea-pig papillary muscles, palmitoyl (DL) carnitine was found to have a positive inotropic effect, with a slow onset of action suggestive of an intracellular site of action, and with a maximal effect of about two-fold at a concentration of 5-10 microM; higher concentrations led to decreased contraction, probably due to increasing detergent-like effects. In isolated fura-2-loaded chick cardiomyocytes, palmitoyl carnitine increased intracellular [Ca2+]; it is proposed that this is the means by which it increases contraction. The main hypothesis above was studied using isolated guinea-pig hearts perfused with either 11.7 mM or 5 mM glucose, and either albumin alone (3%) or albumin bound palmitate (1.5 mM) during low-flow ischaemia (92% reduction in flow) for up to 60 min. With 11.7 mM glucose, the presence of palmitate caused contracture development and increased enzyme release during ischaemia. Contracture also developed when the glucose concentration was reduced to 5 mM in the absence of fatty acid, however, in its presence contracture developed to a greater extent and with increased enzyme release. Long-chain acyl carnitine accumulation was similar in both groups. These studies show that long-chain acyl carnitine accumulation has the potential to induce contracture during ischaemia, although a reduction in glucose availability may also contribute.

The antianginal agent ranolazine is a weak inhibitor of the respiratory complex I, but with greater potency in broken or uncoupled than in coupled mitochondria.

Ranolazine (RS-43285) has shown antianginal effects in clinical trials and cardiac anti-ischaemic activity in several in vivo and in vitro animal models, but without affecting haemodynamics. Its mechanism is thought to mainly involve a switch in substrate utilisation from fatty acids to glucose to, thus, improve efficiency of O2 use; however, its precise molecular target(s) are unknown. In studies to investigate its action further, using isolated rat heart mitochondria, ranolazine was found to weakly inhibit (pIC50 values > 300 microM) respiration by coupled mitochondria provided with NAD(+)-linked substrates but not with succinate. With broken mitochondrial membranes or submitochondrial particles, ranolazine inhibited NADH but not succinate oxidation and with pIC50 values in the lower range of 3-50 microM. Studies with different electron acceptors and respiratory inhibitors indicated that it inhibits respiratory Complex I at a site between ferricyanide and menadione and ubiquinone-1 reduction (i.e. at a similar locus to rotenone). However, unlike rotenone, ranolazine was an uncompetitive inhibitor with respect to ubiquinone-1. Ranolazine inhibition of Complex I was reversible and occurred also with mitochondria from pig, guinea pig, and human heart, and rat liver. Further studies using rat heart mitochondria in different energisation states (i.e. broken, uncoupled, or coupled) showed a 50-100-fold shift to greater potency of ranolazine in the broken compared to the coupled; with the uncoupled it was about 2-fold less potent than the broken. These shifts in potency were not found with rotenone or amytal. Studies with radiolabelled ranolazine showed that it bound to mitochondrial membranes with greater affinity in the broken compared to the coupled or uncoupled conditions. Rotenone displaced radiolabelled ranolazine from its binding site. This property of ranolazine may play some role in its anti-ischaemic activity.

[3H]-lifarizine, a high affinity probe for inactivated sodium channels.

1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions. 5. The potencies of a series of compounds in displacing [3H]-lifarizine from rat cerebrocortical membranes correlated well with their affinities for inactivated sodium channels estimated from whole-cell voltage clamp studies in the mouse neuroblastoma cell line, NIE-115 (r=0.96).6. These results show that [3H]-lifarizine is a high affinity ligand for neuronal sodium channels which potently and selectively labels a site, allosterically linked to toxin binding site 2, associated within activated sodium channels.