Hypertension and diabetes mellitus are closely interrelated and coexist in as many as two-thirds of patients with type 2 diabetes. The consequent risk of such an association is an accelerated development of atherosclerotic cardiovascular disease and nephropathy complications.In choosing an antihypertensive agent, effectiveness needs to be accompanied by favourable metabolic, cardioprotective, and nephroprotective properties. Given the multifactorial nature of hypertension, the approach that has gained widespread agreement is treatment with more than one agent. Agents with different mechanisms of action increase antihypertensive efficacy because of synergistic impacts on the cardiovascular system. Combination therapy allows the use of lower doses of each antihypertensive agent which accounts for the excellent tolerability of combination products.The aim of the present study is to quantify the efficacy of combination therapy of Eprosartan 600 mg respectively Ramipril 5 mg with low-dose Hydrochlorothiazide and Moxonidine on blood pressure levels in patients with essential hypertension and associated diabetes mellitus type 2.The use of monotherapy (Eprosartan or Ramipril) followed by addition of low-dose Hydrochlorothiazide as second agent and of Moxonidine as a third agent will be individualized to the severity of hypertension in the particular patient and to his/her degree of response to current treatment. Cliniortality . Thus, mre < 130/0 mm Hg iThe challenge clinicians are facing is to tighten blood pressure control to less than 130/80 mmHg and to adjust initiation of therapy to the severity of hypertension in the individual patient.This multicenter study will evaluate the efficacy and tolerability of monotherapy, double- and triple- antihypertensive combination therapies in a large spectrum of hypertension & diabetes patient population, as summarised in Table response to that therapy.Table 2 see  specifieThe primary objectives of hypertension management in patients with diabetes are to reduce blood pressure levels to currently recommended target level and thus to reduce the risk of cardiovascular and renal complications without adversely impacting glycemic and lipid control.Previous debate regarding the level of blood pressure reduction that optimizes cardiovascular risk reduction is currently settled. BP goal of < 130/85 mmHg promoted by the JNC-VI guidelines issued 1997  were repA widespread agreement, supported by the above mentioned organizations/societies is in place, regarding the principles governing the use of appropriate antihypertensive drug combinations to maximize hypotensive efficacy while minimizing side effects. Polypharmacy is common place and, with at least one third of patients requiring two or more agents simultaneously, a paradigm shift in the approach of initiating therapy is done by advocating use of two agents in subjects with more severe hypertension . Low-dose thiazide diuretic is favored as one of the two starting agents.In general, monotherapy is likely to be successful in mild hypertensive patients (grade 1 hypertension) without associated major risk factors for CHD. In contrast, patients with type 2 diabetes need more rigorous control of BP in an easier, simpler fashion, given the remarkable complexity of the multiple drug regimens needed to control their comorbid medical problems (e.g., diabetes, obesity, high cholesterol).A large body of evidence derived from a multitude of international trials have demonstrated both the benefit of low-level, goal blood pressure, in terms of prevention of long-term complications and, the need for multiple drug combinations in order to achieve that goal ,18-20. FIn the HOT study , which rGiven the very poor BP control rate, i.e., 11% in patients with hypertension & diabetes, the use of combination therapy is an important therapeutic consideration, as it facilitates quicker and easier attainment of goal BP and should lead to a greater proportion of people with diabetes who achieve BP goal. Initiation of treatment by combination therapy was effectively tested in the VA study at the beginning of the antihypertensive treatment trial era ,26 and rSubjects will be recruited in outpatient clinics/offices of general practitioners and internal medicine/cardiology specialists from the entire spectrum of patients having coexistent hypertension and diabetes mellitus type 2. The upper limit of blood pressure values targeted (≤ 179/109 mmHg) correspond grade 2 hypertension (according to current ESC/ISH and WHO guidelines).Subjects to be recruited are supposed to have both entities  diagnosed since previously, undergoing current antihypertensive treatment and, to be eligible for the study according to the specific inclusion/exclusion criteria described below.The study consists of five distinct phases: Screening (S) (up to seven days), Placebo Run-in phase (two weeks), monotherapy (four weeks, double-blind fashion), double-combination treatment (Eprosartan/HCTZ respectively Ramipril/HCTZ for four weeks, HCTZ open labelled), triple-combination treatment (Eprosartan/HCTZ/Moxonidine versus Ramipril/HCTZ/Moxonidine with HCTZ and Moxonidine open labelled) and Follow-up (two to seven days). Patients allocated to monotherapy will participate in the study for a four weeks period while those starting with double combination therapy will receive medication for a maximum of 12 weeks.The flowchart below captures the main events during the study conduct Fig. .Randomization will be concealed.A stratified randomization will be employed based on the following rules:1. Subjects with blood pressure in range: BP ≥ 130/80 – ≤ 150/90 mm Hg will be randomly allocated to one of the monotherapy arms (Eprosartan or Ramipril). 30 subjects will be recruited for each arm.2. Subjects with blood pressure in the range: BP > 150/90 – ≤ 179/109 mm Hg will be randomly allocated to double-combination therapy (Eprosartan/HCTZ or Ramipril/HCTZ); 190 subjects will be recruited for each arm.The randomization lists will be provided by the Department of Clinical Supplies at Solvay Pharmaceuticals BV with the program ADLS. Patients will be allocated in equal numbers to each sequence. A fixed block size of patients will be used, and only complete blocks of study medication will be provided to the centers.Within each center, randomization numbers will be used in ascending order and patients will be allocated to randomization code numbers in chronological order. The study will be unblinded when all CRFs are in house and the data on the database have been declared clean.The following drugs are to be used in the study:Eprosartan 600 mg, once daily• Ramipril 5 mg, once daily• Hydrochlorothiazide 12.5 mg, once daily• Moxonidine 0.4 mg, once daily• Placebo tablets matching Eprosartan 600 mg and Ramipril 5 mg will be used in the monotherapy, in the double- and in the triple-combination therapy phases. Hydrochlorothiazide tablets will be open labelled in the double- and triple-combination therapy phases. Moxonidine tablets will open-labelled in the triple-combination therapy phase.• Eprosartan and Ramipril and the corresponding placebos will be packaged according to the double-dummy technique. Fig. Patients eligible for participation in the study by the assessment at V2 will be randomized on the basis of the severity of their hypertension and allocated to either monotherapy or to double-combination therapy (described below).Patients with initial blood pressure values in the range ≥ 130/80 – ≤ 150/90 mmHg, will be randomly allocated to one of the monotherapy groups.By the end of the first four-week phase an assessment will be made as to whether patients have reached or not the "goal" hypertension (< 130/80 mmHg).Patients on monotherapy deemed to be responders at the end of four weeks treatment  will terminate their participation in the study and be followed up during the ensuing two to seven days after stopped monotherapy (procedure similar with Follow-up at end of study (V6).Non-responders (according to above mentioned criteria, i. e., with BP still ≥ 130/80 mmHg), will receive double-combination therapy (Eprosartan/HCTZ respectively Ramipril/HCTZ) and will be followed-up for eight weeks (red-dotted line in the flowchart).Patients with initial blood pressure values in the range > 150/90 – ≤ 179/109 mmHg, will be randomly allocated to double-combination therapy. By the end of this first four-week phase an assessment will be made as to whether patients have reached or not the "goal" hypertension (< 130/80 mmHg).Responders will maintain double-combination therapy and will be followed-up for an eight week period (V3 toV5, dotted-line in the flowchart) and retain therapy unchanged.Non-responders will receive triple-combination, as described below.Non-responder patients after four weeks of double-combination therapy will receive triple-combination (Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine) and will be followed-up for an eight weeks period (V3 to V5).After the first four weeks of monotherapy respectively double-combination therapy (V3), patients will be reassessed for compliance, adverse events and supplied with medication for the next eight weeks .During the eight weeks triple-combination therapy all patients will be reassessed for compliance and adverse events (visits V4, V5 and V6). A 12-lead ECG will be performed by S, V3 and V5 while safety laboratory parameters will be performed by S, V2 and finally by visit V5.A Follow-up Visit will be performed on all patients with full physical examination, BP and pulse rate check within the ensuing two to seven days after study end (V6). Further follow-up and optimal treatment will be decided on a case-by-case basis by the physician in charge. Table 3 see  displays1. Males and females aged 40 to 80 years of age. Women of childbearing age will be subject to pregnancy testing and will agree to maintain adequate hormonal contraception.2. Eligible patients should have diagnosed essential hypertension (not controlled with current treatment, i.e., BP ≥ 130/80 – ≤ 179/109 mmHg) and diagnosed associated diabetes mellitus type 2, willing to accept withdrawal of any antihypertensive medication by the time of the Screening visit.A multitude of exclusion criteria, carefully listed in the study protocol, can be summarised in three different groups:1. Ineligibility based on hypertension grade 3 (BP ≥ 180/110 mmHg), any form of secondary hypertension or hypotension (SBP ≤ 90 mmHg).2. Any form of organic heart disease requiring medical treatment that might have hypotensive effect, imply need for invasive investigation or surgery.3. The patient is suffering from a severe concomitant illness related to any body organ or system, likely to affect outcome assessment. Likewise, ineligibility is declared for patient anticipated to have compliance problems, participants in another trial during the past 30 days, pregnancy and lactations and known hypersensitivity to ingredients of any of the employed agents (eprosartan, ramipril, hydrochlorothiazide, moxonidine). In addition, diabetes mellitus type 1 is exclusion criteria.lifestyle change advice regarding relevant diet and physical activity. Visits are to be scheduled in the context of the study . Any antihypertensive medication should be withdrawn latest by Screening visit and will be prohibited during the whole period of ongoing study.The study protocol calls for every patient to be treated optimally by the physician in charge and to receive comprehensive, individualized The study will be conducted in accordance with ICH GCP and the European Directive 2001/20/EC of the European Parliament and of the Council of 4 April 2001  and on the basis of ethical principles laid down in the current revision of the Declaration of Helsinki (Edinburgh 2000). In addition, Solvay Pharmaceuticals GmbH policies and procedures should also be followed.Written consent, involving provision of detailed information regarding the study objectives, design, scope of the intervention, risks and benefits, will be obtained for all patients before initiating any study procedures. Likewise, study documentation is to be subject to the scrutiny of local ethical committees in the two countries participating in the study.goal blood pressure (<130/80 mmHg) at visit V3.The primary objective is to demonstrate the superiority of combination therapy of Eprosartan/HCTZ (600/12.5 mg) versus Ramipril/HCTZ (5/12.5 mg) with the primary parameter of attention being the percentage of patients brought to 0: PE+HCTZ = PR+HCTZNull hypothesis: H1: PE+HCTZ ≠ PR+HCTZ,Alternative hypothesis: HE+HCTZ is the percentage of patients brought to goal blood pressure at visit 3 with Eprosartan/HCTZ and PR+HCTZ is the percentage of patients brought to goal blood pressure at visit V3 with Ramipril/HCTZ.Where PThe primary parameter will be analyzed using the Cochran-Mantel-Haenszel test, controlling for center effects. Statistical significance will be assessed with a two-sided test at 0.05 α level. The confirmative analysis of the primary parameter will be performed on the intent-to-treat patient sample.Secondary efficacy objectives:• To compare the mean change in sitting systolic blood pressure (sitSBP) and sitting diastolic blood pressure (sitDBP) between Eprosartan/HCTZ and Ramipril/HCTZ (V3 vs. V2).• To compare the percentage of patients brought to goal blood pressure by the triple-combination Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine (V5 vs. V3).• To compare the mean change in sitSBP and sitDBP between triple-combination with Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine (V5 vs. V3).• To compare the mean change in sitSBP and sitDBP between monotherapy with Eprosartan vs. Ramipril (V3 vs. V2)• To compare the percentage of patients brought to goal blood pressure at visit V5 between Eprosartan/HCTZ and Ramipril/HCTZ in patients not at goal blood pressure after four weeks of monotherapy.at goal) after four weeks of monotherapy (switched to double combination therapy) (V5 vs.V3); and to compare the mean change in sitSBP and sitDBP as well as the responder rate maintenance in patients who reached goal blood pressure value at the end of first four weeks of double-combination therapy and successively entered an eight weeks follow-up period (V5 vs.V3).• To compare the mean change in sitSBP and sitDBP as well as the responder rate in patients non-responders . The model will include the intercept, treatment and center as fixed effects and the baseline value as covariate. For response rates, the treatment groups will be compared using the Cochran-Mantel-Haenszel test, controlling for center effects. Comparisons of the medication regimens will be reported along with 95% confidence intervals of the relative risk ratios. These analyses will be considered as exploratory.All patients who receive at least one dose of double-blind medication will be assessed for clinical safety and tolerability. Evaluation of safety data will be based on comparisons of patient experience by treatment group. Clinical interpretation of safety will be based on reviews of standard displays of adverse events incidence, pulse rate data, and laboratory test values. Summary statistics of laboratory test values and incidence of adverse events according to treatment and time of onset will be presented.A formal sample size estimation has been done for patients with blood pressure in the range: > 150/90 mmHg and ≤ 179/109 mmHg.goal blood pressure as compared with only 40% in the Ramipril/HCTZ group, a 0.05% two-sided significance level with 80% power to detect the targeted 15% difference will imply the need for 346 patients supposed to complete the four-weeks double-combination therapy phase. Further 35 patients  will be recruited to account for drop-outs.Assuming that 55% of the patients in the Eprosartan/HCTZ group would reach In addition, 60 subjects with BP ≥ 130/80 and ≤ 150/90 mmHg will be randomly allocated to either Eprosartan or Ramipril monotherapy group at visit V2. Inclusion of monotherapy phase with a relatively low number of patients (30 subjects per arm) is justified by the intention to therapeutically target the whole spectrum of patient population having coexistent diabetes mellitus type 2 and mild to moderate hypertension, in whom the agents tested are likely to be effective.Office blood pressure will be determined by Riva-Rocci method with a mercury or a mercury calibrated sphygmomanometer throughout the study. All measurements will be made on the same arm supported at heart level, using the same cuff size and the same equipment. If the patient's arm circumference is > 32 cm, a large blood pressure cuff should be used. Diastolic blood pressure will be measured at the disappearance of Korotkoff sounds phaseV. Measurements should be taken by the same staff member at the particular visits.For an individual patient blood pressure measurements should be performed at 24 hours after the last oral dose, at the same time (± 2 hour) in the morning, between 8 and 10 am. Blood pressure will be measured in the following sequence: after the patient sits quietly for at least 5 minutes, blood pressure will be measured twice at approximately 2-minutes interval. The average of these measurements will be recorded. If the difference between measurements is in excess of 5 mmHg a third reading will be performed and the average value recorded as mean sitting systolic and diastolic blood pressure.Measurements should be performed by the same study assistant using the same device, in each of the centres involved in the study.goal blood pressure to be achieved in these patients. Further, common sense in clinical practice dictates that combination therapies should be tailored to severity of hypertension in the individual patient and that, eventual associated risk factors/comorbidities should be accounted for in the process of treatment decision making.The current evidence base is strongly in favour of combining drugs in order to achieve blood pressure goals, in particular in patients with coexistent hypertension & diabetes. Likewise, there is a widespread agreement in the scientific community as to the Patients with high blood pressure and associated impaired glucose tolerance or overt diabetes mellitus type 2, as a group, are insulin resistant,  glucose For every 5 to 10 mmHg decrease in systolic blood pressure achieved with diuretics, ARBs, ACE inhibitors, beta-blockers or calcium channel blockers in patients with diabetes, there is a 20% to 30% relative risk reduction in cardiovascular events -53.Agents belonging to the nine, most well-known different antihypertensive drug classes produce a similar reduction in systolic and diastolic blood pressure (10–15 and 5–10 mm Hg respectively). Differences in terms of magnitude of blood pressure lowering, as indicated by results from comparative efficacy studies, are usually small . HoweverComparisons between different agents in patients with hypertension & diabetes mellitus type 2, convincingly point to ACE inhibitors and ARBs as being the two classes of antihypertensive drugs that reduce the activity of the renin-angiotensin II system, and should be among the preferred first-step drugs for the treatment of these conditions . AngioteClinical trials carried out world-wide have shown that ACE inhibitors have renoprotective effects ,58 and cIn contrast, in placebo-controlled trials, the ARBs have demonstrated almost no side effects . Both ACUse of low-dose thiazide diuretic (< 25 mg) as a second agent in treatment of patients with hypertension & diabetes is well-documented and widely recommended ,70-73. ISince drug combinations may be required for many years in the age-groups in which type 2 diabetes is most prevalent, there have been calls for the use of agents devoid of adverse effects on carbohydrate and lipid metabolism. It has been suggested that such effects may account for the shortfall in reduction of coronary heart disease observed in clinical trials of diuretics and β-blockers ,81.1 receptors in the medulla, thereby reducing central sympathetic drive and attenuating peripheral vascular resistance. In addition, reduced sympathetic drive results in lower plasma concentrations of catecholamines and renin. Randomised comparative studies show that the efficacy of moxonidine as monotherapy is similar to that of other antihypertensive agents [1 receptor greatly reduces the adverse affects attributable to costimulation of medullary α2-adrenoceptors [Moxonidine stimulates imidazoline-Ie agents . Moreoveoceptors  observedIn clinical studies, moxonidine has been shown to have neutral or beneficial effects on lipid and carbohydrate metabolism ,83. A reOn the available evidence, moxonidine seems to be a logical choice as component of combination treatment of patients with hypertension and associated diabetes mellitus type 2 or impaired glucose tolerance.The poor blood pressure control in patients with hypertension & diabetes in everyday life lies, at least in part, in the emphasis in the evidence-based guidelines of the recent past towards advice on initial, single treatments as well as in their lack of clarity and transparency in recommending pre-specified blood pressure targets ,84-86.goal blood pressure and thereby to minimize long term risk of hypertension/diabetes-related complications [Previous consensual advice that combination treatments expose patients to the increased risk of adverse events has been replaced by good evidence to the contrary: use of several agents combined or of fixed-dose combinations treatments have the potential to bring patients to ications -27,87.blood pressure goal and individualized therapy on the basis of hypertension severity (and addition of a third agent in case of uncontrolled BP with two agents), comparative data to guide clinical practice is still lacking and this applies also to the comparison of ARBs versus ACE inhibitors. The present study attempts to explore this area.Despite the apparent simplicity of the paradigm shift towards clear Pater C, Berrou JP, Luszick J and Beckman K are employees of Solvay Pharmaceuticals.Study concept and design: Pater, Berrou, LuszickDrafting of manuscript: Pater, BhatnagarStatistical expertise: BeckmanTable 2 – Blood pressure-adjusted treatment stratificationClick here for fileTable 3 – Investigations ScheduleClick here for file


Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD344 receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4) in CD34+ stem and progenitor cells.Using a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.The most interesting findings were the high mRNA expression of P2Y So fa, P2Y14) ; the P2X12 and P2Y1, together with the ATP receptor P2X1 [12 antagonist clopidogrel for the prevention of myocardial infarctions in patients with acute coronary syndromes [7 receptor mRNA in monocytes has been observed upon cell differentiation to macrophages [We have previously quantified P2 receptor mRNA expression in platelets (representing megakaryocyte expression), and demonstrated a selective expression of the ADP receptors P2Ytor P2X1 . This isyndromes ,10. Howeyndromes . Circularophages ,14.+ stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory ,2. Few p+ stem and progenitor cells.Our previous studies of P2 receptor mRNA expression in man with real-time PCR has shown a good resemblance with pharmacological and physiological experiments in vascular smooth muscle cells, endothelial cells and platelets ,15. It i1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 could be detected (n = 6). To illustrate expression of the P2 receptors relative to each other the P2Y1 receptor was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y1. Among the P2Y receptor subtypes the P2Y12 and P2Y2 had highest expression  and P2X7(P < 0.01).The most abundant P2X receptor in lymphocytes was the P2X7 in lymphocyte regulation. However, B lymphocytes stimulated with ATP do not undergo the typical increase in permeability up to 900 Da that is typical for the P2X7 receptor. On the other hand, P2X7 mediated effects on Ba2+ and ethidium influx, phospholipase D activity and shedding of L-selectin have been blocked by the P2X7 selective antagonist KN-62 in human lymphocytes [4 receptor was the highest expressed subtype in lymphocytes at the mRNA level. Even though we have demonstrated that more than 90% of the preparation consists of lymphocytes (see methods), it is possible that a small contamination of monocytes may have influenced the results, at least regarding P2X4 receptor mRNA expression, because of its high expression levels in monocytes. P2X4 receptors have indeed been demonstrated at the protein level in human B lymphocytes by confocal immunohistochemistry, in which P2X1, P2X4 and P2X7 were detected at the protein level [4 receptor staining was the most variable of the P2X receptors with weak to moderate levels of staining in a large proportion of cells in three patients and weak levels in only a minority of the cells from the other three patients examined [Selective pharmacological tools to discriminate between P2X receptors are scarce. Nevertheless, several studies have suggested the importance of P2Xphocytes . Thus itin level . Howeverexamined .1 expression was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y1. Among the P2Y receptors, the P2Y2, P2Y13 and P2Y11 had highest expression  and P2X7 (P < 0.01) had lower levels. Thus, unexpectedly the P2X7 receptor was not the highest expressed P2X receptor in monocytes. This is in agreement with patch-clamp experiments suggesting that other P2X receptors are involved [4 receptors, which is supported by our findings [4 and 6–7-fold more for P2X1 and P2X7). A physiological role for all three subtypes can therefore be expected.As showed in Figure involved . Interrefindings . It shou+ stem and progenitor cells are receiving an increasing attention because of their extensive self-renewal and multilineage differentiation ability making them attractive for cellular therapy [+ stem and progenitor cells. We found expression of several P2Y receptors, especially P2Y1 and P2Y2   Figure , n = 3, poptosis . Such ro4 receptor had the highest mRNA expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. The most interesting findings were the high mRNA expression of P2Y12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns have been studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.The P2XThe studies were approved by the local Ethics Committee of the Lund University and were conducted according to the principles of the Declaration of Helsinki.Peripheral blood was drawn from each of 6 healthy volunteers (after informed consent) into heparin vials. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep™ (Axis Shield Poc AS, Oslo, Norway) at 605 g for 30 minutes. The lymphocytes and monocytes thus obtained were washed three times in RPMI 1640 medium with L-glutamine (Gibco/BRL, Life Technologies Ltd, Paisleys, Scotland) and 0.1% human serum albumin (Sigma, St Louise, MO, USA), (medium), and centrifuged each time at 605 g for 5 minutes.6 cells/ml. Flow cytometry (Epics XL-MCL Beckman-Coulter, Florida, USA) analysis on these cells by detection of cell surface CD14 and CD45 showed that approximately 10% of the cells were monocytes. 800 μl of this cell-suspension was plated on a chamber slide 4 well glass slide  at 37°C in an atmosphere containing 5% CO2 and 96% humidity for 1 h in order for the monocytes to adhere. Nonadherent cells were removed by washing three times with medium. Flow cytometry analysis of these nonadherent cells showed that at least 90% were lymphocytes, and were therefore used as source of lymphocytes. The cells attached to the glass slides (<90% monocytes as assessed by flow cytometry) were detached by adding first PBS and then 0.5 mM EDTA-PBS for 3 min in room temperature.The fraction of lymphocytes and monocytes obtained according to this procedure was resuspended in medium with 15% normal human serum (NHS) added to a concentration of 4 × 10+ cells were isolated by 2passages through magnetic columns  by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads . CD34 expression was analyzed by immunostaining with a FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest program (Becton Dickinson) and the purity of isolated populations was reproducibly > 95% [Bone marrow samples were obtained from healthy volunteers (n = 3), after informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation . CD34ly > 95% .Total cellular RNAs were extracted using TRIzol reagent (Gibco BRL, Life Technology) according to the supplier's instructions, dissolved in diethyl-pyrocarbonate (DEPC) treated water and stored at -70°C until used.TaqMan Reverse Transcription Reagents Kit was used to transcribe mRNA into cDNA. Real-time PCR were performed by means of a PRISM 7700Sequence Detector as described previously ,15,28,291, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2X1, P2X4, and P2X7 normalized to GAPDH [T method for the relative quantification of target without running standard curves on the same plate . The amount of target and endogenous reference was determined from the comparative CT method. The target gene normalized to GAPDH was expressed as ΔCT (CT of target gene minus CT of GAPDH). P2Y1 was arbitrarily chosen to be the calibrator in the comparative analysis and is expressed as ΔCTP2Y1 (CT of target minus CT of GAPDH for P2Y1). The normalized calibrated value is given by the equation 2-ΔΔCt, where ΔΔCT is ΔCT -ΔCTP2Y1. To further verify the specificity of PCR assays, the PCR was performed with non-reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown).Previous analysis showed that amplification efficiencies were almost identical for GADPH and the following receptor mRNAs: P2Yto GAPDH ,15. To cUnless otherwise stated, all reagents and drugs were purchased from Sigma Chemical Corp, St. Louis, MI, USA. PCR consumables were obtained from Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA.T values (ΔCT) was performed with a one-way ANOVA, followed by a multiple comparison post test (Tukey's test) using GraphPad InStat version 3.00 (GraphPad Software Inc., USA). Significant differences were considered at P < 0.05 (two-tailed test).Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. n indicates the number of subjects that were tested. Statistical analysis of the normalized CLW designed the study, carried out the RNA isolation and real-time PCR, and wrote the manuscript.+ stem and progenitor cells, and participated in writing the manuscript.SEWJ supervised the isolation of CD34AB supervised the isolation of monocytes and lymphocytes, and participated in writing the manuscript.DE conceived the study, guided throughout the study, and wrote the manuscript.All authors read and approved the final manuscript.


RNA interference (RNAi) is used in the lab to silence virtually any gene. Previously, antisense and ribozymes were successful in the lab, but have been disappointing in the clinic. Will RNAi succeed where these two have not? Science as the “Breakthrough of the Year” in 2002 break out of the lab and lead to novel therapies as well? Pharmaceutical giants are hoping so, and several biotech companies have bet their futures on it, but not everyone is sanguine about the future of RNAi therapy.RNA interference (RNAi) has been called “one of the most has exciting discoveries in biology in the last couple decades,” and since it was first recognized by Andrew Fire et al. in 1998, it has quickly become one of the most powerful and indispensable tools in the molecular biologist's toolkit. Using short double-stranded RNA (dsRNA) molecules, RNAi can selectively silence essentially any gene in the genome. It is an ancient mechanism of gene regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a central role in controlling gene expression in all eukaryotes. In the lab, RNAi is routinely used to reveal the genetic secrets of development, intracellular signaling, cancer, infection, and a full range of other phenomena. But can the phenomenon hailed by the journal At the heart of its promise as a powerful therapeutic drug lies the exquisite selectivity of RNAi: like the fabled “magic bullet,” an RNAi sequence seeks out and destroys its target without affecting other genes. The clinical applications appear endless: any gene whose expression contributes to disease is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer's disease, diabetes, and more.But for all its promise, RNAi therapy is a long way from entering the clinic. While it is a proven wunderkind in the lab, to date no tests have been done in humans, and only the most modest and circumscribed successes have been demonstrated in animals. The road to clinical success is littered with great ideas that have come a cropper along the way, including two other RNA-based therapies, antisense and ribozymes, both of which showed promise at the bench but have largely stumbled before reaching the bedside. Is RNAi also likely to fall short? Or is it different enough to make this third try the charm?To be a successful drug, a molecule must overcome a long set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Then, and even more importantly, it must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells, link up with its intracellular target, and exert its effect; and it must exert enough of an effect to improve the health of the person taking it. And, finally, it must do all this without causing significant toxic effects in either target or nontarget tissues. No matter how good a compound looks in the lab, if it fails to clear any one of these hurdles, it is useless as a drug.For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery, stability, and potency have been the most significant obstacles. “There was a lot of clinical naïveté” in the early days of antisense and ribozymes, according to Nassim Usman, Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado. “Compounds were pushed into the clinic prematurely.” Sirna began as the biotech startup Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions, including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it to cleave specific target RNA molecules see . “The inSimilarly, despite much initial enthusiasm, attempts to develop antisense drugs have been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents translation of the mRNA see . At leasBut as long as patients were getting better, does it matter what the mechanism was? “It doesn't matter if you are a patient, but it does matter if you are trying to develop the next drug,” says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia University College of Physicians and Surgeons in New York City. Stein has researched antisense for more than a decade. “If you get the mechanism wrong, you're not going to be able to do it.”To date, only one antisense drug has received FDA approval. Vitravene, from Isis Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral DNA, though, says Usman, “it's unclear whether it actually works by an antisense mechanism.” Stein expresses a similar skepticism about the mechanism of a second antisense drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells, which is thought to protect them from standard chemotherapy. The FDA is currently reviewing an application for Genasense, based on promising results in the treatment of malignant melanoma.Growing disillusionment with antisense and ribozymes coincided with the discovery of RNAi and the realization that it was a far more potent way to silence gene expression. RNAi uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs) see . siRNAs The extraordinary selectivity of RNAi, combined with its potency—in theory, only a few dsRNAs are needed per cell—quickly made it the tool of choice for functional genomics (determining what a gene product does and with what other products it interacts) and for drug target discovery and validation. By “knocking down” a gene with RNAi and determining how a cell responds, a researcher can, in the course of only a few days, develop significant insight into the function of the gene and determine whether reducing its expression is likely to be therapeutically useful. But does RNAi have a better chance to succeed as a drug than antisense or ribozymes?“The fundamental difference favoring RNAi is that we're harnessing an endogenous, natural pathway,” says Nagesh Mahanthappa, Director of Corporate Development at Alnylam Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is the main reason RNAi is orders of magnitude more potent than either of the other two types of RNA strategies.More potent in the test tube, at least. But stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body's defenses against infection with foreign nucleotides. “For the strongest reasons, you can't get away from this,” says Stein. “The problem is that a charged oligonucleotide will not pass through a lipid layer,” which it must do in order to enter a cell. John Rossi, Director of the Department of Molecular Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based therapies for 15 years, concurs. “The cell doesn't want to take up RNA,” he says, which makes evolutionary sense, since extracellular RNA usually signifies a viral infection. Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or degraded by enzymes.To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa thinks the complexing approach is unlikely to be a simple solution, since drug approval would require independent testing of the lipid delivery system as well. Instead, Alnylam is pursuing backbone modification. “Some minimal modification is going to be necessary” to increase cell uptake and to improve stability in the blood stream, Mahanthappa says. “What we have learned from the antisense field is that even without other delivery strategies, when you administer RNA at sufficient doses, if it's stable, it gets taken up by cells.”“Anything that can be done to increase half-life in circulation would improve delivery,” says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice, protecting them from fulminant hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection into the tail. The RNA got to the liver, silenced Fas, and protected the mice from hepatitis. However, a significant fraction of animals died of heart failure, brought on because the injection volume was about 20% of the mouse blood volume. Such a delivery scheme simply will not work in humans. “Delivery to the cell is still an obstacle,” Lieberman explains. “Unless you really focus on how to solve that problem, you're not going to get very far.”Even assuming delivery problems can be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Mahanthappa says, “The conservative answer is we just don't know. The more aggressive answer is there's no reason to think so.” Rossi isn't so sure. “The target of interest may be in normal cells as well as cancer cells,” he says. “That's where you get toxicity.”But if small RNAs can be delivered to target cells efficiently and without significant toxicity, will they be effective medicines? Usman of Sirna is confident they will be. “If you can get it there, and if it's in one piece, there no doubt in our minds that it will work,” he says. To date, numerous experiments in animal models suggest RNAi can downregulate a variety of target genes effectively. However, there are still two unanswered questions about whether that will translate into effective therapy.The first is whether RNAi's exquisite specificity is really an advantage beyond the bench. “It's unclear whether highly specific drugs give you a big therapeutic effect,” says Cy Stein. For instance, he says, “most active antitumor medicines have multiple mechanisms of action. The more specific you make it, the less robust the therapeutic activity is likely to be.” Rossi agrees: “Overspecificity has never worked,” he says.The second question is what effect an excess of RNA from outside the cell will have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. “We are usurping a natural cell system that is there for some other purpose, for knocking out endogenous gene function,” says Rossi. With the introduction of foreign RNA, will the system continue to perform its normal function as well, or will it become saturated? “That's the big black box in the field,” he says.Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably only likely to be answered in the process of therapeutic development. The first applications are likely to be in cancer (targeting out-of-control oncogenes) or viral infection . To avoid some of the problems of delivery, initial trials may deliver the RNA by direct injection into the target tissue (for a tumor, for instance) or ex vivo, treating white blood cells infected with HIV, for example.Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the rough road it faces. “We haven't solved all the problems, but we know how to proceed to work through them. It's no surprise to us—we've seen this movie before.” Usman expects Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and to have a human clinical trial in progress in 2005. “Without a doubt, there will be an RNAi-based drug within ten years,” Usman predicts.Stein isn't so sure and thinks that too much is still to be learned about RNAi and the body's reaction to it to be confident that RNA-based therapies will ultimately be successful. “The whole field was founded on the belief it was rational, but there are huge gaps in our knowledge, and so you need a bit of luck to be successful,” he says. “I think people are surprised at how complicated it is, but why should it be any other way? It's an intellectual conceit to think that nature is simple.”


Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. Isolation, separation and purification of various types of proteins and peptides, as well as of other specific molecules, is used in almost all branches of biosciences and biotechnologies. Separation science and technology is thus very important area necessary for further developments in bio-oriented research and technology. New separation techniques, capable of treating dilute solutions or solutions containing only minute amounts of target molecules in the presence of vast amounts of accompanying compounds in both small and large-scale processes, even in the presence of particulate matter, are necessary.In the area of biosciences and biotechnology the isolation of proteins and peptides is usually performed using variety of chromatography, electrophoretic, ultrafiltration, precipitation and other procedures, affinity chromatography being one of the most important techniques. Affinity ligand techniques represent currently the most powerful tool available to the downstream processing both in term of their selectivity and recovery. The strength of column affinity chromatography has been shown in thousands of successful applications, especially in the laboratory scale. However, the disadvantage of all standard column liquid chromatography procedures is the impossibility of the standard column systems to cope with the samples containing particulate material so they are not suitable for work in early stages of the isolation/purification process where suspended solid and fouling components are present in the sample. In this case magnetic affinity, ion-exchange, hydrophobic or adsorption batch separation processes, applications of magnetically stabilized fluidized beds or magnetically modified two-phase systems have shown their usefulness.The basic principle of batch magnetic separation is very simple. Magnetic carriers bearing an immobilized affinity or hydrophobic ligand or ion-exchange groups, or magnetic biopolymer particles having affinity to the isolated structure, are mixed with a sample containing target compound(s). Samples may be crude cell lysates, whole blood, plasma, ascites fluid, milk, whey, urine, cultivation media, wastes from food and fermentation industry and many others. Following an incubation period when the target compound(s) bind to the magnetic particles the whole magnetic complex is easily and rapidly removed from the sample using an appropriate magnetic separator. After washing out the contaminants, the isolated target compound(s) can be eluted and used for further work.Magnetic separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube or another vessel. There is no need for expensive liquid chromatography systems, centrifuges, filters or other equipment. The separation process can be performed directly in crude samples containing suspended solid material. In some cases (e.g., isolation of intracellular proteins) it is even possible to integrate the disintegration and separation steps and thus shorten the total separation time . Due to Magnetic separation is usually very gentle to the target proteins or peptides. Even large protein complexes that tend to be broken up by traditional column chromatography techniques may remain intact when using the very gentle magnetic separation procedure . Both thSeparation of target proteins using standard chromatography techniques often leads to the large volume of diluted protein solution. In this case appropriate magnetic particles can be used for their concentration instead of ultrafiltration, precipitation etc. .The purpose of this review is to summarize various methodologies and strategies which can be employed for the isolation and purification of target proteins and peptides with the help of magnetic materials. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. All these information will help the scientists to select the optimal magnetic material and the purification procedure.The basic equipment for laboratory experiments is very simple. Magnetic carriers with immobilized affinity or hydrophobic ligands, magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) or magnetic ion-exchangers are usually used to perform the isolation procedure. Magnetic separators of different types can be used for magnetic separations, but many times cheap strong permanent magnets are equally efficient, especially in preliminary experiments.Magnetic carriers and adsorbents can be either prepared in the laboratory, or commercially available ones can be used. Such carriers are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers or porous glass, or magnetic particles based on the inorganic magnetic materials such as surface modified magnetite can be used. Many of the particles behave like superparamagnetic ones responding to an external magnetic field, but not interacting themselves in the absence of magnetic field. This is important due to the fact that magnetic particles can be easily resuspended and remain in suspension for a long time. In most cases, the diameter of the particles differs from ca 50 nm to approx. 10 μm. However, also larger magnetic affinity particles, with the diameters up to millimetre range, have been successfully used [lly used . MagnetiCommercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used as a polymer matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also available. Examples of magnetic particles used (or usable) for proteins and peptides separation can be found elsewhere -7.Particles with immobilised affinity ligands are available for magnetic affinity adsorption. Streptavidin, antibodies, protein A and Protein G are used most often in the course of protein and peptides isolation. Magnetic particles with above mentioned immobilised ligands can also serve as generic solid phases to which native or modified affinity ligands can be immobilised (e.g., antibodies in the case of immobilised protein A, protein G or secondary antibodies, biotinylated molecules in the case of immobilised streptavidin).2 are used for immobilisation, in some cases magnetic particles are available already in the activated form (e.g., tosylactivated, epoxyactivated etc).Also some other affinity ligands (e.g., nitrilotriacetic acid, glutathione, trypsin, trypsin inhibitor, gelatine etc.) are already immobilised to commercially available carriers. To immobilise other ligands of interest to both commercial and laboratory made magnetic particles standard procedures used in affinity chromatography can be employed. Usually functional groups available on the surface of magnetic particles such as -COOH, -OH or -NHIn the laboratory magnetite  particles can be surface modified by silanization. This process modifies the surface of the inorganic particles so that appropriate functional groups become available, which enable easy immobilisation of affinity ligands . In exceBiopolymers such as agarose, chitosan, kappa carrageenan and alginate can be easily prepared in a magnetic form. In the simplest way the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed is mechanically broken into fine particles . AlternaBasically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide, poly and many others .In another approach used standard affinity or ion-exchange chromatography material was post-magnetised by interaction of the sorbent with water-based ferrofluid. Magnetic particles accumulated within the pores of chromatography adsorbent thus modifying this material into magnetic form ,13. AlteMagnetoliposomes (magnetic derivatives of standard liposomes), either in the original form or after immobilization of specific proteins, have the potential for the separation of antiphospholipid antibodies , IgG antRecently also non-spherical magnetic structures, such as magnetic nanorods have been tested as possible adsorbent material for specific separation of target proteins .Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof material. The racks are constructed for separations in Eppendorf micro-tubes, standard test tubes or centrifugation cuvettes, some of them have a removable magnetic plate to facilitate easy washing of separated magnetic particles. Other types of separators enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from larger volumes of suspensions (up to approx. 500 – 1000 ml). Examples of typical batch magnetic separators are shown in Fig. Flow-through magnetic separators are usually more expensive, and high gradient magnetic separators (HGMS) are the typical examples. Laboratory scale HGMS is composed from a column packed with fine magnetic grade stainless steel wool or small steel balls which is placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic particles are retained within the matrix. After removal the column from the magnetic field, the particles are retrieved by flow and usually by gentle vibration of the column.For work in dense suspensions, open gradient magnetic separators may be useful. A very simple experimental set-up for the separation of magnetic affinity adsorbents from litre volumes of suspensions was described .Currently many projects require the analysis of a high number of individual proteins or variants. Therefore, methods are required that allows multiparallel processing of different proteins. There are several multiple systems for high throughput nucleic acid and proteins preparation commercially available. The most often used approach for proteins isolation is based on the isolation and assay of 6xHis-tagged recombinant proteins using magnetic beads with Ni-nitriloacetic acid ligand . The comMagnetic separations of proteins and peptides are usually convenient and rapid. Nevertheless, several hints may be helpful to obtain good results.Proteins and peptides in the free form can be directly isolated from different sources. Membrane bound proteins have to be usually solubilized using appropriate detergents. When nuclei are broken during sample preparation, DNA released into the lysate make the sample very viscous. This DNA may be sheared by repeated passage up and down through a 21 gauge hypodermic syringe needle before isolation of a target protein. Alternatively, DNase can be added to enzymatically digest the DNA.Magnetic beads in many cases exhibit low non-specific binding of non-target molecules present in different samples. Certain samples may still require preclearing to remove molecules which have high non-specific binding activity. If preclearing is needed, the sample can be mixed with magnetic beads not coated with the affinity ligand. In the case of immunomagnetic separation, magnetic beads coated with secondary antibody or with irrelevant antibodies have been used. The non-specific binding can also be minimised by adding a non-ionic detergent both in the sample and in the washing buffers after isolation of the target.In general, magnetic affinity separations can be performed in two different modes. In the direct method, an appropriate affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting the affinity towards target compound(s) is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilised secondary antibodies, protein A or protein G are used for capturing of the complex. Alternatively the free affinity ligands can be biotinylated and magnetic particles with immobilised streptavidin or avidin are used to capture the complexes formed. In both methods, magnetic particles with isolated target compound(s) are magnetically separated and then a series of washing steps is performed to remove majority of contaminating compounds and particles. The target compounds are then usually eluted, but for specific applications  they can be used still attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA etc.The two methods perform equally well, but, in general, the direct technique is more controllable. The indirect procedure may perform better if affinity ligands have poor affinity for the target compound.In most cases, magnetic batch adsorption is used to perform the separation step. This approach represents the simplest procedure available, enabling to perform the whole separation in one test-tube or flask. If larger magnetic particles (with diameters above ca 1 μm) are used, simple magnetic separators can be employed. In case magnetic colloids (diameters ranging between tens and hundreds of nanometres) are used as affinity adsorbents, high-gradient magnetic separators have usually to be used to remove the magnetic particles from the system.Alternatively magnetically stabilised fluidised beds (MSFB), which enable a continuous separation process, can be used. The use of MSFB is an alternative to conventional column operation, such as packed-bed or fluidised bed, especially for large-scale purification of biological products. Magnetic stabilisation enables the expansion of a packed bed without mixing of solid particles. High column efficiency, low pressure drop and elimination of clogging can be reached ,22.Also non-magnetic chromatographic adsorbents can be stabilized in magnetically stabilized fluidized beds if sufficient amount of magnetically susceptible particles is also present. The minimum amount of magnetic particles necessary to stabilize the bed is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations .Biocompatible two phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation of biologically active compounds, subcellular organelles and cells. One of the disadvantages of this system is the slow separation of the phases when large amounts of proteins and cellular components are present. The separation of the phases can be accelerated by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. This method seems to be useful when the two phases have very similar densities, the volumetric ratio between the phases is very high or low, or the systems are viscous. Magnetically enhanced phase separation usually increases the speed of phase separation by a factor of about 10 in well-behaved systems, but it may increase by a factor of many thousands in difficult systems. The addition of ferrofluids and/or iron oxide particles was shown to have usually no influence on enzyme partioning or enzyme activity ,25.Proteins and peptides isolated using magnetic techniques have to be usually eluted from the magnetic separation materials. In most cases bound proteins and peptides can be submitted to standard elution methods such as the change of pH, change of ionic strength, use of polarity reducing agents (e.g., dioxane or ethyleneglycol) or the use of deforming eluents containing chaotropic salts. Affinity elution (e.g., elution of glycoproteins from lectin coated magnetic beads by the addition of free sugar) may be both a very efficient and gentle procedure.Magnetic affinity and ion-exchange separations have been successfully used in various areas, such as molecular biology, biochemistry, immunochemistry, enzymology, analytical chemistry, environmental chemistry etc -29. TablIn the case of proteins and peptides purifications, no simple strategy for magnetic affinity separations exists. Various affinity ligands have been immobilised on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting the affinity for target enzymes or lectins. Immunomagnetic particles, i.e. magnetic particles with immobilised specific antibodies against the target structures, have been used for the isolation of various antigens, both molecules and cells  and can Magnetic separation procedures can be employed in several ways. Preparative isolation of the target protein or peptide is usually necessary if further detailed study is intended. In other cases, however, the magnetic separation can be directly followed (after elution with an appropriate buffer) with SDS electrophoresis. Magnetically separated proteins and peptides can also be used for further mass spectroscopy characterization ,31. The Enzyme isolation is usually performed using immobilised inhibitors, cofactors, dyes or other suitable ligands, or magnetic beads prepared from affinity biopolymers can be used  are often short-lived and in low abundance. A rapid and sensitive method, based on the immobilization of biotinylated DNA/RNA fragments containing the specific binding sequence to the magnetic streptavidin particles, can be used. The bound DNA/RNA binding proteins are usually eluted with high salt buffer or change of pH .Sc from its conformer which is a cellular protein called PrPC. In fact, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes [Other types of proteins were isolated using specific affinity-based procedures. For example, plasminogen immobilized on magnetic particles was used to separate scrapie and bovine spongiform encephalopathy associated prion protein PrPpurposes ,40.Magnetic separation was also successfully used for the recovery of proteins expressed in the form of inclusion bodies, involving at first chemical extraction from the host cells, then adsorptive capture of the target protein onto small magnetic adsorbents, followed by rapid collection of the product-loaded supports with the aid of high gradient magnetic fields .A new approach for analytical ion-exchange separation of native proteins and proteins enzymatic digest products has been described recently . MagnetiMagnetic solid phase extraction (MSPE) enables to preconcentrate target analytes from larger volumes of solutions or suspensions using relatively small amount of magnetic specific adsorbent. Up to now this procedure was used for preconcentration of low-molecular weight xenobiotics ,43 but uXenopus egg extracts for analyses of the cell mitosis mechanisms [Sometimes the removal of certain proteins will reveal functions involving the depleted proteins or will help in the course of subsequent protein isolation. As an example, Dynabeads have been used to remove involved proteins from chanisms ,45. Rapichanisms .A combination of mechanical cell disintegration and magnetic batch affinity adsorption was used to simplify the isolation of intracellular proteins. Magnetic glass beads were used because of their hardness and rigidity .Dictyostelium discoideum were separated after magnetic isolation of the vesicles that was accomplished by feeding the amoebae with dextran-stabilized iron oxide particles. The cells were broken, the labelled vesicles were magnetically separated and then disrupted to release proteins which were resolved by SDS-PAGE. After „in-gel“ digestion with endoproteinase Lys-C or Asp-N the generated peptides were used for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles [An example of quite different protein purification strategy can also be mentioned. Proteins associated with the endocytic vesicles of vesicles . Analogovesicles .Standard liquid column chromatography is currently the most often used technique for the isolation and purification of target proteins and peptides. Magnetic separation techniques are relatively new and still under development. Magnetic affinity particles are currently used mainly in molecular biology , cell biology and microbiology (separation of target cells) and as parts of the procedures for the determination of selected analytes using magnetic ELISA and related techniques . Up to now separations in small scale prevail and thus the full potential of these techniques has not been fully exploited.It can be expected that further development will be focused at least on two areas. The first one will be focused on the laboratory scale application of magnetic affinity separation techniques in biochemistry and related areas (rapid isolation of a variety of both low- and high-molecular weight substances of various origin directly from crude samples thus reducing the number of purification steps) and in biochemical analysis (application of immunomagnetic particles for separation of target proteins from the mixture followed by their detection using ELISA and related principles). Such a type of analysis will enable to construct portable assay systems enabling e.g. near-patient analysis of various protein disease markers. New methodologies, such as the application of chip and microfluidics technologies, may result in the development of magnetic separation processes capable of magnetic separation and detection of extremely small amount of target biologically active compounds .In the second area, larger-scale  systems are believed to be developed and used for the isolation of biologically active compounds directly from crude culture media, wastes from food industry etc., integrating three classical steps  into a single unit operation . It is nIn the near future quite new separation strategies can appear. A novel magnetic separation method, which utilizes the magneto-Archimedes levitation, has been described recently and applied to separation of biological materials. By using the feature that the stable levitation position under a magnetic field depends on the density and magnetic susceptibility of materials, it was possible to separate biological materials such as haemoglobin, fibrinogen, cholesterol, and so on. So far, the difference of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation may be another way for biological materials separation .It can be expected that magnetic separations will be used regularly both in biochemical laboratories and biotechnology industry in the near future.


Studies conducted in developed countries using economic models show that individual- and household- level variables are important determinants of health insurance ownership. There is however a dearth of such studies in sub-Saharan Africa. The objective of this study was to examine the relationship between health insurance ownership and the demographic, economic and educational characteristics of South African women.The analysis was based on data from a cross-sectional national household sample derived from the South African Health Inequalities Survey (SANHIS). The study subjects consisted of 3,489 women, aged between 16 and 64 years. It was a non-interventional, qualitative response econometric study. The outcome measure was the probability of a respondent's ownership of a health insurance policy.2 test for goodness of fit indicated satisfactory prediction of the estimated logit model. The coefficients of the covariates for area of residence, income, education, environment rating, age, smoking and marital status were positive, and all statistically significant at p ≤ 0.05. Women who had standard 10 education and above (secondary), high incomes and lived in affluent provinces and permanent accommodations, had a higher likelihood of being insured.The χPoverty reduction programmes aimed at increasing women's incomes in poor provinces; improving living environment (e.g. potable water supplies, sanitation, electricity and housing) for women in urban informal settlements; enhancing women's access to education; reducing unemployment among women; and increasing effective coverage of family planning services, will empower South African women to reach a higher standard of living and in doing so increase their economic access to health insurance policies and the associated health services. A health system in any country performs instrumental functions of stewardship (oversight), creation of resources (investment and training), delivering services (provision), and financing (collecting, pooling and purchasing) . UltimatPrior to the 1994 democratic elections in South Africa, the health system was built on the apartheid ideology, which was characterized by racially segregated health services, geographical disparities, fragmentation, duplication and specialized hospital-centred services favouring the urban populations . The heaIn an attempt to overcome the above-mentioned public health challenges, the post-apartheid democratic governments have introduced several reforms in the health sector: creation of a quasi-federal structure with one national and nine provincial departments of health ; establishment of a district health system, including expansion and upgrading of the primary care infrastructure; and health care financing . The latIn spite of the abovementioned policy interventions, primary health care still remains under funded. For example, in 2003/2004 financial year, South Africa spent a total of 36.9 billion Rands (1 Rand = US$6) on health: 61.3% was spent on hospitals curative care, 16.1% on primary health care, 1.8% on HIV/AIDS treatment, 2.3% on nutrition, 4.0% on emergency services, 4.3% on administration, 2.5% on training, 1.6% on support services and 6.1% on other services .According to Doherty and McLeod  and McInIn 2001, there were 146 registered medical schemes  and eight Bargaining Council schemes (i.e. those granted exemption from certain provisions of the Act), all covering a total of 7 million people .The objective of this study was to examine the relationship between individuals' demographic, economic and educational characteristics, and their likelihood of being insured.There are two kinds of risks involved in health care: (i) the risk of becoming ill, with the accompanying loss in the quality of life, cost of medical care, loss of productive time during illness and, in more serious cases, death; and (ii) the risk of total or incomplete or delayed recovery .Welfare economics of uncertainty predicts that individuals would like to insure against both forms of risks. The theory of expected utility, on which this study is based, assumes that each individual strives to maximize the expected value of a utility function; individuals are normally risk-averse, meaning that they have a diminishing marginal utility of income; and health risks for different individuals are basically independent, so that pooling them reduces the risk to the insurer to relatively small proportions .In the South African National Health Inequalities Survey (SANHIS) , the res1) and expected utility without insurance (EU2). We need to analyse the effect of changes in the independent variables on the difference in the level of the expected utility of the two prospects, i.e. EU1 minus EU2. If the difference were equal to zero, the consumer would be expected to be indifferent between the two prospects. However, if the difference were greater than 0, then the risk-averse consumer would be expected to opt for insurance [The potential consumers of insurance are assumed to make decisions based on the magnitude of the perceived difference between the level of expected utility with insurance  for independence was undertaken to test the relationship between health insurance ownership and the individual independent variables. The null hypothesis is that the two variables are independent of one another . The alternate hypothesis is that the two variables are dependent on each other (because there is not a significant difference). Thus, for health rating, the hypotheses are as follows: (i) H0: A person's health insurance ownership status and their health rating are independent or unrelated; and (ii) HA: A person's health insurance ownership status and their health rating are related (dependent on each other).A Chi-Squared test  of 1998.2 Rand. The sub-sample of the households without health insurance had an average income of 853 Rand (STD = 1073) compared to 3172 Rand (STD = 2623) in the group with insurance. Seventy-seven percent of the households with at least one member with health insurance had a monthly income of more than 951 South African Rand, compared to only 28% among the group without health insurance.The average and median household size in the overall sample was five members, with a standard deviation of about 3. The group with health insurance had an average household size of four people (STD = 2.1) compared to 5 people (STD = 2.7) in the group without insurance. Fifty-seven percent of the sub-sample without health insurance had a household size of more than five members compared to 38% among the group with health insurance.The group with health insurance had an average age of 40 years (STD = 12.5) compared to 41 years (STD = 14.1) in the group without insurance. The average age in the whole sample was 40.9 years (STD = 13.6) and the median was 39 years.A) that a person's health insurance ownership status and their health rating are related (dependent on each other).As alluded to in the methods section, a Chi-Squared test for independence was undertaken to test the relationship between health insurance ownership and the individual independent variables. The results are presented in the last column of Table 0: β = 0) about individual regression slope coefficients. The 't' values for individual variables are obtained by dividing their coefficients (e.g. βINCOME) by their standard errors (e.g. SEINCOME). For example, the coefficient for income is 0.0004727 and the standard error is 0.0000339, and given H0: β = 0, the relevant t-value is indeed 13.946 as specified in Table Table INCOME = (βINCOME)/(SEINCOME) = (0.0004727)/(0.0000339) = 13.946t0: β = 0) if the calculated t-value, tk, is greater than the critical t-value, tc, as long as the sign of tk is the same as the sign of the coefficient implied in the alternative hypothesis (HA: β≠0). Otherwise, accept the null hypothesis (H0) that the estimated regression coefficient in question is not significantly different from zero. In the above example, the coefficient of income is statistically significant at 95% level of confidence, based on a two-sided test, since the computed t-value (13.946) is greater than the critical t-value (1.960).The decision rule is: reject the null hypothesis (Hi) on the log of odds of a health insurance policy ownership, holding other explanatory variables constant. The coefficients for environment rating, residence, income, education, age, smoking and marital status are statistically significant at 95% level of confidence, and have positive signs. The latter result implies that an increase in any of these variables spontaneously impacts positively on the log of odds of health insurance policy ownership, holding other factors constant. Contrastingly, the coefficients for household size, alcohol, contraceptive use, age-squared, occupation, employment and health rating are statistically significant, and have got a negative effect on the log of odds of health insurance policy ownership.The coefficient (β) of the estimated binary logit model measures the impact of a one-unit change in an explanatory variable  and health-related quality of life  ,23. The An individual's stock of health determines the total amount of time he/she can spend producing commodities and money earnings . Health The adverse selection problem can be curbed in two main ways without compromising equity objectives, namely: (i) compulsory social health insurance  for a defined population through legislation – not an option for private health insurance which is voluntary by definition; and (ii) the government could step in and provide health insurance to all those at exceptionally high risk (e.g. the elderly and those with chronic diseases) and the poor who cannot afford the premiums. Commercial health insurance firms often curb adverse selection by introducing experience rating, i.e. linking insurance premium to the degree of assessed risk of falling sick (this action may have negative equity implications); and/or subjecting all those who apply for insurance cover to a thorough medical examination .There were three economic variables, namely, income (+), occupation (-) and employment (-), where (.) is the hypothesized sign of the coefficients. The coefficients of the three variables were statistically significant and had the expected signs. High incomes, white-collar occupations and being gainfully employed are significant predictors of health insurance ownership. The proportion of people with health insurance rises considerably as one moves up the household income distribution ladder, with the coverage going from 6.3% of those in the income range 1–950 Rand to over 90.7% among those earning 7600 Rand and above per month. The trend is similar to that reported by Harmon and Nolan  in IrelaThe demographic factors include: age (+), age squared (-) and household size (-), where (.) is the postulated sign of the coefficient. The coefficients for age and age squared were statistically significant at the 5% level. Economic theory predicts that as individuals advance in age, their inherited health stock depreciates at an increasing rate  and they tend to increase investments in health  in an attempt to decrease the rate of depreciation. This is consistent with Grossman's findings  that becThe coefficient for household size variable had a statistically significant negative effect on the likelihood of health insurance policy ownership. This finding is intuitively sensible since any increase in the household size, while holding the income constant, reduces the per capita income.The social factors include: education (+) and marital status (+). The coefficient for education was statistically significant, and had the expected positive sign. Respondents with at least a matriculation (secondary) level of education were two times more likely to be in possession of a health insurance policy than those with a lower level of education. This could be attributed to a positive relationship between a person's: (i) educational level and propensity to acquire skills; (ii) stock of knowledge and his/her market and non-market productivity  and earnMarital status had a statistically significant positive effect on health insurance ownership. Married persons are more likely to have insurance cover than those who are single, separated or divorced. This finding is consistent with the result obtained by Harmon and Nolan , Rhine eThe spatial and environmental factors – residence (+) and environment rating (+) – had a statistically significant effect on health insurance ownership. The respondents living in formal urban settlements or rural white-owned farms had a seven times higher odds of owning a health insurance policy than those living in informal urban settlements or former rural homelands. This could partly be a reflection of the economic well being of the former group.vis-à-vis informal settlements .The respondents who felt that the environment they lived in was good, very good or excellent were twenty-seven times more likely to have a health insurance cover than those who lived in fair or poor environments. This phenomenon may be a reflection of a better socio-economic status of those living in relatively affluent, formal (and cleaner) residential areas The behavioural factors included in the analysis were: contraceptive use (-), alcohol use (-) and smoking (+). The three had a statistically significant effect on the demand for health insurance. The coefficient for contraceptives assumed a negative sign. This implies that the use of contraceptives may not necessarily be linked with individuals' attitudes toward risk.The coefficient for alcohol use also took a negative sign, which implies that those people who drank alcohol were less likely to purchase health insurance. On the contrary, the parameter for smoking assumed a positive sign, meaning that being a cigarette smoker, the probability of a person demanding health insurance coverage increases. In the context of the health insurance market, the latter finding could be a source of concern if it were a signal for the presence of moral hazard. Moral hazard is a potential cost of insurance in which the presence of insurance increases the tendency for losses to occur through careless, irresponsible or perhaps illegal behaviour .For example, the fully insured individuals may embark on risky behaviours, such as smoking, and by so doing expose themselves to a high risk of developing various forms of cancer (throat cancer, lung cancer, etc.). Insurers attempt to control moral hazard by careful underwriting of applicants for insurance and by various policy provisions, such as deductibles  and co-insurance/co-payment  ,29.This study used consumption of contraceptives, alcoholic drinks and cigarettes as proxies for consumers' attitudes toward health risks. The three may not be ideal proxies for risk attitudes; however, there were no better alternatives in the data set. If this were a study primarily designed to analyse the demand for health insurance, it would have been preferable to either proxy risk attitudes using a qualitative scale variable ranging from 1 (extremely risk-averse) to 10 (risk-lover) , directlAppendix), leading to specification bias or omitted variable bias [The main weakness of the current study is that since the data set upon which the analysis was based was gathered for a different purpose , it did not contain insurance-specific attributes, e.g. premiums, co-payments, deductibles, benefits covered and the quality of care in the health facilities where the insured sought care. Thus, we had a situation where important explanatory variables were left out of the estimated regression equation 2  and household socio-economic characteristics (including attitudes toward risk).• The willingness and ability to pay for various forms of health insurance, including voluntary and non-voluntary insurance schemes .• The economic viability of various forms of health insurance, e.g. social health insurance and community-based prepaid schemes.• Design of innovative health insurance schemes; for example, within farmers' cooperative societies , savings• Whether the expansion of private health insurance under the current health care delivery system would yield significant public sector cost savings, and improved targeting of subsidies for the poor and preventive services .• Optimal ways of curbing health insurance problems of moral hazard and adverse selection.• Benefit-incidence analysis of alternative health insurance arrangements.The environment rating, residence, income, education, age, smoking and marital status variables were all found to have a statistically significant (at 95% confidence level) positive relationship with ownership of health insurance schemes. Contrastingly, the other covariates, namely: health rating, age squared, household size, occupation, employment, alcohol use and contraceptive use had a significantly negative relationship with health insurance ownership.There are a number of policy implications of this study:• High incomes, white-collar occupations and being gainfully employed are significant predictors of health insurance ownership. Thus, economic development (or poverty reduction) programmes geared at: (i) improving incomes of the vulnerable segments of the South African population; (ii) reducing involuntary unemployment; and (iii) creating white-collar job opportunities will empower South African women to reach a higher standard of living and in doing so boost their economic access to health insurance policies and the relevant health services.• Policies aimed at ensuring that the majority of South Africans attain a matriculation (i.e. secondary) education level will increase by almost two-fold, the probability of acquiring health insurance.• The self-assessed health status was found to have a statistically negative effect on the demand for health insurance. This implies existence of adverse selection. However, this problem can be reduced through compulsory social health insurance ; or throThe author(s) declare that they have no competing interests.JMK recoded the raw data and participated in the development of the conceptual framework, analysis and drafting of sections of the document. LGS, BN, GMM, RC and TM participated in the development of the conceptual framework and drafting of sections of the document. All authors read and approved the final manuscript.i) and a vector of a household's socioeconomic characteristics (Ri), plus a stochastic error term (ε). The latter component captures errors in model specification (e.g. omission of relevant variables) and errors in data measurement.A binary logit model was used in the analysis of individual household's choice between having no health insurance and having health insurance. We assumed that the expected utility associated with each health insurance option is a function of a vector of its attributes  + ε .................................... (1);ij is the utility that ith household expects to derive from choosing jth health insurance option; j = 1 if a household has health insurance; j = 2 if a household has no health insurance; and X, R and ∈ are as defined above.where: EUth household opts for 'health insurance' if EUi1 > EUi2, prefers 'no health insurance' if EUi1 < EUi2, and is indifferent between the two options if EUi1 = EUi2. Thus, the probability that ith household prefers to have health insurance is: Pi1 = P(EUi1 > EUi2). And, conversely, the probability that ith household prefers not to have health insurance is: Pi2 = P(EUi1 < EUi2).The basic assumption is that the iTo determine the probability of health insurance ownership, the following model was estimated:Pij = (α + β1 HEALTH_RATING + β2 ENVIRONMENT_RATING + β3 RESIDENCE + β4 INCOME + β5EDUCATION + β6 AGE + β7 AGE_SQUARED + β8 RACE + β9 HOUSEHOLD_SIZE + β10 OCCUPATION + β11 EMPLOYMENT + β12 SMOKING + β13 ALCOHOL_USE + β14 CONTRACEPTIVES_USE + β15 MARITAL_STATUS + εi........................ (2)ij = 1 if individual 'i' owns insurance (j = 1) and equals zero otherwise (j = 0); (α) is the intercept term; (β's) are the estimated coefficients; and εi is the stochastic error term. The explanatory variables included in the model are defined in Table where: PThe pre-publication history for this paper can be accessed here:


Published February 15, 2005PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.0020449The art credits were missing from the image accompanying this synopsis. The image caption should read as follows: