In 2002, the World Health Organization published a health system performance ranking for 191 member countries. The ranking was based on five indicators, with fixed weights common to all countries.We investigate the feasibility and desirability of using mathematical programming techniques that allow weights to vary across countries to reflect their varying circumstances and objectives.By global distributional measures, scores and ranks are found to be not very sensitive to changes in weights, although differences can be large for individual countries.Building the flexibility of variable weights into calculation of the performance index is a useful way to respond to the debates and criticisms appearing since publication of the ranking. The World Health Organization recently published a performance ranking of the health systems of its 191 member countries, and intends to update it at regular intervals . It was Publication of the analytical framework and the resulting ranking provoked considerable comment, and a variety of issues concerning the methodology and country positions in the ranking have been raised. A central component of the methodology for measuring overall attainment was the use of fixed weights, common to all countries, to aggregate the five indicators. This feature has been controversial, with some arguing that people in different cultural and social settings value individual health system goals in different ways -13. The In this paper we examine the sensitivity of the attainment scores to alternative weighting schemes that allow weights to vary across countries. These country-specific weights may reveal varying objectives of policy makers or constraints under which they operate. Melyn and Moesen  have refWHO used fixed weights  to aggregate five health system outputs  into a scalar health system attainment index. Overall attainment ranged from 35.7 (Sierra Leone) to 93.4 (Japan) on a [0–100] scale.We propose here an analytical framework that reduces to the WHO fixed-weight methodology as a special case, but that allows varying degrees of freedom for weights to be defined that – in the sense of Melyn and Moesen – implicitly take into account individual country circumstances. For shorthand, we say countries "choose" such weights, which in reality are determined as solution values of a linear program. If the linear program is a fair representation of the objective function of and constraints faced by decision makers, the weights are indeed "chosen", but even if this condition is not necessarily met, the resulting weights may still be of interest.The extent of freedom to choose weights in a linear program can be set by the analyst. The analyst can enforce fixed weights common to all countries, allow countries complete freedom to choose their own weights, or adopt a middle ground in which countries are granted limited freedom to choose weights within bounds thought to be sensible by experts.A generic statement of the performance evaluation problem in primal-dual linear programming format is:In these programs y is a country's output vector, x is its input vector, Y is the sample output matrix and X is the sample input matrix. In the present context y is (5 × 1), Y is (5 × 191), x is (n × 1) and X is (n × 191), with n to be specified below.-1 ≤ 1. Best practice countries have φ-1 = 1, other countries have φ-1 < 1, so φ provides a basis for acomplete ranking of countries on their relative ability to deliver five health system outputs. The dual program seeks a set of nonnegative weights μ,υ attached to a country's outputs and inputs that maximize its attainment. Each country's output weights are normalized by μy = 1, but each is free to select its own set of nonnegative weights.The primal program seeks the maximum radial expansion of a country's outputs, provided that it not exceed the standards established by a convex combination  of best-practice countries in the sample. The optimal value of φ provides a distance measure (i.e. an indication of how far a country has to go) to match best practice as observed in the sample. Since φ ≥ 1, the attainment of a country is evaluated as φIn constructing the overall attainment index, WHO identified five output indicators and no inputs, preferring to treat each country's health system as a "health output management unit". Consequently each country's input vector is represented as a scalar with unit value. Under these circumstances the performance evaluation problem simplifies to:0. The modified primal program seeks the maximum feasible radial expansion of a country's outputs consistent with best practice observed in the sample. The modified dual program seeks a set of nonnegative weights μ for a country's outputs that put it in the best light. A country can be expected to assign relatively large weights to those outputs at which it excels relative to best practice, and relatively low, possibly zero, weights to those outputs at which it lags behind best practice, subject to the normalization μy = 1. where ω = υ + υm(Ymλ - φym) = 0, m = 1, ..., M, so slack in any element of a country's projected output vector φy implies that the country assigns a zero weight to that output. Since it is unreasonable to allow a country to assign a zero weight to any output deemed sufficiently important to have been included in the WHO performance evaluation exercise, it is desirable to restrict weights in some way. This can be accomplished most easily by appending constraints to the dual side of (2) of the form:By complementary slackness, μm ≥ μmym/μy ≥ βm, m = 1, ..., M.     (3)γm) in total output.Restrictions (3) place lower and upper bounds on the relative importance of each output , with lower and upper bounds in (3) set by a modified AHP procedure.The fixed weights used by WHO))  to aggre.In the modified procedure, "expert opinion" was taken to mean the average values of weights arising from population-representative country surveys, each of which included a module on health system goals . Respondm =  and the upper bounds γm = . This specification allows limited freedom to choose. We refer to the four indexes as WHO, LP1, LP2 and LP3, respectively.For each output, the lower bound for calculation of the third index was taken as the minimum of the country average weights, and the upper bound the maximum . CountrySummary statistics of the four attainment indexes appear in Table Rank correlations between pairs of attainment rankings appear in Table Figure Results based on LP1 are unattractive. Over one-third of countries (70 of 191) assign a zero weight to four of the five indicators, and the vast majority of countries assign a weight in excess of 0.9 to either responsiveness distribution or fairness in financial contributions. This means they assign a low or zero weight to population health, the defining goal of the health system, which does not have face validity. In LP1, only Japan, Luxembourg and the United States assign positive weights to all five indicators. Consequently the attainment index is tightly distributed about a very high mean value. The ability to discriminate is sacrificed to freedom to choose, with 75% of countries receiving attainment indexes of 99 or above.Results from LP2 are somewhat more attractive. Nevertheless, when weights are bounded below by 0.1, over three-quarters (147 of 191) of countries assign the minimum weight to four of the five indicators and a 0.6 = 1 - (4 × 0.1) weight to either responsiveness distribution or fairness in financial contributions. The attainment scores are again compressed about a high mean value, and the ability to discriminate is not much improved, with 75% of countries receiving attainment scores of 93 or above. However despite this dramatic compression, the LP2 ranking is globally very similar to the WHO ranking. Eighteen of the countries ranked in the top 20 by WHO appear in the LP2 top 20, and 14 of the countries ranked in the bottom 20 by WHO appear in the LP2 bottom 20.Not surprisingly, the distribution of the attainment scores for LP3 looks even more similar to the distribution of the WHO scores, and has a similar mean and standard deviation. Rank correlation is very high, with only one country falling out of the WHO top 20 and only five countries rising out of the WHO bottom 20. Nonetheless, limited freedom to choose has an important impact on individual countries. The USA, given an ability to emphasize the importance of responsiveness level and responsiveness inequality, and to de-emphasize the importance of health level and inequality in the distribution of health, improves its ranking from #15 to #3. Australia improves from #12 to #7 for similar reasons. Italy, on the other hand, falls from #11 to #18, primarily as a result of the movement of other countries. In LP3, the largest positive changes in ranking are for Mauritius (+25) and Saint Vincent (+22), while the largest negative changes in ranking are for Kazakhstan (-39) and Albania (-36).We began by questioning the appropriateness of the fixed weight approach to aggregating indicators adopted by WHO, on the grounds that fixed weights deny countries at varying stages of development the freedom to choose. We then proposed a sequence of linear programming models that allowed countries varying freedom to choose the weights assigned to their indicators. LP1 allows complete freedom to choose, and generates weights we consider unacceptable, particularly because so many countries give a zero weight to improving health. LP2 allows considerable freedom to choose, but generates many country-specific weights falling outside the range of the within-country means used as bounds in LP3. Clearly, the validity of a procedure that routinely assigns weights out of the range of representative cross-population preferences should be questioned, even without a sophisticated theory of empirical ethics or democratic choice.LP3 applies the AHP procedure to set plausible bounds on weights, and allows limited freedom to choose. It generates a very similar distribution of the attainment index, and a very high linear rank correlation with the WHO ranking. Despite these similarities, we find the LP3 approach intuitively appealing, and are encouraged by its global concordance with the WHO index. However it is fair to ask: if LP3 and WHO generate such similar rankings, why bother? What value does LP3 add? Indeed, individual countries may come to diametrically opposed conclusions about the benefits of LP3 or WHO on the basis of their implied rank changes (e.g. Mauritius vs. Kazakhstan).Howbeit, our first response to the question "why bother" focuses on the distribution of the LP3 weights in comparison to the WHO weights. The WHO weight on responsiveness inequality was 0.125. But the LP3 upper bound of 0.17 is binding on 182 countries, which implicitly want a higher weight on this indicator. The WHO weight on fairness in financial contributions was 0.25. But the LP3 upper bound of 0.30 is binding on 167 countries that want a higher weight. At the other end, the WHO weight on health level is 0.25. But the LP3 lower bound of 0.19 is binding on 170 countries that want a lower weight. The WHO weight on responsiveness level is 0.125. But the LP3 lower bound of 0.12 is binding on 103 countries that want a lower weight. It appears that a majority of countries at all stages of development may implicitly assign greater importance to indicators of health distribution, and less importance to indicators of health level, than the experts whose judgments formed the basis of the original WHO weights.Our second response is more general. By allowing countries limited freedom to choose their weights, LP3 takes a small but nevertheless significant step toward respecting their varying circumstances. While the narrower the bounds on weights, the smaller the step, even the limited freedom embodied in LP3 makes an important difference to some countries.Building in the flexibility of varying weights might be a useful way for WHO to respond to the debates and criticisms appearing since publication of the ranking. We conclude by speculating that a variant of LP3 incorporating information regarding which weights are binding, and in which direction, might yield even greater benefits in terms of respecting individual circumstances.JAL, CJLM and DBE were part of the team at WHO that developed the methods for the world health system performance rankings published by the Organization in 2000.AHP DEA LP (Linear program)LP1 (Linear Program 1)LP2 (Linear Program 2)LP3 (Linear Program 3)WHO JAL developed methods inspired by the benefit-of-the-doubt concept and drafted an earlier version of the manuscript. CAKL adapted and applied the DEA methods described here to health system performance and drafted the initial version of the current manuscript. CJLM conceived the methods for measuring health system performance. DBE coordinated the research at WHO. All authors revised and approved the manuscript.The pre-publication history for this paper can be accessed here:Annex.Graphical interpretation of DEA and conceptual links with value theory.docClick here for file


Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate.Important parameters during recombinant protein production in E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate.During fedbatch cultivation, -1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point.The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h Esherichia coli there seems to be a strong dependence of the productivity and product quality on the limiting substrate feed rate at induction [For several recombinant proteins produced in nduction -8. DiffeOf particular importance for protein translocation processes are the anionic phospholipids but also unsaturated fatty acids. It was reported a great number of times that increased amounts of phosphatidylglycerol could facilitate protein translocation ,13-15. I-1 simultaneously to an optimum in β-lactamase leakage over the outer membrane [-1 unsaturated fatty acids where replaced by cyclic fatty acids [In continuous cultivation it was observed that conditions for establishment of an optimum in phosphatidylglycerol and unsaturated fatty acids could be achieved. An optimum in the accumulation of these compounds occurred at a growth rate of 0.3 hmembrane . It was ty acids  which waty acids ,27. If tThere is thus a strong incentive to select and operate processes which allow the establishment of controlled growth conditions and to understand the specific growth characterises established with respect to the membrane at particular feed rates. Two main techniques are at present available: fedbatch and continuous cultivation. We have previously established data from continuous cultivation  which we-1 and above, the traditional fedbatch concept established early in the former century based on bakers yeast and later on for antibiotics production, is generally used. The feed profile, characterised by this general concept, is initially exponential until the oxygen or heat transfer capacity of the reactor is reached and thereafter constant. This implies that the substrate concentration and the specific growth rate decline with cultivation time after the constant feed profile is adopted. This constitutes the advantage: the declining growth rate allows the cell mass productivity to increase at constant oxygen consumption/heat production. A further advantage of this cultivation mode, in comparison to a standard batch process with substrate overflow and to fedbatch with high exponential feed, is the possibility to avoid inhibitory by-products such as acetate. At a preset time during the feeding process, induction is performed. The drawback is that when the cell mass is very high the substrate consumption is very low and this point might not represent the optimal condition for high product formation, achievement of high product quality or facilitated transport of the protein.The fedbatch technique is used when a high cell density is the primary goal. A variety of feeding strategies can be applied for different research purposes where exponential feed can be used to create conditions that resemble a steady state in all points except for the continuous cell mass accumulation. This technique can however only be used for a few number of generations since the oxygen transfer rate rapidly becomes limiting. In order to reach a high cell density, i.e. cell densities of 50 g, lEscherichia coli.In continuous cultivation the feed/dilution rate is also used to control the growth rate where there is a limit of the chemostat to cultivation at the maximum or near maximum rates when the pH-auxostat is the preferred method . ContinuThe goal of this work is to show how growth rate controls the performance in fedbatch cultivation with respect to the structure of the membrane and to compare these changes to the function of some selected process variables and methods. These data will, in turn, be compared to earlier data derived from continuous cultivation . From thEscherichia coli W3110, a K-12 derived strain, was grown in fedbatch cultivation. The fedbatch concept was characterised by an initial exponential feed, started at time zero, of the growth limiting component, glucose, leading to the establishment of a specific growth rate of 0.6 h-1. This was followed by a constant feed phase where the growth rate declined asymptotically to a value of approximately 0.05 h-1. During this phase the cell mass increases linearly to a cell density of approximately 27 g, l-1, as expected , unsaturated , and cyclic (CFA: C17:cyc). From this figure is evident that the amount of SFA's is the most stable during the declination of growth. There is only a slight reduction of the amount at a growth rate of approximately 0.3 h-1, which is accompanied by a maximum in the amount of unsaturated fatty acids. The unsaturated fatty acids are markedly decreased as the growth rate goes below 0.3 h-1 to 56/70 % of their maximum values, respectively. These maximal values, which coincide with the above mentioned minimum in CL/maximum in PG, thus constitutes a switch-point in the fatty acid and phospholipid metabolism during fedbatch cultivation. At this point there is also a minimum of cyclic fatty acids. The levels of the cyclic fatty acids are then rapidly increasing the lower the growth rate and from its minimum the cyclic fatty acids are dramatically increased by almost 100 % in less than two hours. This occurs although there is a supply of nutrients at all times which is in contrast to earlier literature where it is stated that it is the lack of carbon and energy in the stationary phase which results in cyclic fatty acid formation. During the continuing declination of the growth rate, the cyclic fatty acids continues to increase to a final of three times their minimum value. It should be noted that since the fatty acids of the lipopolysaccharide (LPS) were removed during the sample extraction, the results are only reflecting the inner part of the outer membrane and the cytoplasmic membrane.The fatty acids are shown as a function of the time from constant feed start in Figure The three phospholipids were furthermore examined for their individual fatty acid content. The results are shown in Figure The effects of these membrane changes were investigated on basis of the function of some selected methods and process variables: the mechanical strength of the membrane to ultrasound, the effect of osmotic chock, the shredding of endotoxin to the medium, the leakage to the medium and the amount of cell lysis.β-lactamase, at different time of sonication using the same amount of cell and sonication effect. Figure -1 but from this point on there is continuously increased membrane stability. A comparison to the pattern of unsaturated fatty acid accumulation in Figure The mechanical strength of the membrane during different growth rates was quantified as the release of the periplasmic marker, vice versa. The figure shows that the sucrose addition and the late addition of water results in the major effect on permeabilisation and, for unknown reason, also the initial dilution by Tris-HCl. The addition of lyzosyme, in itself, has no effect which is a result of the fact that it can only penetrate the membrane by the addition of water [Osmotic shock is a frequently used method to release protein in the small scale. The methodology chosen as a model system gives a step-wise permeabilisation of the membrane, which is followed by breakage of the cell wall murein saccculus and formation of sphaeroplasts when lysozyme enters the cell . Severalof water . The effThe release of lipopolysaccharide, LPS, to the medium was measured by means of detection of the presence of a specific inner part of this structure, lipid A, which has properties of an endotoxin. This compound has to be removed in downstream processing of pharmaceutical proteins since it gives a toxic response in patients and must always be proven absent in the final product. It was considered likely that the changes in the underlying phospholipid and fatty acid structure might have large consequences for the release of this component to the medium which was to some extent confirmed by literature studies . In FiguIn Figure -1 where after it slowly declines from 0.50 to a final value of 0.42 g protein, g cells-1.A major concern of this work was the effect of membrane structural changes on protein leakage from the periplasm to the medium. Since it is clear that a periplasmic protein can be present in the medium both by cell lysis and by true leakage from the periplasm, cell lysis was registered as a function of cultivation time. This is shown in Figure β-lactamase, which is constitutively produced from its own promoter. The total β-lactamase production is shown as well as the specific production values in the medium per gram of cells. It is clear that during fedbatch cultivation the total amount of this specific protein follows the total protein accumulation pattern and is constant independent if calculated per cell mass or per total cell protein except for the values at very low growth rate. The β-lactamase value per gram of cells in the medium is thus slightly higher during the first part of the cultivation down to μ = 0.3 h-1 where after the value declines. At very low growth rate there is yet another period of increased specific accumulation. It is realised that this latter period of slowly increased accumulation in the medium coincides with an increased however small lysis in the end of the cultivation. Since the leakage is not increased during this period lysis seems most likely cause for the accumulation. The level of β-lactamase in the medium amounts to approximately 10% of the total β-lactamase production throughout the cultivation as seen from the quotient of β-lactamase in the medium in relation to the total value.In Figure -1 is formed in expense of cyclic fatty acids which are accumulating at low growth rate. Compared to literature data the amount of cardiolipin is approximately 70 % higher than from earlier observations [The conditions during fedbatch leads to a structure, which is approximately constant with respect to phospholipids and saturated fatty acids compared to growth rate. The changes in lipid components arrive from changes in unsaturated fatty acids where the increased values above the maximum at 0.3 hrvations .-1. The high content of cardiolipin is to some extent balanced by high level of unsaturated fatty acids, which compensates the need for flexibility, permeability and movement of compounds within the membrane which growth rate. The growth dependent accumulation and the generally high level of unsaturated fatty acids influence the results of sonication. The high levels above μ = 0.3 h-1 thus supports a weaker membrane, in this respect, while the substitution of unsaturated to cyclic fatty acids reduces the effect at low growth rate. The transport of protein over the membrane is however only slightly higher at higher amounts of unsaturated fatty acids which is implicated by the larger effect of enzyme/water penetration during osmotic chock and the leakage of the model product, β-lactamase, at high growth rate. In total, β-lactamase is however always present in the medium at a level of approximately 10 % of the total production due to that the membrane flexibility caused by unsaturated fatty acid is balanced by the high and constant amount of cardiolipin.The resulting effects on the membrane function are several but are generally and overall characterised by their stability as a function of growth rate. Cell lysis is low at all times i.e. the membrane is not easily breaking-up and this is due to the high content of cardiolipin at all times which is further emphasised by high levels of cyclic fatty acids specifically at growth rates below 0.3 hThere is a very slight increase in cell lysis at the end of cultivation. The reduced capacity of this cell for total and specific protein production at low growth rate, which might be due to a reduced amount of ribosomes , low supContinuous cultivation gives theoretically always the highest productivity due to the reduced shut-down periods for cleaning and refilling. However this technique is rarely used in industrial production of recombinant protein production due to problems of separation of growth and product formation. The pharmaceutical industry will furthermore need a validation protocol, which must be derived for this technique and where the concept of batch reproducibility needs some clarification. There is also a tendency to use techniques which are since long established and to which the personnel are educated. However, sometimes reasons are not well developed and a problem is the lack of data for a scientific choice of production technique.-1) or a low (0.05 h-1) growth rate. The table furthermore indicate if curves, with respect to growth rate, show a trend which reveals the existence of maximum/minimum values of the specific compound. Table In table One of the most significant structural differences between the cultivation techniques is the high content of CL in fedbatch cells in expense of both PG and PE. The formation of large amounts of CL is an obvious cell strategy to save energy and carbon by the release of glycerol to further metabolisation. Since the only difference between the two cultivation techniques is the constant conditions in continuous cultivation and the dynamically changing ones in fedbatch the increased need for carbon/energy must lie in the rapidly changing growth environment.β-lactamase due to the growth dependent changes of this fatty acid.In fedbatch there are also more unsaturated fatty acids accumulated, which develop in expense of saturated fatty acids, which are higher in continuous cultivation. The fatty acid level is also more or less constant in CL, which promotes stability. The conclusion is that cells in fedbatch cultivation interpret the environmental signals in a way that leads to a membrane structure with several features that generally only develop during nutrient exhaust in stationary phase. This is not the case for cells growth in continuous cultivation which develop distinct characteristics at each stable growth rate. This leads to a fedbatch membrane that is rigid compared to cells in continuous cultivation. The overall result is fewer changes in the membrane function due to growth rate. This is represented by constant leakage, constant lysis and an almost constant effect of permeabilisation. The fedbatch membrane shreds also less endotoxin although levels are low for both cultivation techniques. However, the fedbatch membrane is sensitive to sonication, specifically at high growth rate due to the comparatively high amount of unsaturated fatty acids and thus releases more -1. However, this is not allowed to develop in fedbatch to the extent seen in continuous cultivation where a specific growth rate is kept much longer of the process time. The result is that no optimum in the leakage to the medium is clearly established in fedbatch cultivation and the fedbatch process cannot be designed for this since a constant growth rate of 0.3 h-1 cannot be kept while at the same time leading to a high cell accumulation.From the comparative data of table Protein transport is generally considered to benefit from a high content of PG and unsaturated fatty acids. This accumulation leads to high leakage which is verified by the continuous cultivation leakage optimum  and whicThe overall conclusion is thus that if leakage is undesired, fedbatch should not be used but the process could well be run in continuous cultivation at low growth rate. If an increased leakage is preferred this can be optimised in the continuous operation mode principally by change of growth rate but probably also by mutants accumulating a combination of phosphatidylglycerol and unsaturated fatty acids if these are stable enough for prolonged cultivation.The stability of the fedbatch membrane, which is not growth dependent, is the strength of this cultivation technique. The time of cell harvest is thus not critical since the membrane strength and function does not rapidly change which allows for flexibility in operation. The stability of the fedbatch derived cell membrane might thus also by the same reasoning be an advantage in further downstream processing however depending on the chosen unit operations.The merits of the cell characteristics in continuous cultivation are due to the possibility to run the cultivation at a feed rate which is optimal for membrane leakage independent if this is to be small or maximised.-1 and induction should thus take place well above this value. This is a considerable drawback in fedbatch cultivation, which relies on high cell density accumulation leading to low growth rates before induction but easily controlled in continuous cultivation. The coupling of the productivity and even product quality to a specific growth rate further strengthens the implication of using continuous cultivation.Total and specific protein production is lowered at low growth rate and the data points to that recombinant protein production should not be performed below a growth rate of 0.2 hThe conclusion is that the drawbacks of industrial operation of continuous cultivation in the biopharmaceutical industry earlier mentioned should be preferably be solved to explore the benefits that are obviously present with this technique.Escherichia coli W3110 ) [-1, was used. The plasmid pBR322  was transferred to the host cell. This plasmid carries the genes bla and tet where β-lactamase (TEM-1) was used as the model product. The maximum levels were approximately 1 % of the total protein. The medium for the bioreactor and the feed was a mineral salts medium composed of : (NH4)2SO2, 7.0; KH2PO4, 1.6; Na2HPO4 *2H2O, 6.6; (NH4)2-H-citrate, 0.5. Glucose was autoclaved separately and added to bioreactor together with a sterile filtered trace element solution and 1.0 M MgSO4 . A further addition of magnesium, of the same amount, was done at eight hours of feeding. The glucose concentration in the feed was 300 g, l-1. The trace elements solution was composed of (per litre): CaCl2 *2H2O, 0.5g; FeCl3 *6H2O, 16.7 g; ZnSO4 *7H2O, 0.18 g; CuSO4 *5H2O, 0.16g; MnSO4 *4H2O, 0.15 g; CoCl2 *6H2O, 0.18 g; Na-EDTA, 20.0 g.D-rrnE)) , a K12 d600) of 2–3 was reached from which point the fedbatch cultivation was performed. The experiments were carried out in a 14-l bioreactor with a working volume of 8.0 l. The bioreactor was equipped with an air sparger and the air-flow was 0.5–1.0 vvm. By varying of the stirrer speed up to 1000 rpm the dissolved oxygen concentration did never go below (30 ± 2) % air saturation. A pH of 7.0 was kept by addition of 28% (w/w) ammonia solution. Temperature was controlled at 37°C. The cultivations were started as batch cultures with an initial glucose concentration of 2 g, l-1. The feed was started when glucose was almost exhausted. The feed profile consists of two different phases where an exponential feed was followed by a constant feed phase. The exponential feed profile was calculated from the following equation:Four independent fedbatch cultivations were performed in the following manner: inoculum was prepared from a shake flask culture grown overnight at 37°C at 150 rpm. This was transferred into the bioreactor when an optical density at 600 nm (OD0 * eμt-F(t) = Fμ the desired specific growth rate (1/h) and t is the process time (h). The calculation of the initial feed rate, F0, is based on a mass balance at the time of the feed i.e. after the batch growth assuming: pseudo steady state growth during the feed phase, a theoretical yield coefficient (Yx/s) of 0,5 g, g-1 and a negligable amount of the limiting substrate in the reactor.where F is the feed rate (L/h), 0 =(μ *V*X)/(Si*Yx/s)Fx/s is the cell yield on glucose (g/g), X is the cell concentration at F0 (g/L), V is the culture volume (L) and Si is the glucose concentration in the feed (g/L).Where YThe continuous cultivation conditions and data, compared to in the conclusions section, were reported elsewhere . These w-1) by 10 min centrifugation  of 3*5 ml of cell suspension in preweighed test tubes and drying of the pellet overnight at 105°C before weighing. The supernatant from the CDW samples was sterile filtered and used for acetic acid and cyclic adenosine monophosphate, cAMP, analysis. Acetic acid was measured with the enzymatic method from Boehringer Mannheim GmbH (Cat. No 148 261) and cAMP was analysed by an enzyme immunoassay system from Amersham Pharmacia Biotech AB (code RPN 225).The cell growth was followed by optical density at 600 nm. Biomass concentration was determined as cell dry weight  at 55 bar. Data from French press was set as the 100% disruption level and all other values were taken as a percentage of this value.The mechanical strength was estimated by sonication  at constant frequency, cell dry mass, sample volume and acoustic power. Samples were placed in a water bath at +4°C during cell disruption to prevent overheating. The release of -1 cultures were harvested and suspended in 0.3 ml, 200 mM Tris-HCL (pH 8.0), (2) t = 1 min, 0.5 μl, 100 mM EDTA (pH 7.6) was added, (3) t = 2 min, 0.5 ml, 1 M sucrose (pH 8.0) was added thus giving excess of EDTA over Mg2+ ions, (4) t = 3.5 min, egg white lysozyme  was added to a final concentration of 60 μg/ml, (5) t = 4 min, the cell suspension was exposed to a mild osmotic shock by two-fold dilution in water to trigger lysozyme penetration of the outer membrane, and finally (6) t = 8 min, the cell suspension was diluted 11-fold in 10 mM EDTA (pH 7.6). Permeabilisation was recorded by optical density at 450 nm. The samples taken at different growth rate were diluted to the same cell concentration  and the same volume (0.2 ml) was used at the sampling point.Chemical strength was estimated by permeabilisation as described by Witholt et al . The folPhospholipids and fatty acids were determined by the method of Arneborg  where thAS made the fermentations, analyses and data treatments and evaluation. The project was conceived by GL who further participated in the project planning, design and data evaluation. All authors read and approved the final manuscript.


Autism is a behavioral disorder with impaired social interaction, communication, and repetitive and stereotypic behaviors. About 5–10 % of individuals with autism have 'secondary' autism in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. Ninety percent have idiopathic autism and a major gene has not yet been identified. We have assessed the incidence of chromosome abnormalities and Fragile X syndrome in a population of autistic patients referred to our laboratory.Data was analyzed from 433 patients with autistic traits tested using chromosome analysis and/or fluorescence in situ hybridization (FISH) and/or molecular testing for fragile X syndrome by Southern and PCR methods.The median age was 4 years. Sex ratio was 4.5 males to 1 female [354:79]. A chromosome (cs) abnormality was found in 14/421 [3.33 %] cases. The aberrations were: 4/14 [28%] supernumerary markers; 4/14 [28%] deletions; 1/14 [7%] duplication; 3/14 [21%] inversions; 2/14 [14%] translocations. FISH was performed on 23 cases for reasons other than to characterize a previously identified cytogenetic abnormality. All 23 cases were negative.Fragile-X testing by Southern blots and PCR analysis found 7/316 [2.2 %] with an abnormal result. The mutations detected were: a full mutation (fM) and abnormal methylation in 3 [43 %], mosaic mutations with partial methylation of variable clinical significance in 3 [43%] and a permutation carrier [14%].The frequency of chromosome and fragile-X abnormalities appears to be within the range in reported surveys . Limitations of our retrospective study include paucity of behavioral diagnostic information, and a specific clinical criterion for testing.Twenty-eight percent of chromosome abnormalities detected in our study were subtle; therefore a high resolution cytogenetic study with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice when the indication is autism. The higher incidence of mosaic fragile-X mutations with partial methylation compared to FRAXA positive population [50% vs 15–40%] suggests that faint bands and variations in the Southern band pattern may occur in autistic patients. Autism, often referred to as autistic disorder or infantile autism, is a complex behavioral disorder, which, by definition, develops prior to age three. Autism is defined completely on the basis of impairments in social interaction and communication, repetitive and stereotypic behaviors. Recent research has examined autistic traits in a population of twins and found that social impairment actually follows a unimodal distribution without a clear demarcation to separate cases of the disorder   and in other surveys, the common recurring chromosomal abnormality was duplication of the proximal 15q region  receptor subunits that map to the distal, apparently nonimprinted segment of the duplicated region ) ,45. It aulations -31, otheulations -49.Yardin et al 2002  recommenA deletion of 2q37.3 was identified by FISH in a patient with autism and macrocephaly in our study. Wolff et al 2002  also repA de novo deletion of chromosome 3, del(3)(p25), was found in one case with ASD and development delay in our patient pool. A deletion of 3q region was found by Konstantareas & Homatidis 1999 . Genome To date, there have been no reports of 12q deletions in patients with ASD, to the best of our knowledge. For the first time we report an interstitial deletion 46,XY,del(12)(q21.2q23.3) in a patient with ASD, development delay, mental retardation, multiple congenital abnormality, and family history of Down syndrome.46,XY,del(13)(q13.2q14.1)de novo was found in one of our patients' with ASD. Hyperserotonemia in autism is one of the longest-standing biochemical findings. The serotonin 2A receptor gene (HTR2A) on chromosome 13q14q21 is a primary candidate gene in autism. Converging data from recent genome screens also implicates the genomic region containing HTR2A -66. CorrThe recent physical mapping of the serotonin 5-HT(7) receptor gene (HTR7) to 10q23  raises tSo far there has been no observed association or link between chromosome 14 and ASD. Also, the mosaic inversion inv(14)(q11.2q33) [3/20] found in one of our patients is considered a cultural artifact when seen in 1or 2 cells.In this study, a patient with inv(17)(q23q25)de novo, had ASD, hypotonia and developmental delay. Chromosome 17q shows association with autism by genome wide scans and in linkage studies ,66,68. TWe report a patient with 18p deletion due to an unbalanced translocation between 14 and 18. Majority of the reported cases with autism involve deletion of 18q -72. A deOne of the breakpoints in the balanced translocation, 46,XX,t was 1q25, in the present study. A recent report, links D1S1675 that maps to chromosome 1q24 with autism  using obAlthough no cases with X- rearrangements were identified, in this study, the literature on abnormal X chromosome and autism is discussed as it has been cited in multiple cases. An autistic (ICD-10) woman had a translocation, t , a boy wThe typical clinical picture in FRAXA includes mental retardation, macro-orchidism, large ears, and prominent jaw. Within neurons, the FMR protein (FMRP) interacts with mRNA and ribosomes, suggesting a role in regulating protein synthesis . FMRP isA multicenter study in Sweden  found frForty-three percent of FRAX patients had a mosaic mutation in our study. The prevalence of males who carry a full mutation and a permutation is 15–20% -102 amonThe degree of mental retardation seemed not to be influenced by the presence of premutation alleles in some of the cells and a full mutation in the rest of the cells . While MThe molecular mechanism of the FRAX is based on the expansion of a CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. The instability of this CGG repeats containing region is not restricted to the CGG repeat itself but expands to the flanking region as well. de Graaff et al 1996  describeA 6-year old mosaic permutation female carrier had 29,65,80,39 repeats. The autism spectrum disorder in our patient may be due to diminished translational efficiency in FMRP production. Tassone et al (2000)  have sh,116,117.The significance of intermediate alleles found in 7/316 (Average age 4.36 years) of our patients requires further exploration on larger independent samples as they may raise the threshold for important developmental disabilities and/or physical features .The limitations of our retrospective study were a lack of uniform clinical criterion for inclusion and limited behavioral diagnostic information for purposes of dissecting the phenotype. However, for autism diagnosis it may be reliable, as shown in a recent study  which exIn our experience, the incidence of chromosome (3.33%) and fragile-X (2.2%) abnormalities totals to 5.53% in a population of patients with an indication of autism sent for genetic testing. Since, 28% percent of chromosome abnormalities detected in our study were subtle; a high resolution cytogenetic study for ASD patients with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice. The higher incidence of mosaic fragile-X mutations with partial methylation in our ASD population vs incidence of mosaicism in reported populations with fragile X syndrome [50% vs 15–40%], suggests that faint bands and variations in the Southern band pattern may occur rather frequently in fragile X positive autistic patients. The mosaic FRAXA mutation with normal and abnormal or partial methylation may also enhance the threshold for autism spectrum disorder. Careful analysis and high quality gels are required to rule out the type of mutation in patients with autistic traits. FRMP estimates in positive cases would be an extremely useful adjunct especially in mosaic cases. Future studies are necessary to corroborate the high incidence of mosaicism and their role in ASD.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Intra-aortic balloon counterpulsation (IABC) has an established role in the treatment of patients presenting with critical cardiac illnesses, including cardiogenic shock, refractory ischemia and for prophylaxis and treatment of complications of percutaneous coronary interventions (PCI). Patients requiring IABC represent a high-risk subset with an expected high mortality. There are virtually no data on usage patterns as well as outcomes of patients in the Indo-Pakistan subcontinent who require IABC. This is the first report on a sizeable experience with IABC from Pakistan.Hospital charts of 95 patients (mean age 58.8 (± 10.4) years; 78.9% male) undergoing IABC between 2000–2002 were reviewed. Logistic regression was used to determine univariate and multivariate predictors of in-hospital mortality.The most frequent indications for IABC were cardiogenic shock (48.4%) and refractory ischemia (24.2%). Revascularization  was performed in 74 patients (77.9%). The overall in-hospital mortality rate was 34.7%. Univariate predictors of in-hospital mortality included (odds ratio [95% CI]) age (OR 1.06 [1.01–1.11] for every year increase in age); diabetes (OR 3.68 [1.51–8.92]) and cardiogenic shock at presentation (OR 4.85 [1.92–12.2]). Furthermore, prior CABG (OR 0.12 [0.04–0.34]), and in-hospital revascularization (OR 0.05 [0.01–0.189]) was protective against mortality. In the multivariate analysis, independent predictors of in-hospital mortality were age (OR 1.13 [1.05–1.22] for every year increase in age); diabetes (OR 6.35 [1.61–24.97]) and cardiogenic shock at presentation (OR 10.0 [2.33–42.95]). Again, revascularization during hospitalization (OR 0.02 [0.003–0.12]) conferred a protective effect. The overall complication rate was low (8.5%).Patients requiring IABC represent a high-risk group with substantial in-hospital mortality. Despite this high mortality, over two-thirds of patients do leave the hospital alive, suggesting that IABC is a feasible therapeutic device, even in a developing country. Intra-aortic balloon counterpulsation (IABC) has an established role in the treatment of patients presenting with cardiogenic shock -3, refraWe reviewed the charts of 95 patients undergoing IABC at the Aga Khan University Hospital (AKUH), Karachi, Pakistan between January 2000 and December 2002. Patients requiring IABC in the operating room immediately following CABG to assist weaning off cardiopulmonary bypass were excluded from this study. However, those patients who underwent IABP implantation prior to surgery were included. The AKUH is a tertiary care hospital located in the metropolitan city of Karachi that receives a mixture of affluent as well as low and middle income patients and serves the entire city as a referral center for patients requiring high-intensity tertiary care. Variables collected included age, gender, indication for IABC (shock or non-shock), history of diabetes, hypertension, smoking, prior PCI or CABG, left ventricular function, refractory ischemia and treatment (revascularization vs. no revascularization). Cardiogenic shock was defined as a systolic blood pressure (SBP) of < 90 mm Hg for at least 30 minutes (or requirement of inotropes to maintain a SBP > 90 mm Hg) associated with hypoperfusion (decreased urine output or cool extremities) and a heart rate of ≥ 60 beats per minute. Left ventricular (LV) ejection fraction (EF) was assessed by visual estimation. LV function was recorded as normal for an EF of ≥ 55%, mildly impaired for an EF 40–54%, moderately impaired for an EF 26–39% and severely impaired if the EF was ≤ 25%. Heart failure was diagnosed using clinical signs as defined by the Framingham criteria . RefractAll variables were entered into Statistical Package for Social Sciences (SPSS) version 10. Means and standard deviations were calculated for continuous variables and frequencies for categorical variables. Variables were analyzed by simple logistic regression to calculate the unadjusted odds ratios for factors associated with in-hospital mortality. Those variables with a p value of ≤ 0.25 on univariate analysis were entered into the multivariable model and adjusted odds ratios for factors associated with in-hospital mortality were calculated. Finally, the model fit was assessed using the Hosmer-Lameshow test. A p value of < 0.05 was considered significant.Table Table 2 6.09; p = 0.637). In the adjusted analysis, a prior history of CABG did not remain a significant predictor of survival primarily because forty-five out of 46 patients underwent revascularization.In the multivariate analysis Table , the sigWhen age as a risk factor was further analyzed by plotting an ROC curve, an age cut-off of 66.5 years had a high specificity for the outcome of in-hospital mortality . Thus older patients requiring IABC suffer worse outcomes than younger subjects.The overall complication rate related to the device implantation was low. Eight patients (8.5%) developed limb ischemia necessitating removal of the IABP; however, only one of these eight required surgery. There were no significant bleeding complications although one patient developed a hematoma following removal of the device; however this patient did not require blood transfusion or surgical repair of the arteriotomy site.Patients requiring IABC are at high risk for death on account of their critical underlying conditions. Despite this, several data have suggested that IABC can improve morbidity and mortality in specific subsets of patients including those presenting with cardiogenic shock. The use of IABC in developing countries is limited on account of lack of equipment as well as skilled personnel who can insert and manage the device. Ours is the first report on a sizeable experience with IABC from the Indo-Pakistan subcontinent. Our experience is similar to that of other centers in the West. We report a high in-hospital mortality rate in patients undergoing IABC . However, given that nearly half of the subjects had cardiogenic shock at presentation, this mortality rate is reasonably acceptable. Advanced age (over 66.5 years), diabetes and cardiogenic shock at presentation were strong independent predictors of in-hospital mortality, while revascularization  was associated with high odds of survival. The latter finding is consistent with recently reported data from the IABP Benchmark Registry . Of partSeveral limitations of this study should be acknowledged. First, the sample size is fairly small and this is reflected in the relatively wide confidence intervals for the odds ratios. Due to a small sample size, it is difficult to make a comparison of correlates of mortality between subgroups, for example those presenting with cardiogenic shock versus those who did not and those undergoing surgical versus percutaneous revascularization. However, as expected, patients presenting with shock had a significantly higher mortality . Second, the patient group selected may not be representative of other centers in Pakistan given that our institution is a unique tertiary care hospital in the country. Third, our cohort did not contain patients undergoing prophylactic IABC prior to high-risk PCI for indications other than cardiogenic shock. This probably represents practice patterns at our institution whereby, largely due to cost constrains, very high-risk patients (for example those with multivessel disease and/or severe impairment of LV function) are preferentially send for surgery. The cumulative cost of IABC with multivessel stenting far exceeds that of a bypass operation. Only two patients required emergent IABC during PCI in the study period. This may reflect a selection of lower risk patients for PCI at our institution. Fourth, while the survival rate following IABC is nearly 65%, no analysis has been made of the cost effectiveness of this therapy.In conclusion, cardiogenic shock and refractory ischemia are common indications for IABC in a Pakistani setting. Patients requiring an IABP represent a high-risk group with substantial in-hospital mortality. This is consistent with the nature of the presenting illnesses in these patients and is similar to western data. Despite this high mortality, over two-thirds of patients do leave the hospital alive, suggesting that IABC is a feasible therapeutic device, even in a developing country. Age (particularly over 66.5 years), diabetes and cardiogenic shock at presentation are significant predictors of mortality in this group of patients. Revascularization is a significant predictor of survival and complication rates are acceptably low. Larger studies are needed to evaluate which subsets of patients benefit the most from this device and further cost effectiveness analyses are warranted.The author(s) declare that they have no competing interests.FHJ, SAK conceptualized this study and participated in the study design. FHJ performed the statistical analysis. HK, NFM collected the data. KAK, SD, AS were involved in manuscript review. AH, JT and NN participated in manuscript drafting and review. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Between cell divisions the chromatin fiber of each chromosome is restricted to a subvolume of the interphase cell nucleus called chromosome territory. The internal organization of these chromosome territories is still largely unknown.We compared the large-scale chromatin structure of chromosome territories between several hematopoietic chicken cell types at various differentiation stages. Chromosome territories were labeled by fluorescence in situ hybridization in structurally preserved nuclei, recorded by confocal microscopy and evaluated visually and by quantitative image analysis. Chromosome territories in multipotent myeloid precursor cells appeared homogeneously stained and compact. The inactive lysozyme gene as well as the centromere of the lysozyme gene harboring chromosome located to the interior of the chromosome territory. In further differentiated cell types such as myeloblasts, macrophages and erythroblasts chromosome territories appeared increasingly diffuse, disaggregating to separable substructures. The lysozyme gene, which is gradually activated during the differentiation to activated macrophages, as well as the centromere were relocated increasingly to more external positions.Our results reveal a cell type specific constitution of chromosome territories. The data suggest that a repositioning of chromosomal loci during differentiation may be a consequence of general changes in chromosome territory morphology, not necessarily related to transcriptional changes. It is a longstanding observation that chromatin distribution in the interphase cell nucleus varies with the cell type. Flemming described differences in nuclear appearance in 1882 . However-7], the We amended our experimental approach with the determination of the position of a gene locus relative to its chromosome territory. In several previous studies it was observed that a number of active genes located preferentially at the surface of their chromosome territories or even outside -12, whilcLys), which is highly active in macrophages, as a model system to explore the possibility of positional changes during activation of a single gene. cLys does not have co-regulated neighbors. Recently the gene cGas41 was found only 200 bp downstream of the polyA-site of cLys. cGas41 is expressed on a low level in all chicken tissues and cell lines tested, including all cell lines used here .To quantify chromatin dispersal of labeled chromosomes in cells of various differentiation stages, we counted the number of separate, labeled chromatin objects to which the chromosome territories disintegrated at increasing thresholds. In the investigated chicken cell types, chromosome territories of further differentiated cell types disaggregated into more objects. An increase in object number during differentiation may indicate that a significant number of compact chromatin domains with silent genes separate from each other into several, more decondensed, "open" chromatin domains. This would increase the accessibility to transcription factor complexes from the interchromatin compartment by increasing the available chromatin surface area of the chromosome territory. In human lymphoblasts gene rich chromosome 19 territories were found more decondensed than gene poor chromosome 18 territories  and elecMultipotent myeloid precursor cells have the smallest nuclei of the cell types investigated here. Myeloblasts have on average larger nuclei than proerythroblasts. If the observed disaggregation of chromosome territories were based on a nuclear volume increase, the larger myeloblast nuclei should have a stronger dispersion of chromosome territories than proerythroblasts. However, the opposite is true Figure . We thuscLys signal is more exterior than the chromosome 1 territory signal median but in unstimulated macrophages the opposite is true also argues for a cell type specific organization of chromosome territories. A similar observation has been described for a IL-3 induced differentiation of human leukemic K562 cells where the β-globin gene cluster does not change nuclear position but the harboring chromosome 11 territory does . UnfcGas41, which, albeit on a low level, is expressed in all cell types used in this study, including multipotent myeloid precursor cells  and froor cells . FormallWe describe several features of chromosome territory organization that differ between various hematopoietic chicken cell lines. While multipotent myeloid precursor cells had compact chromosome territories, the more differentiated cell types investigated here displayed somewhat disaggregated, diffuse territories. Although nuclear volumes generally are larger in the more differentiated cell types, they do not correlate with the changes in chromosome territory morphology. The chicken lysozyme locus as well as the chromosome 1 centromere is located preferentially in the interior of the chromosome territory in precursor cells and more external in more differentiated cells. Our data suggest that such a repositioning of chromosomal loci during differentiation may be a consequence of general changes in chromosome territory morphology, not necessarily related to transcriptional changes. The radial distribution of chromosomes 1 and 8 also differed between cell types. In summary our data argue for a cell type specific chromosome territory organization in the investigated cell lines.cLys but not the #1 centromere and also other karyotype aberrations, e.g. a trisomy of chromosome 2. All lines were diploid for chromosome 8. Cells were cultured in IMDM supplemented with 2% chicken serum, 8% fetal calf serum and 2 mmol L-Glutamin and maintained at 37°C with 5% CO2. For 3D-FISH experiments cells were cultured on #1.5 glass coverslips (170 μm thick). Except for macrophages, coverslips were pretreated for 35 min with poly-L-lysine . Activation of HD11 was achieved by the addition of 5 μg LPS  per ml medium and subsequent cultivation over night [All cells are from retrovirally transformed chicken cell lines ,57. HD50er night  for 15 mChromosome-specific paint probes for chicken chromosomes 1 and 8 were kindly provided by Dr. Felix Habermann and Dr. Johannes Wienberg, Munich. They were generated by flow sorting of metaphase chromosomes and subsequent degenerated oligonucleotide primed (DOP)-PCR . They prcLys the following amounts of DNA were precipitated for each μl hybridization mix: 2 μl label-DOP-PCR product of an early amplification round of the #1 paint plus 2 μl of a highly amplified paint probe for centromere detection, 50 ng pPoly-III-i Lys, 2.5 μg cot 1 DNA. When paint probes for #1 and #8 were cohybridized, 2 μl label-DOP-PCR product of an early amplification round was used for each chromosome and supplemented with cot1 DNA as above. Denaturation was 5 min at 85°C. Preannealing with the cot 1 DNA was performed for 25 min at 37°C. For 3D FISH, coverslips with cells were denatured in 70% formamide for 3 min at 70°C, and placed immediately in ice-cold 50% formamide/2 × SSC. They were then incubated with 5 μl hybridization mix under a sealed 18 × 18 mm2 coverslip at 37°C for 24 h-72 h. Air-drying was carefully avoided at all steps. Metaphase chromosomes were hybridized as described [All DNA for a given assay was mixed, precipitated and solved in deionized formamide. The same volume of 20% dextransulfat in 2 × SSC was added. In experiments with Detection was performed as described , using r3D image stacks (8 bit) were recorded with a Leica TCS SP confocal laser scanning microscope equipped with an argon  and a HeNe laser (633 nm) . A 100 × N.A. 1,4 oil objective was used to obtain stacks with a voxel size of 0.08 × 0.08 × 0.24 μm. Nuclei with separated homologous chromosome territories were preferably selected for recording. To measure the chromatic aberration, 0.5 μm multi-color latex beads  were fed to activated macrophages. After phagocytosis the cells were fixed and embedded like 3D-FISH preparations. The beads were in the cytoplasma and thus their optical environment was closer to the situation of FISH signals than beads mounted directly on a glass cover slip. The chromatic aberration in x, y and z was corrected before the assignment of signals to categories or distance measurements were performed.The program used for object counting was first applied by Cremer et al. . The ori2. This value reflects the amount of intensity (chromatin) that is enclosed in a given surface area. Chromosome territories with a richly folded surface thus have a rather low value whereas compact, homogeneous territories have a higher value. Most nuclei had zero or few objects at very high threshold levels /(object surface voxels) was measured for all objects and averaged. Object surface voxels are defined as voxels belonging to an object and having at least one of the 26 neighbors not belonging to the object. The unit is 1/μms Figure . TherefocLys and centromere signals with regard to chromosome 1 territories: Image stacks were imported in ImageJ  and eacernet at . The MancLys and the respective centromere were corrected for the chromatic aberration and the distance was calculated.High precision 3D-distance measurements ,36: GravThe quantitative assessment of 3D radial distributions of painted chromosome territories and the measurement of nuclear volumes was performed using a program developed by Dr. Johann von Hase, Heidelberg which is described in detail elsewhere . To deteThe study was designed by SD together with CB and TC. First experiments and development of techniques were performed by RM. S Stadler and VS performed 3D-FISH, microscopy, determination of cLys and centromere 1 localization and high precision distance measurements of all evaluated cells. RM carried out radial distribution statistics and volume measurements. All three were supervised by SD and TC. S Stein programmed and adapted the object counting program, supervised by CC. Object counting and statistical analysis was performed by SD. The manuscript was written by SD with substantial contributions by CB, TC and VS. All authors read and approved the final manuscript.Radial distribution of chromosomes 1 and 8 in nuclei of different cell types. These graphs show the same curves as presented in Figure Click here for file


Some primatologists have argued that to understand human nature we must understand the behavior of apes. In the social interactions and organization of modern primates, the theory goes, we can see the evolutionary roots of our own social relationships. In the genomic era, the age-old question, What makes us human? has become, Why are we not apes? As scientists become more adept at extracting biological meaning from an ever expanding repository of sequenced genomes, it is likely that our next of kin will again hold promising clues to our own identity.Many comparative genomics studies have looked to our more distant evolutionary relatives, such as the mouse and even yeast, to help interpret the human genome. Because the genomes of mice, yeast, and humans have diverged significantly since their last common ancestor—about 75 million years ago for mouse and human, and about 1 billion years ago for yeast and human—there are enough differences between the functional and nonfunctional regions to home in on biologically significant sequences, based on their similarity. Sequences that are similar, or conserved, in such divergent species are assumed to encode important biological functions. These comparative studies have successfully identified and characterized many human genes. And a similar approach comparing primate genomes can help scientists understand the genetic basis of the physical and biochemical traits that distinguish primate species. In this approach, however, rather than looking for genes that are shared across many species, scientists look for those that are unique to a species.One of the primary agents of genome evolution is gene duplication. Duplicated genes provide the raw material for the generation of novel genes and biological functions, which in turn allow the evolution of organismal complexity and new species.  James Sikela and colleagues set out to compare gene duplications between humans and four of our closest primate relatives to find the genetic roots of our evolutionary split from the other great apes. Collecting the DNA of humans, chimpanzees, bonobos, gorillas, and orangutans from blood and experimental cell lines, the researchers used microarray analysis to identify variations in the number of copies of individual genes among the different species. They analyzed nearly 30,000 human genes and compared their copy numbers in the genomes of humans and the four great apes.Overall, Sikela and colleagues found more than 1,000 genes with lineage-specific changes in copy number, representing 3.4% of the genes tested. All the great ape species showed more increases than decreases in gene copy numbers, but relative to the evolutionary age of each lineage, humans showed the highest number of genes with increased copy numbers, at 134. Many of these duplicated human genes are implicated in brain structure and function.The gene changes identified in the study, the authors conclude, likely represent most of the major lineage-specific gene expansions (or losses) that have taken place since orangutans split from the other great apes, some 15 million years ago.  And because some of these gene changes were unique to each of the species examined, they will likely account for some of the physiological and morphological characteristics that are unique to each species. One cluster of genes that amplified only in humans was mapped to a genomic area that appears prone to instability in human, chimp, bonobo, and gorilla. This region, which corresponds to an ancestral region in the orangutan genome, has undergone modifications in each of the other descendent primate species, suggesting an evolutionary role. In humans, gene mutations in this region are also associated with the inherited disorder spinal muscular atrophy. This fact, along with the observation that there are human-specific gene duplications in this region, suggests a link between genome instability, disease processes, and evolutionary adaptation.In their genome-wide hunt for gene duplications and losses in humans and great apes, Sikela and colleagues have highlighted genomic regions likely to have influenced primate evolution. With the impending release of the chimp genome and more primate sequences to follow, scientists can take advantage of both sequence-based and microarray-based genome information to wrest additional insights from our primate cousins and flesh out the details of the human story.


Ultrasound-mediated microbubbles destruction has been proposed as an innovative method for noninvasive delivering of drugs and genes to different tissues. Microbubbles are used to carry a drug or gene until a specific area of interest is reached, and then ultrasound is used to burst the microbubbles, causing site-specific delivery of the bioactive materials. Furthermore, the ability of albumin-coated microbubbles to adhere to vascular regions with glycocalix damage or endothelial dysfunction is another possible mechanism to deliver drugs even in the absence of ultrasound. This review focuses on the characteristics of microbubbles that give them therapeutic properties and some important aspects of ultrasound parameters that are known to influence microbubble-mediated drug delivery. In addition, current studies involving this novel therapeutical application of microbubbles will be discussed. The recent advances in gene therapy and molecular biology have improved the interest in methods of noninvasive delivery of therapeutic agents. Besides the well known application of microbubbles as contrast agents for diagnostic ultrasound, microbubbles have also been demonstrated an effective technique for targeted delivery of drugs and genes -6. DrugsTwo possible strategies for delivering drugs and genes with microbubbles are emerging. The first consists on the ultrasound-mediated microbubble destruction, which is based on the cavitation of microbubbles induced by ultrasound application, and the second is the direct delivery of substances bound to microbubbles in the absence of ultrasound. Different drugs and genes can be incorporated into the ultrasound contrast agents. It has already been demonstrated that perfluorocarbon-filled albumin microbubbles avidly bind proteins and synthetic oligonucleotides . In a siThe mechanisms by which ultrasound facilitates the delivery of drugs and genes result from a complex interplay among the therapeutic agent, the microbubble characteristics, the target tissue, and the nature of ultrasound energy. The presence of microbubbles in the insonified field reduces the peak negative pressure needed to enhance drug delivery with ultrasound. This occurs because the microbubbles act as nuclei for cavitation, decreasing the threshold of ultrasound energy necessary to cause this phenomenon. The results of optical and acoustical studies have suggested the following mechanisms for microbubble destruction by ultrasound: 1- gradual diffusion of gas at low acoustic power, 2- formation of a shell defect with diffusion of gas, 3- immediate expulsion of the microbubble shell at high acoustic power, and 4- dispersion of the microbubble into several smaller bubbles. Cavitation of the bubbles is characterized by rapid destruction of contrast agents due to a hydrodynamic instability excited during large amplitude oscillations, and is directly dependent on the transmission pressure ,18. It h2. However, it was a lower intensity of ultrasound (0.6 W/cm2) that caused more drug delivery with microbubbles. More recently, voltage clamp techniques were used to obtain real-time measurements of membrane sonoporation in the presence of albumin-coated microbubbles (Optison). Ultrasound increased the transmembrane current as a direct result of membrane resistance due to pore formation [The formation of pores in the membranes of cells as a result of ultrasound-induced microbubble cavitation has been proposed as a mechanism for facilitating the drug deposition. Taniyama et al  demonstrormation .Another important therapeutic property of microbubbles is their increased adherence to damaged vascular endothelium. Albumin-coated microbubbles do not adhere to normally functioning endothelium, but their adherence does occur to activated endothelial cells or to extra-cellular matrix of the disrupted vascular wall, and this interaction could be a marker of endothelial integrity . BecauseThe clinical use of viral vectors for gene therapy is limited because viral proteins elicit an immune response within the target tissue , and havTaniyama et al  have alsmyc protooncogene is responsible for the regulation of gene expression involved in the process of intimal hyperplasia that leads to restenosis. Synthetic antisense oligonucleotides, such as those to the c-myc protooncogene, can bind to the messenger ribonucleic acid and inhibit the synthesis of the protooncogenes. Therefore, antisense to c-myc protooncogene can prevent its translation into proteins that may be mediators of the pathologic process of restenosis. These synthetic agents, when administered directly into the vessel, have successfully inhibited restenosis after coronary or carotid injury [myc, 21 pigs had carotid balloon injury performed with an oversized balloon catheter and were randomized to receive intravenous antisense to c-myc bound to PESDA, intravenous antisense alone, or no treatment. The pigs that received antisense bound to PESDA also had transcutaneous 20 kHz ultrasound applied over the carotid wall following injections. The ultrasound targeted group showed a significantly lower percent area stenosis (8 ± 2%) than the two control groups  [Restenosis after vascular balloon injury or stent deployment has been shown to result from neointimal hyperplasia due to smooth muscle cell migration and proliferation. The c-d injury . In 1996d injury  demonstrmyc into coronary arteries using high phase liquid chromatography in pigs. Intravenous PESDA containing anti c-myc was given in the presence or absence of transthoracic 1 MHz ultrasound (0.6 W/cm2). In this study, the authors demonstrated that anti c-myc can be selectively concentrated within a stretch-injured coronary artery segment when given intravenously bound to PESDA. The decrease in neointimal formation following intravenous injection of anti c-myc with PESDA without ultrasound was similar to that observed with higher doses of the same antisense given directly into the coronary artery using an infiltrator delivery system [Since PESDA microbubbles adhere to sites of endothelial injury even in the absence of ultrasound, the efficacy of this therapy in inhibiting coronary restenosis has been evaluated in animals. Porter et al  measuredy system . The basy system ,21,31. Ay system . Since ly system , it is py system , and thuy system . Figure y system  have alsy system .Another innovative application of microbubbles and ultrasound is in the delivery of proteins that induce growth of endothelial cells, such as vascular endothelial growth factor (VEGF). Mukherjee et al  demonstrmyc protooncogene (0.19 ± 0.04 μg/mg), but low-frequency 20 kHz ultrasound significantly increased vascular uptake  when compared to antisense bound to PESDA alone (0.21 ± 0.06 μg/mg). The results of this study suggested that a lower frequency could be better suited to target antisense deposition into major vessels. Because there were minimal differences in peak negative pressure generated by 2 MHz and 20 kHz in this study , the enhanced uptake was attributed to a lower threshold for cavitation with 20 kHz ultrasound frequency.The effect of several ultrasound parameters, including transducer frequency and acoustic power, are known to influence microbubble destruction and, thus, the transfection of genes and drugs. Although the optimal ultrasound parameters for maximizing this process are not known, we will briefly discuss some important aspects. Unger et al  have sho2). The authors found a significant increase in VEGF uptake with the combination of ultrasound and PESDA at all power outputs when compared with controls, but there was a dose-dependent increase in the amount of VEGF uptake with increasing power until 0.6 W/cm2 and a subsequent plateau. Table In another study, the efficacy of ultrasound-mediated delivery of VEGF bound to PESDA into the myocardium of rats was evaluated with an ultrasound frequency of 1.0 MHz at various acoustical outputs (0.2, 0.4, 0.6, 0.8 and 1.0 W/cmHowever, a high peak negative pressure may have detrimental bioeffects. Several investigators have reported on the occurrence of tissue hemorrhage and endothelial cell damage after ultrasound exposure of cultured cells and organs containing air, such as the lungs or the intestine -39. Ay eDr. Jeane M. Tsutsui – declares no competing interests.Dr. Feng Xie – declares no competing interests.Dr. Thomas R. Porter – declares ImaRx Therapeutics, Inc.: Grant support and Consultant; Bristol Myers Squibb Medical Imaging: Grant support; AVI BioPharma, Inc.: Grant support


Early treatment of acute HIV infection with highly active antiretroviral therapy, followed by supervised treatment interruption (STI), has been associated with at least transient control of viremia. However, the durability of such control remains unclear. Here we present longitudinal follow-up of a single-arm, open-label study assessing the impact of STI in the setting of acute HIV-1 infection.Fourteen patients were treated during acute HIV-1 infection and subsequently subjected to an STI protocol that required retreatment if viral load exceeded 50,000 RNA copies/ml plasma or remained above 5,000 copies/ml for more than three consecutive weeks. Eleven of 14 (79%) patients were able to achieve viral loads of less than 5,000 RNA copies/ml for at least 90 d following one, two, or three interruptions of treatment. However, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most individuals. By an intention-to-treat analysis, eight (57%), six (43%), and three (21%) of 14 patients achieved a maximal period of control of 180, 360, and 720 d, respectively, despite augmentation of HIV-specific CD4+ and CD8+ T cell responses. The magnitude of HIV-1-specific cellular immune responses before treatment interruption did not predict duration of viremia control. The small sample size and lack of concurrent untreated controls preclude assessment of possible clinical benefit despite failure to control viremia by study criteria.These data indicate that despite initial control of viremia, durable viral control to less than 5,000 RNA copies/ml plasma in patients following treated acute HIV-1 infection occurs infrequently. Determination of whether early treatment leads to overall clinical benefit will require a larger and randomized clinical trial. These data may be relevant to current efforts to develop an HIV-1 vaccine designed to retard disease progression rather than prevent infection since they indicate that durable maintenance of low-level viremia may be difficult to achieve. Long-term follow-up of 14 patients on HAART during acute infection followed by supervised treatment interruptions shows that most failed to achieve lasting control of viremia. The use of highly active antiretroviral therapy (HAART) can dramatically prolong the life of individuals infected by human immunodeficiency virus 1 (HIV-1) . The sucIn the present study, we performed a detailed longitudinal assessment of the impact of early treatment followed by STIs in patients treated during acute or early HIV-1 infection. The main hypothesis of the study was that early treatment of acute HIV-1 infection followed by STI would lead to immune boosting and subsequent control of viremia without the need for drugs. The primary endpoint was the time to viral rebound above 50,000 copies/ml once or above 5,000 copies for three determinations separated by a week each. The early results of this trial were previously reported, showing that five of eight patients were able to achieve a plasma viral load of 500 copies/ml or less at a median of 6 mo off therapy [The hypothesis of the study was that early treatment of acute HIV-1 infection would confer immunologic maturation and subsequent control of HIV-1 without the need for ongoing drug therapy. Alternatively, if a breakthrough of virus replication was observed, this would provide a boost in HIV-1-specific immunity after reinstitution of antiviral therapy. The primary endpoint was the time to viral rebound to more than 50,000 copies/ml or viral loads above 5,000 copies/ml for three determinations separated by a week each. The secondary objective was to correlate immunologic and virologic parameters with any observed effects including evolution of HIV-1-specific T helper and cytotoxic T lymphocyte responses. The original study protocol, including the patient consent form and the institutional review board approval, can be found in Fourteen patients presenting with acute or early HIV-1 infection were enrolled in this study between July 1997 and January 2000 . Acute HSix of the 14 individuals were investigated in an interim study . These pEntry criteria included treatment with HAART before or shortly after HIV-1 seroconversion, viral suppression on HAART to less than 400 RNA copies/ml for 2 mo, HIV-1 viral load at the time of entry into the study of less than 50 RNA copies/ml, and lack of significant mutations conferring drug resistance ,12. LympHigh- and intermediate-resolution human leukocyte antigen class I typing was performed at a commercial laboratory  by sequence-specific PCR as described .GB virus C (GBV-C) RNA was detected using a two-step nested PCR amplification reaction from whole plasma RNA. Briefly, GBV-C RNA was extracted from plasma using the Qiagen Viral RNA Mini Kit  according to the manufacturer's instructions. Extracted RNA was reverse transcribed using the Qiagen OneStep RT-PCR Kit and amplified by nested PCR; in both steps primers specific for the 5′ UTR of GBV-C were used, as described previously .In order to analyze the chemokine receptor (CCR) 5Δ32 deletion polymorphism, the region spanning the 32-nt deletion was amplified by PCR, and the two alleles were separated by gel electrophoresis . The CCRWe synthesized 410 synthetic peptides 15–20 amino acids long at the Massachusetts General Hospital Peptide Core Facility on an automated peptide synthesizer using Fmoc technology, as described . Peptide6 PBMCs and if the well had at least three times the mean number of SFCs in the three control wells. The dependence of responses on CD8+ T cells was determined by measuring the depletion of CD4+ T cells using the Minimacs cell depletion system . When HIV-1-specific CD8+ T cell responses were detected against adjacent peptides, and therefore might represent targeting of the overlap region, responses to the weaker peptide were excluded for calculations of magnitude and breadth, as previously described [ELISPOT assays were carried out as described previously . Peripheescribed .5 cells) were incubated with baculovirus-derived recombinant p24 protein  at 5 μg/ml for 6 d and then pulsed with 3H thymidine at 1.0 μCi for 6 h before harvesting as previously described [Freshly isolated PBMCs  were able to achieve virologic control to less than 5,000 RNA copies/ml plasma for at least 90 d after one, two, or three treatment interruptions . The perOnce control was achieved, the majority of the patients experienced a subsequent rise in viremia. The median time between cessation of therapy and rebound of viremia  was 17 d .Six of 14 patients (43%) achieved a period of control after stopping therapy for 1 y, but only three of 14 (21%) were able to control viremia off therapy at less than 5,000 RNA copies/ml plasma for more than 2 y. Duration of viremia control during successive treatment interruptions was highly variable, and there was no increase in the sustainability of viral containment during successive STI cycles. The three patients achieving control of viremia for more than 2 y did so during the first (AC-10), the second (AC-02), and the third (AC-14) treatment interruption, respectively A. A pairThese data show that at least transient control of viremia to less than 5,000 RNA copies/ml plasma was achieved in the majority of study participants during at least one of the treatment interruptions, but that durable viral control in participants following treated acute infection occurred infrequently. Moreover, the data do not show a consistent pattern of augmentation of viral control with sequential treatment interruptions.3; range, 492–986), but the effect of treatment interruption was variable, even in those doing well, as defined by sustained low viral loads. Overall, 11 of 14 patients interrupted therapy for at least 12 mo, and these individuals were evaluated regarding the effect of treatment interruption on CD4+ T cell loss . Of the three patients who did not meet viral load criteria for restarting therapy for more than 2 y, one (AC-02) had an increasing CD4+ T cell count of 157 cells/mm3/y, one (AC-10) had a stable CD4+ T cell count (−9 cells/mm3/y) , and one (AC-14) experienced a decline of 344 cells/mm3/y. Comparison with data from the Multicenter AIDS Cohort Study (MACS) showed that the kinetics of CD4+ T cell loss was faster  than in untreated patients with early chronic HIV-1 infection (average loss of −67 cells/mm3/y in patients with a CD4+ T cell count of more than 350 cells/mm3 at baseline). However, CD4+ T cell loss rate was in the same range as what has been described after treatment interruption in chronic HIV-1 infection [3/y; AC-10, +44 cells/mm3/y; and AC-14, −110 cells/mm3/y. When the first 3 mo off therapy were excluded in order to minimize the potential effects of recent treatment on CD4+ T cell number, the rate of change in CD4+ T cell counts during the first year off therapy no longer differed statistically from the MACS data . A possible reason for steep CD4+ T cell slopes may be high CD4+ T cell counts at time of treatment interruption. Comparison with MACS data . The individuals who controlled viremia for a longer time either during the first STI or during any of the treatment interruptions were not different from those who experienced earlier breakthrough as measured by ELISA and Western blot status at initiation of HAART, coreceptor polymorphisms , or the presence or absence of GBV-C coinfection . The only parameter that was predictive of prolonged viral control during the first treatment interruption was a low viremia at time of institution of therapy (p = 0.01): there was a 2.8-fold increase in hazard per order of magnitude increase in viral load. This factor was no longer predictive when the period of longest control of viremia was considered. The time to rebound of viremia (>50 copies/ml or >400 copies/ml) did not correlate with the duration of viral control. Although 11 out of 14 individuals achieved at least transient control of viremia, and three experienced prolonged control, none of these patients possessed the HLA alleles B27 or B57 associated with better disease outcome [Although the study was small, we evaluated clinical and laboratory parameters to see if any was predictive of duration of viral control. Analyses included clinical and laboratory parameters at time of presentation with acute HIV-1 infection, genetic markers associated with different rates of disease progression, and the presence or absence of GBV-C coinfection. All patients presented with symptomatic acute infection. Time between onset of symptoms and institution of therapy did not affect duration of control following STI  (p < 0.05) during the first off-treatment period, reaching a median total magnitude of 2,725 SFCs/106 PBMCs and targeting a median of eight epitopes, and then were sustained when therapy was reintroduced. A further increase in the magnitude and breadth of HIV-1-specific CD8+ T cells was observed in the subsequent off-treatment periods, although these augmentations failed to reach statistical significance. The CD8+ T cell–mediated immune responses emerging during these consecutive cycles of treatment interruption were broadly directed, targeting all structural and most accessory and regulatory HIV-1 gene products (data not shown). However, the magnitude of HIV-1-specific CD8+ T cell responses at the beginning of the first , second , or third  treatment interruptions was not predictive of the time the study participants were subsequently able to stay off therapy according to study criteria.To assess the relationship between the clinical outcome and evolution of HIV-1-specific CD8+ T cells, we longitudinally analyzed the breadth and magnitude of CD8+ T cell responses using an interferon-γ ELISPOT and a panel of 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence. At the beginning of the first STI, HIV-1-specific CD8+ T cells were weak (median of 590 SFCs/106 PBMCs) A and nar6 PBMCs) B. CD8+ Tr = 0.92, p < 0.001) . The increase in CD8+ T cell epitopes targeted during the first treatment interruption was also linearly correlated to the duration until virological failure   C. Simila< 0.001) D. Howevep = 0.72; second, p = 0.12; and third, p = 0.60, respectively). These HIV-1-specific CD4+ T cell responses also tended to rise with reinitiation of therapy, and some of them were very robust, with stimulation indices over 50 detected in several individuals  , or third  treatment interruption was not statistically predictive of the time the study participants were subsequently able to stay off therapy according to study criteria.We next analyzed evolution of lymphoproliferative responses to recombinant HIV-1 p24 Gag protein in order to assess HIV-1-specific CD4+ T cell function. Most individuals had no detectable response at baseline prior to treatment, consistent with prior reports of patients with acute HIV-1 infection . After iividuals E. Simila = 0.43) F, secondAlthough early treatment of acute HIV-1 infection followed by treatment interruptions may enhance control of viremia ,8, the dThe standard immunologic assays and virologic assessments in this cohort revealed considerable heterogeneity among the study participants, and did not show a consistent pattern in duration of viremia control during successive treatment interruptions. Eleven of 14 patients (79%) were able to maintain a viral load of less than 5,000 copies/ml for at least 90 d, but progressive loss of control ensued in the majority of patients and only three patients (21%) were able to maintain control for more than 2 y. These three patients did so during the first (AC-10), the second (AC-02), and the third (AC-14) STI. Clinical, genetic, and immunological parameters did not distinguish these three individuals from the other 11 patients, nor did they predict the duration of control following treatment interruption. Indeed, the longer a patient was off therapy, the stronger and more broadly directed the CD8+ T cell responses became, but these were still not sufficient to maintain prolonged control in most patients. Although three patients did not complete the study as initially intended , the results are not substantially different if these three are censored rather than considered to have failed to control.Loss of viral control in this cohort occurred not only in the presence of strong CD8+ T cell responses, but in most cases also in the presence of virus-specific CD4+ T cell responses, although the CD4+ T cell responses often declined during periods of viremia. In addition, total CD4+ T cell numbers were also monitored and declined in most patients over time, including one of the three patients who were able to maintain low viral loads for at least 2 y. Mechanisms leading to rapid CD4+ T cell loss need to be further studied in future STI trials. Other parameters including chemokine receptor polymorphisms  and GBV-The reasons for progressive loss of control despite augmentation of virus-specific CD4+ and CD8+ T cell responses remain to be defined. In one individual (AC-06), HIV-1 superinfection in the setting of strong and broadly directed HIV-specific cellular immune responses was associated with the loss of viral control, as previously reported . No otheThese data are important in light of other recent data on treatment interruption in both acute and chronic infection. In chronic HIV-1 infection, STI studies showed only marginal, if any, improvements of HIV-1 viremia control following a number of treatment interruptions cycles, despite at least transient increases in HIV-1-specific CD8+ and CD4+ T cell responses ,53,54,55Although durable control of viremia was not achieved, it is noteworthy that the majority of patients were able to achieve transient relative containment of viremia, providing rationale for future studies aimed at further enhancing immune control. Early treatment alone should still be considered an important therapeutic option. Therapeutic vaccinations administered after treated acute HIV-1 infection and before cessation of therapy have given disappointing results thus far , but theAlthough the present study shows progressive viral breakthrough, it was not designed to address whether there might be a change in set point viremia achieved or overall clinical benefit through transient early treatment of acute HIV infection. The definition of failure chosen for this study was a viral load of greater than 5,000 RNA copies/ml plasma, which at the time the study was initiated corresponded to the level of viremia at which treatment was recommended. Larger randomized trials will be needed to determine the potential clinical and virologic benefit of approaches based on STIs. In studies of untreated infection, there is only a 5-fold difference in viremia separating the quartile with the slowest disease progression from the quartile with the most rapid progression , suggestProtocol S1(68 KB DOC).Click here for additional data file.Protocol S2(47 KB PDF).Click here for additional data file.Protocol S3(187 KB PDF).Click here for additional data file.Protocol S4(36 KB PDF).Click here for additional data file.Highly active antiretroviral therapy (HAART), which is used to treat patients with HIV, can have nasty side effects and is expensive. As a result, for the last five years scientists have been trying to determine if it is possible to give patients breaks from taking antiretroviral drugs, without patients' health suffering. It is likely that such a treatment strategy  will not work in patients who have been infected with HIV for a long time. However, until now, the jury was out about whether STI could be of benefit to patients who had only recently been infected with HIV. Some research suggested that if newly infected patients had short “holidays” from taking HAART, it might help to boost their immune system—which in turn might help to keep HIV at bay.The researchers studied 14 patients who had recently been infected with HIV. Patients were treated until their viral load was below the limit of detection by using a very sensitive method. As they were then watched after treatment interruption, most of them were able to maintain a low, though detectable, viral load for some time. The researchers then stopped the treatment and carefully watched the patients over a period of up to five years. If the number of viruses in the patients' blood rose too high, the researchers gave the patients HAART again until the number of viruses fell again. In about half the patients, treatment needed to be restarted within a year because the viruses had started becoming more numerous in the blood. The doctors had hoped that it would take much longer for the number of viruses to reach this level. In other words, giving patients “drug holidays” did not help to keep the virus at bay for long periods of time.It is relatively rare for doctors to be able to diagnose HIV shortly after an individual becomes infected with the virus. Many people have HIV for a long time before they see a doctor. Therefore, even if the STI strategy had worked, it would only have been relevant to a few patients. In addition, the study was small and preliminary, so we have to be careful about reading too much into the results. A limitation in the study is that it did not address whether there might be a long-term clinical benefit despite a gradual increase in viral load.It is important to remember that this was an experimental trial—patients with HIV should not stop taking antiretroviral drugs unless their doctor specifically tells them to do so. Although it looks as though STI may not work in the way everyone had hoped, these results may help scientists develop an HIV vaccine that is designed to keep the disease at bay. Whether early treatment of acute infection has an overall benefit in terms of time-to-development of AIDS or need for long-term treatment after drug discontinuation will need to be answered in larger clinical trials designed to answer these important questions.http://www.aidsinfo.nih.gov/AIDSinfo: http://www.aidsmap.com/AIDSmap: http://www.nlm.nih.gov/medlineplus/aids.htmlMedline Plus AIDS Information: