The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs).Ex-vivo treatment of AVM tissues: Intact AVM tissues were treated with doxycycline for 48 hours. Active and total MMP-9 in the medium were measured. Pilot trial: AVM patients received either doxycycline (100 mg) or placebo twice a day for one week prior to AVM resection. Active and total MMP-9 in BVM tissues were measured.Ex-vivo treatment of AVM tissues: Doxycycline at 10 and 100 μg/ml significantly decreased MMP-9 levels in AVM tissues ex-vivo . Pilot trial: 10 patients received doxycycline, and 4 patients received placebo. There was a trend for both MMP-9 levels to be lower in the doxycycline group than in the placebo group .A clinically relevant concentration of doxycycline decreased MMP-9 in ex-vivo AVM tissues. Furthermore, there was a trend that oral doxycycline for as short as one week resulted in a decrease in MMP-9 in AVM tissues. Further studies are warranted to justify a clinical trial to test effects of doxycycline on MMP-9 expression in AVM tissues. Brain arteriovenous malformations (AVM) represent a relatively infrequent but devastating source of neurological morbidity in relatively young adults . PreventMatrix metalloproteinases (MMPs), a family of proteolytic enzymes, degrade extracellular matrix proteins, cell surface molecules, and other peri-cellular substances . ExcessiThere is an increasing interest in utilizing MMP inhibitors in treating vascular diseases including abdominal aortic aneurysms. Doxycycline is a clinically available antibiotic agent that possesses non-specific inhibitory effects on various MMPs, and for years it has had a well-established safety record in treating infectious diseases.The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline as an MMP inhibitor to decrease MMP-9 activity in AVMs and potentially decrease the rate of spontaneous hemorrhage. This exploratory investigation supports the concept that further studies be conducted to document the ability of tetracycline and its derivatives to decrease MMP-9 levels in AVM nidal tissue. Such a demonstration could provide a firm rationale for proceeding with clinical trials to test the hypothesis that tetracycline and its derivatives are useful to decrease the rate of spontaneous hemorrhage from AVMs in otherwise untreatable patients or in those awaiting interventional treatment.First, we demonstrate that doxycycline can decrease MMP-9 activity in cultured AVM tissues as a proof of the concept. Further, we present results from a pilot clinical study demonstrating effects of oral doxycycline treatment on MMP-9 expression in AVM tissues.After institutional review and informed consent, we obtained AVM specimens after microsurgical resection. AVM nidus was dissected away from any adjacent brain tissue in the operating room and a representative portion of nidus tissue was used for ex-vivo treatment. Ex-vivo culture of AVM tissue was performed using previously described method with modifications .AVM tissues were minced into 1–2 mm fragments and vigorously washed with phosphate buffered saline (PBS). Blood cells dissociated from the tissues were removed by filtration. AVM tissue fragments were placed onto cell culture inserts and immersed in cell culture medium. During the first 24 hours, AVM tissues were incubated in DME H-21 medium containing 10% fetal bovine serum (FBS) to aid in tissue recovery. Following this recovery period, tissue debris and dissociated cells were removed, and AVM tissues were incubated in DME H-21 medium containing 1% FBS for 4 hours. The tissue fragments were equally divided into 12–24 wells. Then AVM tissues were incubated in the medium containing PBS (control) or doxycycline . Each group included 3–5 wells. Size of the tissues dictated a number of treatment groups in each specimen.Medium from each well was collected after 48 hours. Active and total MMP-9 were measured using substrate zymography. Medium was mixed with SDS sample buffer  and separated under non-reducing conditions in a 10% zymogram gel (Invitrogen) containing 0.1% gelatin incorporated as a substrate. Recombinant MMP-2 and MMP-9 proteins (R&D systems) were used as positive controls. After running, the gel was incubated with renaturing buffer (Invitrogen). The gel was then incubated with developing buffer (Invitrogen) overnight at 37°C. The gel was then stained with colloidal blue stain (Invitrogen). Proteolytic bands in the zymogram gels were quantified using Image J Software (NIH).Tissue viability was assessed by measuring the amount of LDH (lactate dehydrogenase) released in the medium according to the manufacturer's instructions . Some of the tissue fragments were embedded in paraffin for histological assessment.Fourteen AVM patients received either 100 mg of doxycycline or placebo twice a day for one week, prior to elective AVM resection. During the AVM resection, AVM tissues were collected, and nidus tissues were frozen in liquid nitrogen. Frozen tissues were stored at -80°C until analysis. Clinical data were collected as previously described .The specimens were homogenized and insoluble materials were removed by centrifugation at 3000 rpm for 5 minutes. We used total MMP-9 ELISA kit (R&D) and active MMP-9 ELISA kit (Amersham).P < .05.Data are presented as mean ± standard deviation. The data for total MMP-9 and active MMP-9 from ex-vivo AVM tissues are presented as a relative expression with control brain samples as 100%. We used ANOVA for comparison, and statistical significance was taken at We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study.AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro.There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability Figure . HoweverTissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues Figure . On day Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9  Figure . In addiAVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery.Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1–10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4.There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9  Figure  &4D. In 14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design.10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group  . In patients with abdominal aortic aneurysm, doxycycline treatment at a conventional dose, 200 mg/day, resulted in a mean plasma concentration of doxycycline at 4.6 μg/ml with a range of 1.3 to 14.4 μg/ml in humans, and there was a corresponding reduction in plasma MMP-9 levels . SimilarOne of the limitations of the ex-vivo study was the relatively short duration of doxycycline treatment. We chose 48 hours treatment to ensure tissue viability and integrity. Although we chose the doxycycline concentrations based on plasma doxycycline levels achieved by a commonly used therapeutic regimen, it is possible that tissue levels of doxycycline might be significantly lower than those of plasma. Furthermore, functional consequences of the reduction of MMP-9 in AVMs need to be examined in animal models or clinical studies. However, at the present time, there is no animal model that approximates recurrent intracranial hemorrhage from AVMs. There are models that can mimic certain aspects of the AVM phenotype. Hyperstimulation of mouse brain with VEGF using adenoviral transduction causes an increase in capillary density, increased MMP-9 activity, and may, in the appropriate genetic background, result in small vascular malformations . DoxycycDoxycycline has been shown in other human vascular diseases to reduce MMP levels. For example, Axisa et al. treated patients undergoing carotid endarterectomy with doxycycline or placebo for 2–8 weeks. Although MMP-1, MMP-3, and MMP-9 were reported to be increased, only MMP-1 had a statistically significant reduction by doxycycline; there was a trend for MMP-9 to be decreased by 22%. Baxter et al. used a much longer treatment period, 6 months, in patients with abdominal aortic aneurysm. They reported a gradual reduction of plasma MMP-9 levels by oral doxycycline over 6 month treatment period (baseline vs 3 months vs 6 months: 119 ± 38 vs 84 ± 33 vs 66 ± 24 ng/ml) .To further explore the feasibility of a clinical application of doxycycline in modifying the clinical course of AVMs, we conducted a pilot clinical trial. This trial was primarily designed to test the effects of a short-term treatment with oral doxycycline on the expression of MMP-9 in AVM tissues. Despite the small sample size and short duration of treatment used in this study, there was a clear trend for one week of oral doxycycline treatment prior to surgical resection to reduce expression of both active and total MMP-9 in AVM tissues. These results indicate the feasibility of oral treatment with tetracycline derivatives in reducing MMP-9 activity in AVM tissues.Despite a large difference in the mean values of MMP levels in our pilot data, variance of MMP levels was high. Accordingly, a sample size of 46 in each group would be needed to demonstrate a difference in active MMP-9, assuming an alpha of 0.05 and power of 80%. However, variance may be reduced by utilizing a longer duration of treatment, as suggested by studies in other diseases ,12. A clThere are some further considerations that may account for our high variance that could be avoided in future studies. Surgical AVM tissues consist mainly from nidal tissues, but they may contain intervening astroglial or neuronal tissues that may not express angiogenic factors to the same extent as nidal tissue. Histological examination of each AVM tissue fragments to confirm a predominance of nidal tissues may yield more homogenous tissue and decrease variability in MMP-9 levels. Some of the homogenized samples underwent a several "thaw-freeze" processes during the preliminary phase of the experiments, which may have increased variability in MMP-9 levels. Embolization treatment may affect production and activation of MMPs. Our previous study, however, showed no association between MMP-9 and embolization treatment in 37 AVM patients . Based oWe used different methods to measure MMP-9 in the medium collected from ex-vivo AVM tissues and homogenized AVM tissues from the pilot clinical trial. We chose zymography, a gel based method detecting enzymatic activity of MMPs, for ex-vivo AVM tissues, because the medium volume that can be used for MMP-9 assay was extremely limited, and our preliminary experiments showed higher sensitivity of zymography compared to ELISA. One of the disadvantages of using zymography is that "gel-to-gel" variability in sensitivity makes it difficult to combine the data obtained from multiple gels. On the other hand, ELISA method allows researchers to assay approximately 40 samples at a time when duplicates are used. In addition, establishing a standard curve, ELISA method can express MMP-9 expression values by the absolute unit such as μg/ml or ng/100 μg protein. Therefore, for the clinical pilot trial, in order to quantitatively compare MMP-9 expression among a large number of samples originally planned, we used ELISA to MMP-9 expression.In summary, results suggest that doxycycline may have a therapeutic potential to reduce MMP-9 activity in AVM tissues and stabilize blood vessels that are prone to rupture. Our findings may provide a basis for a larger clinical trial to study effects of tetracycline derivatives in preventing AVM hemorrhage.The author(s) declare that they have no competing interests.TH, a corresponding author, participated in all aspects of the study and manuscript preparation. MMM participated in study design, experiments, analysis, interpretation of data, and manuscript preparation. JFL participated in experiments, analysis and interpretation of data. MTL participated in study design, analysis, interpretation of data, and manuscript preparation. WLY participated in study design, analysis, interpretation of data, and manuscript preparation.The pre-publication history for this paper can be accessed here:


Malaria takes a heavy toll in Niger, one of the world's poorest countries. Previous evaluations conducted in the context of the strategy for the Integrated Management of Childhood Illness, showed that 84% of severe malaria cases and 64 % of ordinary cases are not correctly managed. The aim of this survey was to describe epidemiological, clinical and biological features of malaria among <5 year-old children in the paediatric department of the National Hospital of Niamey, Niger's main referral hospital.th to October 25th. Microscopic diagnosis of malaria, complete blood cell counts and measurement of glycaemia were performed in compliance with the routine procedure of the laboratory. Epidemiological data was collected through interviews with mothers.The study was performed in 2003 during the rainy season from July 25256 children aged 3–60 months were included in the study. Anthropometrics and epidemiological data were typical of a very underprivileged population: 58% of the children were suffering from malnutrition and all were from poor families. Diagnosis of malaria was confirmed by microscopy in 52% of the cases. Clinical symptoms upon admission were non-specific, but there was a significant combination between a positive thick blood smear and neurological symptoms, and between a positive thick blood smear and splenomegaly. Thrombopaenia was also statistically more frequent among confirmed cases of malaria. The prevalence of severe malaria was 86%, including cases of severe anaemia among < 2 year-old children and neurological forms after 2 years of age. Overall mortality was 20% among confirmed cases and 21% among severe cases.The study confirmed that malaria was a major burden for the National Hospital of Niamey. Children hospitalized for malaria had an underprivileged background. Two distinctive features were the prevalence of severe malaria and a high mortality rate. Medical and non-medical underlying factors which may explain such a situation are discussed. Malaria accounts for 1 in 5 of all children deaths in Africa. Late presentation, misdiagnosis, inadequate management, unavailability or stock-outs of effective drugs are factors that influence high case-fatality rates even among hospital in-patients  is to esNiger is situated in the eastern part of West Africa in the Sahelo-Saharan zone. It is a landlocked country surrounded by Algeria, Libya, Chad, Nigeria, Benin, Burkina-Faso and Mali. The country's climate is a rainy season from June to October and a dry season during the rest of the year. The hottest months are April and May.th of 175 countries with an annual GDP of about $160 per capita (2001). The demographic situation is characterized by a population of 11,544,000 inhabitants (2002) which is expected to double by 2025 with an annual growth rate of 3.5% (1999–2002). The infant and child mortality rate is 255‰ and 63% of the population live below the poverty line ($1 per day per person) [According to the Human Development Index (UNDP 2003), Niger ranks 174The Bamako Initiative of 1987 was adopted by Niger but in a context of extreme poverty, the generalization of the cost recovery may have an impact on the access to health facilities, which should be assessed.Plasmodium falciparum is the predominant species partially associated with Plasmodium malariae and less frequently with Plasmodium ovale, according to geographical variations [Malaria is endemic in Niger with 97% of the population exposed to the risk of malaria , but somriations .Anopheles gambiae and Anopheles arabiensis may play a substantial role in malaria transmission but Anopheles funestus which was no longer found after 1970 seems to have reappeared in several sudano-sahelian zones of Niger [Among the anopheles of epidemiological importance, only of Niger ,11.Resistance to chloroquine was first described in 1991  but few Malaria remains a main cause of morbidity in Niger with an average of 850,000 cases per annum, corresponding to an annual incidence of 80‰ inhabitants, and 33.97% of reported deaths are due to malaria . These eIn Niger, as in many sub-Saharan countries, malaria takes a heavy toll, especially among young children in poor households. An evaluation – conducted in 2003 by the University Research Corporation  in the cA high proportion of deaths in the National Hospital of Niamey (NHN) is related to malaria. The poor quality of medical care makes it difficult to assess the real burden of malaria in terms of mortality and morbidity. Misdiagnosis is likely to be quite common, either in excess or default. The objective of the present prospective study was to describe the epidemiological, biological and clinical features of cases of malaria in children in the paediatric department of the NHN in order to recommend more relevant decision-making and interventions.th to October 25th, which is the peak transmission period. The NHN is the country's main referral hospital and has 852 beds. The paediatric department has two sections: section A (60 beds) for children from 0 to 24 months, and section B (60 beds) for children from 25 months to 15 years of age. A majority of patients come from the urban community of Niamey but some may be referred from all parts of the country.The study was carried out in the NHN during the rainy season of 2003 from July 25Children from 3 months to 5 years of age (60 months) admitted to Paediatric department A or B for presumed malaria were included in the study and treated following procedures normally used in the department: children went through the emergency department where a clinical examination was performed by doctors or by medical students in their final year, venous blood was drawn for biological tests , confirmed cases of malaria were treated intravenously with quinine twice a day for 2–3 days, followed by oral treatment .Upon admission, or within 24 hours, a physician conducted interviews with mothers in order to collect epidemiological data. Information such as age of the parents, number of siblings, the child's rank in relation to siblings, care-seeking previously to hospitalization, delay between the onset of disease and admission to the NHN, and treatment before hospitalization, was collected. The socio-economic background of households was evaluated through the Niamey neighbourhood where children originated from, the educational level of the mother (whether illiterate or not), the occupation of the mother and father, and the existence or non existence of a regular salary.Biological tests were performed along the routine procedures of the NHN's laboratory. Thick and thin blood smears were prepared and stained following standard procedures: Giemsa 10% for 20 minutes  for thick smear, and rapid staining  for thin smear. A thick smear was declared negative only after examination of 200 fields (obj × 100). All smears were checked the following day by a different technician. Parasitaemia was measured on thin smear and expressed as a percentage of parasitised red blood cells. Haematological tests – haemoglobin concentration, hematocrit and complete blood cell count – were performed automatically using a KX21 (Sysmex). The blood glucose rate was performed with a COBAS Mira (Roche) using the glucose oxidase technique .P. falciparum on thick smear and at least one of the following clinical or biological criteria : coma , impaired consciousness (Blantyre >2 and < 5), repeated convulsions (≥ 2/24 hours), prostration, respiratory distress, jaundice, metabolic acidosis (bicarbonates < 15 mmol/L), severe anaemia (Hb < 5 g/dL or Ht < 15%), hyperparasitaemia (parasitaemia > 4% in non-immune patients), macroscopic haemoglobinuria, renal failure, collapse (TAS < 60 mmHg before 5 years of age), abnormal bleeding or pulmonary oedema (X-ray criterion). The conditions in the NHN in 2003 made it impossible to asses acidosis, renal failure (based on creatinine) and pulmonary oedema. Only the following criteria could be considered: coma, impaired consciousness, convulsions, respiratory distress, jaundice, abnormal bleeding, severe anaemia, hyperparasitaemia, hypoglycaemia.According to the 2000 WHO criteria , severe Children were weighed. The index weight-for-age (weight/age ratio) was calculated and analysed with the Epi-Info software, based on the reference population defined by the US National Center for Health Statistics.(NCHS). Malnutrition was defined as a weight/age ratio more than 2 SD below the NCHS's reference population . MalnutrVersion 6 of the program Epi-Info was used for statistical analysis. ANOVA and Kruskal Wallis tests were used for mean comparisons whenever appropriate. Proportions were compared using chi-square tests. Bivariate odds ratio and their 95% confidence intervals were calculated to measure the combination between fatal outcome and different variables. For all tests, a p-value below 0.05 was considered significant.The routine management of the children was not changed. Verbal consent was obtained from the parents after informing them in their native language. The parents were allowed to remove their child from the study at any time during follow-up. All data was entered anonymously into a database and identification numbers were coded. No ethnic data was registered. The protocol was submitted to and approved by the Ministry of Public Health and Endemic Diseases Control's National Malaria Control Program.256 children were recorded over the 3 month survey: 138 males, 118 females (sex ratio = 1.17). The average age was 20.2 months: 3–12 months, n = 103 (40%), 13–24 months, n = 103 (40%) and 25–60 months, n = 50 (20%). The average age of parents was 27 years for the mothers, 39 years for the fathers. The number of living siblings averaged 2.8, and 35% of the children were first born. All children came from the urban community of Niamey, and 241/256 (94 %) from poor neighbourhoods: unsanitary neighbourhoods, near the river or near permanent ponds, some of them without electricity or tap water. The educational level of the mothers was low: 246/256 (96%) were illiterate, and 248/256 (97%) were home workers. Only 58/256 fathers (23%) had a monthly fixed income . The others had unsteady jobs with no stable income.77 children out of 217 documented files for this item (35%) were considered malnourished and 42% dehydrated as a result of clinical examination. The percentage of malnutrition, however, was 58% based on the weight/age ratio, of which 30% were moderate and 28% severe cases of malnutrition. The youngest children (under 2 years old), particularly the 13–24 months age group, which corresponds to the weaning period, were most affected , digestive disorder (51%) and convulsions (19%). These symptoms were often combined  had a positive thick blood smear. The percentage of treated children was the same among children with a negative thick blood smear as among those with a positive one (87% versus 86%). Both groups had quite a similar corrected axillary temperature (38.6° with positive thick smear versus 38.4°).The clinical presentation of children admitted to hospital with a respective prevalence of the various observed symptoms is summarised in Table 3, we found that thrombopaenia was statistically combined with a positive blood smear  . 94 % of children had anaemia (haemoglobin < 11 g/dL). This massive proportion of anaemia was observed in both groups: 95% versus 92% (NS) for "simple anaemia" (haemoglobin < 11 g/dL), and 42% versus 31% (NS) for severe anaemia (haemoglobin < 5 g/dL) (Table 114/132 children with a positive blood smear (86%) met the criteria of severe malaria according to the clinical and biological criteria explored in this study. The most frequent criteria were convulsions (71/114 = 62%) and severe anaemia (55/114 = 48%)  on the biological diagnosis. Non-specific clinical diagnosis followed by a presumptive treatment carried out without any biological confirmation, with an almost total absence of written follow-up in the management of children, are elements showing how difficult it is to diagnose malaria attacks in endemic areas. Both theoretical and practical parameters interfering in the diagnosis of malaria have been reviewed by Rogier .A particularly high percentage of severe malaria cases (86 %) was noted in the study population. Five children aged 6 months or under (one of them aged 3 months) were suffering from severe malaria, confirming the existence of severe malaria among very young children as already pointed out in other studies ,29. The The overall case fatality rate of malaria was 20% among confirmed cases and 21% among severe cases. These values are higher than those observed in other African countries also based on 2000 WHO criteria: 11.9% in Ethiopia , 12% in pfcrt (T76) mutation which confers chloroquine resistance [pfcrt mutation was present in 10/30 samples . These preliminary results need to be completed but such a level of genotypic drug resistance clearly seems insufficient to explain the high mortality rate observed at the NHN. Other implied factors have to be identified.However, such a level of hospital mortality among children who have already received treatment before admission could indicate an increase in the level of chloroquine resistance. Data on resistance is still insufficient in Niger but studies are presently being carried out. Detection of the sistance  was perfOne of them is anaemia. As the prevalence of severe anaemia is very high, the capacity of the blood bank to provide blood in a short time is an important factor in reducing mortality. Transfusion facilities are woefully inadequate in the NHN.This survey conducted at the National Hospital of Niamey during the 2003 rainy season was an opportunity to examine the features of childhood malaria in a referral hospital of one of the world's lowest income countries. One of the dominant features in the NHN is that malaria affects an underprivileged population already affected by malnutrition. Only 52% of presumed cases are parasitologically confirmed. The rate of severe cases is high (86%) with two main clinical presentations: severe anaemia in less than 2-year old children and neurological form between 2 and 5-year old children. The mortality rate (21% among severe cases) is higher than established by most previously published data.The study highlights several underlying factors that contribute to such an alarming situation:(1) Patient factors: poor socio-economic background leading to multiple difficulties in getting access to healthcare , and also the persistence of traditional beliefs interfering with care seeking behaviour.(2) Provider factors: the recovery of medical care costs, although essential for the financial survival of the hospital, leads to multiple difficulties for the patient as seen above. Healthcare quality standards are also too low at all levels of the health organization, not just because of technical deficiencies (e.g. deficiency in intensive care units or in transfusion supplies) but also because of the inadequate training and attitudes of health-care workers.All these factors need to be taken into account in order to find ways of improving the management of malaria in children. A recent awareness initiative is under way at the NHN, and several initiatives are being taken towards a gradual involvement of the hospital in a quality-oriented policy. But such local interventions will not reach their goal if extra efforts are not made at all levels to fight against disadvantages in the access to health facilities.FGA was an initiator of the study and took part in its development, supervised laboratory staff, took part in the analysis of the data, and wrote this article. EA was an initiator of the study, took part in its development, coordinated the clinical study, took part in the analysis of the data and in the writing of the paper. VL and MG took part in the development of the study, collected clinical and epidemiological data, and analysed the data. MLI conducted molecular tests in the Institut Pasteur de Madagascar. HK and HB supervised medical and nursing staff in the paediatric department. All authors have read and approved the final manuscript.


Using expression pattern criteria, we identified 360 genes with preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or cis-element analysis tool to identify clusters of cis-elements with compositional similarity among multiple genes. Several clusters contained genes that shared 5–6 cis-elements that included ETS and zinc-finger binding sites. cis-Elements AP2 EGRF ETSF MAZF SP1F ZF5F and AREB ETSF MZF1 PAX5 STAT were shared in a thymus-expressed set; AP4R E2FF EBOX ETSF MAZF SP1F ZF5F and CREB E2FF MAZF PCAT SP1F STAT cis-clusters occurred in activated T-cells; CEBP CREB NFKB SORY and GATA NKXH OCT1 RBIT occurred in stimulated lymph nodes.Gene clusters, formed based on similarity of expression-pattern across either all tissues or the immune tissues only, had highly significant associations both with immunological processes such as chemokine-mediated response, antigen processing, receptor-related signal transduction, and transcriptional regulation, and also with more general processes such as replication and cell cycle control. Within-cluster gene correlations implicated known associations of known genes, as well as immune process-related roles for poorly described genes. To characterize regulatory mechanisms and cis-elements of genes with similar patterns of expression, we used a new version of a comparative genomics-based This study demonstrates a series of analytic approaches that have allowed  the implication of genes and regulatory elements that participate in the  differentiation, maintenance, and function of the immune system.  Polymorphism or mutation of these could adversely impact immune system  functions. This[) database of human and mouse gene expression [High-throughput gene expression profiling provides a powerful approach to the investigation of relative transcriptional activity as a function of biological differentiation across a variety of cells and tissues. Published examples that probe a wide variety of distinct, differentiated materials include the Human Gene Expression (HuGE) Index database  and the pression . These rpression . To do tCis-element clusters that are conserved in pairs of orthologs are strong predictors of regulatory regions within mammalian genes [cis-regulatory elements that occur in coordinately regulated genes of the immune system and may play a role in controlling their expression during development or mature cell function. Several of the modules identified through this approach contain cis-elements whose biological relevance has been experimentally validated in previous studies. Other computationally identified modules from this immunomic database have not been studied in detail, but the results and a tool to analyze them further, are provided at the website [an genes ,20,21. W website . Taken tOur goal is to identify genes, which are essential for the differentiation, maintenance, and function of the immune system, and their associated regulatory elements. Polymorphisms or mutation in these might underlie well-known variation among individuals in effectiveness of their immune response. Mouse immune genes were identified from our gene expression database constructed using the 8638 element microarray and probed with mRNA prepared from 65 normal adult and fetal tissues. We chose to select relevant genes by collecting those expressed above a threshold value rather than by statistical analysis of variance. Given the small number of replicates and the large number of comparisons being made, we would not have enough statistical power to detect differentially expressed genes by using traditional statistical tests with appropriate specificity. In addition, with the reduced specificity of statistical tests, the biologically non-significant, but somewhat reproducible differences in gene expression will obscure changes that are of biologically significant magnitude, but vary from replicate to replicate. Expression of genes is not discontinuous from tissue to tissue, but varies quantitatively over a wide range. The threshold to distinguish expressed from non-expressed genes was set to identify the hundred or so most highly expressed gene in each relevant tissue.[Genes were considered to be "immune genes" if they were more highly expressed in one or more of 6 immune tissues  than in most other normal adult and fetal mouse tissues. 680 genes were identified where the amount of cDNA hybridized from one or more immune tissues was 3 or more times greater than hybridization of cDNAs from the reference whole mouse Figure . To incr and NCBI. The rem to find Hierarchical tree clustering of the 360 sequences and 65 normal adult and fetal tissues was carried out by Pearson correlation using the log of the average of the relative expression ratio for each gene as measured in replicate arrays Figure . While t[[Mcm2 through Mcm7; the DNA polymerases and primase, Pola2, Cdc6, Prim1; the processivity factor Pcna, and cyclin E1, Ccne1. They play a role in regulation of chromosomal replication in many types of cells [A putative function could be assigned to 298 expressed sequences  based on and MGI [ approvedof cells . In the H2-Aa, H2-Ab1, H2-DMa, H2-Eb1, H2-K, H2-L, H2-Oa, H2-Ob, H2-Q7, and B2m  as part of the lymphocytes' response to cytokines. The product of Rac2 is a G protein that participates in the cascade of kinases leading to activation of TFs. Chemokines are a family of small proteins that activate cells such as lymphocytes as part of the host response to infection. Genes that encode the chemokines  and chemokine receptors (Cxcr4 and Ccr2)  as predicted by computational and biochemical studies. Experimentally identified, conserved, regulatory elements within the promoter of MHC class I genes include: an enhancer A element (two NFKB sites), an interferon-stimulated response element (ISRE), site α (cAMP-response element), enhancer B (inverted CCAAT), CCAAT, and TATA elements [cis-elements in the promoters of H2-K and its human ortholog, HLA-A, are shown in Figure HLA-A, computationally identified binding sites previously found by biochemical analyses include: IRFF, CREB, ECAT, PCAT, TBPF and two NFKB. The enhancer-A element of the MHC class I promoter encompasses two NFKB binding sites and plays an important role in the constitutive and cytokine-induced expression of MHC class I genes. Our IRFF site is the reported ISRE and can bind interferon regulatory factor 1 to activate MHC class I transcription. Site α of the MHC Class I promoter corresponds to our CREB binding site and plays an important role in regulation of expression of Class I genes. Our PCAT and ECAT sites include sequences consistent with the CCAAT site and our TBPF is a TATA binding site, as reported in the MHC class I promoter immediately upstream of the transcription start site [Regulatory modules predicted by comparative analyses of DNA sequences must be validated by genomic footprinting and other biochemical techniques, which prove that the predicted elements . Computaart site . Computaart site ; the EGRart site ; and AhRart site . We concLocally developed programs, TraFaC and CisMols, were used to identify and display putative regulatory modules in genes grouped by patterns of expression. The algorithms use a moving 200 bp window to scan regions of DNA for specific sequences characteristic of TF binding sites Figure .Cluster analysis of genome wide expression data from microarrays permits the grouping together of genes with similar patterns of expression across cells, tissues or experimental conditions. Clustering of genes by patterns of expression was first applied on a large scale to yeast , where cEts1 and Tcf12, chromatin matrix associated protein Smarcf1 (recently renamed Arid1a), the ATP-binding cassette transporter Abcg1 (transports peptides during antigen processing), the 2'-5'-oligoadenylate synthetase Oasl2 that is induced by interferon, and the histocompatibility antigen H2-K that plays a role in antigen presentation and processing. Sequences of both the mouse gene and its human ortholog were available for seven genes . The 3 kb upstream regions of all 7 genes were compared to identify modules of shared cis-elements. The search criteria were limited by (1) requiring modules to contain at least 3 TF binding sites, one of which is a lymphoid element (see this list of lymphoid elements in Methods), (2) to be evolutionarily conserved, that is, to occur within the phylogenetic footprints in the aligned mouse-human orthologs, and (3) to be located within 3 kb upstream and 100 bp downstream of the first bp of exon 1 (transcription start). Examples of modules of cis-elements are shown in Table Arid1a, Abcg1 and Sgpl1 are most similar to one another. They also have the most similar patterns of expression across tissues, when clustered in hierarchical trees. One module, AP2F EGRF MAZF SP1F ZBPF, contains 5 cis-elements within a 200 bp window and is present within 3 kb upstream of transcription start in Arid1a, Abcg1 and Sgpl1. The conserved modules containing multiple transcription factor binding sites  with the other genes.K-cluster set 15 contained 14 genes. While these genes had similarities in patterns of expression across a group of 15 tissues, their most prominent shared characteristic was preferential expression in thymus. They were diverse in function. For instance, the group comprised transcription factors cis-elements immediately upstream of the transcription start site (promoters) of specific individual genes: Arid1a, Abcg1, and Zfp162. Elements were required to be within 500 bp of transcription start to be shown in Figure Arid1a, Abcg1 and Zfp162.Figure CD72 found on B-cells, the transcription regulators Irf5 and Icsbp1, the tyrosine kinases Hck, Stk10, and Lyn that are a part of the intracellular signaling cascade, the mitogen activated protein kinases Map3k1 and Map4k1 that participate in the very earliest steps of induction of new gene expression after lymphocytes are exposed to antigen, and the ATP-binding cassette transporters Abca7 and Tap1 of the type that transport peptides during antigen processing. Other less well-characterized genes in these sets may have functions similar to the genes that are better annotated. Sequences of 11 genes from Set 7 and their human orthologs were examined for the presence of clusters of TF binding sites, at least one of which is a lymphoid element, as defined in Methods. The 11 genes shared relatively few clusters of TF binding sites. There were 7 clusters shared by 3 genes. The largest cluster contained 6 elements, AP2F CDEF EGRF SP1F ZBPF ZF5F and was shared by Irf5 and Stk10. There were 21 clusters shared by 2 genes and containing 3 to 6 TF binding sites. The composition and location of these are shown in images from CisMols  and 8 in liver  served as negative controls, i.e. not tissues that play a critical role in lymphocyte differentiation or the immune response. The MCM family and Myc are involved in replication of DNA and chromosomes. The TNF and TNFR families of genes encode receptors and ligands that couple directly to signaling pathways for cell proliferation, survival and differentiation [Prss16 encodes a thymus specific protease which is specifically expressed by epithelial cells in the thymic cortex and plays a role in T-cell development and, perhaps, in susceptibility to autoimmunity [Hdac7a encodes a histone deacetylase. Members of the Hdac family of genes modify histones and play a role in the regulation of expression of genes such as those functioning in the cell cycle, apoptosis, and transcription [In addition to searching for potential regulatory regions within sets of genes clustered by similarities of patterns of expression across sets of tissues and within regions immediately upstream of exon 1, we also sought to identify genes characterized by high expression in specific immune tissues. It is not known whether clustering by pattern of expression across tissues and/or grouping by high expression in specific tissues (or neither) will be a useful way to group genes for computational identification of regulatory elements and regulatory regions. It is clear, however, that although modules of ntiation . Prss16 immunity . Hdac7a cription . Cxcl9 icription .cis-elements that they contain. When modules of genes highly expressed in thymus, stimulated lymph nodes, or activated T-cells were compared with one another and to modules of genes expressed in muscle and liver, it is clear that the composition (cis-elements) of modules are not unique to a specific gene. However there is some evidence of unique arrangements of elements within modules. There are also cis-elements that are not commonly shared. For example, the individual cis-elements HESF, HAML, MYT1 and P53F were not found in modules of genes other than those highly expressed in thymus. Likewise, E2FF was only present in modules of activated T-cells, but clearly does not play a unique role in the immune system. Members of the E2F family of TFs are key participants in cell proliferation, apoptosis, and differentiation [Mcm2, Mcm5, Mcm6, Mcm7, and Myc. These genes are highly expressed in proliferating cells generally, an example of which is the activated T-cell.The results of the above approach are shown in Table ntiation . E2FF iscis-elements  in highly expressed genes are listed in Table Abca1, C1qg, Abcg1 and Sgpl1; module AP2F EGRF HESF MAZF SP1F ZBPF was present in Sgpl1 and Abcg1. Of the modules highly expressed in stimulated lymph nodes, AP2F CDEF EGRF SP1F ZBPF ZF5F was present in Stk10 and Irf5; module GATA HOXF NKXH was present in Stk10 and Irf5. Of the modules highly expressed in activated T-cells, E2FF EBOX ETSF MAZF SP1F ZF5F was present in Kpnb1 and Mcm6; module BCL6 CREB E2FF STAT was present in Icsbp1 and Tnfrsf4.Regulatory modules, which have been proved biologically to regulate expression of genes, contain multiple TF binding sites, much as is shown in Figures LCP2 and PARVG) cause severe immunodeficiency disease. Examples of other diseases caused by mutations in these 32 genes were B- and T-cell malignancies, autoimmune disorders, and reduced viral or bacterial resistance. Of the 320 genes removed from the list of 680, 37 had orthologs with gene symbols listed in OMIM. 4 genes were expressed in lymphocytes and mutation in one, Bruton's tyrosine kinase, causes agammaglobulinemia. Mutations in other genes caused disorders of coagulation, red cells, or granulocytes, rather than the immune system. We conclude that the list of 360 immune genes includes a higher percentage of genes preferentially expressed in immunocompetent tissues and with more specific immune-related functions than does the full list of 680 sequences expressed in immune tissues, but also with expression in non-immune tissues.Individual differentiated biological states can be characterized by gene expression profiling. Large-scale comparisons of profiles of cells, tissues, and developmental stages have the potential to identify a wealth of coordinately regulated groups of genes that reflect the interplay of their functional relationships and transcriptional control mechanisms. We have built a database comprised of the mRNA expression profiles of 65 normal adult and fetal C57BL/6J mouse tissues using the Incyte Mouse GEM1, 8638 element, clone set. Using microarray analysis, 680 sequences were identified that were highly expressed in one or more of 6 immune tissues. Many were also expressed in certain other tissues. Some of these other tissues were organs such as heart, kidney, and brain which do not normally contain lymphocytes in large numbers and do not play a role in the immune response. Others, such as intestine and lung, interface with the external environment, contain significant numbers of lymphocytes, and can mount an immune response. The 680 expressed sequences were filtered to remove 320 that were expressed in "non-immune" brain or heart or kidney. This resulted in a list of 360 expressed sequences called "immune genes" that were less broadly expressed in tissues without immune function than were the 680. Mutations and polymorphisms in both the 680 expressed sequences and the 360 immune genes have a significant chance of specifically affecting immune function. We predict this will be more common with changes in the 360 immune genes. We tested this by comparing reports of disease causing mutations in the 360 immune genes with those reported for the 320 genes that were more broadly expressed (Online Mendelian Inheritance in Man). Of the 360 mouse immune genes, 32 had an ortholog with gene symbol in OMIM and17 had annotations that described a function clearly linked to development or function of the immune system. Mutations in 2  the Incyte set of 8638 genes probably contains representative cDNAs from 25% or less of all mouse genes; (2) genes that are essential to immune function, but are expressed at similar levels in immune and other tissues will not be included in the immune set; and (3) a gene with a very low level of expression will be missed, if cDNA made from its RNA is not present in sufficient quantity to give a signal on the microarray. Genes may be included or excluded in error because of the large number of genes screened for expression with a limited number of replicates. Incyte cDNA microarrays are no longer manufactured and no Incyte arrays or public databases are available to check expression of our immune genes in other species. There are two relevant publicly available Novartis gene expression databases , which cis-element analyses  certain combinations of TF binding sites are phylogenetically conserved and appear to be reused across genes when specific patterns of expression are required. Cis-elements from the same family have a high probability of interacting with similar groups of transcription factors, although they will not necessarily be in the same position relative to the transcription start site. We have identified genes and putative regulatory modules that play a role in the differentiation, maintenance, and function of the immune system. These results serve to advance both our understanding of normal gene and immune system function and also to identify genes and regulatory regions whose mutation or polymorphic variation lead to immunologic disease.Using comparative genomics-based, nalyses , as described previously for our group ,47. Rela[Genes on the Incyte array were identified by NCBI GenBank accession and systematic numbers and by gene symbol, where available. For those sequences that could not be assigned a gene symbol, sequence homologies to known mouse genes were sought using MouseBLAST , BLAT 1, MGD 2323, and L with Ens sequence. Confirm[[The GO and MGI databases were searched for annotations of the immune genes, using Stanford SOURCE . For gen, Mouse G, and Loc[. A functcis-acting regulatory sequences in genes that were coordinately regulated, we first selected groups of genes based on their expression patterns in different immune tissues. The complete genomic sequences (with flanking upstream and downstream regions of 40 kb) of the selected genes and their orthologs were extracted from the Ensembl/UCSC human and mouse databases [[[[[[cis-regulatory regions of each of the different groups of genes from mouse and human to identify known TF binding sites. The CisMols analyzer [To identify putative consensus atabases ,13. Wheratabases . Repeat tabases [. Conservabases [[ or Genombases [[[ servers ases [[[[ and Advases [[[[[ programsanalyzer  permits JJH and BJA were primarily responsible for the design, coordination and conduct of the study. AGJ and AG were responsible for regulatory region analyses and software development. AGJ was responsible for ortholog analysis and novel ortholog assignments. JJH, BJA and AGJ drafted the manuscript and figures and JJH, BJA and AGJ contributed editorial revisions. SK, SW and CE were responsible for generating, quality assurance, and initial assembly of the gene chip data. JDK provided purified lymphoid cells, read the manuscript and provided comments and discussion. All authors read and approved the final manuscript.List and annotation of 360 expressed sequences ("immune genes").Click here for fileFASTA sequences and the corresponding coordinates on the human and mouse genome assemblies (May 2004) of the promoter regions used in the analysis and displayed in figures 2 and 4.Click here for fileUsing K-means clustering in GeneSpring (Version 4.2.1), 160 annotated genes, were divided into distinct sets based on similarity of expression patterns across 15 tissues. Tissues were given equal weight, the number of clusters was set at 20, and similarity was measured by standard correlation.Click here for fileFASTA sequences and the corresponding coordinates on the human and mouse genome assemblies (May 2004) of the promoter regions used in the analysis and displayed in figures 2 and 4.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileCisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them.Click here for fileTissue lists used in the generation of microarray profile data.Click here for file


Many patients with several concurrent medical conditions (multimorbidity) are seen in the primary care setting. A thorough understanding of outcomes associated with multimorbidity would benefit primary care workers of all disciplines. The purpose of this systematic review was to clarify the relationship between the presence of multimorbidity and the quality of life (QOL) or health-related quality of life (HRQOL) of patients seen, or likely to be seen, in the primary care setting.Medline and Embase electronic databases were screened using the following search terms for the reference period 1990 to 2003: multimorbidity, comorbidity, chronic disease, and their spelling variations, along with quality of life and health-related quality of life. Only descriptive studies relevant to primary care were selected.Of 753 articles screened, 108 were critically assessed for compliance with study inclusion and exclusion criteria. Thirty of these studies were ultimately selected for this review, including 7 in which the relationship between multimorbidity or comorbidity and QOL or HRQOL was the main outcome measure. Major limitations of these studies include the lack of a uniform definition for multimorbidity or comorbidity and the absence of assessment of disease severity. The use of self-reported diagnoses may also be a weakness. The frequent exclusion of psychiatric diagnoses and presence of potential confounding variables are other limitations. Nonetheless, we did find an inverse relationship between the number of medical conditions and QOL related to physical domains. For social and psychological dimensions of QOL, some studies reveal a similar inverse relationship in patients with 4 or more diagnoses.Our findings confirm the existence of an inverse relationship between multimorbidity or comorbidy and QOL. However, additional studies are needed to clarify this relationship, including the various dimensions of QOL affected. Those studies must employ a clear definition of multimorbidity or comorbidity and valid ways to measure these concepts in a primary care setting. Pursuit of this research will help to better understand the impact of chronic diseases on patients. With technological advances and improvements in medical care and public health policy, an increasingly large number of patients survive medical conditions that used to be fatal. As a result of this phenomenon, and parallel to the aging of the population, a growing proportion of primary care patients presents with multiple coexisting medical conditions. From available data, it was estimated that 57 million Americans had multiple chronic conditions in 2000 and that this number will rise to 81 million by 2020 .chronic conditions; they also proposed some qualifiers to better classify the type of multimorbidity . Unfortunately, much confusion still exists in the literature, where the 2 terms often seem to be used interchangeably. For the purpose of this paper, the term "multimorbidity" will be used according to Van den Akker and colleagues' definition and the focus will be solely on chronic diseases.The terms "comorbidity" and "multimorbidity" have been used to describe this phenomenon. Feinstein  originalPrevious reports on multimorbidity or comorbidity have documented that this phenomenon influences outcomes in many areas of health care -19. OutcQuality of life (QOL) is an outcome measure that is increasingly being used to evaluate outcomes in clinical studies of patients with chronic diseases -26. QOL Little is known about the impact of multimorbidity on QOL of primary care patients , althougFor this review, we consulted Medline and Embase electronic databases for the reference period 1990 to 2003. Figure Quality of Life Research and Health and Quality of Life Outcomes journals. We also screened references from key articles retrieved (hand searching).To identify studies pertinent to the primary care setting, the following search terms were used: general practice, family practice, family medicine, family physician, and primary health care. A parallel strategy was used to identify all descriptive studies, regardless of the context of care, and the results were then combined. For the initial screening, the search was restricted to studies on human subjects, published in French or English. To be complete, we directly searched the One researcher (LL) performed the initial screening. Any ambiguous findings were discussed with the lead investigator (MF) and a consensus was reached.For the purpose of this systematic review, we selected original, cross-sectional, and longitudinal descriptive studies that had evaluated the relationship between multimorbidity or comorbidity and QOL or HRQOL as the main outcome of interest. As stated earlier, we focused on the population of patients seen, or likely to be seen, in the primary care setting including members of the general population and residents of nursing homes and home healthcare facilities. We also selected original descriptive studies that had examined the relationship between multimorbidity or comorbidity and QOL or HRQOL as a secondary outcome.Figure Before being included in the synthesis, the quality of each article selected was critically analyzed. For this assessment, we devised a scale in which points were assigned for study parameters indicative of good quality . Using this scale Table , 2 reseaFigure Of the 7 studies that featured QOL as a primary outcome -42, 5 haAll studies came to the same conclusion, namely that there is an inverse relationship between the number of medical conditions and QOL or HRQOL. This association may be affected by the patient's age or gender. Whereas multimorbidity mostly affects physical dimensions of QOL or HRQOL ,37,41, dIn each study, investigators relied on simple count of chronic diseases from a limited list to measure multimorbidity. The chronic conditions included in this list varied among the studies, and no attempt was ever made to assess or account for the severity of each condition. Furthermore, 5 of the 7 studies did not consider psychiatric comorbidity, either because the illnesses considered did not include psychiatric diagnoses or because patients presenting with psychiatric diagnoses were excluded from the QOL evaluation. In most cases, the diagnostic information was obtained by a questionnaire that was completed by a nurse or a doctor or sometimes self-administered. One study assessed comorbidity via chart review.To measure QOL, a variety of scales were used. Most studies (5/7) used tools from the Medical Outcomes Study i.e., the Short-Form-36 Health Survey (SF-36) and Short-Form-20 Health Survey (SF-20). However, the Nottingham Health Profile (NHP) was used in one study and the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) was used in another. Although the number of domains explored varied from one study to the next, the measuring instruments used have excellent psychometric properties and validity.The 4 studies associated with the highest quality scores explored only a limited number of potential confounders, namely age ,38, gendOf the 23 studies that evaluated the relationship between multimorbidity or comorbidity and QOL as a secondary outcome measure -65, mostAs with the results from the main outcome studies, we found an inverse relationship between the number of medical conditions and the QOL relating to physical domains in all studies. However, the relationship between multimorbidity and QOL relating to psychological or social domains was less clear. Some investigators reported an effect of multimorbidity on these domains in patients with 3 or more diagnoses , while oAs in the main outcome studies, tools from the Medical Outcomes Study, including the SF-36 (17 studies), SF-20 (3 studies), and Short-Form-12 Health Survey (SF-12) (1 study), were used to evaluate QOL in most of these studies. However, the NHP was used in one study and the Quality of Well-Being Scale (QWB), in another. In the majority of studies, all of the QOL domains were explored.Although this systematic review confirms the inverse relationship between multimorbidity and QOL, it also raises some important questions. First, the relative lack of studies in primary care evaluating the association between multimorbidity and QOL or HRQOL is surprising given the number of patients who suffer from multiple concurrent chronic conditions. Although the existence of this association makes logical sense, it still has to be demonstrated and thoroughly studied to find ways of improving care for specially affected patients. Thus, the pressing question may not be whether there is an association but rather how strong is the association and what factors are responsible for it? Identifying these factors may contribute to better care for the affected patients. There is a clear need for further studies to address these issues.Ultimately, multimorbidity has the potential to affect all domains of QOL. However, the influence of multimorbidity on the social and psychological dimensions of QOL is much less clear than its influence on the physical domains. It is noteworthy that several studies showed a significant decline in social and psychological dimensions of QOL in patients with 3, 4, or more concurrent diagnoses. What does this finding mean? Is there any bias that can explain this difference, or is it related to a certain capacity for adaptation? Are there other factors associated with this finding? All of these questions have yet to be answered.All the studies examined were cross-sectional in nature. The effect of multimorbidity may vary over time. Some medical conditions may improve while others worsen resulting in various effects on QOL. Therefore, cross-sectional studies may not capture the real effect of multimobidity on QOL and predict the direction of change over time.The absence of a uniform way of defining and measuring multimorbidity is of special concern and may explain some of the variability in our results. In most of the studies we evaluated, investigators had used only a simple list of diseases to identify concurrent medical conditions in patients, providing very incomplete information. Furthermore, the numbers and types of medical conditions in these lists varied among the studies, precluding comparisons.Given the urgent need for conceptual clarity, Van den Akker and colleagues' definition of multimorbidity should be refined and advanced to achieve a common understanding. A distinction must be made between simple and complex chronic diseases. Treated hypothyroidism (simple) and ischemic heart disease (complex) obviously do not have the same impact on QOL. Moreover, the influence of single-organ versus multi-organ diseases needs to be appropriately weighed. Additional factors to be considered when defining multimorbidity include the severity of the conditions and the presence or absence of associated pain.The use of self-reported diagnoses in many studies is another methodological limitation that may have introduced error. Patients may confuse symptoms and minor ailments with more significant disease states or may forget to report important diagnoses that are still active. Self-reporting may even be completely inaccurate in the presence of psychosomatic disorders. Conducting a chart review, clinical interview or using any specific standardized method may be a better way to obtain data related to diagnoses.Another methodological limitation of most of the studies evaluated was their failure to consider the influence of psychiatric comorbidity. This was either because psychiatric diagnoses were not included in the lists of disease states or because patients presenting with psychiatric diagnoses were excluded from QOL assessment. Given the importance of psychiatric conditions in primary care practice with a prevalence of more than 20% , this liQOL tends to decrease with age , whereasOnly a few of the studies evaluated had explored the effect of gender. Furthermore, their results were contradictory, with gender being more detrimental to the QOL of women in some cases ,58 and mLittle has been reported about the effects of other potential confounding variables , although these factors are recognized as having an impact on QOL -71. A feacute conditions, the time since the diagnosis of important chronic conditions, and the duration and prognosis of health problems are among factors that may explain some of the variability in QOL or HRQOL.Many other factors should be explored in this regard. For example, the presence of coexisting In light of the findings of this systematic review, further research is needed to clarify the relationship between multimorbidity and QOL. The early work will certainly be conceptual and theoretical. The resultant conceptual clarity would benefit both researchers and practitioners. How do we define and how should we measure multimorbidity are among the first questions to be addressed. More descriptive studies, which take into account the influence of multiple potential confounders, can then be conducted. Multivariate analyses will help control for the effects of these confounding variables. The effects of age and gender also need to be further explored, with reference to established norms. Although there is still a need for cross-sectional studies, longitudinal studies are also needed to identify changes in the relationship between multimorbidity and QOL over time.The main limitation of a systematic review is its inability to include all of the relevant literature. We realize that some articles may have been missed during the search stage. However, our review of a huge number of abstracts generated by different strategies improved the sensitivity of the search. Obviously, the absence of a keyword for multimorbidity is a limitation. However, we found that in the majority of cases in which the term "multimorbidity" was used to search, the term "comorbidity" also appeared in the list of keywords. Adding the term "chronic disease" also helped to circumvent the problem. Restricting the search to articles published in French or English is another limitation.This systematic review focused on the relationship between the presence of several chronic coexisting medical conditions and QOL or HRQOL in a primary care setting. However, the studies evaluated had important limitations due to the lack of a uniform definition for multimorbidity or comorbidity, the absence of assessment of disease severity, the use of self-reported diagnoses, and the frequent exclusion of psychiatric diagnoses. The potential impact of important confounding variables was also neglected. In light of these observations, it seems clear that further studies are needed to clarify the impact of multimorbidity on QOL or HRQOL and its various dimensions . A clear understanding of this relationship will ultimately help both researchers and primary health care professionals to deliver more comprehensive care.MF was responsible for the conception and design of this systematic review and was also involved in the literature review. In addition, he was responsible for critically assessing the evaluated articles and drafting this manuscript. He takes responsibility for the integrity of the work as a whole and provided final approval of this version of the manuscript.LL provided a major contribution to the literature review and critical appraisal of the identified articles. She also participated in the drafting of this manuscript and gave final approval of this version.CH participated in both the conception and design of this review. She also contributed by critically revising this manuscript and gave final approval of this version.AV participated in the design of this review. He also made an important contribution in critically revising the manuscript and gave final approval of this version.ALN participated in the drafting of the manuscript and made an important contribution by critically revising it. He also gave final approval of this version.DM participated in the drafting of the manuscript and made an important contribution by critically revising it. She also gave final approval of this version.


Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown.TBC-Sec7).Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains. Guanine Nucleotide Exchange Factors (GEFs) are obligatory components of signaling cascades regulated by small GTP-binding proteins . Their biochemical activity is to stimulate the dissociation of the tightly bound GDP nucleotide from the small G protein in response to cellular signals. Thereby, they favor the binding of the more abundant cellular GTP, organizing the active conformation of the small G protein which can recruit its effectors (reviewed in ). Each sbona fide ArfGEFs . Thus aiewed in ). Large iewed in , Arabidoiewed in  and Sacciewed in , the BIGiewed in  and yeasiewed in . An addiiewed in . Analysiiewed in ). Most miewed in ,14,18, eiewed in , and GNOiewed in .The domain architecture of non-catalytic regions of ArfGEFs, hence their contribution to specific aspects of the build-up of the Arf response, is essentially not established except for those ArfGEFs with domains found in other classes of cellular regulators. The known domains include membrane-interacting PH domains in the CYH (reviewed in ), EFA6  and possParamecium tetraurelia alveolate. Sequence comparisons combined with secondary structures and splicing patterns analysis identifies five novel domains that are conserved between BIG and GBF subfamilies, thus unifying them as a higher order subfamily with a probable common ancestor. Our analysis of Sec7-domain containing sequences from Paramecium also introduces a novel subfamily of ArfGEFs unique to alveolates, which we call TBS (TBC-Sec7).Here we investigate the domain architecture in the BIG and GBF subfamilies, including all sequences from fully annotated eukaryotic genomes and our novel annotation of Sec7-containing proteins from the The BIG and GBF subfamilies are the only ArfGEFs subfamilies common to all eukaryotes  and the Homology Upstream of Sec7, ~170 aa) domains – and three in its C-terminus -the HDS1 , HDS2 (~160 aa) and HDS3 (~120 aa) domains , from which unrooted phylogenetic trees can be built  domain  but appears to lack the specialization into the BIG and GBF subgroups, has the same ArfGEF distribution as plants or features a second ArfGEF subfamily. We thus searched the newly sequenced genome from n Figure , which wThe identification of a conserved modular architecture in all GBG subfamily members suggests that the mechanistic basis for their activation of Arf is likely to follow a similar scenario. Candidate functions for the conserved domains include oligomerization, the collection of input signals, membrane localization, regulation of the exchange activity, scaffolding of Arf proteins to their downstream effectors, not excluding signaling to partners outside the Arf pathways. Dimerization has been reported in the BIG subgroup for BIG1, which forms heterodimers with the highly homologous BIG2 ArfGEF , and in Paramecium tetraurelia genes were identified with the BLAST algorithm using genome sequence data from Genoscope [Tetrahymena thermophila genome sequencing project server [Protein sequence databases were searched with amino acid sequences from human BIG1, human GBF1 and Arabidopsis GNOM using the BLAST algorithm . Paramecenoscope  and manuenoscope . Tetrahyt server . Arabidot server , rice set server . Splice t server . Multiplt server  with deft server ,41. Relit server  using a t server . Phyloget server . Non-strt server .Suppressor of ypt. DCB: Dimerization/Cyclophilin Binding; HUS: Homology Upstream of Sec7; HDS: Homology Downstream of Sec7; TBC: Tre/Bub2/Cdc16; SCF: Skp1/Cull1/F box.GEF: Guanine nucleotide exchange factor. CYH: cytohesins/ARNO; EFA: Exchange Factor for Arf6; FBS: F-Box/Sec7; TBS: TBC/Sec7; GBF: Golgi-associated BFA-resistant guanine nucleotide exchange Factor; BIG: BFA-Inhibited Guanine nucleotide exchange factor; GBG: GBF/BIG Gefs; SYT1: B.M. and V.B. carried out sequence and phylogenetic analysis. A.J. participated in the domain analysis. J.Co. annotated Paramecium sequences. D.S. and P.M. performed splicing pattern analysis. N.K. and G.J. analyzed the distribution of large ArfGEFs in plants. J.Ch. conceived and coordinated the study and wrote the manuscript.


Toxoplasma gondii is associated with slow reaction and poor concentration, whilst infection with Coxiella burnetii may lead to persistent symptoms of fatigue.It has been suggested that infection with T. gondii and C. burnetii was assessed.425 farmers completed the Revised Clinical Interview Schedule (CIS-R) by computer between March and July 1999 to assess psychiatric morbidity. Samples of venous blood had been previously collected and seroprevalence of T. gondii and 31% were positive for C. burnetii. Infection with either agent was not associated with symptoms reflecting clinically relevant levels of concentration difficulties, fatigue, depression, depressive ideas or overall psychiatric morbidity.45% of the cohort were seropositive for Toxoplasma gondii or Coxiella burnetii is associated with neuropsychiatric morbidity, in particular with symptoms of poor concentration or fatigue. However, this is a relatively healthy cohort with few individuals reporting neuropsychiatric morbidity and therefore the statistical power to test the study hypotheses is limited.We do not provide any evidence that infection with  Toxoplasma gondii and Coxiella burnetii may lead to long term neuropsychiatric morbidity. More specifically, T. gondii infection is hypothesised to be associated with slow reaction and poor concentration [C. burnetii infection is reported to be associated with persistent symptoms of fatigue for up to ten years following exposure [T. gondii is common in the UK population as a whole (40% to 50%) whereas exposure to the rickettsia-like C. burnetii leading to Q fever is relatively rare in urban populations [It has been suggested that infections with the zoonoses ntration ,2 whilstexposure -5. Exposulations -8.T. gondii and C. burnetii and current psychiatric morbidity measured by the Revised Clinical Interview Schedule (CIS-R).We have examined data previously collected from a farm-based occupational cohort recruited in three areas of England to test these hypotheses. Furthermore, given the high risk of suicide amongst farmers as an occupational group, we alsoA representative cohort of 606 farmers, farmworkers and family members has been recruited since 1991 in three areas of England to investigate occupational risk factors for zoonoses. A randoThe computer-administered version of the CIS-R was used to assess the prevalence of symptoms of neurotic psychopathology in the week prior to interview. The CISAs reported previously, the prevalence of clinically relevant levels of each of these neurotic symptoms was relatively low. FifteenToxoplasma gondii at Hereford PHL using the Eiken latex agglutination test (cut off titre 1/32). Seroconverters were confirmed by PHLS Toxoplasma Reference Laboratory at Swansea PHL using the dye test and by IgM ELISA. Serum IgG specific antibody levels for Coxiella burnetii phase II antigen were estimated at Bristol Public Health Laboratory (PHL) using an indirect immunofluorescence test. Serum with a reciprocal titre of 32 or more were taken as positive. Seroprevalence data for T. gondii and C. burnetii were available for 370 and 422 individuals respectively.At enrolment, 10 ml of venous blood was taken from all subjects. Samples were screened for All analyses allowed for clustering by farm holding in the survey data . Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using logistic regression and were adjusted for sex and age in ten year bands.T. gondii and 31% (95% CI 26% to 36%) were positive for C. burnetii. Forty six individuals were seropostivie for both. Exposure to either infection did not differ significantly between men and women . T. gondii seroprevalence increased significantly with age . C. burnetii seroprevalence was not associated with age .In total, 45% (95% CI 40% to 50%) of the cohort were seropositive for T. gondii were not more likely to report symptoms reflecting clinically relevant levels of fatigue, concentration difficulties, depression, depressive ideas or overall psychiatric morbidity than those who were seronegative [Exposure to both % to 50%), whereas% to 50%).This survey benefited from using the CIS-R as a standardised assessment suitable for lay interviewers in assessing minor psychiatric disorder in an occupational setting. The computerised CIS-R assessment provides an easy, quick, inexpensive yet thorough assessment which is acceptable to interviewees and also helps to eliminate observer bias.T. gondii or C. burnetii and who subsequently developed symptoms might have been more likely to drop out of the cohort either before recruitment or before this more recent survey . If this were the case we might underestimate the strength of association between exposure and psychiatric outcome. Finally we have no indicator of duration of infection for those individuals who are currently seropositive for either infectious agent which might affect the observed association if a long lag time is required between exposure and presentation of symptoms.The prevalence of psychiatric symptoms was lower than expected in this cohort of farm workers, indicatToxoplasma gondii or Coxiella burnetii is associated with neuropsychiatric morbidity, in particular with symptoms of poor concentration or fatigue.We do not provide any evidence that infection with T. gondii – Toxoplasma gondiiC. burnetii – Coxiella burnetiiCIS-R – Revised Clinical Interview ScheduleOR – odds ratio95% CI – ninety five percent confidence intervalThe authors declare that they have no competing interests.HT completed the statistical analyses and drafted the manuscript, DT and RS conceived of the PHLS Farm Cohort study and participated in its design and coordination, GL developed the psychiatric assessments and advised in their use, AS advised on the study hypotheses under investigation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Hyperlipidemia secondary to protease inhibitors (PI) may abate by switching to anti-HIV medications without lipid effects.An open-label, randomized pilot study compared changes in fasting lipids and HIV-1 RNA in 104 HIV-infected adults with PI-associated hyperlipidemia  who were randomized either to a regimen in which their PI was replaced by abacavir 300 mg twice daily (n = 52) or a regimen in which their PI was continued (n = 52) for 28 weeks. All patients had undetectable viral loads (HIV-1 RNA <50 copies/mL) at baseline and were naïve to abacavir and non-nucleoside reverse transcriptase inhibitors.3 in the abacavir-switch and PI-continuation arms, respectively. At week 28, the abacavir-switch arm had significantly greater least square mean reduction from baseline in total cholesterol , LDL-cholesterol , and triglycerides  than the PI-continuation arm, with no differences in HDL-cholesterol . A higher proportion of patients in the abacavir-switch arm had decreases in protocol-defined total cholesterol and triglyceride toxicity grades, whereas a smaller proportion had increases in these toxicity grades. At week 28, an intent-to treat: missing = failure analysis showed that the abacavir-switch and PI-continuation arms did not differ significantly with respect to proportion of patients maintaining HIV-1 RNA <400 or <50 copies/mL or adjusted mean change from baseline in CD4+ cell count. Two possible abacavir-related hypersensitivity reactions were reported. No significant changes in glucose, insulin, insulin resistance, C-peptide, or waist-to-hip ratios were observed in either treatment arm, nor were differences in these parameters noted between treatments.At baseline, the mean total cholesterol was 243 mg/dL, low density lipoprotein (LDL)-cholesterol 149 mg/dL, high density lipoprotein (HDL)-cholesterol 41 mg/dL, and triglycerides 310 mg/dL. Mean CD4+ cell counts were 551 and 531 cells/mm3, substituting abacavir for hyperlipidemia-associated PIs in combination antiretroviral regimens improves lipid profiles and maintains virologic suppression over a 28-week period, and it simplifies treatment.In hyperlipidemic, antiretroviral-experienced patients with HIV-1 RNA levels <50 copies/mL and CD4+ cell counts >500 cells/mm Protease inhibitors (PIs) used as components of highly active antiretroviral therapy (HAART) have been well documented to reduce both the morbidity and mortality associated with HIV infection ,2. Howevn = 46), 46 mg/dL with nelfinavir (n = 21), 31 mg/dL with indinavir (n = 26), and 4 mg/dL with non-PI-containing antiretroviral regimens (n = 28)  400 copies/mL) and the Roche PCR assay Amplicor HIV-1 MONITOR UltraSensitive Version 1.0 (LLOQ 50 copies/mL) . Virologic failure was defined as HIV-1 RNA >1000 copies/mL on two occasions at least 1 week apart. CD4+ cell counts were determined by flow cytometry at baseline, and at weeks 4, 12, 20, and 28.Frequency and severity of all clinical and laboratory adverse experiences were assessed at each visit. A cardiovascular disease risk factor assessment was conducted at baseline only. Body mass index (BMI) and waist-to-hip ratio (WHR) were measured at baseline and at weeks 4, 8, 12, 20 and 28. Three types of waist measurements were conducted: mid, minimal, and umbilicus. Insulin resistance measures (C-peptide and fasting insulin to glucose ratio) were assessed at baseline and at weeks 4, 8, 12, 20, and 28. Leptin and lactate were measured at baseline and week 28.Diagnosis of possible abacavir-related hypersensitivity reaction was to be considered if the following multi-organ signs and symptoms appeared in a patient following initiation of abacavir: fever, rash, gastrointestinal symptoms , lethargy, or malaise with or without concomitant respiratory symptoms , musculoskeletal symptoms , headache, paresthesia, and edema. No rechallenge of abacavir was permitted in patients developing this syndrome. The definition and usual clinical presentation of abacavir-related hypersensitivity has been defined previously ,25.Adherence was assessed by the Patient Medication Adherence Questionnaire-7 (PMAQ-7) . The PMAHealth-related quality of life (QOL) was evaluated using the Medical Outcomes Study 36-item Short Form Health Survey (SF-36) , which pt-test with a two-sided significance level of 0.05, assuming a dropout rate of 20%. This sample size calculation is based on data from NZTA4002, in which the standard deviation of total cholesterol in the nelfinavir/zidovudine/lamivudine group at baseline was 39.5 mg/dL.A sample size of 80 patients per treatment arm was deemed necessary for 80% power to detect a difference of 20 mg/dL in fasting total cholesterol between groups using a two-group The primary efficacy population was the intent-to-treat (ITT) population, which consisted of all patients who were randomized into the study. The safety population consisted of all randomized patients who received at least one dose of study drug. Analysis of covariance (ANCOVA) was used to compare the least squares means (LSM) of the two treatment groups. The LSM represented the mean value adjusted for the average value of the covariate from both treatment groups. LSMs of change from baseline in serum lipids , apolipoprotein B, apolipoprotein E, leptin, C-peptide, glucose, insulin, and lactate were reported. The ANCOVA model included terms for treatment group, baseline value of the variable of interest, PI strata and gender.Two types of analyses were performed with HIV-1 RNA data: the ITT: observed analysis and the ITT missing equals failure analysis (ITT: M = F). In the ITT: observed analysis, only available assessments were used , regardless of whether the patient was still receiving their original therapy. In the ITT: M = F analysis, all missing values were considered as failure. Proportions of patients with HIV-1 RNA <400 copies/mL and <50 copies/mL were compared between treatment groups with a 95% confidence interval (CI) on the difference between proportions.P value of 0.05 was considered statistically significant.Differences in domain scores to the SF-36 and the PMAQ-7 were compared using the Wilcoxon rank sum test. Differences between treatment arms in the incidence of treatment-related adverse events by body system were evaluated by Fisher's Exact test. A 3 in the abacavir-switch and PI-continuation arms, respectively. About two-thirds of the patients were HIV Category A. The abacavir-switch and PI-continuation arms were well matched with respect to mean baseline HIV-1 RNA levels , total cholesterol (244 vs 241 mg/dL), LDL-cholesterol (149 vs 149 mg/dL), VLDL-cholesterol (68 vs 56 mg/dL), HDL-cholesterol (39 vs 42 mg/dL), triglycerides (340 vs 280 mg/dL), CAD risk factors , stavudine (36% vs 42%), lamivudine (32% vs 40%), and zidovudine (6% vs 2%).Of 104 patients enrolled in the study, 52 were randomized to the abacavir-switch arm and 52 to the PI-continuation arm . No patient required efavirenz intensification of their treatment regimen.A similar number of patients withdrew prematurely from the study for the reasons delineated in Table P <0.001), LDL-cholesterol , LDL direct-cholesterol , VLDL-cholesterol , triglycerides , apolipoprotein B , and apolipoprotein E  versus the PI-continuation arm. Over the study period, no significant differences were observed between the abacavir-switch or PI-continuation arms with respect to LDL subfractions (particle size A and major class) or HDL-cholesterol . The abacavir-switch arm showed a trend for greater LSM reduction from baseline in free fatty acids .In the ITT analysis, the abacavir-switch arm experienced greater reductions in total cholesterol Figure  and LDL-P = 0.016). A higher proportion of patients in the abacavir-switch arm had shifts to lower toxicity grades as compared to the PI-continuation arm, whereas a lower proportion in the abacavir-switch arm had shifts from baseline in cholesterol to more severe toxicity grades  goal value (<200 mg/dL) in the abacavir-switch arm (198 mg/dL); however, it remained above this value in the PI-continuation arm (230 mg/dL). The LSM LDL-cholesterol level at week 28 was 133 mg/dL in the abacavir-switch arm and 153 mg/dL in the PI-continuation arm   Figure . A higheP = 0.508; 95% CI ), and <50 copies/mL in 62% [32/52] and 75% [39/52] of patients in these respective groups .In the ITT: M = F analysis, no differences were observed between the abacavir-switch and PI-continuation treatment arms at any time during the study regarding proportions of patients achieving undetectable HIV-1 RNA. At week 28, HIV-1 RNA was <400 copies/mL in 69% [36/52] and 77% [40/52] of patients in the abacavir-switch and PI-continuation arms, respectively  . Three patients (6%) in the abacavir-switch arm were protocol-defined virologic failures. However, one of these patients remained on a 2-NRTI/abacavir regimen after withdrawing from the study and his viral load declined. The second of these patients was noncompliant and discontinued his antiretroviral treatment prior to the confirmation blood draw for virologic failure. The third virologic failure in the abacavir-switch arm had a history of prior dual nucleoside therapy for >1 year.No differences were observed regarding time to virologic breakthrough .At baseline, the mean CD4+ cell counts were 551 and 531 cells/mmP ≤ 0.027 at each week) and in the Physical Effects domain for all visits (P < 0.05) except those at weeks 4 and 28. In answer to the PMAQ-7 questions "it is easy for me to take my medicines at the time I am supposed to" and "my medicines are convenient to take", more patients in the abacavir-switch arm answered "definitely true" at week 28 . In the SF-36, the week 28 QOL scores did not differ between treatment arms for most domains, except Vitality  and Health . There were no differences between treatment arms with respect to any health care utilization parameter.PMAQ-7 results showed no apparent differences between the abacavir-switch and PI-continuation arms in overall adherence at week 28 (90% vs 94%) or in the Social Support, Adaptability, Knowledge and Attitudes, and Memory and Recall domains. However, the abacavir-switch arm had significantly higher scores in the Scheduling and Timing domain for all study visits (P = 0.002) and non-site specific adverse events (P = 0.003) were observed in the abacavir-switch arm. Treatment-related adverse events reported by >5% of patients in the abacavir-switch arm included nausea (12/50 [14%]), fatigue (6/50 [12%]), diarrhea (4/50 [8%]), and depressive disorders (3/50 [6%]). Two patients (4%) in the abacavir-switch arm experienced possible abacavir-related hypersensitivity reactions. Adverse events leading to treatment withdrawal in 7 patients in the abacavir-switch arm included possible abacavir-related hypersensitivity reaction (2), mild nausea (2), mild shortness of breath/tachycardia (1), mild disorientation (1), and combination of mild diarrhea, facial edema/numbness and malaise (1). Hyperlipidemia led to treatment withdrawal in 1 patient in the PI-continuation arm. No differences between the two treatments were observed in body weight, waist-to-hip ratio, BMI, lactate, leptin, glucose, insulin, or C-peptide.Substitution of PIs with abacavir in HAART regimens was generally well tolerated. No significant differences between treatment arms were observed for cardiovascular, endocrine/metabolic, hepatobiliary tract/pancreatic events, lower respiratory events, musculoskeletal, neurology, psychiatric, or skin adverse events, although more gastrointestinal (3), substituting abacavir for hyperlipidemia-associated PIs in HAART regimens improves lipid profiles and maintains virologic suppression over a 28-week period. The lipid findings are consistent with those of other studies in which abacavir was substituted for PIs in HAART regimens [fasting lipids rather than lipids in a non-fasted state (which may confound results). Thus, true differences between abacavir and PI-containing regimens could be determined, and lipid criteria established by the NCEP ATP III could be applied [The results of this study indicate that in hyperlipidemic, virologically suppressed, immunocompetent antiretroviral-experienced patients . A similar phenomenon was observed over 48 weeks in another PI-to-abacavir switch study, CNA30017 . As no cMaintenance of virologic suppression and increases in CD4+ cell count over the 24-week period following the switch to abacavir were expected in view of the lack of change in these surrogate markers previously observed in abacavir-switch studies conducted over 48 weeks to 1 year ,29.The abacavir-containing regimen was generally well-tolerated, and the type and incidence of adverse events (notably fatigue and nausea) and rare occurrence of suspected abacavir-related hypersensitivity reactions were consistent with what has been reported in other clinical trials . New advResults of the PMAQ-7 indicated significantly better Scheduling and Timing scores in the abacavir-switch arm. Significant improvement in this PMAQ-7 dimension was similarly reported at 24 weeks in the PI-to-abacavir switch study, COL30305 . ImproveSwitching to abacavir is just one of several switch strategies that have been investigated to date in an attempt to remedy hyperlipidemia in patients receiving PI-containing HAART. It is acknowledged that the ideal candidate for this switch strategy is a patient started on triple therapy, where pre-existing abacavir resistance is unlikely ,32. Anot3, they were highly immunocompetent and may not be representative of typical HIV-infected patients presenting to their physician with PI-associated hyperlipidemia. The study did not evaluate the influence of NRTI background drugs on lipid changes because the patients remained on the same baseline NRTIs. This could have affected the results because stavudine is known to elevate cholesterol and triglyceride levels [This study had several limitations. As patients in this study had mean CD4+ cell counts >500 cells/mme levels , wherease levels . This sh3 at 12 months). However, the abacavir-switch arm had a significantly lower incidence of adverse events than the efavirenz-switch and nevirapine-switch arms , a lower incidence of neuropsychiatric adverse events than the efavirenz group , and significantly fewer cases of discontinuation of study drug due to adverse events . Overall, NEFA, like our study, indicated that abacavir was an appropriate drug to substitute for PIs in HAART regimens as long as patients were virologically suppressed pre-switch and minimally antiretroviral-experienced. Our study differed from NEFA in that all participants in ESS40003 had PI-associated hyperlipidemia at baseline  and because our study used a less stringent definition of virologic failure .Further valuable clinical information could have been acquired from this study had switch agents in addition to abacavir been used as comparators. In the only study of this type that has been conducted to date – the Nevirapine-Efavirenz-Abacavir (NEFA) Study – a significantly lower proportion of stabilized HAART recipients (HIV-1 RNA <50 copies/mL for = 6 months) switching from a PI to abacavir developed fasting plasma triglycerides >400 mg/dL and plasma cholesterol >240 mg/dL at 12 months compared to treatment groups in which patients switched from a PI to efavirenz or nevirapine . Kaplan-3, substituting abacavir for hyperlipidemia-associated PIs in HAART regimens improves lipid profiles and maintains virologic suppression over a 28-week period, and it simplifies treatment.In conclusion, in antiretroviral-experienced patients with HIV-1 RNA <50 copies/mL and CD4+ counts >500 cells/mmThe author(s) declare that they have no competing interests.JEH, PHK and VCW conceived the study design, PHK, MGS, EDJ, AR, JFO, VCW, JWS reviewed and approved study design, VCW provided the statistical methods for the study and performed the statistical analysis of the results, EH, VCW, JHW, JWS wrote, reviewed and edited the protocol, GEP drafted manuscript and evaluated lipid data previously published in antiretroviral studies described in Background and Discussion, JWF, JEH, PHK, MGS, EDJ, AR, JFO, JEH, JWF, JHW, JWS, ADS-C, VCW reviewed and edited the manuscript, ADS-C, JWF, PHK, MGS, EDJ, AR, JFO enrolled study subjects, ADS-C, JWF, JEH, JHW monitored the study, ADS-C, JWF, JEH, JWS, VCW evaluated the clinical data from the study, ADS-C set up the study at study sites and JEH contributed to secure funding.The pre-publication history for this paper can be accessed here: