Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure. Cells may be heterologous (different species), allogeneic , or autologous . Autologous cells are preferred because they will not evoke an immunologic response and thus the deleterious side effects of immunosuppressive agents can be avoided.The goal of tissue engineering is to repair organ pathologies such as those acquired congenitally or by cancer, trauma, infection, or inflammation. It is based upon the foundations of cell transplantation and materials science. Tissue can be engineered 1) 2 to one that covers a surface area of >4000 m2  within 8–14 weeks. These studies indicate the possibility of collecting autologous bladder cells from human patients, expanding them in culture, and returning them to the human donor in sufficient quantities for reconstructive purposes [The ideal autologous cells can often be found within the organ itself. These cells (committed precursors) may be isolated, expanded and transplanted back into the same patient, thus representing an autologous transplantation resource. Previously, urothelial cells could be grown in the laboratory setting with only limited expansion. Several protocols were developed over the last 20 years which identified the undifferentiated cells and kept them undifferentiated during their growth phase -4. Usingpurposes -11. Majoi.e. pancreas, liver). Stem cells are envisioned as being an alternate source of cells from which the desired tissue can be derived. Human embryonic stem cells (HESC) can be derived from discarded non transferred embryos and have the advantage of being pluripotential  and able to self-renew indefinitely. However, their clinical application is limited because they represent an allogeneic resource and thus their use would require high dose immunosuppressant therapy.While autologous cells are recognized as the ideal transplantation resource, many patients with end-stage organ disease are unable to yield sufficient cells for expansion and transplantation. Furthermore, some primary autologous human cells cannot be expanded from particular organs (New stem cell technologies such as somatic cell nuclear transfer (therapeutic cloning) and parthenogenesis offer an exciting alternative to create an inexhaustible supply of ESC that can differentiate into all cell types of the embryo, while not being rejected by the patient's immune system. Although many tissues have been created with ESC, they are not used clinically because of an inability to control differentiation. Hence, their ability to form multiple tissue types also becomes their limitation. New genomics and bioinformatics technologies have and will continue to offer new insights into the understanding of ESC growth and differentiation and their application to engineering tissues. In the near future, these new technologies will allow for the generation of an unlimited supply of any cell type in the body.ex vivo, ESC can potentially be the ideal resource for tissue engineering because of two fundamental properties, 1) the ability to self-renew indefinitely, and 2) the ability to differentiate into all three germ layers.The political and ethical controversy surrounding stem cells began in 1998 with the creation of HESC derived from discarded, non-transferred human embryos. The HESn = 7,334) are expected to survive the freeze/thaw process. From this, 25% are expected to develop to blastocyst stage . If one assumes a 15% efficiency rate for establishment of a HESC line from blastocysts , it Somatic cell nuclear transfer (SCNT) entails the removal of an oocyte nucleus followed by its replacement with a nucleus derived from a somatic cell obtained from that patient. Activation with chemicals or electric shock stimulates cell division up to the blastocyst stage at which time the inner cell mass is isolated and cultured, resulting in ESC. This approach is distinct from reproductive cloning because the blasotcyst is not transplanted back to the uterus. Hence, development does not proceed beyond the 100 cell stage. This process also differs from fertilization since no sperm is used in this process. The resulting ESC are perfectly matched to the patients immune system and no immunosuppressants would therefore be required to prevent rejection.adult somatic cell using nuclear transfer [While interest in the field of nuclear cloning remains high since the birth of Dolly 1997), the first successful nuclear transfer was actually reported over fifty years ago by Briggs and King . Cloned 97, the ftransfer , goats [transfer ,22, micetransfer , and pigtransfer -27.A better understanding of the differences between reproductive cloning and therapeutic cloning may help alleviate some of the controversy surrounding these technologies ,29. BannWhile promising, somatic cell nuclear transfer technology has certain limitations requiring further improvement before it can be applied widely in clinical practice. Currently, the efficiency of the overall cloning process is quite low as the majority of embryos derived from animal cloning do not survive after implantation -35. In pIt must be noted that abnormalities have been found in liveborn clones including macrosomia with an enlarged placenta ("large-offspring syndrome") , respiraBos taurus) [in vivo growth. After 12 weeks cloned renal cells were harvested, expanded in vitro, then seeded onto biodegradable scaffolds. The constructs (consisting of cells + scaffolds) were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth.We applied principles of both tissue engineering and therapeutic cloning in an effort to produce genetically identical renal tissue in an animal model ( taurus) . Bovine ex vivo renal replacement devices are known to be life-sustaining, there are obvious benefits for patients with end-stage kidney disease if such devices could be implanted long-term without the need for an extracorporeal perfusion circuit or immunosuppressive drugs.The kidney is a complex organ with multiple cell types and a complex functional anatomy rendering it one of the most difficult organs to reconstruct ,47. PrevCloned renal cells were seeded on scaffolds consisting of three collagen-coated cylindrical polycarbonate membranes figure . The endChemical analysis of the urine-like fluid  revealed that the implanted renal cells possessed filtration, reabsorption, and secretory capabilities. Histological examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli- and tubule-like structures. A clear continuity between glomeruli, tubules, and the polycarbonate membrane was noted that allowed the passage of urine into the collecting reservoir figure . ImmunohAs previous studies have confirmed bovine clones harbor mitochondrial DNA (mtDNA) of strictly oocyte origin -57, the in vivo and cytotoxic T lymphocytes expansion in vitro [in vivo and Elispot analysis of interferon-gamma secreting T-cells in vitro. Both analyses revealed that the cloned renal cells showed no evidence of T-cell response, suggesting that rejection will not necessarily occur in the presence of oocyte-derived mtDNA  is production of offspring by a female with no genetic contribution from a male and without meiotic chromosome reduction. The process is common reproductive strategy among insects such as aphids, flies, ants, and honeybees, but is also known to occur in vertebrates including lizards, snakes, fish, birds, and amphibians. The first demonstration of artificially-stimulated parthenogenesis in vitro was made by Jacques Loeb (1899), who was able to activate oocytes from sea urchins and frogs by pricking them with a needle or by changing the ambient salt concentration. Pincus (1939) demonstrated parthenogenetic activation of mammalian eggs using temperature and chemical stimuli. Thus far, parthenogenetic activation of eggs has been studied in a variety of mammals including mice, goats, cows, monkeys, and humans. Plachot et al. described parthenogenesis in humans by examining 800 human oocytes and showed that 12 activated parthenogenetically and four underwent normal cleavage[Parthenogenesis  to elevate Ca2+ levels transiently, followed by application of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-dimethylaminopurine). Success rates and viability appear to be organism dependent. Mouse parthenotes are capable of developing beyond the post-implantation stage in vivo [Callithrix jacchus) have only been shown to implant [2n polar body under the zona pellucida and a 2n protonucleus in the cytoplasm. After chemical activation to mimic the effects of sperm penetration on changes in cellular Ca2+ gradient, the cell fails to complete meiosis II. Instead, the second polar body is never extruded, resulting in a diploid protonucleus derived from two sets of sister chromatids. These chromatids then begin to undergo mitosis resulting in a parthenote manifesting uniparental disomy. Although the derivation of embryonic-like stem cells from oocytes  is relatively inefficient (perhaps due to complexities of genomic imprinting), when they are differentiated into adult tissues, they appear fully functional.There is no confirmed example of  in vivo ,67; porc in vivo ; primate implant . The rea implant ,71. Inde implant . In mostin vitro in an undifferentiated state for up to 14 months. In vitro, they have been differentiated into cardiomyocyte-like cells, smooth muscle, beating ciliated epithelia, adipocytes, several types of epithelial cells, as well as dopaminergic and serotoninergic neurons. Almost all of these neurons express TUJ1 (beta-tubulin III), and up to 25% of the TUJ1+ cells co-express tyrosine-hydroxylase. This latter enzyme marker is considered diagnostic for catecholaminergic neurons . Furthein vivo, since injection of monkey PSC into immunocompromised mice induces formation of benign teratomas containing tissue derivatives from all three germ layers  including cartilage, muscle, bone, neurons, skin, hair follicles, and intestinal epithelia [These observations are recapitulated pithelia ,75. Of ppithelia . The reain vivo tumorigenic event) the de facto acceptance of experiments using teratoma tumor tissue lends some legitimacy to experimentation on parthenotes. These contradictions await reconciliation in a comprehensive ethical framework.To be sure, parthenotes are not free from ethical controversy and are viewed by some in society as artificial entities that in some sense represent 'tampering with nature.' Since a parthenote is analogous to a mature ovarian teratoma , and NP that were further differentiated for 8 days (PSC-neurons). We have identified numerous targets such as receptors and ligands present at each of these distinct time points, and are modifying our culture system in order to improve the quality and quantity of differentiation. Furthermore, we are comparing the gene expression profiles of PSC derived neurons to gene expression profiles of reference neurons. Not only will this provide new insight into the type of neurons that may be generated, but it offers clues into what our stem cell derived neurons might be lacking. We can then go back to the culture system and try to target these specific genes/signaling pathways.Further study of stem cell genomics will give additional insight into pluripotentiality. An understanding of pluripotentiality might allow for a somatic cell to be de-differentiated into an intermediate stage, which could then be expanded, differentiated and transplanted back into the patient. We are presently characterizing the genetic signature of pluripoteniality by analyzing gene expression among primate stem cells derived from a variety of methods . By identifying "stemness" genes by comparing undifferentiated stem cells to their differentiated counterpart, and comparing this to stem cells of different origins, a core set of pluripotential target genes may be mapped. Of particular interest are the 1,075 genes that are similarly down-regulated in IVF derived human ESC and monkey PSC. Furthermore, we have detected paternally imprinted genes in our HESC but not in our PSC data sets. From this we conclude that paternal imprinting might not be necessary for pluriopotentiality.Our systems biology approach incorporates the fields of genomics, cell biology, nuclear transfer, and materials science, and utilizes personnel who have mastered the techniques of bioinformatics, cell harvest, culture, expansion, transplantation, as well as polymer design essential for the successful application of these technologies. Experimental efforts are currently underway involving virtually every type of tissue and organ of the human body. Various tissues are at different stages of development with some already being used clinically, a few in pre-clinical trials, and some in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a viable therapeutic option for those who require tissue replacement or repair.


A review of "On Intelligence", a book exploring brain function by entrepreneur Jeff Hawkins and science writer Sandra Blakeslee Is Michael Moore liberal America's Rush Limbaugh? If so, is he filling a much needed, or a much lamented, gap in turning issues that are really cast in pastel shades into Day-Glo relief? In this hale monograph, Jeff Hawkins (rendered by Sandra Blakeslee) plays exactly this role for theoretical neuroscience. As a pastel practitioner myself, but furtively sharing many of Hawkins' prejudices and hunches about computational modelling in neuroscience, I am caught between commendation and consternation.Hawkins is an engineer, entrepreneur, and scientist who founded and led the companies Palm and then Handspring. He created, against what must have been considerable obstacles, the first widely successful PDA, and continued the development of this platform. He has thus amply earned a bully pulpit. The autobiographical segments of this book detail that, throughout his career, he has been interested in understanding how the brain works, using his substantial knowledge and intuition about the architecture and design of conventional computers as a counterpoint.More recently, Hawkins has generously put his money where his ideas about mentation dictate, founding the Redwood Neuroscience Institute and also funding various conferences and workshops. The institute is dedicated to ‘studying and promoting biologically accurate mathematical models of memory and cognition.’Despite its youth, the Institute already has attracted notable attention as a centre for theoretical neuroscience. Hawkins' quest, and-depending on which statements of the book you read-its endpoint (‘… a comprehensive theory of how the brain works … describ[ing] what intelligence is and how your brain creates it’) or just its tipping point , are the subject here.There are really three books jostling inside the covers. One is the (highly abbreviated) autobiography. The history of modern computing is very brief and  very glorious, and this story is most entertaining. Don't miss the wonderfully faux naive letter from Hawkins to Gordon Moore asking, in 1980, to set up a research group within Intel devoted to the brain. That Hawkins prospered in clear opposition to accepted wisdom is perhaps one of the key subtexts of the book.The second, and rather less satisfying, book is about the philosophy of mind and the history of artificial intelligence and neural network approaches to understanding the brain and replicating cognition. With respect to the fields of artificial intelligence and neural nets, the text seems rather to be fighting yesterday's battles. The importance of learning, flexibility in representation and inference, and even decentralisation of control has been more than amply recognised in the inexorable rise of probabilistic approaches in both fields.With respect to the philosophy of mind, there seems to be something of an enthusiast's disdain for the niceties of philosophical pettifogging, even arguing by assertion. The discussions at the end on creativity and consciousness all seem a bit gossamer. The book is somewhat careless about functionalism, a key doctrine for computational theorists about how brains give rise to minds. According to this doctrine, at least roughly, it is the functional roles of, and functional interactions among, the physical elements of brain that matter, and not their precise physical nature. If you can capture those functional aspects correctly, for instance, in a computer program, then you can (re-)create what's important about mental states. Functionalism licenses a form of inquiry into the computational jobs played by structures in the brain. However, although formally agreeing that ‘there's nothing inherently special or magical about the brain that allows it to be intelligent,’the book slips into statements such as ‘brains and computers do fundamentally different things,’which are, at best, unfortunate shorthand.The book is a little apt to sneak plausible, but misleading, claims under the radar. Just to give one instance, it compellingly compares a six year old hopping from rock to rock in a streambed with a lumbering robot failing to do the same task. However, this is a bit unfair. One of Hawkins' self-denying ordinances is to consider the cortex pretty much by itself. As aficionados of the cerebellum  would be quick to point out, the singular role for the cortex in such graceful behaviour is rather questionable.The third book is what I think is intended to be the real contribution. This contains a (not wholly convincing) attempt to conceptualise the definition of intelligence in terms of prediction rather than behaviour, and then to describe its possible instantiation in the anatomy (and mostly only the anatomy) of the cortex.To situate Hawkins' suggestions, it is instructive to consider current models of how the cerebral cortex represents, and learns to represent, information about the world without being explicitly taught. Being a popular account, the book fairly breezes by these so-called unsupervised learning models see , in whicFirst, where does the structure in the inputs come from? For the sake of concreteness, think of the input as being something like movies on a television screen. Movies don't look like white noise, or ‘snow’, because of their statistical structure. For instance, in movies, pixel activities tend to change rather slowly over time, and pixels that are close to each other on the screen tend to have relatively similar activities at any given time. Neither of these is true of white noise. More technically, movies constitute only a tiny fraction of the space of all possible activations of all the pixels on your screen. They  have a particular statistical structure that the cortex is supposed to extract.What is the cortex supposed to do with this structure? The idea is that the cortex learns to model, or ‘parameterize’, it. Then, the activities of cortical cells over time for a particular input, for example, a particular face in a movie, indicate the values of the parameters associated with that face. Thereby the cortical activities represent the input. The parameters for a face might include one set for its physical structure , another set for the expression, and yet others, too.Cortical representations are thus intended to reflect directly the statistical structure in the input. Importantly, for inputs such as movies, this structure is thought to be hierarchical and, concomitantly, to provide an account of the observed hierarchical structure of sensory cortical areas. One source of hierarchical structure in movies is the simple fact that objects (such as the faces) have parts (such as eyes and cheeks) whose form and changes in form over time are interdependent. Another source of hierarchical structure is that the same face can appear in many different poses, under many different forms of illumination, and so on. Pattern theory , one of How does the cortex do all this? Of course, some fraction of this structure was built in over evolution. However, the unsupervised learning tradition concentrates on ontogenic adaptation, based on multiple presented input movies. An additional facet of the lack of supervision is that this adaptation is taken as not depending on any particular behavioural task.Finally, what does this process allow the cortex to do? The whole representational structure is intended to support inference. Crudely, this involves turning partial or noisy inputs into the completed, cleaned-up patterns they imply, using connections between areas in the cortical hierarchy. Construed this way, probabilistic inference actually instantiates a very general form of computation. Crucially, over the course of the development of unsupervised learning methods, it has been realised that the best way to approach the extraction of input structure, and inference with it, is through the language and tools of probability theory and statistics. The same realisation has driven substantial developments in artificial intelligence, machine learning, computer vision, and a host of other disciplines.We can now return to the book. Hawkins compactly sums up his thesis in the following way. ‘To make predictions of future events, your neocortex has to store sequences of patterns. To recall appropriate memories, it has to retrieve patterns by their similarity to past patterns . And finally, memories have to be stored in an invariant form so that the knowledge of past events can be applied to new situations that are similar but not identical to the past.’In fact, to take the latter points first, the sort of auto-associative storage and recall to which Hawkins refers is a theoretically and practically hobbled version of unsupervised learning's probabilistic inference. Invariance is closely related to the deformations we described above in the context of pattern theory.Unsupervised learning has certainly paid substantial attention to sequences of inputs and prediction, and to some good effect. For instance,  speech recognition programs are based on a probabilistic device called a hidden Markov model, which is a key element in a wealth of unsupervised learning approaches to prediction. However, despite heroic efforts, these modelling methods are incapable of capturing the sort of complex structure seen in inputs such as natural languages. They fail on phenomena like long-distance dependencies, for example, the agreement between the cases of subjects and verbs, which are rife. This does tend to offer a vaccine against Hawkins' otherwise infectious optimism.Once place in which Hawkins goes beyond existing unsupervised learning models is in an extension to actions and control, and in an ascription of parts of the model to cortical anatomy. The hierarchical conception of cortex here goes all the way down to primary motor cortex . This allows auto-associative recall of sequences of past inputs and outputs to be used to specify actions that have formerly been successful. The discussion of this possibility is, unfortunately, rather brief. Central issues are omitted, such as the way that planning over multiple actions might happen. Also, the way that value is assigned to outcomes to determine success or failure is not discussed. The latter is widely believed to involve the neuromodulatory systems that lie below the cortex and that the book's cortical chauvinism leads it cheerfully to ignore.By contrast, the book has a rather detailed description of how the model should map onto the anatomy of the cerebral cortex. Like many unsupervised learning modellers, Hawkins is a self-confessed ‘lumper’. He ignores huge swathes of complexity and specificity in cortical structure and connections in favour of a scheme of crystalline regularity. Though this will doubtless irk many readers  constitute a terrible waste of cortical representational power, or, worse, (b) interfere with, or warp, the parameterization of the aspects of the input that are important, making it harder to extract critical distinctions. The book does not address this issue, relying on there being enough predictive power to capture any and all predictions, including predictive characterisation of motor control.Second, although our subjective sense is that we build a sophisticated predictive model of the entire sensory input, experiments into such phenomena as change blindness  show thiAs a final example of a spur to insomnia, unsupervised learners worry that To sum up, in terms of the adage that genius is 1% inspiration and 99% perspiration, the book's enthymematic nature suggests that not quite enough sweat has been broken. Were it 1% inspiration and 99% aspiration, though, then the appealing call to arms for a new generation of modellers should more than suffice.


The Depression Network Study (DeNt) is a multicentre study designed to identify genes and/or loci linked to and/or associated with susceptibility to unipolar depression in Caucasian families. This study presents the method and socio-demographic details of the first 470 affected sibling pairs recruited from 8 different sites in Europe and the United States of America.th edition (DSM-IV) or the International Classification of Diseases 10th edition (ICD-10) criteria for recurrent unipolar depression of moderate or severe degree and who had at least one similarly affected sibling were eligible for the study. Detailed clinical and psychological assessments were undertaken on all subjects including an interview using the Schedules for Clinical Assessment in Neuropsychiatry. Blood samples were collected from all participants to extract DNA for linkage analysis.Probands fulfilling either the Diagnostic and Statistical Manual 4The different sites used different recruitment strategies depending on local health care organisation but despite this there was remarkable similarity across sites for the subjects recruited. Although the Bonn site had significantly older subjects both for age of onset and age at interview, for the sample as a whole, subjects were interviewed in their mid-40s and had experienced the onset of their recurrent depression in their 20s. Preliminary genome screening was able to include 929 out of the 944 subjects (98.4%) typed at 932 autosomal and 544 X chromosome markersThis paper describes the methodology and the characteristics of the subjects from the 414 families included in the first wave of genotyping from the multi-site DeNT study. Ultimately the study aims to collect affected sibling pairs from approximately 1200 families. Genetic risk factors are well established for major affective disorders and a recent twin study has suggested that unipolar depression has a stronger genetic influence than was previously thought. McGuffin and colleagues  have estThe majority of studies suggest a relative risk to siblings (λs) of affective disorder is in the region of 3 .The inheritance of unipolar depression is complex and involves an inter-play of genetic and environmental factors. For unipolar depression these include certain types of severe and threatening life events such as events associated with humiliation or loss ,6.Despite an excess of females to males of about 2 to 1 for unipolar depression, the heritability in a clinically ascertained sample was the same in men and women  while otMost previous linkage studies have been carried out in families identified by a bipolar proband and where unipolar and bipolar relatives are frequently grouped together into a broad definition of affective disorders. Most such studies have focussed on multiple affected extended pedigrees on the assumption that there may be a sub-set segregating a gene of major effect. This approach has been successful in complex disorders such as early onset Alzheimer's disease and breast cancer. However, consistent evidence of major gene effects in bipolar disorder has not been forth-coming. In addiSibling pairs affected with recurrent unipolar depression were recruited from 8 clinical sites: Aarhus, Denmark; Bonn, Germany; Dublin, Ireland; Lausanne, Switzerland; St Louis, USA and London, Cardiff and Birmingham, UK. In addition, where available, parents of the affected sibling pairs were also included in the study.th edition operational criteria (DSMIV) [th edition operational criteria (ICD10), for unipolar depression [Subjects were identified from psychiatric clinics, hospitals, general medical practices and from volunteers responding to media advertisements. Caucasian subjects over the age of 18 were included if they had experienced 2 or more episodes of unipolar depression of at least moderate severity separated by at least 2 months of remission as defined by the Diagnostic and Statistical Manual 4 (DSMIV)  or the Ipression . ProbandSubjects were also excluded if they experienced psychotic symptoms that were mood incongruent or present when there was no evidence of a mood disturbance. Other exclusion criteria were intravenous drug use with a lifetime diagnosis of dependency; depression occurring solely in relation to alcohol or substance abuse or depression only secondary to medical illness or medication, and a clear diagnosis of bipolar disorder, schizophrenia, schizo-affective disorder or acute or transient psychotic disorders in first or second-degree relatives.All subjects were interviewed using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) ,16. ItemAll interviewers from each site attended a 4-day SCAN training course in the UK. Each site also undertook further inter-rater reliability meetings regularly and annually all interviewers from all sites took part in a joint inter-rater reliability exercise.All sites obtained ethical approval for the DENT study within their own countries and institutions. All study participants gave written informed consent for participation in the study.In addition to the SCAN interview all study participants completed the Eysenck Personality Questionnaire  and a deAt the time of the SCAN interview interviewers obtained 25 ml of whole blood that was collected in 37.5 ml (EDTA containing) monovettes. In addition drops of blood were placed on a Guthrie blood spot card. The blood samples were labelled with a bar code, gently mixed and stored frozen upright in a -20 degree centigrade freezer pending DNA extraction.All phenotypic information from interviews and questionnaires was coded by assigning a number to each subject, and removing any personal identifying information. The same codes were used on the blood sample tubes using a bar code system. The phenotypic information was first entered on an EXCEL spread sheet after which a data file was created using Statistical Procedures for the Social Sciences (SPSS) version 10 for Windows for the statistical analyses.All the interviewers from all sites took part in a joint inter-rater reliability exercise (in English) involving both audio-taped interviews with study subjects and videotaped interviews with actors, role-playing a depressed subject. Item by item kappa statistics for SCAN items, were calculated comparing each interviewer's ratings against AF's "master" rating. A mean item by item kappa coefficient across all the sites of 0.77 (range 0.63 – 0.89) was obtained indicating a substantial level of inter-rater agreement.For inclusion in the first part of the linkage analysis, 944 affected subjects were genotyped from the 8 study sites as follows: Aarhus 48, Birmingham 146, Bonn 110, Cardiff 126, Dublin 154, Lausanne, 56, London 111 and St Louis 193. The age at interview and age of illness onset by gender of the subjects recruited at each site are shown in Table Mean age at interview for both sexes combined for each site were as follows: Aarhus 44.13 years (standard error of the mean (SEM)1.5), Birmingham 48.44 years (SEM 1.1), Bonn 51.46 years (SEM 1.2), Cardiff 44.06 years (SEM 0.9), Dublin 43.32 years (SEM 1.0), Lausanne 47.21 years (SEM 1.3), London 45.67 years (SEM 1.0), St Louis 47.14 years (SEM 0.9). These mean age differences were statistically significant : F = 6.26 degrees of freedom (df) 7, 936, p < 0.001. Tukey Post hoc test: Dublin, Cardiff, Aarhus, London, St Louis, Lausanne, Birmingham < St Louis, Lausanne, Birmingham, Bonn).Mean age at illness onset for both sexes combined per site were as follows: Aarhus 22.67 years (SEM 1.3), Birmingham 24.30 years (SEM 0.8), Bonn 27.28 years (SEM 1.2), Cardiff 23.47 years (SEM 0.9), Dublin 22.27 years (SEM 0.8), Lausanne 24.85 years (SEM 1.4), London 22.08 years (SEM 1.1), St Louis 18.17 years (SEM 0.8). These mean age differences were statistically significant .However, there were no significant differences between sites for the numbers of men and women recruited .Of the 944 subjects, 670 (71%) were female and 274 (29%) were males and hence, the female/male ratio was 2.45:1.Mean age at interview for all female subjects was 45.40 years (SEM 0.5) and for all males subjects was 45.69 (SEM 0.8). There were no significant gender differences for age at interview The mean age of illness onset for depressed male subjects was 22.61 years (SEM 0.7) compared to 22.52 years (SEM 0.4) for depressed female subjects. There was no significant sex difference for age of onset..Fifty five percent of male subjects and 61 % female subjects were living with a partner (married or cohabiting), while 45 % male subjects and 39% female subjects were living alone . Female subjects were significantly more likely to be living with a partner compared to male subjects. (Chi squared test = 26.89 df = 1 p < 0.001).There were 295 female and 117 male probands, 335 female and 144 male siblings and 40 female and 13 male parents included in the total sample. There were no significantly differences for the gender of probands, siblings or parents .The mean age at interview for probands was 45.94 years (SEM 0.6) and for siblings was 45.80 years (SEM 0.5). There were no significant differences for age at interview between probands and their siblings Probands gave a mean age of illness onset of 20.22 years (SEM 0.6) while siblings reported a mean age of onset of 21.04 years (SEM 0.6). Again these differences were not statistically significant There were also no significant differences between probands and their siblings for marital status; 161 probands and 170 siblings were living alone while 242 probands and 290 sibings were living with a partner (chi squared test = 0.81 df = 1 p = ns).Genotyping was carried out by DeCode and the results checked for mis-specified relationships by the programs RELPAIR and Graphical Representation of Relationships (GRR) at the Institute of Psychiatry. RELPAIR compares the multipoint probability of the data conditional on the possible relationships, while GRR calculates the IBS mean and SD for each pair and plots these values, representing each type of relative pair using a different colour. Decisions about each problem pair were made on the basis of the results from both programs, although where there was discrepancy between the programs the GRR results were used.To check genotypes with Mendelian and other pedigree errors the PEDSTAT and MERLIN programs were used.These data cleaning processes resulted in 929 individuals being genotyped at 932 autosomal markers and 44 X chromosome markers. Success rates for the autosomal markers were above 61% and for 90% were above 86%. For the X chromosome the success rate was above 66%. For individuals the genotyping success rate was above 73% for autosomal markers and 61% for the X chromosome.The Depression Network study has recruited affected sibling pairs and some of their parents from 7 European and 1 North American site for a linkage analysis of recurrent unipolar depression. Because of differences in local service organisation, different recruitment strategies have been employed at the different sites. This may account for the significant differences for age at interview and age at illness onset between sites. The Bonn site recruited the oldest sibling pairs, both in terms of when subjects were interviewed and also when their illnesses had commenced. The Bonn subjects had a mean age at interview of 51.46 years compared to the Dublin subjects whose mean age at interview of 43.31 years was the youngest. Similarly the Bonn subjects mean age at illness onset was 27.28 years compared to a mean age of illness onset nearly a decade earlier for the St Louis subjects (18.17 years). It is noteworthy however that the St Louis sample included several large affected sibships. Subjects from families where there are many affected relatives may have a more genetic form of the disorder that might be contributing to an earlier age of onset.Despite these inter-site differences, the results show that there are also considerable similarities across the sites for the subjects recruited. Subjects have been mainly interviewed in their mid 40s and have experienced the onset of their recurrent depression in their early to mid 20s. Consequently subjects had on average around 20 years of history of episodes of depression when interviewed.As expected the study has shown the same preponderance of female to male subjects as many other studies with a gender ratio of around 2.45:1 [We would also not expect to find any significant differences between probands and siblings in terms of gender, age at interview, age at illness onset or marital status, which the results show is the case. Indeed for the purpose of finding genes for depression we would require siblings to have experienced similar forms of the illness.Although some subjects were excluded following genotyping due to errors that could not be reconciled, this preliminary genome linkage screen was able to include 929 subjects (98.4%) genotyped at 932 autosomal and 544 X chromosome markers. The results of the whole genome screen will be presented in due course.The Depression network study is the first co-ordinated international collaboration of its kind on the genetics of depression and one of the largest ever neuropsychiatric linkage study collection to use a uniform methodology to define and describe the phenotype. Despite taking place across eight sites and in six different countries good inter-rater agreement has been achievable as has good comparability of data.The study has been designed to overcome the difficulties that have been encountered in linkage studies of other psychiatric disorders such as schizophrenia and bipolar disorder. These started out optimistically with the assumption that genes of large effect would exist in at least some multiply affected families. However, after over a decade of contradictory findings and non replications, there is now consensus that such families are very rare or perhaps nonexistent. Rather it seems likely that common familial psychiatric disorders result from the combined effect of multiple genes none of which is either necessary or sufficient to cause the condition . ConsequThis study was funded by 3 year research grants to each participating site from Glaxo Wellcome Research and Development.AF & PMcG were overall study Principal Investigators (PIs), conceived the study and were co-ordinators of the study design, diagnostic reliability and data analysis. AF trained the interviewers from all the sites and wrote the paper. SB, LM and JP obtained the funding from Glaxo Wellcome, recruited site PIs and oversaw the quality of data collection, handling and analysis. GB analysed the genotyping data. The following authors were the individual site PIs in charge of all aspects of subject recruitment and data quality locally: OM Aarhus site, NC, LJ and IJ (joint) Birmingham site, MR and WM (joint) Bonn site, AK and MO (joint) Cardiff site, MG Dublin site, MP Lausanne site AF and PMcG (joint) London site and TR St Louis site. All authors have read and approved the contents of the final manuscript.The pre-publication history for this paper can be accessed here:


Neo-adjuvant chemotherapy is an integral part of multi-modality approach in the management of locally advanced breast cancer and it is vital to predict the response in order to tailor the regime for a patient. The common final pathway in the tumor cell death is believed to be apoptosis or programmed cell death and chemotherapeutic drugs like other DNA-damaging agents act on rapidly multiplying cells including both the tumor and the normal cells by following the same common final pathway. This could account for both the toxic effects and the response. Absence or decreased apoptosis has been found to be associated with chemo resistance. The change in expression of apoptotic markers (Bcl-2 and Bax proteins) brought about by various chemotherapeutic regimens is being used to identify drug resistance in the tumor cells. A prospective clinical study was conducted to assess whether chemotherapy induced toxic effects could serve as reliable predictors of apoptosis or response to neo-adjuvant chemotherapy in patients with locally advanced breast cancer.50 cases of locally advanced breast cancer after complete routine and metastatic work up were subjected to trucut biopsy and the tissue evaluated immunohistochemically for apoptotic markers (bcl-2/bax ratio). Three cycles of Neoadjuvant Chemotherapy using FAC regime  were given at three weekly intervals and patients assessed for clinical response as well as toxicity after each cycle. Modified radical mastectomy was performed in all patients three weeks after the last cycle and the specimen were re-evaluated for any change in the bcl-2/bax ratio. The clinical response, immunohistochemical response and the drug-induced toxicity were correlated and compared.Descriptive studies were performed with SPSS version 10 and the significance of response was assessed using paired t-test. Significance of correlation between various variables was assessed using chi-square test and coefficient of correlation calculated by Pearson correlation coefficient.There was a statistically significant correlation observed between clinical, immunohistochemical response (bcl-2/bax ratio) and the drug-induced toxicity.Responders also had significant toxicity while non-responders did not show significant toxicity following neoadjuvant chemotherapy. The chemotherapy-induced toxicity was observed to be a cost effective and reliable predictor of response to neo-adjuvant chemotherapy. Neo-adjuvant chemotherapy (NACT) is an integral part of multi-modality approach in the management of locally advanced breast cancer (LABC). It is required both for the local control  and distant or systemic control -5. In thApoptosis is a closely regulated form of active cell death defined by characteristic biochemical and morphological criteria. A large number of anti-cancer agents with widely differing modes of action have been demonstrated to induce apoptosis in vitro, suggesting this as a significant final common pathway for exerting their clinical effects. Mechanisms that suppress apoptosis may be important in the development of intrinsic and acquired resistance to cytotoxic drugs .It was suggested more than 20 years ago that apoptosis might account for much of the spontaneous cell loss (known from kinetic studies) to occur in many tumors and its extent often is enhanced by well-established modalities such as chemotherapy, irradiation and hormone ablation. However, during the past few years, advances in the understanding of the control of apoptosis at the molecular level has extended its potential oncologic significance far beyond the mere provision of a mechanistic explanation for tumor cell deletion. In particular, the discovery that the products of certain proto-oncogenes can regulate apoptosis has opened up exciting avenues for future research .The protean effects of various neoplastic agents on synthesis of DNA, RNA, and inhibition of synthesis which may or may not lead to cell death, requires only that some critical concentrations of active drug or metabolite be present in a cell. Proliferating normal cells may therefore be subject to the same detrimental effects of chemotherapy experienced by neoplastic tissue and successful chemotherapy dictates that recuperative abilities of normal tissues are greater than those of cancer. The two tissues generally most adversely affected by antineoplastics are the hemopoetic cells of the bone marrow and the epithelium of the aero digestive tract as a result of high growth fractions and short cycling times of the cells. The ability of the cancer patients to perform normal activities and function is also recognized both as a determinant of how well the patient may respond to chemotherapy and an index of the general toxicities of the anti cancer agents.A variety of anti-cancer drugs have been shown to induce extensive apoptosis in rapidly proliferating "normal" cell population and the tumors alike. Thus enhanced apoptosis is also likely to be responsible for many of the adverse effects observed following chemotherapy . ApoptosApoptosis is a regulated phenomenon capable of being inhibited and activated. Indeed there is evidence that stimulation of some cells by trophic cytokines or increase in their levels of expression of Bcl-2 proto-ontogeny can greatly increase their resistance to the apoptosis-inducing effects of anticancer drugs. Thus Bcl-2 proto-ontogeny expression may be implicated in the development of resistance of tumors to therapeutic agents and may contribute to tumor growth and perhaps to ontogenesis by allowing the inappropriate survival of cells with DNA abnormalities .Deregulated expression of the Bcl-2 protein represents the best-known example of a potent blocker of apoptosis. Over expression of Bcl-2 has now been shown to protect a wide variety of cell types from induction of apoptosis by many different anticancer agents. Several homologues of Bcl-2 protein have also been shown to act as inhibitors of apoptosis, including Bcl-Xl and others as apoptotic proteins such as Bax. In vitro data suggest that it is the relative ratios of anti-apoptotic and pro-apoptotic proteins that determine the likelihood of cells to undergo apoptosis in response to chemotherapeutic drugs in vivo test bed to further confirm the clinical relevance of these observations. The clinical response or the absence of response along with the toxic effects observed could well help predict the outcome with a particular chemotherapeutic regime and facilitate planning of an alternate regime for better response [The increasing use of pre-operative chemotherapy (PCT) in breast cancer offers an response -8.It is vital to assess the response to NACT in order to tailor the regime for a particular patient to predict the intrinsic or acquired chemo resistance. DNA-damaging agents such as chemotherapeutic drugs can induce apoptosis and increased resistance to chemotherapeutic agents, which has been found to be associated with decreased capacity to undergo apoptosis -9. CentrThe clinical response along with complete pathological response (CPR) is still considered a surrogate marker of response against which all other predictive markers are compared. The need to have a reliable and inexpensive predictor of response in a third world scenario can not be over emphasized since majority of patients present relatively late and the resources are limited and scarce ,3. Since1. To assess the clinical and immunohistochemical response to NACT in patients with LABC.2. To correlate immuno-histochemical (apoptotic markers i.e. Bcl-2/Bax ratio) and clinical response to the drug induced toxicity.3. To ascertain whether the drug induced toxicity could be utilized as a reliable indicator and predictor of response to neoadjuvant chemotherapy.50 FNAC proven cases of locally advanced breast carcinoma according to AJCC (American Joint Committee On Cancer) classification were included in the studyA thorough clinical and ultrasonographic examination (USG) of all the patients including the opposite breast was performed to stage the disease accurately. A core biopsy using a tru-cut needle was performed for immuno-histochemical estimation of the apoptotic markers i.e. base-line Bcl-2/Bax ratio before initiating the chemotherapy. Routine and metastatic work up was done including complete blood examination , chest radiograph, ECG (Echocardiography when ECG had a positive finding), liver function tests, Bone Scan, USG abdomen, KFT (Kidney function tests).2, adriamycin -50 mg/m2, 5-fluorouracil-600 mg/m2) at an interval of three weeks. Before each cycle the patient was clinically and sonologically examined for the breast tumor size, axillary lymph node status & appearance of systemic metastasis. All patients were given the same antitoxicity treatment according to a standardized unit protocol including adequate hydration and inject able antiemetics before initiating the chemotherapy.Patients were subjected to three cycles of FAC regime  solution and incubated with blocking antibodies  at 37°C. Sections were washed with TBS solution. Incubation with avidin-biotin complex (ABC) was done at 37°C for one hour and washed with TBS solution. 3,3 Diaminobenzidine tetra hydrochloride solution applied for 3–5 min. Counter-staining with haemotoxylin solution done for 3–5 min. Sections were washed with distilled water, air dried and mounted using DPX mountant.For Bax, positive controls were taken as germinal centers of the lymphoid follicles and normal breast tissue and negative control was taken as the test slide without primary antibody. For Bcl-2, positive controls were the mantle zone of lymphoid follicles and the negative controls were the test slides without primary antibody.The pattern of positive staining for bcl-2 and bax was cytoplasmic, granular.The primary antibodies for bcl-2 and bax were procured from DAKO.Bax-Rabbit Anti-Human code no. A 3533.Bcl-2 Monoclonal Mouse Anti-Human code no. M 0887.Dilution for both was 1: 40.The results were interpreted on the basis of two criteria:(1) Percentage of cells showing immune bodies; <5%: score = 0, 5–25%: score = 1, 25–75%: score = 2, >75%: score = 3(2) Intensity of staining; mild: score = 1, moderate: score = 2, intense: score = 3.Since there was a strong correlation between the intensity of staining and percentage of cells showing immune bodies, the percentage of cells showing immune antibodies alone was considered for calculating the bcl-2/bax ratio"."Descriptive studies were performed with SPSS version 10. The significance of response assessed using paired t-test. Significance of correlation between various variables assessed using chi-square test and coefficient of correlation was calculated by Pearson correlation coefficient.50 cases of locally advanced breast cancer were included in this study. Mean age of the patients was 45.5 years (range: 28 to 71 years) and 53.3% patients were pre-menopausal. Size of the tumor was measured clinically as well as by ultrasound and the patients were subdivided into four groups: <5 cm(0%), 5–8 cm(56.6%), 8–10 cm(26.6%), >10 cm(16.6%). According to the axillary lymph node status the patients were divided into three groups: N0 (0%), N1 (33%), N2 (67%).Objective clinical response was defined as more than 50% reduction in the tumor size after three cycles of neoadjuvant chemotherapy. Immuno-histochemical response was defined as decrease in the Bcl-2/Bax ratio.Clinical response including the reduction in the tumor size and axillary lymph node status was observed in 70% of patients and was found to be statistically significant p < 0.0001). There were no patients in the No group and 29.4%of the N1 patients were down staged to N0 while70.6% remained N1. In patients with N2 disease 7.7% were down staged to N0 status while 46.2% were downstaged to N1 status and 46.2% did not show any response. Immuno-histochemical response was observed in 60% and was also found to be statistically significant (p = 0.008). Correlation between immuno-histochemical and clinical response was also found to be statistically significant (p < 0.0001) [Table . There wAcute vomiting was observed in 63.3% patients. 81% clinical responders had vomiting (p = 0.002) and 78% immunohistochemical responders also had vomiting which was statistically significant (p = 0.04). Alopecia was observed in 86% clinical responders (p = 0.000) and 94% immuno-histochemical responders (p = 0.000), which was also significant. Leucopoenia was observed in only 14% and 17% of clinical and immuno-histochemical responders respectively and was found to be an insignificant predictor of response in the present study. When multiple toxicities were correlated with the clinical and immuno-histochemical response, 46.7% of patients had both acute vomiting and alopecia. 67% clinical responders (p = 0.001) had both vomiting and alopecia.72% immunohistochemical responders (p = 0.001) had both vomiting and alopecia.A significant positive correlation was observed between the presence of vomiting (r = +0.558), alopeciar = +0.802) and response to neoadjuvant chemotherapy. A significant negative correlation was observed between the absence of side effects and poor response to neoadjuvant chemotherapy in 65% of patients after two courses of FAC . NACT haesection -13.Introduced by Kerr et al (1972) to describe characteristic morphological changes seen during programmed cell death . It is d(1) Chromophin condensation(2) Membrane blebbing(3) Appearance of apoptotic bodies(4) Fragmentation of genomic DNACertain biochemical and genetic events have been identified that are associated with multiple cell types including mammary epithelium. These include the DNA fragmentation via end nuclease activation and cleavage of intracellular proteins, expression of bcl-2 family members, tumor suppressor gene p-53 directed events, proto-oncogene activation and activation of transmembrane receptor signaling pathways such as tumour necrosis factor ,17-22. AThe heterogeneous nature of breast cancer has resulted in overwhelming interest in search for prognostic markers to identify patients who might benefit most from the therapeutic modalities available.Assessment of apoptosis and individual components of apoptotic pathway are therefore relevant in determining prognosis in a particular patient . DNA damBcl-2 is anti-apoptotic in function, whereas bax is proapoptotic and it is the interaction between the two that determines the likelihood of a tumor to undergo cytotoxic drug mediated regression. Therefore any increase in bcl-2 or decrease in bax will push the balance towards chemo resistance and an increase in bax or decrease in bcl-2 will result in increased apoptosis -30.It was observed in a study conducted by Kymionis et al  that incIn the present study the clinical response in terms of reduction of tumor size and immuno-histochemical response in terms of change of bcl-2/bax ratio correlated significantly with the drug induced toxicity following NACT.The time course of various toxic manipulations depends on the drug, its dose and frequency of administration, intrinsic characteristics of the tissue of interest and any local circumstances . There are few general rules however like mucosal toxicities of pain, erythema, ulceration etc. occur 3–10 days after the administration of most offending drugs. Bone marrow effects can be manifestated a few days later averaging 7–14 days. The recovery of normal functioning tissues in both cases is well under way 4–5 days after the zenith of the toxic effects.Alopecia may involve the scalp or the whole body can present within 2 weeks of the drug dose or be a progressive cumulative event. Other cumulative toxicities seen only after administration of a certain quantity of drug over some length of time include cardiomyopathy from anthracyclins.An alkylating agent belonging to the nitrogen mustard subgroup. It is inactive as such produces few acute effects and is not locally damaging. It is transformed in to active metabolites like aldophosphamide and phospharimide mustard in the liver, which then produces the wide variety of antitumour action. The prominent side effects are alopecia and cystitis, which are caused by another metabolite acrolein.It is a pyrimidine antagonist and is converted in the body to the corresponding nucleotide, 5-fluro-2-deoxy-uridine monophosphate, which inhibits thymidine synthetase and blocks the conversion of deoxyuridilic acid to deoxythymidilic acid. Selective failure of DNA synthesis occurs due to non-availability of thymidylate. Flourouracil may itself get incorporated in to nucleic acids and this may contribute to its toxicity. Even resting cells are affected, though multiplying cells are more susceptible like the cells in the gastrointestinal tract (GIT) and bone marrow.It is an anti tumor antibiotic and is capable of causing breaks in the DNA strands by activating topoisomerase II and generating quinone type free radicals. They have mutagenic potential. Maximum action is exerted at S phase, but toxicity is usually exerted in G2 phase. Cardiotoxicity is a unique side effect. Rapidly multiplying cells are more susceptible therefore it also acts on cells of GIT, bone marrow in addition to the tumor cells.Leucopenia has not been observed to be a frequently encountered chemotherapy induced toxicity using commonly used regimen in most of the studies -24. ThisThe rapidly proliferating normal and tumor cells are more susceptible to the action of chemotherapeutic agents, which could explain the significant correlation observed between the effects and the toxicity in the present study. There was a strong correlation observed between the immunohistochemical response (bcl-2/bax ratio), clinical response and drug toxicity. This indirectly indicates a correlation between chemotherapy induced apoptosis and the toxicity and therefore like apoptotic markers, chemotherapy induced toxic effects along with objective clinical response could serve as reliable and cost-effective indicators or predictors of response to NACT in patients with LABC. While many biological markers are in use and many are under trial to tailor the chemotherapy for a particular patient, most of these markers including apoptotic markers or p-glycoprotein etc. are not very frequently available and are expensive for a third world cancer set up. Thus the chemotherapy-induced toxicity along with clinical response may be utilized as a cost effective and reliable predictor of response to NACT in patients with LABC. This would also serve as an intermediate end point in determining drug sensitivity for adjuvant treatment, especially when adjuvant therapy is planned with the same regimen as induction chemotherapy. This can also help in planning an alternative regime in non-responders.None declared.CM, the principal and the corresponding author was the Supervisor and the Chief surgeon who performed and standardized surgery on the patients and designed the study.• VS participated in the designing of the study, performed the statistical analysis and was the first surgical assistant and Senior Postgraduate in charge of the cases in the study.• JP, Postgraduate surgical resident was the second assistant in charge of the cases and participated in the sequence alignment.• AL, Resident surgery participated in the data processing and statistical analysis.• SS was in charge of the molecular genetic studies at the Tumor biology lab ICMR.• AB participated in the genetic studies and data processing.• All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Strictly defined, evolution is a change in the gene pool, or total set of genes, of a given population over time. Genetic changes that increase the fitness of an organism—that is, increase survival or fertility—are more likely to be retained, through natural selection, and passed on to succeeding generations. In the classic case of Darwin's finches, different ecological niches exerted different selective pressures on an original population, and resulted in 14 different species, each sporting a beak uniquely adapted to harvesting particular available food sources.When it comes to microbial evolution, an ecological niche often takes the form of a host. If the microbe is a pathogen, its presence might trigger strong selective pressure from the host's immune system, precipitating an evolutionary two-step between microbe and host. Hosts with strong immune defenses can typically tolerate relatively virulent pests: conversely, ill-defended hosts die, which is bad news for the parasite. When the myxoma virus first infected a population of European rabbits in Australia in 1950, the virus was particularly lethal. Over time, less virulent strains were selected for—killing off your habitat would be an unsustainable fitness cost by most standards—and the rabbits developed resistance.In keeping with evolutionary theory, host immunity should affect the evolution of parasite virulence. Though theory predicts that immunity could potentially heighten virulence, there's no evidence that this is true. Being able to predict how natural selection will act on, and thus shape, virulence is vital for developing effective public health policies—and desperately needed vaccines—to deal with the ever growing roster of rapidly evolving pathogenic threats.Plasmodium in the mouse. Mackinnon and Read first directly injected two groups of mice with infectious parasites: “immunized” mice, which had been exposed to Plasmodium and then treated with the antimalarial drug mefloquine, and “naïve” mice, which had not. Parasites were serially transferred twenty times via a syringe from one mouse host to another. The virulence and infectiousness of the respective strains were evaluated by introducing the strains into another set of immunized and naïve mice.To investigate whether immune system defenses escalate pathogen virulence, Margaret Mackinnon and Andrew Read studied the malarial parasite As theoretically predicted, parasites evolved in the immunized mice were indeed more virulent than parasites evolved in the naïve mice. But what if the parasites were first transmitted through their natural vector, the mosquito, rather than through a syringe? Would they be as virulent? Interestingly, infection was not as severe after mosquito transmission. But parasites evolved in the immunized mice retained a higher level of virulence than those evolved in the naïve mice. This means that immunity accelerates the evolution of virulence in malaria, even after mosquito transmission, making them more dangerous to nonimmunized hosts.How does immune selection create more virulent pathogens? One possibility is that even though many parasites die in immunized hosts, those that “win the race to the syringe”—or the mosquito—are likely genetically equipped to stay ahead of advancing immune system defenses.It's not entirely clear why selection would favor more virulent parasites, but since the virulent strains showed no problems transmitting infection to new hosts, it's likely that such strains would spread throughout an immunized population. While mosquito transmission likely plays a significant role in virulence evolution—it clearly reduced virulence here—the molecular mechanics of this effect are also mostly speculative at this point. Many questions remain, but these results make a strong case that vaccine development aimed at protecting individuals against infectious pathogens would do well to consider the evolutionary implications, or increased pathogen virulence could be an unintended consequence.


Although the effects of resistant starch (RS) on postprandial glycemia and insulinemia have been extensively studied, little is known about the impact of RS on fat metabolism. This study examines the relationship between the RS content of a meal and postprandial/post-absorbative fat oxidation.14C]-triolein with breath samples collected hourly following the meal, and gluteal fat biopsies obtained at 0 and 24 h. RS, regardless of dose, had no effect on fasting or postprandial insulin, glucose, FFA or TAG concentration, nor on meal fat storage. However, data from indirect calorimetry and oxidation of [1-14C]-triolein to 14CO2 showed that addition of 5.4% RS to the diet significantly increased fat oxidation. In fact, postprandial oxidation of [1-14C]-triolein was 23% greater with the 5.4% RS meal than the 0% meal (p = 0.0062).12 subjects consumed meals containing 0%, 2.7%, 5.4%, and 10.7% RS . Blood samples were taken and analyzed for glucose, insulin, triacylglycerol (TAG) and free fatty acid (FFA) concentrations. Respiratory quotient was measured hourly. The 0%, 5.4%, and 10.7% meals contained 50 μCi [1-These data indicate that replacement of 5.4% of total dietary carbohydrate with RS significantly increased post-prandial lipid oxidation and therefore could decrease fat accumulation in the long-term. Resistant starch (RS) is any starch that is not digested in the small intestine but passes to the large bowel for fermentation . RetrogrAcute human studies describe variable postprandial glycemic and/or insulinemic responses to RS ingestion. In general, it is accepted that RS consumption lowers postprandial glucose concentrations marginally and postprandial insulin concentrations markedly. Many groups report a decrease in postprandial glycemic or insulinemic responses to RS ingestion relative to digestible starch (DS) consumption -7, whereMany studies have examined the relationship between RS ingestion and postprandial metabolite and hormone concentrations. Fewer studies have documented the effect of RS on lipid metabolism. In humans, five weeks of RS feeding lowered fasting cholesterol and triglyceride concentrations and postprandial plasma insulin concentrations relative to digestible starch (DS) feeding ,13. It hIt is possible that strong physical association between RS and dietary lipid may slow the absorption, and thereby increase the oxidation, of dietary lipid. Currently, there is no evidence pertaining to the dose-response relationship for RS ingestion  and postprandial glycemia, insulinemia, fat oxidation, or meal fat storage. It is important that these parameters be defined before designing and conducting long-term, prospective RS feeding studies.No difference in fasting or postprandial insulin, glucose, FFA, or triglyceride concentration was observed between any of the RS doses examined Figure .Overall, the dose of RS in the meal had a significant influence on ΔRQ (respiratory quotient) values . Meal fat oxidation at the 5.4% RS dose was significantly higher than both the 0% (p = 0.0062) and 10.7% doses (p < 0.0001). Separate tests at 6 h or 24 h following the test meal gave comparable results  suggest that the inclusion of 5.4% RS in the meal elevated postprandial fat oxidation. Unexpectedly, this effect was lost if the dose was increased to 10.7% RS.Similarly, the oxidation of -triolein  suspended in olive oil and the tests were conducted as 24 h inpatient stays at the GCRC. The fat tracer was fed as a triglyceride (glycerol tri [1-14C]oleate) rather than a FFA (eg. [1-14C]oleate) in order to reflect any change in the absorption of triglyceride FFA which might be due to a strong physical association with RS thereby slowing absorption. At hourly intervals following the meal, then at 8, 10, 12, 14 and 24 hours, breath samples were collected via exhalation through a tube with a one-way valve into scintillation vials containing 2 mmol benzethonium hydroxide (to trap 2 mmol CO2), 1 ml methanol, and 1 mg phenolpthalene as a pH indicator. Gluteal fat biopsies were collected by aspiration through a 14 g stainless steel needle at baseline and 24 h after ingestion of the test meal. All breath and fat samples were assayed for the presence of 14C (as described below). For these 24 h tests, subjects received 30% of daily energy needs at each of breakfast, lunch, and dinner, with the remaining 10% of calories received in an evening snack. The timing of meals/snacks was kept constant over all tests. All food was provided by the GCRC on an inpatient basis and the macronutrient content of each meal was the same as that of the test meal. Only the test breakfast contained RS during these 24 h tests, all other meals were composed of standard, commercially available products.In three of the test meals , the bread product in the test meal was spiked with 50 μCi [1-All glucose, FFA, and TAG assays were conducted by the GCRC Core Laboratory using an automated Cobas Mira Plus . Serum insulin measurements were also performed by the GCRC Core Laboratory using a human insulin RIA kit .Fat samples, frozen in liquid nitrogen and stored at -80°C until processing, were incubated in 450 μl Solvable  at 50°C for 12 h before the addition of 100 μl 30% (v/v) hydrogen peroxide (for sample bleaching). Fat samples were counted in Aquasol  whereas breath samples were counted in Scintisafe 30%  using a Beckman LS6500 scintillation counter . After scintillant was added, all samples were kept in the dark at room temperature for 48 h before being counted to reduce chemiluminescence.et al. .24h/g tissue weight) - (dpmbaseline/g tissue weight) × 1/specific activityμg fat stored/g fat tissue = 2)/vCO2 (min.ml) = (dpmt - dpmbackground) × 1/vCO2 =counts from sample (dpm/mol CO2/ min.mldpm.mol CO2/ min.ml × 0.446 (as 1 ml CO2 = 0.446 mol)dpm/min = dpm.mol COg fat oxidized = AUC(dpm/min) × 1/specific activity2 is the rate of CO2 production as assessed during indirect calorimetry. t is sample time (min). AUC is the incremental area under the curve.where vCOAll statistical analyses were performed using the statistical analysis software SAS, version 8.1  with a significance level of p = 0.05 and p = 0.01 for interaction terms. All results are presented as mean ± SEM, except for subject characteristics which are described as mean ± SD. To investigate each of the outcomes  we used a mixed model with fixed effect terms for RS DOSE, TIME and the interaction of the two, RS DOSE*TIME. Subjects were included as random effects. The interaction term was not significant for any of the outcomes tested so an additive model was used to test the overall effect of RS DOSE and the differences between doses. To test the effects of RS DOSE at different TIMES, a model that included RS DOSE, TIME and RS DOSE*TIME was used. The repeated measures nature of the study design was taken into account by using the covariance structures available in SAS PROC MIXED. For example, measurements within a subject are assumed to be more highly correlated than between subjects, and within a particular treatment, within a subject, the measurements are assumed to be more correlated. Measurements closer in time to one another were modeled with an autoregressive, or AR(1) covariance structure.RS, resistant starch; DS, digestible starch; TAG, triacylglycerol; FFA, free fatty acid; FFM, fat free mass; SCFA, short-chain fatty acids; GCRC, General Clinical Research Center; RQ, respiratory quotientJanine Higgins and Ian Brown are listed as inventors on RS patents filed by Penford Australia Limited. Both Drs. Higgins and Brown are listed as inventors on these patents as they have intellectual property ownership of some of data used in these but receive no financial benefit.JH conceived of the study design and was responsible for overall study coordination, conducting patient visits, data analysis, and manuscript preparation. DH was responsible for patient scheduling, day-to-day study coordination, conducting patient visits, and data entry. WD contributed to the study design and manuscript preparation, and conducted patient physical examinations and fat biopsies. IB contributed to the study design, selection of RS foods, and assisted with manuscript preparation. MB conducted all statistical analysis. DB contributed to the study design and manuscript preparation, and conducted patient visits, patient physical examinations and fat biopsies. All authors read and approved the final manuscript.Individual meal (a) and total fat oxidation (b) in response to the RS content of a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults.Click here for fileIndividual area under the glucose curve vs. meal (a) and total fat oxidation (b) in response to a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults. Data from all three test meals  is shown. The relationship between area under the glucose curve and fat oxidation remains the same (i.e. no relationship) when represented as individual doses or, as in this plot, for all doses .Click here for fileIndividual area under the glucose curve vs. meal fat oxidation in response to a 0% (a) or 5.4% (b) RS test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in healthy adults. Data from individual test meals is shown.Click here for fileIndividual area under the insulin curve vs. meal (a) and total fat oxidation (b) in response to a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults. Data from all three test meals  is shown. Click here for file


Familial hypercholesterolemia is a human monogenic disease caused by population-specific mutations in the low density lipoprotein (LDL) receptor gene. Despite thirteen different mutations of the LDL receptor gene were reported from Russia prior to 2003, the whole spectrum of disease-causing gene alterations in this country is poorly known and requires further investigation provided by the current study.Forty-five patients with clinical diagnosis of FH were tested for the apolipoprotein B (apoB) mutation R3500Q by restriction fragment length analysis. After exclusion of R3500Q mutation high-sensitive fluorescent single-strand conformation polymorphism (SSCP) analysis and automatic DNA sequencing were used to search for mutations in the LDL receptor gene.We found twenty one rare sequence variations of the LDL receptor gene. Nineteen were probably pathogenic mutations, and two  were considered as neutral ones. Among the mutations likely to be pathogenic, eight were novel , and eleven  have already been described in other populations. None of the patients had the R3500Q mutation in the apoB gene.Nineteen pathogenic mutations in the LDL receptor gene in 23 probands were identified. Two mutations c.925-931del7 and L380H are shared by St.-Petersburg population with neighbouring Finland and several other mutations with Norway, Sweden or Denmark, i.e. countries from the Baltic Sea region. Only four mutations  were recurrent as all those were found in two unrelated families. By this study the number of known mutations in the LDL receptor gene in St.-Petersburg area was increased nearly threefold. Analysis of all 34 low density lipoprotein receptor gene mutations found in St.-Petersburg argues against strong founder effect in Russian familial hypercholesterolemia. Familial hypercholesterolemia (FH) OMIM #143890) is one of the most common monogenic human diseases. It is inherited as an autosomal dominant trait with the prevalence of the heterozygous form conventionally considered to be about 1 of 500 in most populations 43890 is . FurtherThe spectrum of LDL receptor mutations varies between different human populations and more than 900 mutations in the LDL receptor gene have been characterized worldwide -9. A recPatients with FH were recruited from two lipid clinics of St.-Petersburg, namely Institute of Human Brain and Institute of Experimental Medicine. The clinical diagnosis of familial hypercholesterolemia was based on the following criteria: highly elevated plasma total cholesterol and LDL-cholesterol, presence of tendon xanthomata, corneal arcus or both, and positive family history of myocardial infarction and hypercholesterolemia with at least one first-degree relative affected. Forty-five probands fulfilling at least two of three criteria listed above were selected for the study. Full data of patients including their lipid data are given in the Discussion section  in exon 2 that allows a simple detection of this mutation. Restriction enzyme test was performed for the proband and her 4 descendants  that was identified by means of SSCP and verified by sequencing in probands from two unrelated families. SSCP patterns on silver-stained gels differ strikingly in patients with and without the mutation. The presence of the mutation was confirmed in the son of the proband by SSCP.In heterozygotes a deletion of 41 nucleotides at c.1291-1331 in exon 9 (mutation FsV409:S423X) results in occurrence of specific heteroduplexes. Besides, a PCR fragment of smaller size as compared to the normal allele is revealed in silver-stained polyacrylamide gels  were found to be recurrent, i.e. all of those were found in two apparently unrelated families. Together with the data obtained earlier ,38 our rThe author(s) declare that they have no competing interests.FMZ, MYM, VIG, PHN, GGN, AS performed cloning, SSCP, sequencing and familial analysis, BML, VOK, DD, ADD selected patients, VBV and OF participated in the study design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Sign-language comprehension activates the auditory cortex in deaf subjects. It is not known whether this functional plasticity in the temporal cortex is age dependent. We conducted functional magnetic-resonance imaging in six deaf signers who lost their hearing before the age of 2 years, five deaf signers who were >5 years of age at the time of hearing loss and six signers with normal hearing. The task was sentence comprehension in Japanese sign language.The sign-comprehension tasks activated the planum temporale of both early- and late-deaf subjects, but not that of hearing signers. In early-deaf subjects, the middle superior temporal sulcus was more prominently activated than in late-deaf subjects.As the middle superior temporal sulcus is known to respond selectively to human voices, our findings suggest that this subregion of the auditory-association cortex, when deprived of its proper input, might make a functional shift from human voice processing to visual processing in an age-dependent manner. Sign languages involve the use of the hands and face, and are perceived visually -3. Usinget al.  observed [et al.  found thPerformance on the JSL comprehension task was similar across the groups F2, 14) = 1.279, P = 0.309, one-way ANOVA). The patterns of activity evoked during the sign-comprehension task in the hearing signers and the deaf groups are shown in Figure 4 = 1.2794 = 1.279The onset of deafness is related to language acquisition. Prelingual deafness occurs before spoken language is learned. Hearing people generally learn their first language before 5 years of age; hence, prelingual deaf individuals are either deaf at birth or became deaf prior to developing the grammatical basis of their native language, which is usually before the age of 5 years. Postlingual deafness is the loss of acoustic senses, either suddenly due to an accident or as a gradual progression after native-language acquisition . Hence, The posterior STS was activated in all groups during sign comprehension, which is consistent with the proposed neural substrates that subserve human movement perception . The posBoth the early- and late-deaf groups showed activation in the planum temporale, whereas hearing signers did not. Anatomically, the anterior border of the PT is the sulcus behind Heschl's gyrus and the medial border is the point where the PT fades into the insula. The posterior border of the PT involves the ascending and descending rami of the Sylvian fissure . Functioet al. [It has been shown that non-linguistic visual stimuli (moving stimuli) activate the auditory cortex in deaf individuals, but not in hearing subjects ,16. McSwet al.  showed tet al.  revealedet al. ,20 that et al.  indicateet al. .The middle STS, anterior to the Vpc line, was activated more prominently in the early- than the late-deaf subjects. This difference is probably not related to linguistic processes, as both early- and late-deaf subjects are equally capable of learning JSL with the same amount of training. The middle STS region is presumably the area that is selective to human voice processing . This arper se but rather to vocal features that carry nonlinguistic information [Considering that the STS voice-selective area is not sensitive to speech ormation , the funThe results of the present study suggest that in early-deaf subjects, non-auditory processing, such as that involved in the perception and comprehension of sign language, involves the under-utilized area of the cortex that is thought to be selective to the human voice (middle STS). This indicates that the sensitive period for the establishment of human voice processing in the STS might be during the first 2 years of life.The subjects comprised six early-deaf signers (mean age: 22.8 ± 3.1 years), five late-deaf signers (mean age: 34.4 ± 16.2 years) and six hearing signers  and a standard birdcage head coil. Each volume consisted of 11 slices, with a slice thickness of 8 mm and a 1-mm gap, which covered the entire cerebral cortex. The time interval between two successive acquisitions of the same image was 3,000 ms, the echo time was 50 ms and the flip angle was 90 degrees. The field of view was 22 cm. The digital in-plane resolution was 64 × 64 pixels. For anatomical reference, T1-weighted images were also obtained for each subject.x, y and z axes.The first three volumes of each fMRI session were discarded because of unstable magnetization. The remaining 40 volumes per session were used for statistical parametric mapping  implemented in Matlab  ,27. Follt statistic (SPM{t}).Statistical analysis was conducted at two levels. First, the individual task-related activation was evaluated. Second, the summary data for each individual were incorporated into the second-level analysis using a random-effects model to make inferences at a population level. The signal was proportionally scaled by setting the whole-brain mean value to 100 arbitrary units. The signal time course for each subject was modeled using a box-car function convolved with a hemodynamic-response function and temporally high-pass filtered. Session effects were also included in the model. The explanatory variables were centered at zero. To test hypotheses about regionally-specific condition effects , estimates for each model parameter were compared using the linear contrasts. The resulting set of voxel values for each contrast constituted a statistical parametric map (SPM) of the a priori hypothesis that there would be more prominent activation in the early- than late-deaf subjects, we focused on the temporal cortex, which was anatomically defined in standard stereotaxic space [t} was set at P < .001 without a correction for multiple comparisons.The weighted sum of the parameter estimates in the individual analyses constituted 'contrast' images that were used for the group analysis. Contrast images obtained via individual analyses represent the normalized task-related increment of the MR signal of each subject. To examine group differences  in activation due to the sign-comprehension task, a random-effect model was performed with the contrast images (1 per subject) for every voxel. Using the ic space . The thrNS carried out the fMRI studies, data analysis and drafted the manuscript. HY and TO conducted the MR imaging. MY, TH and KM prepared the task materials. YY and HI participated in the task design and coordination. All authors read and approved the final manuscript.


Falls are common among community-dwelling elderly people and can have a considerable impact on quality of life and healthcare costs. People who have sustained a fall are at greater risk of falling again.We replicated a British randomised controlled trial which demonstrated the effectiveness of a multidisciplinary intervention programme to prevent falls.The objective is to describe the design of a replication study evaluating a multidisciplinary intervention programme on recurrent falls and functional decline among elderly persons at risk. The study consists of an effect evaluation, an economic evaluation and a process evaluation.The programme is aimed at community-dwelling elderly people aged 65 years or over who have visited an accident and emergency department (A&E department) or a general practitioners' cooperative (GP cooperative) because of a fall.The design involves a two-group randomised controlled trial. Participants are followed for twelve months after baseline. The intervention programme consists of a detailed medical and occupational therapy assessment with referral to relevant services if indicated. People in the control group receive usual care.The main outcome measures of the effect evaluation are number of falls and daily functioning. The economic evaluation will be performed from a societal perspective. A process evaluation will be carried out to evaluate the feasibility of the intervention programme. This article describes the design of a replication of a randomised controlled trial (RCT) evaluating the effectiveness and cost-effectiveness of a multidisciplinary intervention programme to prevent further falls among elderly people at risk.Publishing the design and protocol of a study before results are available is important for several reasons. A published protocol allows easier comparison between what was originally intended and hypothesised and what was actually done , and it About one-third of people over the age of 65 fall at least once a year . People Since preventing falls has been a matter of interest for years, many programmes aimed at preventing falls have been developed and evaluated. Unfortunately, many of these have turned out to be ineffective . HoweverBecause details of the status of the participants, the context of the intervention and the content and presentation appear to be critical, it has been recommended to re-evaluate effective intervention programmes in different healthcare systems . We therThe main objective of our current study is to evaluate the effects of a multidisciplinary intervention programme on recurrent falls and functional decline among elderly persons who have visit a general practitioners' cooperative (GP cooperative) and/or an accident and emergency department (A&E department) because of a fall. This objective has resulted in the following research questions.• Is a multidisciplinary intervention programme more effective than usual care in preventing new falls and functional decline among community-dwelling elderly people who visit a GP cooperative and/or A&E department at a hospital because of a fall?• Is the multidisciplinary intervention programme cost-effective compared to usual care when assessed from a societal perspective?Besides the effect evaluation and economic evaluation, a process evaluation is being carried out to assess the feasibility and applicability of the intervention programme for those receiving and implementing the intervention.Figure We have taken various measures to ensure blinding in the data collection process. Questionnaires are collected anonymously and sorted by number. Follow-up measurements by phone are contracted out to an independent call centre, whose operators are unaware whether the subjects have been allocated to the intervention or the control group.The study design and protocols were approved by the Medical Ethics Committee of the University Hospital and University of Maastricht.Various studies have been conducted to assess the effectiveness of programmes to prevent falls. Although most studies were aimed at the general population of elderly people, details of the status of the participants appear to be critical ,6. SeverRecruitment of subjects took place at the local GP cooperative and the A&E department of the University Hospital in Maastricht. The Maastricht GP cooperative is a group of GPs from practices in the town of Maastricht and the surrounding area who have founded a non-profit organisation to provide care for their own patients after hours . The MaaThe following inclusion criteria were used: age 65 years or older, community-dwelling, having visited the A&E department or GP cooperative at the University Hospital Maastricht for the consequences of a fall, and living in Maastricht or its surrounding area. People were only allowed to enter the programme after completing and returning an informed consent form. Exclusion criteria were: not able to speak or understand Dutch, not able to complete questionnaires or interviews by telephone, cognitive impairment  ,13, longSample size calculations were based on the expected effects of the intervention on the main outcome measure, the percentage of people sustaining a fall during one year of follow-up. The study by Close et al.  found thTo adapt the programme developed by Close et al.  to the DThe final programme includes a medical and occupational therapy assessment resulting in recommendations. The medical assessment consists of an examination performed by a geriatrician, a geriatric nurse and a rehabilitation physician to identify and address risk factors for falling. The examination includes a comprehensive general examination, but in addition focussed on a more detailed assessment of visual acuity, stereoscopic vision, mobility, balance, cognition, affect, use of medication and examination of feet and footwear. Recommendations or indications for referral resulting from this examination are sent to the patient's GP. After the medical assessments, an occupational therapist visits the patients to identify possible risk factors for falling in the home environment. The therapist makes recommendations regarding adaptations to the home environment, assistive devices, home care and behavioural change. Recommendations by the occupational therapist are sent directly to the subjects themselves and to their GPs. As stated before, the intervention period is scheduled to last for a maximum of 3.5 months after the baseline measurement.An important addition to Close et al.'s protocol  is the cPeople in the control group receive usual care. At present, no guidelines exist for the systematic assessment of the underlying causes of an injurious fall presented at an A&E department or GP cooperative in the Netherlands. In usual care, medical risks and other risk factors such as environmental hazards in the home and patients' risk behaviour are not systematically registered and addressed by hospital physicians, specialists or general practitioners. Moreover, no systematic attention is currently being paid to the specific consequences of an injurious fall for the daily functioning of individual patients in their unique situation. We placed no restrictions on co-interventions.The primary outcome measures of the effect evaluation are number of falls and daily functioning. Number of falls is subdivided into three separate measures: the percentage of elderly people sustaining a fall during the one-year follow-up period, recurrent falls during follow-up , and injurious falls during follow-up . Falls are recorded continuously by means of a fall calendar during the twelve-month follow-up period. Subjects are called monthly to report their falls as recorded on the fall calendar relating to the previous month.We decided to measure daily functioning by means of the Frenchai Activity Index (FAI) , in contOur secondary outcome measures are: recuperation from the fall, health complaints, perceived health measured by means of the first two items of the RAND-36 , ADL andBesides the primary and secondary outcome measures, we assess some background variables which are considered to be predictors, confounders or effect modifiers. The following personal characteristics are assessed: age, sex, marital status, living alone and socio-economic status. In addition, we assess the circumstances and causes of the falls reported at the GP cooperative and/or A&E department, the consequences of the falls , the tyThe economic evaluation in our study is being performed from a societal perspective, which implies that all costs and outcomes are taken into account if possible. The economic evaluation will be a combination of a cost-effectiveness and a cost-utility analysis. The primary outcome measure for the cost-effectiveness analysis will be the percentage of people sustaining a fall during one year of follow-up. As mentioned above, falls are recorded by means of a calendar. Within the cost-utility analysis, the effects are measured in terms of generic health-related quality of life descriptions, measured according to the standard Dutch version of the EuroQol (EQ 5-D)  in self-We will assess programme costs, healthcare costs and patient and family costs. All costs are measured by means of a cost diary , in whicThe process evaluation involves assessing the extent to which the intervention programme is performed according to protocol, the nature of the recommendations made to the participants, participants' compliance with these recommendations and the opinions of participants, physicians and therapists about the intervention programme and recommendations. Data on these topics are collected using the following methods: structured registration forms for the medical and occupational parts of the intervention programme; self-administered evaluation forms filled in by the participants after the medical intervention; interviews by telephone with the participants six weeks or longer after the recommendations are sent and interviews with all participating physicians and therapists at the end of the intervention period.Data will be primarily analysed according to the intention-to-treat principle, i.e., including all participants with valid data, regardless of whether they received or did not receive the intervention. Subsequently, the results of the intention-to-treat analysis will be compared with the results of an on-treatment analysis, to assess whether protocol deviations have caused bias. Participants with documented deviations from the study protocol  will be excluded from this on-treatment analysis.Comparability between the intervention and control groups will be assessed at baseline to check for differences between the two groups. Outcome at four and twelve months will be compared between the intervention and control groups by both univariate and multivariate techniques. We will use multivariate analysis to adjust for possible differences in baseline scores and background variables between the intervention and control groups. Dropouts and losses-to-follow up will be described.The economic evaluation will involve calculating cost-effectiveness and cost-utility ratios. The additional costs and additional benefits of the intervention programme compared with usual care will be examined by calculating incremental cost-effectiveness and cost-utility ratios. These incremental ratios represent the difference in mean costs between the intervention and usual care groups in the numerator and the difference in mean effects in the denominator.Since the recruitment period is only 14 months, and the follow-up period is also relatively short (12 months), it is unlikely that there will be substantial differences between costs made by and for patients who started in the first part of the recruitment period and those who started in the last part. Therefore, discounting of costs is not required. Finally, a sensitivity analysis will be done to assess the generalisability of the assumptions made in the costing process. This sensitivity analysis, which involves calculating the upper and lower limits of the confidence interval of cost and effect variables, will allow us to explore and quantify the uncertainty not related to sampling variations.The process evaluation will mainly be analysed by means of descriptive techniques.Recruitment of eligible subjects commenced in December 2002 and ended in February 2004, resulting in a total of 333 eligible subjects being included in the trial. Randomisation resulted in the allocation of 166 participants to the intervention group and 167 to the control group. Of the 333 persons recruited, 105 (32 %) are male and 228 (68 %) are female.The follow-up period is to end in May 2005. Results on the effects of the programme will be available in 2006 and will be published in the relevant journals.Although the intervention has been subject of earlier research, this study will provide new information about the effectiveness in the Dutch situation. Furthermore, the results of the economic evaluation can provide information about the cost-effectiveness of the intervention and the effects on quality of life. In case of shown effectiveness and cost-effectiveness, we aim to implement this intervention into usual healthcare.The author(s) declare that they have no competing interests.All author's read and approved the final version of the manuscript.MH is the investigator and performed most of the writing of this manuscript.JH supervises the planning of the project and wrote the study design.JD supervises the planning and methodological aspects of the project.SA supervises the economic evaluation.HC supervises the clinical part of the project.JE is the principal investigator of the study.The pre-publication history for this paper can be accessed here: