The consistent finding of higher prevalence of hypertension in US blacks compared to whites has led to speculation that African-origin populations are particularly susceptible to this condition. Large surveys now provide new information on this issue.Using a standardized analysis strategy we examined prevalence estimates for 8 white and 3 black populations .The range in hypertension prevalence was from 27 to 55% for whites and 14 to 44% for blacks.These data demonstrate that not only is there a wide variation in hypertension prevalence among both racial groups, the rates among blacks are not unusually high when viewed internationally. These data suggest that the impact of environmental factors among both populations may have been under-appreciated. Population surveys in the US from early in the last century have consistently documented higher blood pressures and related cardiovascular sequelae in blacks compared to whites ,2. The eInternational comparative studies on hypertension have been seriously limited by the absence of a valid method of standardization. In the last decade, however, high quality population surveys have been conducted in a wide range of populations that used either careful internal standardization or sufficiently comparable methods -15. We rBlack populations were drawn from the International Collaborative Study on Hypertension (ICSHIB) and the National Health and Nutrition Survey III ,16. A prThe surveys that formed the basis of ICSHIB were conducted in localized communities by door-to-door screening ,7. In sund BP from the clinic visit was used to create the mean for the age-gender groups, except for England where the 2nd home BP was used. Hypertension was defined as BP ≥ 140/90 mmHg or current use of antihypertensive medication.The examination methods have been reported in detail previously ,7,16. InBP, body mass index (BMI), and hypertension prevalence were calculated for 5-year age-gender groups and aggregated as the primary data file. To achieve maximum overlap we restricted the analysis to 35–74 years for age-specific estimates of BP and hypertension prevalence, and 35–64 years for age-adjusted results. In the US NHANES whites and blacks were analyzed separately with the appropriate weighting for population size. As previously reported, the prevalence estimates obtained for US blacks from ICSHIB were virtually identical to those from NHANES [The age-averaged BPs and BMIs are presented for each survey, by gender, in Table Hypertension prevalence, which accounts for the effect of treatment, follows a similar pattern although the east-west gradient among the African-origin groups is more consistent Table . Among tThe only etiological factor on which standardized information was available was obesity, measured by its proxy BMI. The correlation between average BMI and hypertension prevalence was 0.6 (p < 0.01), all populations combined. Within the black populations the same correlation was observed between mean BMIs and hypertension prevalence (r = 0.6). Among whites, however, the relationship was weaker (r = 0.3). Of course, since obesity will be correlated with many other aspects of lifestyle, it is difficult to infer whether weight gain itself is playing a less important role in determining the variation among white populations. The contrasts noted above in hypertension prevalence by gender are consistent with the relative excess of obesity in women compared to men among Jamaicans and US blacks ,6.Comparisons of BP distributions across populations are made difficult by the requirement of comparability of the survey methods. In the last two decades, however, adoption of standardized protocols along with rigorous training have greatly improved the quality of epidemiological studies of hypertension ,17-19. AThe data presented here demonstrate a two-fold variation in prevalence of hypertension in both European- and African-origin populations. The prevalences are similar in blacks in the US and whites in Europe, although important gender differences are apparent. Although not a systematic sample, the populations that are included generally reflect the characteristic social setting in which these groups are found around the world. Summed across all groups, the white populations on average have a substantially higher burden of hypertension. This result can be attributed in large part to the inclusion of several black samples from developing countries where risk factors for hypertension are presently at a lower level. In the only head-to-head comparison within the same survey, US blacks have a prevalence that is 50% higher than among whites. Data from the UK, including the national survey, also demonstrate higher BPs and more hypertension among blacks of Caribbean and African descent -27. On tAre these findings merely artifactual, reflecting either methodological error or the sampling process? The most unexpected features of the data presented here are the high rates of hypertension in Europe, when contrasted to whites in Canada and the US. These results have been reported in greater detail in an earlier publication . It is bThe primary purpose of this analysis was to provide descriptive results and very limited information was available on factors that might explain the findings we observed. The gradient among the black populations is consistent with the transition to an industrialized lifestyle and is thereby collinear with most known risk factors . BMI is Treatment guidelines and practice patterns vary widely among these countries -19. WideIf the North American-European contrasts are occurring in genetically homogeneous populations, large environmental influences must be at work that are not apparent on the surface. A similar process could be taking place across the social environments into which persons of African origin are assorted within societies such as the US and the UK. The debate over inherent susceptibility cannot be resolved with these data since neither the genetic nor the environmental influences can be held constant, allowing a test of the relative influence of the other factor. In fact, the question of inherent susceptibility is probably non-testable under any circumstances -37. WhilThe author(s) declare that they have no competing interests.RC, KWM, AL and JB were responsible for study concept, design and supervision. RC, KWM, JB, SG, MJ, AA, TF MK, PP, BS and MT were involved in data acquisition. RC, KWM, JB, MJ, PP and AL were responsible for analysis and interpretation of data. RC and KWM drafted the manuscript. RC, JB, SG, AL, AA, TF, MJ, MK, PP, BS and MT were involved in critical revision of the manuscript for important intellectual content. Statistical expertise was provided by MJ. Administrative, technical and material support was provided by KWM, MK, and BS.The pre-publication history for this paper can be accessed here:


Analyses of genetic data at the level of haplotypes provide increased accuracy and power to infer genotype-phenotype correlations and evolutionary history of a locus. However, empirical determination of haplotypes is expensive and laborious. Therefore, several methods of inferring haplotypes from unphased genotypic data have been proposed, but it is unclear how accurate each of the methods is or which methods are superior. The accuracy of some of the leading methods of computational haplotype inference  are compared using a large set of 308 empirically determined haplotypes based on 15 SNPs, among which 36 haplotypes were observed to occur. This study presents several advantages over many previous comparisons of haplotype inference methods: a large number of subjects are included, the number of known haplotypes is much smaller than the number of chromosomes surveyed, a range in values of linkage disequilibrium, presence of rare SNP alleles, and considerable dispersion in the frequencies of haplotypes.In contrast to some previous comparisons of haplotype inference methods, there was very little difference in the accuracy of the various methods in terms of either assignment of haplotypes to individuals or estimation of haplotype frequencies. Although none of the methods inferred all of the known haplotypes, the assignment of haplotypes to subjects was about 90% correct for individuals heterozygous for up to three SNPs and was about 80% correct for up to five heterozygous sites. All of the methods identified every haplotype with a frequency above 1%, and none assigned a frequency above 1% to an incorrect haplotype.All of the methods of haplotype inference have high accuracy and one can have confidence in inferences made by any one of the methods. The ability to identify even rare (≥ 1%) haplotypes is reassuring for efforts to identify haplotypes that contribute to disease in a significant proportion of a population. Assignment of haplotypes is relatively accurate among subjects heterozygous for up to 5 sites, and this might be the largest number of SNPs for which one should define haplotype blocks or have confidence in haplotype assignments. Very rapid and inexpensive methods exist for determining the genotype of diploid organisms at single nucleotide polymorphisms (SNPs). Unfortunately, these high-throughput methods do not provide direct information on which SNP alleles at multiple sites coexist on the same chromosome. Instead, computational methods must be employed to infer the set of SNP alleles that are cosegregating on a single chromosome, referred to as haplotypes. However, the inference of haplotypes from phase-unknown data is computationally difficult, partly due to the fact that the number of possible haplotypes roughly increases as a power of 2 with each additional SNP.Interest in the accurate inference of haplotype structure from unphased genotypic data has increased tremendously in recent years for several reasons. Relative to analysis of single polymorphisms, haplotypes can greatly improve one's ability to infer the evolutionary history of a DNA region ,2. AdditThere are three principal computational approaches to inferring haplotypes from unphased SNP data. The most commonly used approach is implementation of the expectation-maximization (EM) algorithm . This meThe accuracy of computational haplotype inference improves as the magnitude of linkage disequilibrium (LD) among sites increases . Gene coThe 15 sites studied by Horan et al.  span 535Increasing sample size improves the accuracy of inferred haplotypes. In Horan's study  308 chroThe distribution of haplotype frequencies also influences the accuracy of haplotype inference in two ways. First, the presence of some haplotypes at comparatively high frequency increases the chances that those haplotypes can be unambiguously inferred from homozygotes, allowing the alternative haplotype to be inferred with high confidence in compound heterozygotes. Second, the presence of some haplotypes at near-zero frequency allows truly nonexistent haplotypes to be accurately estimated as having zero frequency. The empirical haplotype frequencies in this study exhibit considerable dispersion. Two haplotypes are relatively common . The latter analyses were performed to better approximate the characteristics of data sets that are typically collected in genetic epidemiological studies. Although the presence of rare alleles and haplotypes improves the accuracy of haplotype inference, sites with a low frequency minor allele are often ignored due to their reduced usefulness in mapping disease loci and the assumption that such loci will contribute little to population-wide predisposition to disease. Very little difference was observed in the accuracy of haplotype inference between the two data sets.Assignment of haplotypes to individuals was very accurate by all methods Table . ApproxiF. As measured by the mean squared error (MSE) the implementation of the program Phase that ignored linkage disequilibrium among sites gave marginally lower accuracy for the full data set and for the data set composed of higher frequency alleles, but the magnitude of the MSE was small for all methods and spanned only about a two-fold difference between the best and worst value. PL-EM successfully identified the largest number of correct haplotypes, but this success rate was accompanied by the burden of the highest number of incorrect haplotypes inferred. Indeed, the aggregate frequency of incorrect haplotypes inferred by PL-EM was about 1% higher than for the other methods. This observation may have practical value for the analysis of unphased genotypic data. PL-EM may be slightly advantageous if the analytical goal of identify the largest number of correct haplotypes is much more important than minimization of the number of incorrect haplotypes inferred, which may be the case in studies of functional genetics. However, if minimization of the number of incorrect low-frequency haplotypes is more important, as will usually be the case in genetic epidemiological studies, PL-EM may not be the optimal method. Unfortunately, none of the methods is clearly superior in minimizing the number of incorrect haplotypes inferred.Estimation of haplotype frequencies was also highly accurate, and there was no meaningful difference in accuracy among the methods as measured by the similarity index, IImportantly, none of the methods failed to identify haplotypes with frequencies above 1%. Conversely, no incorrect haplotype was assigned a frequency greater than 1%. Indeed, the aggregate frequency of incorrect haplotypes was ≤ 3.7% by all methods. These results are reassuring in two respects. First, it appears unlikely that any of the methods will fail to identify a haplotype that is a major contributor to disease risk within a study population. Second, it also is unlikely that an incorrect haplotype will be implicated as a significant disease risk.It has been noted previously ,18 that k, where k is the number of heterozygous sites. For this reason, the difficulty of correctly assigning haplotypes to subjects increases dramatically as those subjects become heterozygous at more sites. Therefore, the error rate for assigning haplotypes was evaluated based on the number of sites at which subjects were heterozygous. Up to 3 heterozygous sites, the error rate is below 10%. For 4 heterozygous sites the error rate is about 15% and exceeds 20% only when 6 sites are heterozygous. Phase was an odd exception to this pattern due to an unusually high error rate for four heterozygous sites, despite the lowest error rates for five and six heterozygous sites. On the assumption that an error rate not much larger than 10% is desirable for a genetic study, it appears that computationally assigned haplotypes for subjects heterozygous at more than four SNPs should be viewed with extreme caution. Similarly, there is a current effort to define haplotype blocks in the human genome to facilitate genome-wide scans for disease loci with a minimum number of sites that must be genotyped. If the results for this gene can be generalized, it would appear unwise to define haplotype blocks based on more than 4–5 SNPs.The number of possible haplotypes compatible with an individual's unphased genotype is 2All of the implementations of the EM and Bayesian methods of haplotype inference had high accuracy. Therefore, if this data set is representative of other SNP genotyping studies one can have high confidence in the assignment of haplotypes and estimation of haplotype frequencies produced by any one of the programs. Each method identified every haplotype with a frequency greater than 1%. Therefore, it is unlikely that any of the methods would fail to identify a haplotype contributing to disease risk in a significant proportion of a population. Conversely, no incorrect haplotype was assigned a frequency greater than 1%, indicating a low probability of an incorrect haplotype being identified as a significant disease risk factor. Assignment of haplotypes was very accurate for subjects heterozygous for up to three SNPs, and was at least 80% accurate for up to five heterozygous sites. This suggests that haplotype blocks should perhaps be defined based on no more than five sites and that this might be the practical limit at which one can have confidence in the assignment of haplotypes to subjects.The empirically determined set of haplotypes from Horan et al.  were kinThe full data set of Horan et al.  includesPairwise D' , the linThe accuracy of haplotype inference was examined by several metrics. The mean squared error (MSE)  is definpek and ptk are the inferred and empirically determined frequencies for the kth haplotype, and h is the number of haplotypes. IF and IH were proposed by Excoffier and Slatkin [IF is another measure of how closely the inferred and empirical haplotype frequencies correspond and is given bywhere  Slatkin . IF is aIF ranges from 0 to a maximum value of 1 when the frequencies match perfectly. IH compares the number of haplotypes inferred to the number actually known to occur and ranges from 0 to 1 (complete correspondence between inferred and true). IH is defined aswhere the variables are defined as above. mtrue is the number of haplotypes known to occur, mest is the number of inferred haplotypes with frequency ≥ 1/(2n), and mmissed is the number of known haplotypes that were not inferred. The error rate [where ror rate  is the p


Microglia make up the innate immune system of the central nervous system and are key cellular mediators of neuroinflammatory processes. Their role in central nervous system diseases, including infections, is discussed in terms of a participation in both acute and chronic neuroinflammatory responses. Specific reference is made also to their involvement in Alzheimer's disease where microglial cell activation is thought to be critically important in the neurodegenerative process. A role for immune responses, involving antigen presentation and immune-response-generating cytokines, in neurodegenerative diseases such as Alzheimer's disease was recognized for a decade before the term neuroinflammation came into widespread use ,2. A PubMany researchers now consider this innate immune response in the brain to be a potentially pathogenic factor in a number of CNS diseases that lack the prominent leukocytic infiltrates of adaptive immune responses, but that do have activated microglia and astrocytes, i.e., neuroinflammation.The idea that neuroinflammation is detrimental implies that glial cell activation precedes and causes neuronal degeneration , a sequeIn the following, we provide some basic definitions and discussion to more precisely define the idea of neuroinflammation as a CNS tissue response to injury, and the notion of neuroinflammation as a pathogenic factor in neurodegenerative diseases.Inflammation is a reaction of living tissues to injury . The disBefore "neuroinflammation" became a commonly used term, neuroscientists spoke of "reactive gliosis" in describing endogenous CNS tissue responses to injury. Reactive gliosis specifically referred to the accumulation of enlarged glial cells, notably microglia and astrocytes, appearing immediately after CNS injury has occurred. In contrast to glial reactivity, which suggests a largely passive response to injury; glial activation implies a more aggressive role in responding to activating stimuli: activated glial cells release factors that act on and engender responses in target cells analogous to the responses of activated immune cells in the periphery. Activation of immune cells in the periphery leads to leukocyte infiltration of tissues, but this is notably absent in the brain unless there has been destruction or compromise of the blood brain barrier ,8. In thIn limited, acute reactions to injury, in the absence of blood-brain barrier breakdown, there is the subtler response of the brain's own immune system, composed largely of rapid activation of glial cells. These responses represent the other end of the spectrum of CNS injury, where limited neuronal insults trigger glial cell activation without breakdown of the blood brain barrier and without concomitant leukocytic infiltration. This form of "pure" glial response occurs in neuronal injury caused by either loss of afferents  or loss Although such specific responses might, in a strict sense, be included in the term "neuroinflammation," neuroinflammation as generally used and understood applies to more chronic, sustained cycles of injury and response, in which the cumulative ill effects of immunological microglial and astrocytic activation contribute to and expand the initial neurodestructive effects, thus maintaining and worsening the disease process through their actions.The concept of chronic inflammation (as opposed to acute inflammation) is more relevant in the context of understanding CNS disease (as opposed to CNS injury), as the very term "disease" implies chronicity. Chronic multiple sclerosis is, of course, an unequivocal and long-recognized example of an inflammatory brain disease. Although the underlying cause(s) of multiple sclerosis have not been elucidated, it is probably safe to say that the persistent injurious stimulus that accounts for neuroinflammation in multiple sclerosis is a myelin-related protein that has escaped self-tolerance and become an autoimmunogen. Consistent with the chronic persistence of this autoimmunogen is a persistent accumulation of blood-derived mononuclear leukocytes in the CNS parenchyma, a phenomenon that is similar to what is found in other autoimmune diseases such as rheumatoid arthritis or polymyositis.Infections are another group of diseases that are classically recognized as inflammatory in nature, with meningeal, perivascular, or even parenchymal infiltrates of peripheral leukocytes. There are, however, exceptions. Rabies is a disease in which the peripheral immune response is slow and inadequate, and in which classic inflammatory changes are less striking than those found in other viral encephalidites. Babes, in 1897 , describvia myelomonocytic cells: monocytes, perivascular cells, and microglia [Today, the most important example of a chronic brain infection is human immunodeficiency virus (HIV). Chronic HIV encephalitis is characterized by the same nodules of activated microglia that Babes described in rabies. HIV enters and persists in the CNS icroglia . HIV infPrion diseases represent another chronic infectious CNS disease that is not accompanied by leukocytic infiltrates. Microglial activation, again, appears to be the most prominent inflammatory component of prion diseases ,14, althNeurodegenerative diseases – particularly Alzheimer's disease, but also amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease – lack the prominent infiltrates of blood-derived mononuclear cells that characterize autoimmune diseases. On the other hand, there is abundant evidence that many substances involved in the promotion of inflammatory processes are present in the CNS of patients with such neurodegenerative diseases. By far the bulk of this body of evidence is related to studies in Alzheimer's disease . What diβ-Amyloid precursor protein βAPP functions as a neuronal acute-phase, injury-response protein. For instance, there is excessive expression of βAPP, accompanied by microglial activation and cytokine expression, after traumatic head injury . With heChronic microglial activation is an important component of neurodegenerative diseases, and this chronic neuroinflammatory component likely contributes to neuronal dysfunction, injury, and loss (and hence to disease progression) in these diseases. The recognition of microglia as the brain's intrinsic immune system, and the understanding that chronic activation of this system leads to pathologic sequelae, has led to the modern concept of neuroinflammation. This vision of microglia-driven neuroinflammatory responses, with neuropathological consequences, has extended the older vision of passive glial responses that are inherent in the concept of "reactive gliosis."Aβ: β-amyloid peptideβAPP: Aβ precursor proteinCNS: central nervous systemHIV: human immunodeficiency virusMS: multiple sclerosisNone declaredWJS conceived this review, wrote the initial draft, modified this with the comments of REM and WSTG, and wrote the final draft. REM and WSTG contributed particularly to the sections on infections and on Alzheimer's disease. All authors read and approved the final version.


The development of an HIV vaccine remains one of the most difficult challenges confronting biomedical research today. A new international collaboration shares its plan to address the challenge In June 2003, an international group of scientists proposed the creation of a Global HIV Vaccine Enterprise . The autThe major difficulties encountered in the development of an HIV vaccine are scientific, not organizational.The motivation behind the proposal for a Global HIV/AIDS Vaccine Enterprise was the recognition that development of an HIV vaccine remains one of the most difficult challenges confronting biomedical research today ,3. FortuThe Global HIV/AIDS Vaccine Enterprise represents a novel paradigm to seek and identify international agreement on the critical roadblocks for developing an HIV vaccine and on creating a shared scientific plan that addresses those roadblocks see . The Ent• More new HIV infections and AIDS deaths occurred in 2004 than in any prior year –3. A vac• Development of an HIV vaccine is one of the world's most difficult and important biomedical challenges.• Harnessing new scientific opportunities for HIV vaccine development will require an effort of a magnitude, intensity, and design without precedent in biomedical research.• The Global HIV Vaccine Enterprise is an alliance of independent organizations committed to accelerating the development of a preventive HIV/AIDS vaccine based on a shared scientific plan.• The scientific strategic plan was developed with the collaboration of over 140 scientists and other participants from 17 countries and several international organizations.• The plan identifies critical unanswered scientific questions along the critical path for vaccine discovery, from antigen design to the conduct of clinical trials.• Novel vaccine candidates need to be designed to induce high levels of broadly reactive and persistent immune responses against HIV strains circulating in different parts of the world.• Standardization and validation of high-throughput laboratory assays conducted under GLP will allow comparison of results from different vaccines, which is a linchpin of rational decision making in vaccine development.• The Enterprise will encourage decision makers to establish clear and transparent processes to identify and prioritize the most promising vaccine candidates.• The Enterprise will seek to engage the best researchers who are willing to work in a highly collaborative manner and to dedicate the majority of their efforts to solve the fundamental roadblocks in HIV vaccine development.• To mount an accelerated global search for a safe and effective HIV/AIDS vaccine, annual funding for such research should double—to US$1.2 billion per year.• Several founding partners of the Enterprise have already committed, or are planning to commit, new funding to support the proposed Enterprise activities, and to create a culture of mutual accountability for the effective implementation of the scientific strategic plan.• Enterprise activities are guided by an international Coordinating Committee, supported by different technical expert groups, including representatives from funders and implementers of HIV vaccine R&D.It must be emphasized, however, that the major difficulties encountered in the development of an HIV vaccine are scientific, not organizational, and arise directly from the complexities of HIV and AIDS. “Small science” should not be replaced with “big science.” Both approaches must be undertaken. Creation of research environments that support the creativity both of individual investigators and of larger, collaborative efforts will accelerate the scientific breakthroughs needed to successfully develop a safe and effective HIV vaccine.In August 2003, the authors of the Enterprise proposal invited a group of leading scientists, public health experts, and policy makers to meet at the Airlie House in Warrenton, Virginia, United States, to refine the vision for the Enterprise. The Airlie group agreed that the Global HIV/AIDS Vaccine Enterprise should be developed as an alliance of independent organizations committed to accelerating the development of a preventive vaccine for HIV/AIDS through implementation of a shared scientific strategic plan, mobilization of additional resources, and greater collaboration among HIV vaccine researchers worldwide .The subsequent initial planning phase of the Enterprise involved leading government research agencies, private industry, non-governmental organizations, and funders involved in HIV vaccine research and development (R&D) activities, including the Bill & Melinda Gates Foundation (BMGF), the International AIDS Vaccine Initiative (IAVI), the National Agency for Research on AIDS of France (ANRS), the United States National Institutes of Health (NIH), the United Nations Joint Programme on HIV/AIDS (UNAIDS), the World Health Organization (WHO), and the Wellcome Trust. The Enterprise is expected to grow with time and include additional organizations and research groups willing to contribute to the implementation of its scientific strategic plan. A Steering Committee composed of representatives from several of the founding organizations provided guidance and coordination, with the BMGF serving as interim Secretariat.Six Working Groups involving more than 120 participants from 15 countries, the WHO, and UNAIDS were formed to develop the scientific plan of the Enterprise. These Working Groups met from January to April 2004, identified critical unanswered questions, and proposed actions to address them. In May 2004, the Steering Committee of the Enterprise analyzed the recommendations from the Working Groups and identified the scientific priorities for initial action.Several common themes emerged from the Working Groups. There was clear agreement on the key scientific challenges, as well as strong consensus that the HIV vaccine field has progressed to a point where it should be possible to answer some of the persistent questions more definitively. To meet these challenges, the Working Groups called for enhanced access to reagents and technologies, adequate resources, and strengthened human capacity in several key areas, especially in developing countries, where clinical trials need to be conducted. There was also agreement that the present way of doing business, which centers primarily on individually led research groups or networks, needs to be supplemented by establishing focused, collaborative structures and providing access to common standards and technologies, which would enable comparison of data and candidate vaccines. This would, in turn, support a rational process for decision making to advance candidate vaccines through the different phases of evaluation.One immediate goal is to design HIV candidate vaccines that consistently induce potent, broadly reactive, persistent neutralizing antibodies, as well as memory T cells that suppress viral replication and prevent escape of virus from immune control ,7. AdditThe current state of the art suggests a two-pronged strategy to accelerate the development of a safe and effective HIV vaccine. One component should center on candidate vaccines already in the pipeline, nearly all of which are designed primarily to induce T cell responses. In some animal models these T-cell-inducing candidate vaccines suppress post-infection viremia and prevent or delay HIV disease, rather than prevent infection ,13. In sThe second component should address critical gaps in scientific knowledge through carefully designed, focused, coordinated, and well-supported approaches. The fruits of this work will be a clearer understanding of what properties are needed for a successful vaccine and how to design candidates that incorporate those properties.Scientific areas in which a more collaborative and organized Enterprise approach will be beneficial include the following: vaccine design based on the characteristics of recently transmitted viruses, evaluation of immune correlates of protection in animal models, and design of novel candidates vaccines that induce neutralizing antibodies and T cell immune responses.Identifying which T cell candidate vaccine is most promising has become an urgent priority.Strategically, vaccines that are designed based on recently transmitted viruses hold the best hope of inducing relevant immune responses against currently circulating strains. Recent data suggest that the subset of viral strains that are sexually transmitted has unique genetic and antigenic properties, including greater susceptibility to neutralization than the bulk of circulating virus . While sTo address these issues, a representative number of virus strains derived from recently infected individuals representing those populations who will participate in vaccine efficacy trials, including populations in developing countries, should be obtained. These virus isolates should be subjected to a comprehensive genetic and biologic characterization, together with an analysis of host immune responses and the genetic background of those populations participating in the clinical trials.This continuous and ongoing effort will require a multidisciplinary global approach, linking investigators who are conducting epidemiological and cohort studies , laboratory scientists working on the virology and immunology of acute/early infection and on the genetic characterization of affected human populations, vaccine designers and manufacturers, and clinical trialists. In addition, systems for data management and analysis that will facilitate the rapid translation of new information into improved vaccine designs need to be developed.Nonhuman primate models of AIDS offer opportunities to evaluate potential correlates of immune protection. While a particular immunization strategy that works in animal models may or may not predict protection in humans, important insights into potential immunologic mediators of protection would result from such studies. Several experimental vaccines induce varying degrees of protection against simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus in rhesus macaques. In particular, studies using models in which a very high level of protection from acquisition of infection was achieved are needed, i.e., immunization with live attenuated SIV and attenuation of SIV infection by short-term antiretroviral treatment administered immediately after SIV inoculation ,17.To facilitate this process, assays for many different immune responses to SIV and chimeric simian/human immunodeficiency virus need to be standardized, validated, and made available to different research groups. Likewise, agreements need to be reached on those monkey challenge models that most closely resemble HIV transmission and infection in humans. Large numbers of animals will be needed to achieve statistical significance for experimental findings , which iThere is increasing agreement that a successful vaccine needs to induce both humoral and cell-mediated immunity. Development of immunogens capable of inducing antibodies that neutralize primary HIV isolates from all genetic subtypes and regions of the world remains the most difficult challenge in the field of HIV vaccinology ,20. SuccGiven the importance of these endeavors and the uncertainty as to what path will lead to success, multiple intersecting approaches need to be explored, including, for example, the design, production, and evaluation of (1) envelope proteins that stably reveal neutralization epitopes that may be only transiently exposed during viral entry into target cells, (2) immunogens that contain rigid, stable epitopes that mimic the portion or portions of envelope recognized by broadly neutralizing monoclonal antibodies, (3) modified envelope proteins that better expose existing relevant epitopes, and (4) molecules that resemble a stabilized version of the mature envelope trimer on the virion surface. These are examples of current approaches being explored, some or all of which may prove ineffective. Additional novel ideas need to be proposed and explored.To achieve the above objectives, new tools and technologies such as those able to detect rare, broadly neutralizing monoclonal antibodies through large-scale screening of human sera will have to be developed. In addition, the very limited existing capacity to translate structural information into stable immunogen products needs to be expanded.Nearly all current vaccine candidates in the clinical pipeline are T-cell-inducing vaccines, e.g., poxvirus recombinant vectors, adenoviral vectors, DNA constructs with or without adjuvants, and lipopeptides. The ongoing effort to evaluate these products and to develop new ones is considerable . IdentifThe identification and optimization of promising candidates will require (1) defining clear, transparent processes for decision making, (2) establishing agreement on vaccine characteristics upon which decisions should be based, (3) developing and using validated assays to assess those parameters, to allow for preclinical and clinical comparison among candidates, and (4) establishing closer coordination and data-sharing among product developers, which will accelerate the availability of critical information needed to identify and further develop the most promising candidates.Development of an HIV vaccine remains one of the most difficult challenges confronting biomedical research today.Research is also needed to develop improved novel T-cell-inducing candidate vaccines, especially those that avoid or otherwise circumvent anti-vector immune responses , and thoComparison of results from preclinical and clinical studies is the linchpin of rational decision making regarding further development of vaccine candidates. Therefore, the initiation of approaches that will permit valid comparisons is crucial.Progress to standardize and validate a limited number of T cell assays has been made within the laboratories of vaccine developers and within some partnering research networks. This approach now needs to be more broadly applied and extended to the analysis of neutralizing antibody responses. A robust infrastructure that develops, expands, and ensures broad access to quality assay technologies will allow valid comparison of data across trials and networks worldwide.In order to achieve this goal, the following are required: (1) a decision-making process to select a set of robust assays, standardized and validated across laboratories, for measuring vaccine-induced immune responses in humans and animals; (2) wide availability of common reagents ; (3) capacity for developing novel assays and reagents of potential value and for their translation to preclinical and clinical settings; (4) “core” laboratories that run selected assays and serve as a reference laboratory for satellite laboratories ; (5) satellite laboratories located at or very near clinical trial sites to carry out a range of activities such as processing blood, storing and shipping specimens, and conducting basic immunological evaluation, and to participate in other Enterprise-organized activities such as acute/early infection studies; (6) an ongoing global quality assurance function encompassing all participating core and satellite laboratories and covering both routine safety as well as immunologic and virologic assessments; and (7) transfer of research assays and, when and where feasible, validated endpoint assays to satellite labs, including the necessary training activities.In addition, new assay development has failed to keep pace with current understanding of the biology of the immune system and recent advances in technology. A more active program of applied research and assay development is needed to explore new concepts that would advance technical abilities and provide a better understanding of the immune responses generated by HIV vaccines.Two assays are currently used for the primary evaluation and enumeration of antigen-specific T cells: Interferon-γ ELISPOT and multiparameter flow cytometry. The ELISPOT assay was initially developed to measure CD8+ T cell responses. Several observations in both mice and humans have indicated that protective immune responses will likely require stimulation of both CD4+ and CD8+ T cell effector and memory functions; it is unlikely that induction of Interferon-γ-secreting T cells alone correlates with protective immunity . TherefoDifferent laboratories use different assays to measure antibodies that neutralize HIV and related viruses, SIV and chimeric simian/human immunodeficiency virus. These assays vary technically, but the most widely accepted assays measure reduction in virus infectivity in cells that express the receptors necessary for virus entry. Assays that offer the greatest value are those that are validated, amenable to high throughput, low in cost, readily transferable, and that can be performed according to GLP guidelines.The ability to measure the magnitude and breadth of neutralization against diverse HIV strains is essential to evaluating responses generated by candidate HIV vaccines. Only with multiple strains of virus can neutralization breadth be ascertained in a meaningful way. Standard panels of HIV strains are in early stages of development. Expansion or extension of current standardization and validation activities, production and provision of necessary reagents, and access to quality assurance programs are needed to ensure worldwide comparability of assay results . The strAs more HIV candidate vaccines enter the pipeline, current capacity will be rapidly exhausted.A major obstacle to designing a suitable global virus panel is the paucity of information on neutralization serotypes. There is general agreement that if a reasonably small number of neutralization serotypes exist, their identification would guide the creation of an optimal panel of isolates for neutralizing antibody assays and the design of polyvalent immunogens. Although there is some controversy as to whether HIV-1 neutralization serotypes exist, the magnitude of benefit that would result if serotypes were identified warrants establishment of a neutralization serotype discovery program that employs the latest technologies.Manufacture of vaccine candidates for large clinical trials and to meet eventual worldwide demand requires the development of processes for producing consistent, active vaccine batches on a large scale. Development of these bioprocesses must be integrated with analytical work , incorporate validated assays, and be applicable to the manufacture of sufficient vaccine to meet global needs after licensure. These processes are typically individually developed as a candidate vaccine advances from early clinical testing to late-stage evaluation and licensure. Worldwide expertise and capacity for this bioprocess development work is already limiting and exists almost exclusively in the private sector. As more HIV candidate vaccines enter the pipeline, current capacity will be rapidly exhausted.The initial priority is to identify or establish one or more dedicated HIV vaccine bioprocess and analytical development groups that bring together the skill set and capacity to manufacture different promising candidates for clinical trials. The bioprocess development groups would also help train people and transfer manufacturing skills in whole or in part to manufacturing sites around the world. This training program would address the acute shortage of bioprocess experts.At a later stage, building, acquiring, or contracting facilities to carry out bioprocess and analytical work and to produce several different types of candidate vaccines should be considered. Such facilities would further assist in transferring manufacturing technology to other production facilities, preferably in one or more developing countries. Decisions about which candidates a facility undertakes would be made through a well-defined, comprehensive evaluation process. The facilities could eventually be expanded to provide production capacity to launch a vaccine for public health use, should no manufacturer be available to produce the vaccine quickly upon licensure.As a growing number of HIV candidate vaccines begin to move through the clinical trials pipeline, the gap between existing global capacity and future requirements for conducting large efficacy trials has grown in magnitude and urgency, especially in developing countries. This gap in developing countries must be addressed through (1) increasing the quantity and quality of research staff, (2) establishing sustainable research facilities to support trials, and (3) expanding access to large, well-defined populations of uninfected people at high risk of HIV infection.The acute shortage of qualified personnel is a major bottleneck to the conduct of clinical trials in developing countries.The recommended solutions take a long-term view and are aimed at building site capacity rather than preparing for specific trials. Sites should not be confined to conducting HIV vaccine trials but should be positioned to contribute to other research of public health importance to the community and the country, including, for example, other areas of HIV research  and/or other diseases. Additional field trial sites must be developed to be able to conduct planned and anticipated efficacy trials. Sites should be selected in a strategic, data-driven manner, and should demonstrate the ability to recruit and retain large numbers of HIV-negative volunteers from populations with substantial HIV incidence. New efficacy trial sites should be developed in regions with emerging epidemics rather than only in areas with already-established disease. “Early-warning systems” must be available to identify these newly emerging sub-epidemics. Defining optimal methods for collection of HIV incidence data from populations at potential efficacy trial sites is essential. Whenever possible, efficacy trial sites should be linked to (1) academic medical centers to enhance research capacity and help train clinical researchers, (2) accredited local and regional laboratory facilities to provide infection endpoint and safety assessments, and (3) centers that can provide appropriate care and treatment to trial participants.The acute shortage of qualified personnel is a major bottleneck to the conduct of clinical trials in developing countries with severe or rapidly emerging HIV epidemics. Development of intellectual capacity at these sites should emphasize (1) expanding research training opportunities for personnel in the broad range of topics required to conduct high-quality clinical research, (2) establishing and adequately supporting long-term career paths for such individuals, and (3) fostering political and social environments locally and nationally that support the conduct of clinical research. Building HIV scientific and operational expertise at clinical trial sites should be linked to other HIV/AIDS research activities .Site development must include strategies to develop or enhance existing capacity to deliver health care, including HIV prevention, care, and treatment, to the local community participating in clinical trials. Provision of, or referral to, basic clinical services such as voluntary counseling and testing and diagnosis and treatment of sexually transmitted infections will be essential.In addition, site development should include building skills that are ancillary but critical to the actual conduct of clinical trials, such as educating communities, building community partnerships, managing site finances, and piloting applications through regulatory decision-making processes.The Enterprise must address a number of problems that currently impact the review of HIV vaccine trial protocols and that could delay future decisions regarding product licensure in developing countries. Most regulatory challenges arise from the fact that regulatory approvals are granted at the national level, but many developing countries lack the expertise, well-defined processes, clear delineation of authority, and/or other system components needed to make regulatory decisions expeditiously. As a result, new products are often licensed in these regions based on prior approval in the US or Europe and/or endorsement by the WHO. Under these circumstances, data specific to developing country populations  often do not enter into the decision making. The absence of defined pathways to approve products targeting a country's needs when a product is not also submitted to regulators in the US or Europe remains another obstacle. The Enterprise process has identified these action-item priorities: (1) harmonize and exchange information needed by regulatory bodies within the differing legal frameworks of different countries, (2) facilitate regulatory decision making, possibly using regional approaches for conducting reviews and making recommendations, (3) build regulatory capacity, (4) perform risk/benefit evaluations in the context of differing epidemic dynamics and country needs and resources, (5) identify and remove potential scientific impediments to rapid regulatory decision making, and (6) address ethical issues that interface with regulatory decision making, such as ensuring informed consent and defining the degree to which trial participants should receive a standard of care that is higher than others in their community.Given the Enterprise focus on stronger collaboration, data sharing, and use of common materials and reagents, an intellectual property (IP) framework that facilitates this “enabling environment” is crucial for success. While IP issues may arise throughout the vaccine development process, at present the top priority is to stimulate early stage research and vaccine design by increasing scientific freedom to operate and sharing of data and biological materials.Specific areas for further consideration include: (1) minimizing restrictions on freedom of operation, perhaps by early stage covenants not to litigate and followed by later stage agreements based on true valuations of IP; (2) sharing of information , materials, expertise, trade secrets, and platform technologies in a protected and secure manner while also remaining in compliance with national laws devised to prevent monopolies and insider trading; (3) recognizing the contribution of different countries to HIV vaccine development through approaches that assure affordable access to successful vaccines; and (4) maximizing access to essential technologies and inventions.On October 21, 2004, a group of participants from 16 countries, the European Commission, UNAIDS, and the WHO met to finalize the scientific plan and to discuss how to formulate specific actions.Participants noted that the structure of an activity should depend on several factors, including, for example, the degree to which the activity can be predefined, the degree to which the creativity of academic researchers needs to be harnessed, and the mechanisms available to the funding organization.A number of options were discussed, with consensus as to those that would fit various scientific priorities.First, networks of focused consortia and real or virtual centers are well suited to systematically address many of the major scientific roadblocks identified in this plan. These consortia or centers would link to each other to ensure a comprehensive, systematic approach, sharing information so that each can be as productive as possible, and also to share reagents and procedures so that data among groups can be compared and, where possible, merged for analysis . The speSecond, a global system of central laboratories linked to satellite laboratories that work together (using GLP) would provide a range of standardized functions, help ensure the quality of clinical research, and enable comparison of data from different trials . TogetheThird, a number of contract laboratories capable of developing, acquiring, storing, and distributing common reagents will prove critical to the success of collaborative research and development projects, and to ensuring reagent quality. These reagents could include (1) peptides, antisera/antibodies, and viral isolates for immune assays, including a standard panel of virus strains and sera representative of the global genetic and immunologic variability of HIV, and (2) additional broadly neutralizing monoclonal antibodies, especially from non-clade B viruses, to facilitate elucidation of the motif or motifs they recognize. These contract laboratories would be expected to work very closely with and enable the work of Enterprise consortia, centers, immune assessment laboratories, and clinical sites.Fourth, a network of Clinical Research Training Centers in developing countries could work collaboratively to ensure development of quality trial sites. These centers would (1) conduct or facilitate training of trial site personnel in activities that are generic to the conduct of clinical trials, as well as those specific for HIV vaccine trials, for example, an HIV vaccine fellowship program for developing country scientists; (2) coordinate and work together with other Enterprise consortia or centers, such as those established to characterize acute/early infection in developing country settings or to prepare a standard panel of HIV strains representative of currently circulating viruses; and (3) share standard operating procedures, vaccine development plans, and strategies for engaging and ensuring community and political support.Fifth, a network of individuals and companies with manufacturing experience, particularly process development expertise, could link to consortia, centers, and others involved in vaccine development to provide development and manufacturing expertise to facilitate the advancement of improved HIV vaccine candidates.The above structures are proposed to address the initial Enterprise scientific priorities. Additional consultative groups, reference and centralized facilities, and other mechanisms may be needed to facilitate collaborative work and strengthen the global capacity for the conduct of HIV vaccine research and development as the field progresses.Different implementing and funding agencies will need to work in close collaboration to ensure harmonious implementation of the scientific plan. Initial actions should focus on the areas of vaccine discovery and standardization of laboratory assays, which are considered critical for the success of the Enterprise and the eventual development of a safe and effective HIV vaccine. Activities to address recommendations in the areas of product development and manufacturing, clinical trials capacity, regulatory considerations, and IP issues should be launched after these initial components of the plan are under way.Regardless of timing, each scientific endeavor needs to outline specific strategies to ensure information exchange and capacity building among the collaborating partners and institutions. The funding mechanisms employed  will depend on the task to be accomplished and the needs and capabilities of each funding organization. In the spirit of coordination, collaboration, and transparency promoted by the Enterprise, two or more partners may jointly support one or more activities, taking care to avoid duplication in the use of their respective resources. When a research area is jointly funded, all communication regarding goals, research plans, progress, obstacles, etc., should be openly and transparently shared among all stakeholders—funders, project managers, and researchers.As an alliance of independent entities, the Global HIV/AIDS Vaccine Enterprise will be challenged to carry out three essential functions. One is to continue regular scientific assessments. The scientific priorities outlined in this paper will need to be monitored, re-evaluated, and updated. An evolving scientific plan must reflect lessons learned, new opportunities, and the influence of new scientific findings and new technologies. Revised versions of the scientific plan must be made fully and publicly available. The second essential function is to establish global processes. To optimize progress across a large and complex set of activities at the global level, standards, performance criteria, and processes for data sharing, communication, and convening must be established. The Enterprise will convene fora to address policy issues such IP, clinical trials, site development, and regulatory hurdles. And the third essential function is shared accountability. The partners in this alliance will need to create a culture of mutual accountability for the effective implementation of the scientific strategic plan. Since the Enterprise is not a single organization, a shared “way of doing business” is one of its most important defining traits. Articulating an explicit set of “working principles” is therefore crucial to the identity and smooth functioning of the Enterprise.For the Enterprise as a whole the following conditions apply: (1) the central task is to develop and implement an ambitious scientific plan with the necessary scale, balance and sequence of activities, and structure to carry it out; (2) the plan must focus on critical roadblocks that would benefit substantially from global collaboration while fostering continued R&D by individuals, small groups, and individual networks; (3) the incentives holding the alliance together will include collaborative arrangements and structures that give people the resources, necessary critical mass, centralized facilities, common reagents, assays and technologies, and data they need to effectively remove critical roadblocks; (4) all activities will reflect the commitment to create an environment that maximizes the ability of participants to share data and biological materials, e.g., through the use of common standards for measurements and appropriate IP arrangements; and (5) the Enterprise also commits to working for rapid global access to a successful vaccine.For participating investigators and organizations, key principles include (1) the willingness and desire to work in an open, collaborative fashion, sharing data and reagents in a collegial fashion, with the appropriate balance between productive competition and effective collaboration, and (2) the willingness and ability to devote the majority of their time to tackling these problems within a focused environment, completely committing to solve the problems at hand.The implementation of the scientific plan of the Enterprise will be overseen and supported by the organizational structure described in The Coordinating Committee will facilitate all aspects of the Enterprise's activities. This committee consists of representatives of the Enterprise founders as well as additional scientific leaders selected from inside and outside the field of HIV vaccine research and development. The committee will develop procedures for term rotation and inclusion of new members, to ensure appropriate representation of all relevant partners, and will engage external stakeholders for advice, expertise, and assistance, appointing technical expert groups as needed. A Secretariat will provide logistical and administrative support to the Coordinating Committee and Enterprise partners. The BMGF will serve as Interim Secretariat until a permanent Secretariat is established.The road to success will be a bumpy one.The Funders Forum will be an open forum of sovereign, independent funding organizations, starting with a nucleus of those who already embrace the principles of the Enterprise and who are actively supporting or intend to support and fund HIV vaccine research and development. Members of the Funders Forum will be high-level decision makers within the ranks of funding organizations and governments, as close as possible to the source of resources. Since the Enterprise is not a discrete organization with a pool of money, funders will support specific areas using their own mechanisms, according to their own practices and policies, and following Enterprise principles. The scientific plan will provide guidance that may help funders better align existing resources but, more importantly, will facilitate the efficient and focused application of new resources as they become available. Multiple funders who wish to support a single Enterprise-defined project could form collaborative agreements, memoranda of understanding, or other forms of written agreement among themselves to outline their respective roles and responsibilities; address IP, program management, oversight, and other issues; and establish mechanisms for communication and conflict resolution. The funders with greatest flexibility could provide incentives for sharing reagents and data, and linking projects together, e.g., by supporting the additional work that nationally or regionally funded laboratories would need to undertake in order to participate in a global network, or by supporting a program to develop and share reagents.In some cases, funders may wish to support an implementing organization that will take responsibility for managing the project and reporting back to the funder and other stakeholders. In other cases, funders may have the capability and capacity to play a substantial role in facilitating the project. In still other cases, funders may have the capability to assume a leadership role in overseeing the conduct of the activity, particularly in cases where the activity is well defined in advance.In addition, an Annual Stakeholders Forum will be organized to bring together the broader community of scientists, policy makers, public health officials, and community representatives involved in the search for an HIV/AIDS vaccine. This meeting will serve as a forum to (1) update the broader community on Enterprise activities and progress, and (2) provide the community with a mechanism for feedback and dialog.Global expenditures on HIV vaccine research and development in 2002 were tentatively estimated to be on the order of US$624–670 million, the large majority (67.3%) provided by the public sector, followed by the philanthropic sector (17.4%) and industry (15.3%). An analysis of how those funds have been invested revealed that the large majority (43.1%) is being used in preclinical research activities, followed by clinical trials (28.2%), basic research (20.7%), cohort development and clinical trial infrastructure (6.5%), and vaccine education, advocacy, and policy development (1.4%) .The largest funder of HIV vaccine research and development activities has been the NIH, with almost US$350 million in 2002. The NIH budget for HIV vaccine research has grown from less than US$50 million in 1996, to an estimated US$514.6 million for 2005, corresponding to 17.6% of the NIH total HIV-related research budget for 2005.The Enterprise Coordinating Committee will analyze the additional financial requirements to fully implement the scientific plan of the Enterprise, and the Enterprise Secretariat will explore options to leverage these funds from the public and private sector. Initial estimates by Enterprise partners suggest that US$1.2 billion per year, or double the current expenditures on HIV vaccine research and development, will be needed. Although this amount may appear unrealistic at present, it would represent only a fraction of the total global expenditures in response to the AIDS pandemic and a very reasonable investment in view of the enormous social, political, and economic consequences of the pandemic. However, it is essential that the proposed increase in funding for HIV vaccine R&D be additional to existing AIDS expenditures, and not at the expense of current prevention, treatment, and care efforts.The founding partners of the Enterprise, including the NIH, the BMGF, and the Wellcome Trust have already committed, or are considering committing, resources towards new initiatives that will begin to enact portions of the Enterprise scientific plan over the next six to nine months. Each funder will utilize their own funding processes and will align the design, scope, and scale of programs to those laid out in this plan. For example, the NIH National Institute of Allergy and Infectious Diseases will establish the Center for HIV Vaccine Immunology, which will target several scientific priorities identified here.As a sign of global recognition of the importance of better, more strategic coordination in the search for an HIV vaccine, the “Group of Eight” leading industrialized nations in June 2004 endorsed the goals of the Enterprise and agreed to review progress in implementation at its 2005 summit meeting in the United Kingdom . LikewisWith almost 5 million new HIV infections and 3 million AIDS deaths occurring every year worldwide, the development of a safe, effective, and accessible HIV vaccine represents one of the most urgent global public health needs. This global emergency led to the proposal to harness the power of science to find a definitive solution to one of the most catastrophic health problems of our time. The Global HIV/AIDS Vaccine Enterprise has evolved over the past 18 months from a concept proposed in a scientific journal by a cadre of researchers to a global consensus concerning the major scientific roadblocks facing HIV vaccine development, a strategic approach to address those roadblocks, and guiding principles for the plan's implementation in a manner and degree commensurate with the challenges at hand. Several organizations have already embraced the Enterprise concept and are moving to tackle portions of the scientific plan. Still, much more remains to be done. The road to success will be a bumpy one requiring the energy, commitment, and action of a wide number of government and non-governmental organizations globally. Recognizing the enormity of the roadblocks as well as the potential benefits of a safe and effective HIV vaccine, it is essential that many more organizations and agencies contribute additional expertise and resources and work together as a global community in a cooperative, collaborative, and transparent manner to fully implement the Enterprise scientific plan.


Umbilical metastasis is one of the main characteristic signs of extensive neoplastic disease and is universally referred to as Sister Mary Joseph's nodule.A 59-years-old Caucasian female underwent liver transplant for end stage liver disease due to hepatitis C with whole graft from cadaveric donor in 2003. After transplantation the patient developed multiple subcutaneous nodules in the umbilical region and bilateral inguinal lymphadenopathy. The excision biopsy of the umbilical mass showed the features of a poorly differentiated papillary serous cystadenocarcinoma. Computed tomographic scan and transvaginal ultrasonography were unable to demonstrate any primary lesion. Chemotherapy was start and the dosage of the immunosuppressive drugs was reduced. To date the patient is doing well and liver function is normal.The umbilical metastasis can arise from many sites. In some cases, primary tumor may be not identified; nonetheless chemotherapy must be administrated based on patient's history, anatomical and histological findings. Metastases to the umbilicus are universally referred to as Sister Joseph's (or Sister Mary Joseph's) nodule. Etiology is related to the presence of primary malignant disease in the abdominal cavity or occasionally in the chest and/or breast -5. HistoAlthough skin metastasis is rare and range between 5% and 9%, it is estimated that 1% to 3% of abdomino-pelvic tumors metastasize to the umbilicus ,3,5. TheSerous papillary cystadenocarcinoma has been documented previously as a cancer responsible for Sister Joseph's nodule because of the metastasis to the regional lymph nodes -9. Herei2 I.V) once a week for 16 weeks. To date, after 6 months of follow-up, the patient is doing well, the liver function is normal and tacrolimus serum level is 1 ng/ml. A recent whole body CT-scan showed regression of iliac and inguinal lymph nodes involvement.A 59-year-old Caucasian woman was admitted to our hospital for progressively enlarging but painless mass in the region of the umbilicus. The umbilical mass of approximately 5 cm in diameter and has developed two months after liver transplantation. The nodule was firm with irregular borders, wine-red shade and fixed to the surrounding tissues. The overlying skin was not ulcerated. She never smoked, consumed alcohol or used oral contraceptive pills. The gynecological history was unremarkable. The patient underwent a liver transplantation for end stage liver disease (ESLD) secondary to hepatitis C in mid of 2003. Prior to the transplant, her serum alpha-fetoprotein  and CEA concentration were measured and both were within normal limits. The transplant procedure and immediate post-transplant period were unremarkable. The post-transplant immunosuppressive regimen consisted of Tacrolimus (6 mg/day) and steroids (prednisone 10 mg/day). Two months after transplantation, during routine follow-up, physical examination revealed multiple subcutaneous satellite nodules in close proximity to the umbilical region and lower abdominal wall. There was bilateral inguinal lymphadenopathy. A plain anterior/posterior chest x-ray was normal. Ultrasound of the abdominal cavity showed a 4 × 4 cm mass below the umbilicus and additional smaller nodules confined to the lower abdominal wall. Paraaortic lymph nodes were not identified. The echogenicity of the mass consisted of alternative hyper- and hypoechoic with poorly defined edges. However, the general condition of the patient was good and the laboratory test showed: hemoglobin level 9.6 g/dl, total bilirubin 0.6 mg/dl, conjugated bilirubin 0.1 mg/dl, Alkaline Phosphates 273 U/l, GGT 40 U/L, AST 36 U/L, and ALT 56 U/L. There was serological evidence of past hepatitis C virus infection. The biopsy of the umbilical mass showed features characteristic for a poorly differentiated serous papillary cystadenocarcinoma. An extended resection of the umbilical and paraumbilical metastasis was performed. In addition, extensive investigation to detect the primary neoplastic origin was initiated. Tumor marker evaluation consisted of: CEA 0.22 [normal 0–3.7 ng/ml], alpha feto protein 1.49 , CA125 27.73 , CA 19.9 was 8.61 , CA 15.3 was 22.48 . Multiple tumor marker measurements were obtained and each time similar results were reported. A computed tomographic (CT) scan was unable to demonstrate any lesion that could be considered a primary source of the metastasis to the umbilicus. Trans-vaginal ultrasonography showed a normal uterus and ovaries; ultrasonographic examination of thyroid and breast did not show any pathological changes as well. In spite of laborious efforts, the tumor's primary site was not found at that time. However, considering the histological results, the umbilicus metastasis could be attributed almost entirely to the ovarian cancer stage 4, since the main primary site in females are the ovaries. In order to prevent further tumor extension and expansion, tacrolimus was reduced from 6 mg/day to 1.5 mg/day. In addition, chemotherapy with Taxol was initiated  declare that they have no competing interests.FP: conception and design, interpretation of data, drafting the article and revisingEA: interpretation of data, drafting the article and revisingSDD: acquisition of data and revisingNM: interpretation of data, design and coordination and helped to draft the manuscriptGB: collection of data, design and coordination and helped to draft the manuscriptRM: collection of data, design and coordinationMM: acquisition of data and drafting the articleTJ: drafting the article and interpretationFR: acquisition of data and designMC: interpretation of data, drafting the articleUV: drafting the article, revising and supervision


Annotating genomes remains an hazardous task. Mistakes or gaps in such a complex process may occur when relevant knowledge is ignored, whether lost, forgotten or overlooked. This paper exemplifies an approach which could help to ressucitate such meaningful data.2-terminal sequence allowing to reannotate a wrongly annotated ornithine carbamoyltransferase as a putrescine carbamoyltransferase, (iii) identification of conserved motifs allowing to distinguish unambiguously between the two kinds of carbamoyltransferases, and (iv) comparative study of the gene context of these different sequences.We show that a set of closely related sequences which have been annotated as ornithine carbamoyltransferases are actually putrescine carbamoyltransferases. This demonstration is based on the following points : (i) use of enzymatic data which had been overlooked, (ii) rediscovery of a short NHWe explain why this specific case of misannotation had not yet been described and draw attention to the fact that analogous instances must be rather frequent. We urge to be especially cautious when high sequence similarity is coupled with an apparent lack of biochemical information. Moreover, from the point of view of genome annotation, proteins which have been studied experimentally but are not correlated with sequence data in current databases qualify as "orphans", just as unassigned genomic open reading frames do. The strategy we used in this paper to bridge such gaps in knowledge could work whenever it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context. As a consequence of the deluge of completely sequenced genomes belonging to a large array of species, one can expect to identify many homologues of enzymes which have been previously well studied at the experimental level. This seems to be the general rule and the public sequence databanks (DDBJ/EMBL/GenBank) are now inundated by putative amino acid sequences which have been annotated uniquely by the widely used two-step process : (1) detection of a homologous relationship by a pairwise sequence similarity search at the level of primary structure and (2) inference of functional similarity from this detected homology.However, the opposite might be true. For various reasons one can either miss or misinterpret the actual function of a putative protein when annotating by homology, resulting in a wrong function transfer. Several studies have already emphasized this point . On tde novo pyrimidine biosynthesis and ornithine carbamoyltransferase  which plays a crucial role in both anabolism and catabolism of arginine.Our group ,5) is pr is pr5])In a recent study of the phylogeny of the 245 available OTCases (paper in preparation), we confirmed the existence of two families, OTC alpha and OTC beta, previously proposed on the basis of phylogenetic studies ))27]). InThe strategy we used to identify such orphan sequences could work in any other case where it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context. Note that we incidentally used such a strategy to annotate the genes encoding a putative agmatine deiminase in the genomes listed in Fig. bona fide sequences from specific sequencing groups sites  sequences were collected from the public databases SwissProt, TREMBL and TREMBLNEW . To facinstitute  and Sangnstitute ) using eRooted phylogenetic trees were derived from multiple alignements of ATCases and OTCases using two different approaches. (1) New sequences were manually added and aligned to the previously published  multipleE. faecalis is the putrescine carbamoyltransferase. BL carried out the phylogenetic analyses, the gene context study and drafted the manuscript which was further improved (and approved) by all authors.NG dug up the "ancient" data on putrescine carbamoyltransferase, contributed his knowledge about carbamoyltransferases and made important additions to the manuscript. YX brought essential informations about the genetics and biochemistry of the enzymes involved in arginine metabolism and their evolution. DGN participated in the collection of new carbamoyltransferase sequences and their manual alignment and identified which sequence of


The web-based collection of images is housed on the ASC website, which went live on November 5 The second edition of the Bethesda 'blue book' atlas maintains an easy to read format with bulleted morphologic criteria and half-page color illustrations. The content has been divided into chapters based on the major 2001 Bethesda System interpretive categories. Highlights of the new edition include: (1) incorporation of liquid based cytomorphologic criteria and images; (2) new sections addressing ancillary testing, educational notes and recommendations, computer assisted interpretation and anal-rectal cytology; and (3) inclusion of sample reports, references, and detailed legends for images. Overall, the second edition has tripled in size from the original version, with a total of 186 images of which 90% are new images and 40% are from liquid based specimens. Some are classic examples of an entity while others have been selected to illustrate interpretive dilemmas or "borderline" cytomorphologic features that may not be interpreted in the same way by all cytologists.In parallel with production of the Bethesda book atlas, the ASC-NCI task force has also developed a Bethesda web-based collection of images. Approximately 350 images, (40% of which are from liquid based specimens) along with linked explanatory notes, including all the images in the published atlas, can be viewed on this site. The website is user friendly and has several search modalities for viewing the images including searching by Bethesda terminology, atlas chapter headings, keyword(s), or preparation type. It also allows for individual self-assessment by participating in a "self test" in which viewers can compare their interpretation to other participants' responses.Step 1: individual Bethesda forum group members (32 participants) reviewed and selected images for their chapter from among those in the first edition of the atlas and new submissions; and Step 2: the images selected from Step 1 were reviewed individually  by 13 task force members and scored on a scale of 1–5 for agreement with interpretation and quality of image. In all over 1000 images were reviewed of which 186 images were selected for the atlas and an additional 163 for the website.The image selection process for the book atlas and website involved a multistage review: A subset (n = 77) of the book atlas images were posted as "unknowns" on the Internet from mid July to mid September 2003 as part of a study – the Bethesda Interobserver Reproducibility Project (BIRP). The site was open to the cytopathology community to view the images and provide their interpretations. Immediately after submitting their response, participants were able to view a histogram of the distribution of results submitted by all prior participants for that image. Over 600 cytologists from around the world participated in BIRP. Summary histograms for each of the 77 images can be viewed on the Bethesda atlas website (select BIRP images from the left menu). Preliminary BIRP results presented at the ASC annual meeting in Orlando in November 2003 showed that Negative for Intraepithelial Lesion or Malignancy (NILM) and Low Grade Squamous Intraepithelial Lesion (LSIL) reference images attained the highest concordance scores, while glandular abnormalities demonstrated the most splay in distribution of interpretations. BIRP analyses are ongoing and further results should be available in 2004.ASC Bethesda 2001 Task Force:Ritu Nayar (Chair), Diane Solomon  George Birdsong (Adequacy), Jamie Covell (Glandular Lesions), Ann Moriarty , Dennis O'Connor , Marianne Prey (Computer assisted interpretation), Steve Raab (Ancillary testing), Mark Sherman , Sana Tabbara , Tom Wright (Squamous lesions), Nancy Young (Non-neoplastic findings).ASC Consultants: ASC 2002/2003 Presidents: Diane Davey and Dave Wilbur.Information Technology representatives: Mike Montgomery (NCI) and Brandon Winbush (Northwestern University), Aquilent .The second edition of the 'blue book' atlas can be ordered through Springer-Verlag (1-800-SPRINGE) for $34.95.


Saccharomyces cerevisiae by starvation for nutrilites is a well-documented phenomenon of an unknown mechanism. We have previously shown that the polymerase delta proofreading activity controls spontaneous mutagenesis in cells starved for histidine. To obtain further information, we compared the effect of adenine starvation on mutagenesis in wild-type cells and, in cells lacking the proofreading activity of polymerase delta .Mutagenesis induced in the yeast + revertants accumulated at a very high rate on adenine-free plates so that their frequency on day 16 after plating was 1.5 × 10-4 for wild-type and 1.0 × 10-2 for the Exo- strain. In the Exo- strain, all revertants arising under adenine starvation are suppressors of the original mutation, most possessed additional nutritional requirements, and 50% of them were temperature sensitive.Adepol3-01 mutation leads to a further 66-fold increase of the rate of mutations. Our data suggest that adenine starvation induces genome-wide hyper-mutagenesis in the Exo- strain.Adenine starvation is highly mutagenic in yeast. The deficiency in the polymerase delta proofreading activity in strains with the  In some experiments, SD was solidified with 1.5% Noble Agar  (see below). We named the SDNA medium containing all the nutrilites required by the strain SDNA+ade. When adenine was not included we named it SDNA-ade.+ papillae throughout the experiment . On the day 14, the Ade+ revertants as well as Ade- survivors isolated on SDNA-ade on day 14 were tested for the presence of ts mutations. To obtain Ade- survivors, Ade+ colonies were marked and then adenine was added to two wells of SDNA-ade plates seeded with 1,000 cells/plate on day 14. Dishes were incubated for 6 days more and previously non-marked colonies were isolated on YEPD. Colonies were then tested for their Ade- phenotype on SDNA-ade.AdeTo detect ts mutants, cells from a fresh culture were point inoculated by a needle on YEPD plates, which were incubated for several days at 28°C. Colonies were then replica plated on two YEPD dishes: one of them was incubated at 28°C while the other one at 37°C for two days. The first replica was always incubated at 37°C. A strain was considered to be a ts mutant if in two days it was able to grow at 28°C but not at 37°C. The strains CG379-3-29(LR) and de3-01-CG were included as controls.S. cerevisiae was purified using the NucleoSpin® Tissue kit (Macherey-Nagel).Genomic DNA from ADE5,7 locus (from 490 bp upstream of the start codon to 473 bp downstream of the stop codon) was amplified from genomic DNA of the CG379-3-29(LR) strain using primers ADE5-F1 (5'-CAAAAGTAGAAGACCCCC-3') and ADE5-R1 (5'-CCATTCATCAATTACGG-3'). The PCR reaction was performed according to standard protocol, using the proofreading-proficient Pfx DNA polymerase (Invitrogen). The PCR produced a DNA fragment of 3383 bp, which was purified from an agarose gel by the High Pure PCR Product Purification Kit (Roche). The nucleotide sequence of both strands of the purified DNA fragment was determined by the Sanger method [The ADE5,7 gene in the Saccharomyces Genome Database [The sequence of the amplified fragment from the CG379-3-29(LR) strain was compared with the sequence of the Database  by the BDatabase .ADE5,7 alleles in Ade+ revertant strains were obtained by PCR amplification, using purified genomic DNA as template and the primers ADE5-F4 (5'-CCG TAA ACA TAG GAA TCG-3') and ADE5-R4 (5'-TTG TAC GAG ATT GTT ACC-3'). PCR, performed as previously described, generated a 398 bp long DNA fragment, encompassing the ade5,7 mutation in the CG379-3-29 strain. The purified DNA products were sequenced using the same primers, ADE-F4 and/or ADE5-R4.DNA fragments of the + revertants and AA, AL, NB and GM studied mutagenesis during starvation. YIP constructed the pol3-01 strain, participated in the design of the study and writing the manuscript. EC performed sequencing of the ade5-1 allele and molecular analysis of Ade+ revertants. NB, GM and YIP coordinated the study. AA, AL, EC participated in the design of the study.AA performed molecular analysis of AdeAll authors read and approved the final manuscript.


Primary scientific and medical literature is written for academic communities and the public good -- so should be governed by copyright licenses that permit the full range of uses that could benefit stakeholders in research. This is the third installment in a series of editorials on the implications of open-access publishing for established publishing practices and stakeholders in scientific and medical research.The questions, tensions, and social concerns surrounding copyright and the Internet are very different for scientific and medical literature than for other kinds of easily reproducible digital works. Peer-reviewed publications are often the sole tangible products of the tremendously time-consuming and expensive process of conducting primary research in biology and medicine. Who should own the primary research articles that are the culmination of years of work by scientists and staggering financial investments by governments, universities, and tax-exempt foundations? What uses should the documents' owners permit?In practice, academic authors typically assign full copyrights to their articles to the publishers of the journals in which the works appear. Scientists do not benefit financially from the transaction; indeed, they often subsidize the cost of their articles' publication in the form of page charges, color charges, and other fees levied by publishers. The prestige associated with publishing in a selective journal is sufficiently valuable that scientists are generally willing to abdicate the legal rights to their own work without remuneration.www.creativecommons.org), for example, allow copyright holders to mark their work with freedoms—to permit a work's reproduction for any noncommercial purpose  or for any purpose at all provided that the original authorship is properly attributed (the Attribution License).In recent years, however, several technological and legal innovations have led a growing number of scientists to begin to question the sagacity of this arrangement. The advent of electronic publishing and the Internet itself have made technically possible a slew of novel uses of primary research papers. Simultaneously, the traditional “all rights reserved” copyright license has been supplemented by a variety of alternative licenses—of equal legal validity and available at no charge to anyone who wants them—that allow copyright holders to prevent some uses of a work without permission, but to authorize others. Different licenses created by the nonprofit organization Creative Commons  When a document is “freely available,” someone who comes across it may be permitted to do nothing more than read it online on a publisher's Web site; the right to use the article in any other way is typically granted only at the publisher's discretion. When a document is open access, however, a wide range of additional uses are perpetually and irrevocably allowed— from the reproduction and distribution of the paper by a professor for his or her students to the archiving of the paper in a searchable online repository available to anyone in the world with an Internet connection, and more.One implication of the variety of copyright licenses now widely available is that the right to use an article in one way or another is largely independent of its accessibility online. A paper that is touted as “freely available” or “free access” is very different from one that is “open access.” . The CCAL also ensures that institutions are permitted to archive not only articles written by their own faculty, but all other works published under the same legal terms as well, thereby facilitating their permanent accessibility and preservation. For example, the LOCKSS (“Lots of Copies Keep Stuff Safe”) program (http://lockss.stanford.edu/projectdescbrief.htm), an ongoing project to support libraries' efforts to “create, preserve, and archive local electronic collections,” is viable only insofar as institutions are permitted to store information themselves, rather than access it exclusively via publishers' Web sites. Many collaborative projects between libraries and publishers have been complicated by legal constraints, including the stipulation that archives remain “dark,” or inaccessible to users, until any commercial incentive for restricting access to articles has been exhausted—clearly a suboptimal arrangement for researchers, and one that is unnecessary for collections of works governed by the CCAL.The Creative Commons Attribution License (CCAL), which governs this editorial and all other content in One of the truly revolutionary implications of open-access articles, however, is that we simply do not know the full range of their potential applications. They are available for any use that any entrepreneur can envision, so long as the authors of the papers are properly credited. The only certainty, then, is that the utility of open-access research articles will be limited solely by the imagination of those that are inspired by the possibilities—rather than by legal constraints.PLoS Biology and license their works under nonexclusive terms that reserve only some—rather than all—rights. There are several common objections, generally leveled by publishers, against this practice. For example, it is sometimes argued that the traditional copyright arrangement in scientific publishing protects against uses of articles that authors would object to—while the CCAL permits such uses and renders authors helpless to prevent them.Authors retain the copyright to all articles in To the extent that the uses in question are for academic or archival purposes, such as those discussed above, it is certainly true that the CCAL permits practices that “all rights reserved” licenses do not. Indeed, the expanded range of legitimate academic uses of articles is among the primary selling points of the CCAL in the context of scientific publishing. To the extent that the ostensibly objectionable uses are commercial, the problem is easily remedied with the Creative Commons Noncommercial License, which prohibits commercial reuse of a work without the copyright holder's consent.PLoS has chosen, for reasons both philosophical and pragmatic, to permit the commercial use of works we publish. As a matter of principle, all of our policies reflect the view that scientific publishers are service providers and should not themselves restrict the potential applications of the largely publicly funded work in their journals. More concretely, if a commercial enterprise is interested in repackaging the articles that PLoS has published, we are loath to prevent an author's work from wider distribution. Any risk that a company will use an article for a purpose its author would be uncomfortable with is, in our view, substantially outweighed by the benefits of allowing—not on a case-by-case basis, but across the board—the reproduction of the article for inclusion in online encyclopedias, or for distribution in countries in which Internet access is unreliable, or, indeed, for creative uses we hope to inspire by making primary research articles legally available to commercial interests.Another recurring objection to the copyright arrangement that PLoS employs is that authors are inappropriate copyright holders because they are ill-equipped to protect their own works against plagiarism, misattribution, and other misuse. Most scientists, however, have enough familiarity with cases of plagiarism in their own field to know that their strongest protection against mis- or nonattribution is derived not from the threat of prosecution for copyright infringement, but from community standards of conduct. Furthermore, among the benefits of open-access articles is the fact that their full texts, rather than just their abstracts, are searchable—which, as any teacher knows, makes plagiarism much easier to detect.Beyond plagiarism and misattribution, it is not clear what uses of primary research articles authors would actually want to prevent . Scientists do not receive royalties for their published work. The more widely their articles are read and cited, the more their professional reputations are bolstered. Certainly, research articles have a wide range of uses that publishers typically object to— and indeed often file suit over—such as their compilation in coursepacks by copy shops. Those applications, however, tend not to constitute “misuse” in many authors' eyes.There is no question that the licensing arrangement PLoS employs is relatively novel—and therefore untested over the long term—in biomedical publishing. However, it hardly takes a radical understanding of the interests of authors and users of primary research articles to conclude that the open-access terms of copyright promise substantial benefits for both groups.What, then, can scientists do to encourage other publishers to follow suit and strike similar legal arrangements with authors as a matter of course? One answer is to “vote with your submissions;” that is, authors should submit their work preferentially to journals with copyright and licensing practices that genuinely serve their interests. Another equally important action for scientists is to raise the issue with their professional societies. Scholarly associations exist, among other reasons, to serve the needs of their members—and society members should actively urge their society journals to employ the CCAL or a similar license for their research articles.Scientific and medical literature is different from fiction or movies or music. The United States government invests more than $28 billion per year in the National Institutes of Health alone to fund research in biology and medicine. The scientists who conduct that research and the research paid for by other public-minded institutions in the United States and abroad have an affirmative moral obligation to share the knowledge they create—not just with students and faculty at elite Western universities, but with everyone who could use it and build upon it. When authors publish their work in journals with restrictive copyright practices, it becomes illegal (often for even the authors themselves) to store primary research articles in many archives or include them in coursepacks or use them for other responsible purposes. Those obstacles to sharing knowledge can be avoided without legislative intervention, however, if scientists and publishers alike embrace a legal paradigm for disseminating new discoveries that maximizes their utility.