In 1995, the first complete bacterial genome sequence was published. Now, nearly 200 bacterial genomes have been completed, and a new one hits the scientific press most weeks. This burgeoning industry is not just scientific “stamp collecting,” however. Having all these genome sequences may provide useful clues about why some bacteria cause human disease, how to control their spread, and how to treat the infections caused by them. By comparing genome sequences, scientists can learn much more about what makes a bacteria tick than they can learn from a single sequence.Derrick Fouts and his colleagues have taken this comparative approach with  Campylobacter . Infection with a  Campylobacter  species is one of the most common causes of human bacterial gastroenteritis. In the US, 15 out of every 100,000 people are diagnosed with campylobacteriosis every year, and with many cases going unreported, up to 0.5% of the general population may unknowingly harbor  Campylobacter  in their gut annually. Diarrhea, cramps, abdominal pain, and fever develop within 2–5 days of picking up a pathogenic  Campylobacter  species, and in most people, the illness lasts for 7–10 days. But the infection can sometimes be fatal, and some individuals develop Guillain-Barré syndrome, in which the nerves that join the spinal cord and brain to the rest of the body are damaged, sometimes permanently.Campylobacteriosis is usually caused by  C. jejuni , a spiral-shaped bacterium normally found in cattle, swine, and birds, where it causes no problems. But the illness can also be caused by  C. coli  (also found in cattle, swine, and birds),  C. upsaliensis  (found in cats and dogs), and  C. lari  (present in seabirds in particular). Disease-causing bacteria generally get into people via contaminated food, often undercooked or poorly handled poultry, although contact with contaminated water, livestock, or household pets can also cause disease.In 2000,  C. jejuni  was the first food-borne pathogen to be completely sequenced, but we still know little about how  Campylobacter  species cause disease. In their search for clues, Derrick Fouts and coworkers have completely sequenced the genome of  C. jejuni  strain RM1221 (isolated from a chicken carcass) and compared it with the previously sequenced  C. jejuni  strain NCTC 11168 and with the unfinished sequences of  C. coli  strain RM2228 (a multi-drug-resistant chicken isolate),  C. lari  strain RM2100 (a clinical isolate), and  C. upsaliensis  strain RM3195 (taken from a patient with Guillain-Barré syndrome).The researchers describe numerous differences and similarities between these different  Campylobacter  strains and species. For example, there are major structural differences between the genomes caused by the insertion of new stretches of DNA. Some of these pieces of DNA may carry genes that improve bacterial virulence or fitness, so their presence could help to explain the different biological behaviors of these strains. There are also major variations in the genes responsible for synthesis of molecules that are important for the interaction of  Campylobacter  with the environment. Such differences could underlie the host specificity of the different species.Differences between the  Campylobacter  species in genes that are likely to be involved in aspects of bacterial virulence, such as adherence, motility, and toxin formation, are all detailed by Fouts et al., who also describe a new putative  Campylobacter  virulence locus. Further work is needed to relate these genomic differences to functional differences, but this detailed comparative genomic analysis provides the core blueprint for this important family of human pathogens. And in doing so, it lays the foundation for the development of new ways to monitor and control  Campylobacter  in the food chain and in human infection.


In the elderly, many alterations of both innate and clonotypic immunity have been described. Alterations to the immune system in the elderly are generally viewed as a deterioration of immunity, leading to the use of the term immunosenescence. However, although many immunological parameters are often notably reduced in the elderly, retained function of both innate and clonotypic immunity in the elderly is tightly correlated to health status. Recognising the important role of the immune system in ageing, over the last few years, journals oriented towards gerontology and geriatric sciences have increasingly published articles dealing with the immunology of ageing, but a specialised journal in this area does not exist.  Immunity & Ageing  is a new Open Access, peer reviewed journal that aims to cover all the topics dealing with innate and clonotypic immunity which are relevant to ageing. The journal will provide an opportunity to focus on this topic, which is emerging as one of the critical mechanisms of ageing. Furthermore, as an online, Open Access journal,  Immunity & Ageing  will promote immediate accessibility to research, which is generally not possible for articles published in printed journals. We hope this forum, concentrating on the themes of ageing and immunology with a strong focus on human studies, will create a new perspective for viewing a world that is inevitably becoming older.Immunity & AgeingImmunity & Ageing

Immunity & Ageing  is a new Open Access, peer reviewed journal that aims to provide a forum for articles examining ageing from an immunological point of view. During the past century, humans have gained more years of average life expectancy than in the last 10,000 years; we are now living in a rapidly ageing world. The sharp rise in life expectancy, coupled to a steady decline in birth rates in all developed countries, has led to an unprecedented demographic revolution characterized by an explosive growth in the number and proportion of older people. The number of people aged 60 years or older exceeded 635 million in 2002, and is expected to grow to nearly 2 billion by 2050. The proportion of people aged 60 and over stands about 1 in 4 in many Western European countries as well as in Japan. Should the present trend continues, this ratio is expected to reach 1 in 3 by 2050 [ 1 ]. Among the aged, the oldest old (>85) make up the fastest growing category. As access to medical care improves worldwide, the rate of population ageing will accelerate. If global communications is making the world "young and fast", then global ageing is surely "maturing and slowing" it. In any case, these epidemiological facts underscore the importance of studies on successful and unsuccessful ageing and necessitate the prompt spread of knowledge about ageing in order to satisfactorily decrease the medical, economic and social problems associated with advancing years.

Immunity & Ageing  is a new Open Access, peer reviewed journal that aims to provide a forum for articles examining ageing from an immunological point of view.Immunity & AgeingDuring the past century, humans have gained more years of average life expectancy than in the last 10,000 years; we are now living in a rapidly ageing world. The sharp rise in life expectancy, coupled to a steady decline in birth rates in all developed countries, has led to an unprecedented demographic revolution characterized by an explosive growth in the number and proportion of older people. The number of people aged 60 years or older exceeded 635 million in 2002, and is expected to grow to nearly 2 billion by 2050. The proportion of people aged 60 and over stands about 1 in 4 in many Western European countries as well as in Japan. Should the present trend continues, this ratio is expected to reach 1 in 3 by 2050 [ 1 ]. Among the aged, the oldest old (>85) make up the fastest growing category. As access to medical care improves worldwide, the rate of population ageing will accelerate. If global communications is making the world "young and fast", then global ageing is surely "maturing and slowing" it. In any case, these epidemiological facts underscore the importance of studies on successful and unsuccessful ageing and necessitate the prompt spread of knowledge about ageing in order to satisfactorily decrease the medical, economic and social problems associated with advancing years.Ageing Ageing is a post-maturational process that, due to a diminished homeostatic capacity and increased vulnerability, reduces responsiveness to environmental stimuli and is generally associated with an increased predisposition to illness and death. At the beginning of the 19 th  century, mortality was described as increasing exponentially with respect to progression through the lifespan [ 2 ]. This trend, also described in invertebrates, persists: in Western countries the mortality rate increases 25 times more rapidly in individuals over 60 years old compared to people aged 25–44. Causes of death in aged people are increased compared with individuals between 25 and 44 years old: cancer 43-fold, pneumonia and influenza 89-fold, heart disease 92-fold and stroke and chronic lung disease greater than 100-fold [ 3 ]. Thus far, to understand ageing mechanisms, much attention has been paid to gene mutations in invertebrates and caloric restriction in rodents. However, these data suggest a key role for immunity in the survival of the elderly because susceptibility to these diseases depends at least in part on optimal immune function [ 4 , 5 ]. So, a better understanding of the ageing immune system may provide the most important clues for slowing the inevitable decline associated with the passage of time.
Ageing
Ageing is a post-maturational process that, due to a diminished homeostatic capacity and increased vulnerability, reduces responsiveness to environmental stimuli and is generally associated with an increased predisposition to illness and death. At the beginning of the 19 th  century, mortality was described as increasing exponentially with respect to progression through the lifespan [ 2 ]. This trend, also described in invertebrates, persists: in Western countries the mortality rate increases 25 times more rapidly in individuals over 60 years old compared to people aged 25–44. Causes of death in aged people are increased compared with individuals between 25 and 44 years old: cancer 43-fold, pneumonia and influenza 89-fold, heart disease 92-fold and stroke and chronic lung disease greater than 100-fold [ 3 ]. Thus far, to understand ageing mechanisms, much attention has been paid to gene mutations in invertebrates and caloric restriction in rodents. However, these data suggest a key role for immunity in the survival of the elderly because susceptibility to these diseases depends at least in part on optimal immune function [ 4 , 5 ]. So, a better understanding of the ageing immune system may provide the most important clues for slowing the inevitable decline associated with the passage of time.Immunity in ageing In the elderly, many alterations in innate and clonotypic immunity have been described and viewed as deleterious, hence the term immunosenescence. In 1969, Roy Walford published his landmark book, "The Immunologic Theory of Aging", and first coined the term immunosenescence [ 6 ]. Significantly, most of the areas that he pioneered during his illustrious research career remain the "hot" areas of current gerontological research. On the other hand, immunosenescence is a complex process involving multiple reorganizational and developmentally regulated changes, rather than simple unidirectional decline of the whole function [ 7 , 8 ]. However, some immunological parameters are commonly notably reduced in the elderly and, reciprocally good function is tightly correlated to health status [ 4 , 5 ].
Immunity in ageing
In the elderly, many alterations in innate and clonotypic immunity have been described and viewed as deleterious, hence the term immunosenescence. In 1969, Roy Walford published his landmark book, "The Immunologic Theory of Aging", and first coined the term immunosenescence [ 6 ]. Significantly, most of the areas that he pioneered during his illustrious research career remain the "hot" areas of current gerontological research. On the other hand, immunosenescence is a complex process involving multiple reorganizational and developmentally regulated changes, rather than simple unidirectional decline of the whole function [ 7 , 8 ]. However, some immunological parameters are commonly notably reduced in the elderly and, reciprocally good function is tightly correlated to health status [ 4 , 5 ].Innate immunity in ageing The process of maintaining life for the individual is a constant struggle to preserve his/her integrity. This can come at a price when immunity is involved, namely systemic inflammation [ 9 ]. Inflammation is not a negative phenomenon per se: it is the response of the immune system to the invasion of viruses or bacteria and other pathogens. The problem is that in the course of evolution the human organism was set to live 40 or 50 years. Today, however, the immune system must remain active for longer. This very long activity leads to a chronic inflammation that slowly but inexorably damages all the organs: this is the typical phenomenon linked to ageing and it is considered the major risk factor for age-related chronic diseases, such as osteoporosis, sarcopenia, type 2 diabetes, Alzheimer's disease and atherosclerosis, though progression seems also dependent on the genetic background of individuals [ 4 , 5 , 8 , 10 , 11 ]. Emerging evidence suggests that pro-inflammatory genotypes are related to unsuccessful ageing, and, reciprocally, controlling inflammatory status may allow a better chance of successful ageing [ 4 , 5 , 8 , 12 ]. In other words, age-related diseases are "the price we pay" for an active immune system that defends us thorough out life, but also has the capacity to harm us later, as its fine tuning becomes compromised [ 13 ]. In fact, our immune system has evolved to control pathogens, so pro-inflammatory responses are likely to be evolutionarily programmed to resist fatal infections with an increased resistance to pathogens. Thus, inflammatory genotypes are an important and necessary part of the normal host responses to pathogens in early life, but the overproduction of inflammatory molecules might also cause immune-related inflammatory diseases and eventually death later. Therefore, low responder genotypes might better control inflammatory responses and age-related disease development, resulting in an increased chance of long life survival in a facilitory environment with reduced pathogen load and medical care, such as might be present in Western societies [ 12 , 14 - 16 ].
Innate immunity in ageing
The process of maintaining life for the individual is a constant struggle to preserve his/her integrity. This can come at a price when immunity is involved, namely systemic inflammation [ 9 ]. Inflammation is not a negative phenomenon per se: it is the response of the immune system to the invasion of viruses or bacteria and other pathogens. The problem is that in the course of evolution the human organism was set to live 40 or 50 years. Today, however, the immune system must remain active for longer. This very long activity leads to a chronic inflammation that slowly but inexorably damages all the organs: this is the typical phenomenon linked to ageing and it is considered the major risk factor for age-related chronic diseases, such as osteoporosis, sarcopenia, type 2 diabetes, Alzheimer's disease and atherosclerosis, though progression seems also dependent on the genetic background of individuals [ 4 , 5 , 8 , 10 , 11 ]. Emerging evidence suggests that pro-inflammatory genotypes are related to unsuccessful ageing, and, reciprocally, controlling inflammatory status may allow a better chance of successful ageing [ 4 , 5 , 8 , 12 ]. In other words, age-related diseases are "the price we pay" for an active immune system that defends us thorough out life, but also has the capacity to harm us later, as its fine tuning becomes compromised [ 13 ]. In fact, our immune system has evolved to control pathogens, so pro-inflammatory responses are likely to be evolutionarily programmed to resist fatal infections with an increased resistance to pathogens. Thus, inflammatory genotypes are an important and necessary part of the normal host responses to pathogens in early life, but the overproduction of inflammatory molecules might also cause immune-related inflammatory diseases and eventually death later. Therefore, low responder genotypes might better control inflammatory responses and age-related disease development, resulting in an increased chance of long life survival in a facilitory environment with reduced pathogen load and medical care, such as might be present in Western societies [ 12 , 14 - 16 ].Clonotypic immunity in ageing Recently, longitudinal studies have shown that a cluster of immunological parameters can be used to evaluate the expectation and quality of life, i.e. the immunological risk phenotype [ 17 , 18 ]. Senescence of clonotypic immunity is most likely principally a result of alterations to T cells. Lifelong and chronic antigen load seems to be the major driving force of immunosenescence, which impacts on human lifespan by reducing the number of virgin antigen-non experienced T cells, and, simultaneously, fills the immunological space with expanded clones of memory and effector, antigen-experienced T cells [ 18 - 20 ]. Gradually, the T cell population shifts to a lower ratio of naïve cells to memory cells, the thymus pumps out fewer naïve T cells with age and those T cells remaining, especially the CD8+ subset, also show increased oligloclonality with age [ 4 ]. Thus, the repertoire of cells available to respond to antigenic challenge from previously unencountered pathogens is reduced. In addition, older organisms are often overrun by memory cells that carry a single type of T cell repertoire, i.e. clonal expansion. Thus, the memory cells from old individuals might recognize a limited set of antigens despite being plentiful in number. Many of the clonal expansions crowding an elderly person's immune system result from previous infections by persisting viruses [ 18 , 19 ]. In contrast to T cells, no evidence for a loss of B cell function has been found as neither the total number of B cells or immunoglobulin secreting cells have been shown to be profoundly decreased with age. However, the B-cell repertoire is influenced by ageing during an actual immune response, where the spectrum of expressed immunoglobulin genes, as well as the frequency of somatic mutations, affects the quality, though not necessarily the quantity, of the antibody response, which is highly relevant in clinical practice [ 7 , 21 ].
Clonotypic immunity in ageing
Recently, longitudinal studies have shown that a cluster of immunological parameters can be used to evaluate the expectation and quality of life, i.e. the immunological risk phenotype [ 17 , 18 ]. Senescence of clonotypic immunity is most likely principally a result of alterations to T cells. Lifelong and chronic antigen load seems to be the major driving force of immunosenescence, which impacts on human lifespan by reducing the number of virgin antigen-non experienced T cells, and, simultaneously, fills the immunological space with expanded clones of memory and effector, antigen-experienced T cells [ 18 - 20 ]. Gradually, the T cell population shifts to a lower ratio of naïve cells to memory cells, the thymus pumps out fewer naïve T cells with age and those T cells remaining, especially the CD8+ subset, also show increased oligloclonality with age [ 4 ]. Thus, the repertoire of cells available to respond to antigenic challenge from previously unencountered pathogens is reduced. In addition, older organisms are often overrun by memory cells that carry a single type of T cell repertoire, i.e. clonal expansion. Thus, the memory cells from old individuals might recognize a limited set of antigens despite being plentiful in number. Many of the clonal expansions crowding an elderly person's immune system result from previous infections by persisting viruses [ 18 , 19 ].In contrast to T cells, no evidence for a loss of B cell function has been found as neither the total number of B cells or immunoglobulin secreting cells have been shown to be profoundly decreased with age. However, the B-cell repertoire is influenced by ageing during an actual immune response, where the spectrum of expressed immunoglobulin genes, as well as the frequency of somatic mutations, affects the quality, though not necessarily the quantity, of the antibody response, which is highly relevant in clinical practice [ 7 , 21 ].Immunity & Ageing . Why do we need an Open Access journal? Considering the paramount function of the immune system during ageing, journals oriented towards gerontology and geriatric sciences are now publishing an increasing number of articles dealing with immunology and ageing, but a specialised journal in this area does not exist.  Immunity & Ageing  was conceived to cover all topics relevant to immunity and ageing in an interdisciplinary manner. It is clear that immunological mechanisms are involved in process and manifestation of ageing in many systems. An example is displayed by the increase in serum and tissue levels of circulation pro-inflammatory cytokines which is accompanied by typical cellular ageing phenomena such as telomeric loss, oxidative damage, DNA defects, accumulation of advanced glycosylation products, cellular loss and others. These defects may be stimulators of cytokine secretion, and subsequently, cytokines are released into the systemic circulation. This results in a slowly progressive endo-crinosenescence and neurosenescence. The different factors may influence each other in form of a vicious spiral [ 22 ]. The journal will provide an opportunity to focus the topic of immunology of ageing, which is emerging as one of the critical mechanisms of ageing which aspects should get to all scientists physicians and other professions who are involved in the different inter-related disciplines, and the bridge that is emerging between these fields. Furthermore, as an online, Open Access journal,  Immunity & Ageing  will promote "immediate" accessibility to this fast moving area of research, which is generally not possible for articles published in printed journals. From a modern perspective, electronic publishing is obviously attractive for its speed, easy global access and low cost, which all present considerable advantages over print and are seen as attractive factors by authors, readers and publishers. Open Access appears to provide exactly what people would like: rapid publication for authors, free access to information for readers, and inexpensive, global availability to readers for publishers [ 23 ]. We believe knowledge should belong to those who want it and access should not be unjustly and unacceptably expensive or difficult.  Immunity & Ageing  will take advantage of the Open Access policy to make peer-reviewed information widely and almost immediately available. We hope this forum, focussing on the themes of ageing and immunology, will create a new perspective for looking at a world that is inevitably becoming older. Creating and pushing forward a research knowledge base in  Immunity & Ageing  provides a unique opportunity to dissect out some parameters which might make health span equate with increasing lifespan.
Immunity & Ageing . Why do we need an Open Access journal?
Immunity & AgeingConsidering the paramount function of the immune system during ageing, journals oriented towards gerontology and geriatric sciences are now publishing an increasing number of articles dealing with immunology and ageing, but a specialised journal in this area does not exist.  Immunity & Ageing  was conceived to cover all topics relevant to immunity and ageing in an interdisciplinary manner. It is clear that immunological mechanisms are involved in process and manifestation of ageing in many systems. An example is displayed by the increase in serum and tissue levels of circulation pro-inflammatory cytokines which is accompanied by typical cellular ageing phenomena such as telomeric loss, oxidative damage, DNA defects, accumulation of advanced glycosylation products, cellular loss and others. These defects may be stimulators of cytokine secretion, and subsequently, cytokines are released into the systemic circulation. This results in a slowly progressive endo-crinosenescence and neurosenescence. The different factors may influence each other in form of a vicious spiral [ 22 ].Immunity & AgeingThe journal will provide an opportunity to focus the topic of immunology of ageing, which is emerging as one of the critical mechanisms of ageing which aspects should get to all scientists physicians and other professions who are involved in the different inter-related disciplines, and the bridge that is emerging between these fields.Furthermore, as an online, Open Access journal,  Immunity & Ageing  will promote "immediate" accessibility to this fast moving area of research, which is generally not possible for articles published in printed journals. From a modern perspective, electronic publishing is obviously attractive for its speed, easy global access and low cost, which all present considerable advantages over print and are seen as attractive factors by authors, readers and publishers. Open Access appears to provide exactly what people would like: rapid publication for authors, free access to information for readers, and inexpensive, global availability to readers for publishers [ 23 ]. We believe knowledge should belong to those who want it and access should not be unjustly and unacceptably expensive or difficult.  Immunity & Ageing  will take advantage of the Open Access policy to make peer-reviewed information widely and almost immediately available. We hope this forum, focussing on the themes of ageing and immunology, will create a new perspective for looking at a world that is inevitably becoming older. Creating and pushing forward a research knowledge base in  Immunity & Ageing  provides a unique opportunity to dissect out some parameters which might make health span equate with increasing lifespan.Immunity & AgeingImmunity & AgeingImmunity & Ageing


Background In Down syndrome patients several metabolic abnormalities have been reported, some involving the lipid metabolism. The level of LDL in plasma is the major determinant of the risk of vascular disease. There appear to be no studies on the LDL receptor in Down syndrome patients.
Background
In Down syndrome patients several metabolic abnormalities have been reported, some involving the lipid metabolism. The level of LDL in plasma is the major determinant of the risk of vascular disease. There appear to be no studies on the LDL receptor in Down syndrome patients.Methods Flow cytometric methods for measuring the LDL receptor in peripheral blood mononuclear cells (PBMC) can identify patients with hypercholesterolemia. We applied this method in 19 old patients with Down syndrome and 23 healthy controls.
Methods
Flow cytometric methods for measuring the LDL receptor in peripheral blood mononuclear cells (PBMC) can identify patients with hypercholesterolemia. We applied this method in 19 old patients with Down syndrome and 23 healthy controls.Results Down syndrome patients had high levels of triglycerides and low levels of HDL, and high levels of CRP. We also found a down-regulation of LDL receptor expression.
Results
Down syndrome patients had high levels of triglycerides and low levels of HDL, and high levels of CRP. We also found a down-regulation of LDL receptor expression.Conclusions Down syndrome patients show no increase in the frequency of cardiovascular disease. The low incidence in cardiovascular disease despite the low level of HDL, high levels of CRP and reduction of LDL receptor expression lead to the conclusion that either these are not risk factors in these patients or that other risks factors – not yet identified – are considerably lower.
Conclusions
Down syndrome patients show no increase in the frequency of cardiovascular disease. The low incidence in cardiovascular disease despite the low level of HDL, high levels of CRP and reduction of LDL receptor expression lead to the conclusion that either these are not risk factors in these patients or that other risks factors – not yet identified – are considerably lower.

Introduction Several studies have discussed the psychological and intellectual problems, immunological deficiencies, and early aging of Down syndrome (DS) patients. Several metabolic abnormalities have been reported, some involving the lipid metabolism [ 1 ]. Apart from some contradictory studies in the past, there are only few investigations of the cholesterol fractions in DS patients. Therefore, it must be concluded that the low prevalence of coronary artery disease in individuals with DS cannot be explained by their cholesterol fractions. Mortality statistics of these patients showed practically no deaths due to advanced atherosclerosis [ 2 ], and similarly, pathological studies have detected no increase in atherosclerosis – or even a complete absence of atherosclerotic changes [ 3 ]. In children [ 4 ] and also adolescents [ 5 ] with DS low levels of high-density lipoprotein (HDL) have been reported and recently, we have learned much about the vasoprotective HDL cholesterol [ 6 ]. Anyway DS remains a disease in which atherosclerosis is rare [ 7 ]. Measurements of LDL receptor expression are also necessary to fully characterize the functional status of the low-density lipoprotein (LDL) pathway which substantially influences LDL levels in plasma, and its discovery constituted a major biological advance by providing molecular explanations of hypercholesterolemia. The plasma LDL level is the major determinant of the risk of vascular disease. We analyzed, also, C reactive protein (CRP), a cardiovascular risk factors coded by genes lying on Chromosome 21. Flow cytometric methods for measurement of LDL receptor on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia [ 8 ]. Data in uremic patients suggest that a defect in LDL receptor function in PBMC may be due to a decrease in LDL receptor expression, which could contribute to the aberrant lipoprotein metabolism [ 9 ]. We therefore investigated LDL receptor expression on uninduced PBMC, particularly T lymphocytes because they express more LDL receptors than monocytes [ 10 ]. Since the progression of atherosclerosis is age-dependent, LDL receptor interactions are important in lipid plaque formation and T cells are present in early atherosclerotic lesions, interacting with LDL through the LDL receptor [ 11 ], we studied LDL expression on T lymphocytes in a group of old patients with DS.
Introduction
Several studies have discussed the psychological and intellectual problems, immunological deficiencies, and early aging of Down syndrome (DS) patients. Several metabolic abnormalities have been reported, some involving the lipid metabolism [ 1 ]. Apart from some contradictory studies in the past, there are only few investigations of the cholesterol fractions in DS patients. Therefore, it must be concluded that the low prevalence of coronary artery disease in individuals with DS cannot be explained by their cholesterol fractions. Mortality statistics of these patients showed practically no deaths due to advanced atherosclerosis [ 2 ], and similarly, pathological studies have detected no increase in atherosclerosis – or even a complete absence of atherosclerotic changes [ 3 ].In children [ 4 ] and also adolescents [ 5 ] with DS low levels of high-density lipoprotein (HDL) have been reported and recently, we have learned much about the vasoprotective HDL cholesterol [ 6 ]. Anyway DS remains a disease in which atherosclerosis is rare [ 7 ].Measurements of LDL receptor expression are also necessary to fully characterize the functional status of the low-density lipoprotein (LDL) pathway which substantially influences LDL levels in plasma, and its discovery constituted a major biological advance by providing molecular explanations of hypercholesterolemia. The plasma LDL level is the major determinant of the risk of vascular disease. We analyzed, also, C reactive protein (CRP), a cardiovascular risk factors coded by genes lying on Chromosome 21. Flow cytometric methods for measurement of LDL receptor on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia [ 8 ]. Data in uremic patients suggest that a defect in LDL receptor function in PBMC may be due to a decrease in LDL receptor expression, which could contribute to the aberrant lipoprotein metabolism [ 9 ].We therefore investigated LDL receptor expression on uninduced PBMC, particularly T lymphocytes because they express more LDL receptors than monocytes [ 10 ]. Since the progression of atherosclerosis is age-dependent, LDL receptor interactions are important in lipid plaque formation and T cells are present in early atherosclerotic lesions, interacting with LDL through the LDL receptor [ 11 ], we studied LDL expression on T lymphocytes in a group of old patients with DS.Methods Blood samples were drawn from 19 old DS patients (male, average age 55 years) and 23 healthy individuals (male, average age 55 years) without dyslipidemia or any family history of coronary heart disease, no smokers or drunkers, with a Body Mass Index (BMI) < 25. Lipid measurements are given in Table  1 . Plasma C reactive protein (CRP) concentration form DS and control was evaluated by LANIA (Latex Agglutination Nephelometric Immunoassay) technique (Biolatex, Spain). Samples were diluted 1:36 and results were calculated automatically by IMMAGE system. The minimum detectable concentration was 0.4 mg/dl. Table 1 Cholesterol fractions in old patients with Down syndrome and healthy subjects. Means ± SD. Healthy subjects Down syndrome Total cholesterol 150 ± 19.64 152 ± 28.79 Triglycerides 55.9 ± 21.46 104.5 ± 50.2 HDL-cholesterol 48.4 ± 10.5 40.6 ± 4.24 LDL-cholesterol 88.3 ± 17.2 89 ± 24.4 None had been treated with lipid-lowering drugs before blood sampling. This study was conducted in accordance with the Declaration of Helsinki, 1975, amended in 1983. Blood, collected in tubes containing EDTA, was cooled to 20°C and diluted 1:1 with Hank's buffered saline solution (HBSS, Biochrome, Biospa, Milan, Italy). PBMCs were prepared under sterile conditions, using Ficoll-Hypaque (Pharmacia Biotech, Milan, Italy) and diluted blood was layered in a centrifuge tube and centrifuged for 40 min at 400  g , 20°C. The interface containing the PBMCs was isolated, and the cells were washed three times in HBSS and resuspended in RPMI-1640 (Biochrome, Biospa, Milan, Italy) with L-glutamine (290 mg/L), penicillin (100,000 U/L), streptomycin (100 mg/L) and 100 mL/L human lipoprotein-deficient serum (HLPDS) to a final concentration of 10 6  cells/mL. Tissue culture flasks were placed in ice-water for 60 min in the dark to reduce cell adhesion. PBMCs were removed by flushing with ice-cold HBSS (4°C) and washed twice in ice-cold HBSS with 20 mL/L HLPDS. The cell number was adjusted to 0.3 × 10 6  cells/mL, and 100-μL aliquots of cell suspension were pipetted into polypropylene tubes and placed in ice-water. Cells were incubated with 1.5 μg of monoclonal mouse anti-human LDL receptor-specific antibody, clone C7 (Amersham Life Science, Milan, Italy), for 30 min in the dark at 4°C. After this the cells were washed twice in ice-cold HBSS with 20 mL/L HLPDS, and incubated with 3 μL of fluorescein isothiocyanate (FITC, Dako Cytomation, Milan, Italy) for 30 min in the dark at 4°C. Cells were then incubated with 1 μL of R-phycoerythrin (RPE)-conjugated monoclonal antibody CD3-RPE or IgG 1 isotype-RPE for T lymphocytes. The flow cytometry measurements were done in a FACScan flow cytometer (Becton Dickinson, Milan, Italy) equipped with a 15 mW, 488 nm, air-cooled argon laser and linked to a computer with CellQuest software. Forward scatter (FSC) and side scatter (SSC) were adjusted to exclude debris and dead cells. FITC emission was measured at 530 nm (FL1) and RPE emission at 585 nm (FL2); compensation was set using FITC-conjugated C7 (C7 FITC)-labeled cells (FL2-FL1) and CD3-RPE-labeled cells (FL1-FL2). Means were compared by the unpaired  t -test or one-way analysis of variance (ANOVA). Data are presented as means ± SD. Differences were considered statistically significant at  p  < 0.05.
Methods
Blood samples were drawn from 19 old DS patients (male, average age 55 years) and 23 healthy individuals (male, average age 55 years) without dyslipidemia or any family history of coronary heart disease, no smokers or drunkers, with a Body Mass Index (BMI) < 25. Lipid measurements are given in Table  1 . Plasma C reactive protein (CRP) concentration form DS and control was evaluated by LANIA (Latex Agglutination Nephelometric Immunoassay) technique (Biolatex, Spain). Samples were diluted 1:36 and results were calculated automatically by IMMAGE system. The minimum detectable concentration was 0.4 mg/dl.Cholesterol fractions in old patients with Down syndrome and healthy subjects. Means ± SD.Healthy subjectsDown syndromeTotal cholesterolTriglyceridesHDL-cholesterolLDL-cholesterolNone had been treated with lipid-lowering drugs before blood sampling. This study was conducted in accordance with the Declaration of Helsinki, 1975, amended in 1983.Blood, collected in tubes containing EDTA, was cooled to 20°C and diluted 1:1 with Hank's buffered saline solution (HBSS, Biochrome, Biospa, Milan, Italy). PBMCs were prepared under sterile conditions, using Ficoll-Hypaque (Pharmacia Biotech, Milan, Italy) and diluted blood was layered in a centrifuge tube and centrifuged for 40 min at 400  g , 20°C. The interface containing the PBMCs was isolated, and the cells were washed three times in HBSS and resuspended in RPMI-1640 (Biochrome, Biospa, Milan, Italy) with L-glutamine (290 mg/L), penicillin (100,000 U/L), streptomycin (100 mg/L) and 100 mL/L human lipoprotein-deficient serum (HLPDS) to a final concentration of 10 6  cells/mL.gTissue culture flasks were placed in ice-water for 60 min in the dark to reduce cell adhesion. PBMCs were removed by flushing with ice-cold HBSS (4°C) and washed twice in ice-cold HBSS with 20 mL/L HLPDS. The cell number was adjusted to 0.3 × 10 6  cells/mL, and 100-μL aliquots of cell suspension were pipetted into polypropylene tubes and placed in ice-water. Cells were incubated with 1.5 μg of monoclonal mouse anti-human LDL receptor-specific antibody, clone C7 (Amersham Life Science, Milan, Italy), for 30 min in the dark at 4°C. After this the cells were washed twice in ice-cold HBSS with 20 mL/L HLPDS, and incubated with 3 μL of fluorescein isothiocyanate (FITC, Dako Cytomation, Milan, Italy) for 30 min in the dark at 4°C. Cells were then incubated with 1 μL of R-phycoerythrin (RPE)-conjugated monoclonal antibody CD3-RPE or IgG 1 isotype-RPE for T lymphocytes.The flow cytometry measurements were done in a FACScan flow cytometer (Becton Dickinson, Milan, Italy) equipped with a 15 mW, 488 nm, air-cooled argon laser and linked to a computer with CellQuest software. Forward scatter (FSC) and side scatter (SSC) were adjusted to exclude debris and dead cells. FITC emission was measured at 530 nm (FL1) and RPE emission at 585 nm (FL2); compensation was set using FITC-conjugated C7 (C7 FITC)-labeled cells (FL2-FL1) and CD3-RPE-labeled cells (FL1-FL2).Means were compared by the unpaired  t -test or one-way analysis of variance (ANOVA). Data are presented as means ± SD. Differences were considered statistically significant at  p  < 0.05.tpResults Table  1  shows cholesterol fractions of DS patients and healthy controls. DS total cholesterol and LDL did not differ from controls ( p  = 0.8 and  p  = 0.9 respectively). Blood levels of CRP were higher in DS than in controls, as illustrated in Figure  1 (Controls 1.3 ± 0.3; DS = 5.7 ± 4.6 mg/L, p < 0.01). A regression analysis of data shows non relationship among CRP and cholesterol-related molecule levels. Figure 1 Levels of C reactive protein (CRP) in plasma from children with DS and age matched controls. Data are presented as mean ± S.D. Triglycerides were higher, and HDL lower in DS patients ( p  < 0.01 and  p  < 0.05). Our data also show that the expression of LDL receptor on T lymphocytes was down-regulated in DS patients (Table  2  and Figure  2 ). Table 2 Mean fluorescence intensity (MFI, %) of LDL receptor expression in healthy subjects and old patients with Down syndrome. Means ± SD. Healthy subjects Down syndrome MFI (%) 196.76 ± 20.54 139.87 ± 13.32 Figure 2 Mean fluorescence intensity (MFI) of LDL receptor expression in healthy subiects (bold line) and Down syndrome old subjects (fine line). Mean intensity fluorescence (MIF) of LDL receptor expression was significantly different in DS patients and healthy controls ( p  < 0.0001) (Table  2 ).
Results
Table  1  shows cholesterol fractions of DS patients and healthy controls. DS total cholesterol and LDL did not differ from controls ( p  = 0.8 and  p  = 0.9 respectively). Blood levels of CRP were higher in DS than in controls, as illustrated in Figure  1 (Controls 1.3 ± 0.3; DS = 5.7 ± 4.6 mg/L, p < 0.01). A regression analysis of data shows non relationship among CRP and cholesterol-related molecule levels.ppTriglycerides were higher, and HDL lower in DS patients ( p  < 0.01 and  p  < 0.05). Our data also show that the expression of LDL receptor on T lymphocytes was down-regulated in DS patients (Table  2  and Figure  2 ).ppMean fluorescence intensity (MFI, %) of LDL receptor expression in healthy subjects and old patients with Down syndrome. Means ± SD.Healthy subjectsDown syndromeMFI (%)Mean intensity fluorescence (MIF) of LDL receptor expression was significantly different in DS patients and healthy controls ( p  < 0.0001) (Table  2 ).pDiscussion Patients with DS who reach adolescence nowadays have a nearly normal life expectancy thanks to better medical care. When they die at a later age, cardiovascular diseases are less common than in the general population and they have even been proposed as "an atheroma-free model" [ 3 ]. Our results concerning cholesterol fractions suggest that DS patients should have a cardiovascular disease risk, if conclusions valid for the general population can be transferred to this category of patients. Although serum lipoprotein profiles cannot explain the lower prevalence of cardiovascular disease in individuals with DS, our triglyceride and HDL findings are in line with published figures. Similar findings were reported by other authors but in young patients [ 4 , 5 ]. Multiple factors are responsible for atherosclerosis, such as dietary habits but still the unexplained decline of LDL receptor expression with aging contributes importantly to borderline-high levels and cannot be ignored. For example the loss of estrogen-stimulated LDL receptor synthesis after menopause is an important contributor to elevated cholesterol in postmenopausal women. In addition, several genetic defects inherited singly appear to be causes of moderate hypercholesterolemia [ 10 , 12 ]. Generally defects of LDL receptor expression are associated with a high risk of premature atherosclerosis. In the elderly LDL receptor uptake is unexpectedly increased, and LDL receptor regulation and expression and serum LDL composition seem abnormal. There may also be alterations to the lipid metabolism of immune system cells during aging [ 13 ]. In our series, a reduction in LDL receptor expression was not correlated with high LDL serum levels or total cholesterol. Moreover, lipophospholipid (LPC) is generated by hydrolysis of phosphatidylcholine which is present in LDL; LPC may promote the start of an immune response and atherosclerosis may be the most extreme demonstration of this immune regulation pathway [ 14 ]. LPC may be a potent super-regulator of T-cell activation by inflammation at sites of tissue damage and in the early stages of atherosclerosis. Interestingly, DS patients show no increase in their frequency of cardiovascular disease. These conditions may be explained by mild immune defects in the syndrome, mainly involving macrophages and/or T H 1 lymphocytes responses [ 15 ]. Alternatively, a over expression of atherosclerotic protective factors -yet unknown- maight be present in Down syndrome. As we reported earlier, the low incidence of cardiovascular disease in these patients and the high-risk factor of oxidatively modified LDL (oxLDL) [ 16 ] with – in this study – the low level of HDL, high levels of CRP and reduction of LDL receptor expression, lead to the conclusion that in this group of "healthy old" DS subjects, classical biochemical risk factors for atherosclerosis have been detected but risks, probably, are considerably lower.
Discussion
Patients with DS who reach adolescence nowadays have a nearly normal life expectancy thanks to better medical care. When they die at a later age, cardiovascular diseases are less common than in the general population and they have even been proposed as "an atheroma-free model" [ 3 ]. Our results concerning cholesterol fractions suggest that DS patients should have a cardiovascular disease risk, if conclusions valid for the general population can be transferred to this category of patients. Although serum lipoprotein profiles cannot explain the lower prevalence of cardiovascular disease in individuals with DS, our triglyceride and HDL findings are in line with published figures. Similar findings were reported by other authors but in young patients [ 4 , 5 ].Multiple factors are responsible for atherosclerosis, such as dietary habits but still the unexplained decline of LDL receptor expression with aging contributes importantly to borderline-high levels and cannot be ignored. For example the loss of estrogen-stimulated LDL receptor synthesis after menopause is an important contributor to elevated cholesterol in postmenopausal women. In addition, several genetic defects inherited singly appear to be causes of moderate hypercholesterolemia [ 10 , 12 ]. Generally defects of LDL receptor expression are associated with a high risk of premature atherosclerosis. In the elderly LDL receptor uptake is unexpectedly increased, and LDL receptor regulation and expression and serum LDL composition seem abnormal. There may also be alterations to the lipid metabolism of immune system cells during aging [ 13 ].In our series, a reduction in LDL receptor expression was not correlated with high LDL serum levels or total cholesterol. Moreover, lipophospholipid (LPC) is generated by hydrolysis of phosphatidylcholine which is present in LDL; LPC may promote the start of an immune response and atherosclerosis may be the most extreme demonstration of this immune regulation pathway [ 14 ]. LPC may be a potent super-regulator of T-cell activation by inflammation at sites of tissue damage and in the early stages of atherosclerosis.Interestingly, DS patients show no increase in their frequency of cardiovascular disease.These conditions may be explained by mild immune defects in the syndrome, mainly involving macrophages and/or T H 1 lymphocytes responses [ 15 ]. Alternatively, a over expression of atherosclerotic protective factors -yet unknown- maight be present in Down syndrome. As we reported earlier, the low incidence of cardiovascular disease in these patients and the high-risk factor of oxidatively modified LDL (oxLDL) [ 16 ] with – in this study – the low level of HDL, high levels of CRP and reduction of LDL receptor expression, lead to the conclusion that in this group of "healthy old" DS subjects, classical biochemical risk factors for atherosclerosis have been detected but risks, probably, are considerably lower.List of abbreviations Down syndrome (DS); high-density lipoprotein (HDL); C reactive protein (CRP); Body Mass Index (BMI); low-density lipoprotein (LDL); peripheral blood mononuclear cells (PBMC); lipophospholipid (LPC).
List of abbreviations
Down syndrome (DS); high-density lipoprotein (HDL); C reactive protein (CRP); Body Mass Index (BMI); low-density lipoprotein (LDL); peripheral blood mononuclear cells (PBMC); lipophospholipid (LPC).Competing interest The author(s) declare that they have no competing interests.
Competing interest
The author(s) declare that they have no competing interests.