Two contradictory hypotheses have been proposed to explain the relationship between allergic conditions and malignancies, the immune surveillance hypothesis and the antigenic stimulation hypothesis. The former advocates that allergic conditions may be protective against development of cancer, whereas the latter proposes an increased risk. This relationship has been studied in several case-control studies, but only in a few cohort studies.The association between allergic conditions and risk of developing leukemia, Hodgkin's disease, non-Hodgkin's lymphoma and myeloma was investigated in a cohort of 16,539 Swedish twins born 1886–1925. Prospectively collected, self-reported information about allergic conditions such as asthma, hay fever or eczema was obtained through questionnaires administered in 1967. The cohort was followed 1969–99 and cancer incidence was ascertained from the Swedish Cancer Registry.Hives and asthma tended to increase the risk of leukemia . There was also an indication of an increased risk of non-Hodgkin's lymphoma associated with eczema during childhood (RR = 2.3, 95% CI 1.0–5.3).In contrast to most previous studies, our results do not indicate a protective effect of allergic conditions on the risk of developing hematological malignancies. Rather, they suggest that allergic conditions might increase the risk of some hematological malignancies. An association between allergic conditions and cancer risk has been the subject of several epidemiological studies. Both positive and negative associations have been observed and two hypotheses have been formulated to explain such relationships. First, the immune surveillance hypothesis, which proposes that allergic conditions may lead to a decreased risk of malignancy by enhancing the ability of the immune system to detect and eliminate malignant cells . EvidencMost previous studies are retrospective case-control studies. Recall bias may have influenced the results of retrospective case-control studies that have asked for past allergic conditions after diagnosis of malignancy -5,7,8, aThe purpose of the present study was to investigate the influence of allergic conditions on the risk of developing leukemia, malignant lymphoma and myeloma in a well-established cohort of Swedish twins, and to see whether the results support the immune surveillance hypothesis or the antigenic stimulation hypothesis. An important strength of this study is that information about allergic conditions and confounding factors has been collected prospectively, and therefore the exposure is not subject to differential misclassification.The study base consists of a cohort of twins followed by the Swedish Twin Registry, which was established in 1961 when questionnaires were sent to all 25,778 individuals in a same-sexed twin pair who were born between 1886 and 1925 and were both living in Sweden in 1961. A response from both twins in a twin pair was required for inclusion in the Swedish Twin Registry  . After 1th and 90th percentiles; 46 and 71 years, respectively). The cohort was followed from January 1, 1969 until diagnosis of a hematological malignancy, death or end of the study (December 31, 1999), whichever came first. Cancer incidence and date of death were ascertained by record linkage to the Swedish Cancer Registry and the Swedish Cause of Death Registry, respectively. In the Swedish Cancer Registry, leukemia is coded according to ICD-8 during the investigated period, while all other malignancies are coded according to ICD-7. We identified 324 subjects with hematological malignancies; 10 cases of HD (ICD-7 201), 112 cases of NHL (ICD-7 200.0–200.3, 202.0–202.4), 75 cases of myeloma (ICD-7 203) and 134 cases of leukemia (ICD-8 204.0–207.9). The leukemias consist of 3 cases of ALL, 67 cases of CLL, 31 cases of AML, 7 cases of CML and 26 cases of unspecified leukemia.The twin cohort was used as a population-based cohort without considering twin status. The present study includes the 16,539 individuals (7,167 men and 9,372 women), who responded to the questionnaire mailed in 1967, and who were still alive and not previously diagnosed with a hematological malignancy January 1, 1969. The median follow-up time was 23 years for men and 27 years for women. The median age at baseline was 56 years , "Have you ever had hay fever, rose fever or allergic rhinitis (characterized by running nose, watery and itching eyes when you do not have a cold)?" (No/Yes), "Did you have eczema when you were a baby?" (No/Yes), "Did you at times later in life have eczema-like skin conditions?" (No/Yes), Do you know the name of the skin lesion you have or have had? (No/Yes, psoriasis/Yes, hives (urticaria) allergic rash/Yes, contact eczema/Yes, eczema in knee or elbow fold/Yes, allergic eczema/Yes, others: specify name). There were no questions about dates when symptoms first started or ended, and no information about treatments used. In the questionnaire from 1963 responses to two questions about asthma and eczema were included. The subjects could mark if they had had asthma or eczema from a list of 17 diseases or the alternative that they hadn't had any of the given diseases. The answer from 1963 was used only if a subject had failed to answer the corresponding question in the 1967 questionnaire. Each medical condition was analyzed separately. In addition we combined the different conditions in order to achieve larger numbers of individuals and thereby obtain more precise results. First, a variable for eczema was created, requiring at least one positive answer for childhood eczema or allergic eczema. Then, a general group of allergic conditions was created, combining the positive answers for eczemas with positive answers on the questions of ever having had asthma or hay fever.All analyses were adjusted for age at enrolment and sex. We have controlled for confounding from alcohol consumption (g/month), level of education, and smoking habits (non-smokers, former smokers, current smokers). Adjustment for these factors did not affect the risk estimates in the majority of analyses, and changed the magnitude of the effect at the most 6% in a few instances. Therefore, the presented results are only adjusted for age and sex.We estimated the RR and its 95% CI of each hematological malignancy through Cox's Proportional Hazards Model, (SAS program PHREG, SAS Institute, Cary, North Carolina). To ensure that confidence intervals were not erroneously narrowed due to dependencies within twin pairs we performed analyses that adjusted variance estimates for correlated outcomes. We accomplished this through the use of a SAS macro that stems from the same theoretical background -24 and yOur results showed either increased risks or risks close to unity for hematological malignancies following allergic conditions. For leukemia, we found an increased risk associated with hives, and an indication of elevated risk associated with asthma, although with wide confidence intervals Table . For leuThe risk estimates for myeloma were generally close to or below unity Table . The numOur results suggested that allergic conditions are risk factors for hematological malignancies, and gave support to the antigenic stimulation hypothesis. Thus, the results are in concordance with most previous cohort studies ,15,18,19per se on the immune system may be difficult. This study has focused specifically on how allergy influences the risk of developing hematological malignancies.A major strength of the present cohort study is that information about allergic conditions and confounding factors has been collected prospectively. Therefore the exposure is not subject to differential misclassification, which is in contrast to retrospective case-control studies where recall bias may be a problem ,3,9 and Another strength is that the study is based on the Swedish Twin Registry, which is a unique resource allowing for an unusually long period of follow-up. In our study, 31 years of follow-up was possible. The cohort has been followed continuously in the Population Registry and the Cause of Death Registry during the study period, and therefore loss to follow-up is unlikely to be a problem. The Swedish Twin Registry is considered a study base representative of the general population of Sweden and has been used in many epidemiological studies -27. AnotOne limitation of the study is the small number of exposed cases, and therefore random variation cannot be excluded as an explanation for our findings and for the same reason no stratification for calendar time was performed. Another limitation in the study is that there may be non-differential misclassification of the malignancies. The study period covers 31 years, and during this time diagnostic practices may have changed. In particular, some cases previously diagnosed as HD are now likely to be classified as NHL . This tyDifferential misclassification of exposure is unlikely as there is no reason to believe that reporting exposure should differ between subjects subsequently (years later) diagnosed with a cancer, and those who are not. Non-differential misclassification of exposure is likely to affect the results, but cannot explain increased risks since it would dilute the effect estimates towards unity. The allergic conditions are self-reported and not diagnosed by a physician. However, these self-reported conditions have been used in an earlier study of brain tumors, where some support for the postulated hypothesis that allergic conditions are associated with a decreased risk of developing glioma was found . Also, tWe found an increased risk of NHL among individuals with eczema during childhood. In the literature, there are only few studies concerning an association between eczema and NHL. In one study, a history of eczema was associated with an increased risk of NHL . In seveIn our material subjects with hives showed an increased risk of leukemia, especially after exclusion of CLL. Several other studies have also found an increased risk of AML  and otheClearly, these conflicting results indicate that this area needs to be investigated further. Allergic conditions, like asthma and hay fever, are increasing and it is of great importance to clarify if and how they are connected to hematological malignancies. The contradictory findings may have many explanations, e.g. that different immunological mechanisms may be involved in different types of asthma, that the pathogenesis is likely to be different even in seemingly similar hematological malignancies, and that new forms of pharmacological therapy may influence not only the outcome of asthma but also the risk of developing cancer. To solve these problems, large prospective epidemiological studies on individuals with clinically strictly defined allergic conditions, including data on pharmacological treatment and severity of disease, need to be combined with information about morphologically defined hematological malignancies, including subtyping with techniques from modern molecular biology.In summary, findings from our cohort study suggest that chronic antigenic stimulation from allergic conditions might increase the risk of some hematological malignancies.ALL = Acute lymphoblastic leukemia; AML = Acute myeloid leukemia; CLL = Chronic lymphocytic leukemia; CML = Chronic myeloid leukemia; HD = Hodgkin's disease; NHL = Non-Hodgkin's lymphoma; RR = Relative risk; CI = Confidence Interval.The author(s) declare that they have no competing interests.KCS has been the principal investigator, contributed to the planning of the study, performed the statistical analysis and drafted and coordinated the writing of the manuscript. LH participated in the planning of the study and the writing of the manuscript. JS contributed to the writing of the manuscript. MF carried out the study design and contributed to the writing of the manuscript. All authors contributed to the interpretation of results, have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


This review article is aimed at comparing the results of histopathological and clinical imaging studies to assess coronary atherosclerotic plaques in humans. In particular, the gap between the two techniques and its effect on the understanding of the pathophysiological basis of coronary artery disease is critically discussed. Amongst the clinical approaches ultrasound imaging is one of the more promising technique to understand dysfunction. The need is to compare morphopathological counterpart to have a correct pathophysiological interpretation.In four reviews, morphopathology of the main cardiovascular disorders, in relation to the status of art of clinical imaging will be presented. The aim is to recall the pathological anatomy to stimulate ultrasound experts to further sharpen their technology till "histological" perfection.The present first review concerns the coronary atherosclerosis since the current dogma of "unifying theory" assumes that the acute coronary syndromes, namely unstable angina, myocardial infarct and sudden death, are caused by atherosclerotic plaque rupture or "explosion" with occlusive thrombus formation preceded by intramyocardial emboli. An assumption which implies to discover a clinical imaging able to show when a coronary atherosclerotic plaque becomes vulnerable i.e. prone to rupture.The risk of the latter has been correlated with large lipid core (atheroma in our definition), thin fibrous cap (< 65 μm) covering atheroma. Therefore any imaging should have a 50 μm resolution to identify a fibrous cap prone to rupture, 100 μm or 150 μm thickness being respectively at low or minimal risk. Matrix-digesting enzymes released from inflammatory cells (monocytes, macrophages, T-cells, B-cells, neutrophils, mastcells) may contribute to plaque rupture. An attractive approach since, despite many years of preventive and therapeutical attempts, coronary heart disease (CHD) remains the main cause of death and morbidity in advanced societies and selects people at the top of their work skillness and productivity.The first question is whether ultrasound imaging may or may not discriminate extent and morphology of plaque variables seen within the intima.physiological intimal thickening [Morphology of the atherosclerotic plaque has been described in textbook and articles -4. In coickening -5 which The first conclusion is that such intimal thickening is a physiologic structural respons to hemodynamics and not the initial phase of atherosclerosis as claimed by some authors.In contrast to the uniformely diffuse physiological intimal thickening, the atherosclerotic one is focal and protrudes within the lumen which is progressively reduced. In order to quantitatively study this progression, we sampled systematically in each heart the first tract of the main left trunk (LCA), left descending (LAD) and circumflex (LCX) branches, right coronary artery (RCA), posterior descending branch (PD) and the middle tract of LAD and marginal and posterior tracts of RCA. These selected tracts correspond to the sites where atherosclerotic changes generally occur. The coronary arterial sampling was performed in 100 fatal cases of acute myocardial infarct without other diseases and not undergone invasive techniques; 208 cases of sudden and unexpected coronary death (SUD) which occurred in apparently normal people, acting their usual life, without resuscitation attempts and autopsy findings limited to coronary atherosclerosis of any degree, myocardial necrosis or scar, with or without cardiac hypertrophy: 50 cases with chronic angina pectoris who died within 25 day after coronary by-pass surgery; and 97 normal subjects who died by accident without pathological findings at autopsy but coronary atherosclerosis. In a total of 3,640 coronary sections the following variables were quantified:Lumen reduction calculated in percent of the normal diameter measured in normal coronary arteries and branches injected by plastic substance under pressure. Measurement often referred to the cross-sectional area within the internal elastic membrane may result in severe stenosis despite a normal lumen since the atherosclerotic plaque may enlarge the cross-section.1. Shape of residual lumen: concentric if encircled by pathological intima or semilunar when an arch of the wall was normal.2. Length calculated in number of segments involved by the plaque, all extramural coronary arteries being sistematically cross-sectioned at 3 mm interval.3. 4. Intimal and tunica media thickness measured in microns.Atherosclerotic changes within the intima: fibrosis alone, basophilia i.e. proteoglycan accumulation, atheroma or lipoprotein/cholesterol material, calcification, vascularization, hemorrhage, adventitial and intimal lymphocytic infiltration. All these variables were expressed in percent of the total intima but vascularization calculated in number of vessels found.5. Amongst 1,519 sections without lumen reduction with an intimal physiological thickness less of 300 μm we never found subendothelial or internal lipoprotein/cholesterol infiltration or deposit (fatty streaks), monocytes or macrophages or foam cells, platelet aggregates, fibrin-platelet thrombi or inflammatory elements. A similar negative finding was observed in 1,315 coronary sections with a lumen reduction less than 69% and pathological intimal thickness less than 600 μm. In all these sections we were unable to demonstrate an intimal fissuration. We must emphasize that in selecting our material cases of familial hypercholesterolemia were excluded.In general the atherosclerotic intimal variables increased in frequency and extent in parallel with the lumen reduction and pathological intimal thickeness with the exception of proteoglycan accumulation less found in stenoses >90% and intimal thickness >2000 μm. Of 990 sections with calcification, 488 (49%) had mild stenosis, calcification being severe in 162 (33%). This finding indicates that calcification per se does not necessarily means a severe lumen reduction. The less frequent variable was intimal hemorrhage mainly seen in plaques located in a vessel tributary to an acute infarct.When different groups of CHD and normal people were matched, an excess of atheroma, hemorrhage, calcification and adventitial/intimal lymphocytic inflammation was observed in AMI group while a significant defect was present in normal subjects with the same degree of stenosis. In synthesis, two other main findings are worth of mention: 1) proteoglycan accumulation is a relatively late event which occurred in the deep layer of the intima near to the tunica media and below the fibrous cap of a plaque. Lipoprotein/cholesterol plus macrophages (foam cell) and/or calcium salts appeared only in this proteoglycan pool in agreement with their interaction with glycosaminoglycans Fig. ; 2) advemyohyperplastic plaque is totally different from the hypercholesterol plaque obtained experimentally by hypercholesterol diet in animals free of atherosclerosis or observed in a small group of patients with familial hypercholesterolemia. In literature too often the hypercholesterol plaque is taken as a model of an atherosclerotic plaque in man [This study induced the recognition of two types of coronary atherosclerotic plaque: one, which belongs to the general population, (including CHD patients) and starts as nodular intraluminal proliferation of smooth muscle cells followed by elastic tissue hyperplasia and final substitution by fibrous tissue. Subsequently, a deep proteoglycan pool forms and becomes a deposit of lipoprotein/cholesterol and/or calcium salts. The recurrence of these phenomena explains the radial, circumferential, longitudinal progression of the coronary plaque resulting in increasing lumen reduction. This type of e in man . In timiThe recurrent basic changes in myohyperplastic plaque  explain the various intimal aspects amongst different plaques and different tracts of the same plaque Fig. . A synopDifferent echocardiographic techniques have been employed in the attempt to provide adequate visualization of coronary arteries Table . Howeverready to go for clinical use. In particular, definition of plaque composition seems not enough reproducible to provide an alternative independent standard to quantitative histology [ICUS image analysis has been extensively used for determining plaque composition -13. Howeistology .New technical development based on noninvasive molecular imaging ,16, suchThe working hypothesis is to stabilize the atherosclerotic plaque by increasing the thickness of the fibrous cap or by regression of atheroma burden (1). However, present imaging techniques (coronary cineangiography, angioscopy, contrast magnetic resonance, contrast echocardiography, nuclear scintigraphy, etc.) cannot provide adequate clinical evaluation of plaque vulnerability. In particular, ICUS is unable to provide discrimination between physiological and pathological intimal thickening and to define the shape of plaque, i.e. concentric or semilunar. In fact, amongst 2121 coronary sections at the site of maximal lumen-diameter reduction the stenosis was concentric in 70% of the cases . Furthermore, in 408 CHD patients the maximal stenosis in each single case was less than 69% in 68, 70%, in 67, 80% in 109 and >90% in 164. These data mean that the residual lumen ranged from 900 to less than 50 μm and catheter of 1500 μm in most instances must break the plaque, being the residual lumen too small. Therefore, shape and contour of a plaque can be altered with a misleading higher frequency of semilunar stenosis giving an erroneous support to the questionable concept of vessel wall remodelling following an atherosclerotic plaque formation.According to the previous data the main conclusions are: 1. The natural history of coronary atherosclerotic plaque in the general population, inclusive of CHD patients, is different from plaques obtained by experimental hypercholesterol diet or found in familial hypercholesterolemia. Most data refer to the latter as a valid model for the human plaque. In particular, fatty streaks is not the starting change of the myohyperplastic atherosclerotic plaque; 2. Emphasis is given to a "macrophagic inflammation" as source of proteolytic and/or thrombogenic moleculae causing plaque rupture. However, macrophagic reaction belongs to a repair process to digest necrotic or extraneous material rather than typical elements of an inflammatory process, as lymphocytes, neutrophils. The assumption that on increased number of labelled macrophages may indicate a risk of rupture is questionable in human coronary myohyperplastic plaque.In the present review we have discussed the behaviour and meaning of components of the human coronary atherosclerotic plaque to emphasize the inconsistency of the current myths:1. Experimental hypercholesterol model and correspondent human conditions do not represent the natural history of atherosclerosis in coronary arteries in the general population.2. Physiological intimal thickening can not be interpreted as starting point of the atherosclerotic process.3. Fatty streak does not represent the early atherosclerotic lesion.4. Calcification is not synonymous of severe stenosis.5. Hemorrhage is not consequent to endothelial fissuration.6. Prevention of macrophage "inflammation" as source of substances able to disrupt the fibrous cap allowing rupture and thrombosis as well as identification of the thickness of fibrous cap to diagnose a vulnerable plaque may have little, if any, sense. Rupture and thrombosis may be secondary phenomena and not the cause of an acute coronary syndrome.7. Degree and number of severe coronary plaques do not predict onset, course, complications and death in CHD.Prof. Giorgio Baroldi contributed to the conception and organization of this review and to the final comments.Dr. Riccardo Bigi and Dr. Lauro Cortigiani summarized the use of ultrasound techniques in atherosclerotic plaque imaging


Leishmania, requires a Th1 type immune response, whereas a dominant Th2 response leads to exacerbated disease. Experimental human zinc deficiency decreases Th1 but not Th2 immune response. We investigated if zinc and copper levels differ in different clinical forms of leishmaniasis, and if these trace metals might be involved in the immune response towards the parasite.The process of elimination of intracellular pathogens, such as Leishmania humoral and cellular immune response were evaluated by quantifying specific plasma IgG, lymphoproliferation and cytokine production, respectively. Plasma levels of Cu and Zn were quantified by atomic absorption spectrophotometry.Blood was collected from 31 patients with either localized cutaneous (LCL), mucosal (ML) or visceral (VL) leishmaniasis, as well as from 25 controls from endemic and non-endemic areas. Anti-Leishmania IgG (Spearman r = 0.65, p = 0.0028). Cu/Zn ratios were highest in patients with deficient cellular (VL<<LCL<ML) and exacerbated humoral (VL>LCL>ML) immune response. Ex vivo production of parasite-induced IFN-γ was negatively correlated to plasma Cu levels in LCL (r = -0.57, p = 0.01). In vitro, increased Cu levels inhibited IFN-γ production.A significant decrease in plasma Zn was observed in all three patient groups (p < 0.01 for LCL and ML, p < 0.001 for VL), as compared to controls, but only VL (7/10) and ML (1/7) patients displayed overt Zn deficiency. Plasma Cu was increased in LCL and VL (p < 0.001) but not in ML, and was strongly correlated to anti-1. Zn deficiency in VL and ML indicate possible therapeutic administration of Zn in these severe forms of leishmaniasis. 2. Plasma Cu positively correlates to humoral immune response across patient groups. 3. Environmentally or genetically determined increases in Cu levels might augment susceptibility to infection with intracellular pathogens, by causing a decrease in IFN-γ production. Leishmania, transmitted through the bite of the sand fly vector. Several Leishmania species are able to cause a wide spectrum of clinical manifestations, ranging from the mild cutaneous form, the disfiguring mucosal form and the life-threatening visceral form, also known as kala-azar. In Brazil, Leishmania (L.) braziliensis causes cutaneous and mucosal disease, L. amazonensis causes cutaneous and, sporadically, visceral disease, while L. chagasi is exclusively associated with visceral disease. The clinical outcome of infection thus not only depends on the species involved, but also on the patient's immunocompetence. In recent years, a protective immune response against intracellular pathogens, such as Leishmania, Listeria and mycobacteria, has been defined as type 1 (Th1), whereas protection against extracellular pathogens, such as helminths, requires a type 2 (Th2) response. The murine model of experimental leishmaniasis has been instrumental for the elaboration of the Th1/Th2 paradigm, inasmuch as the preferential action of Th1 (IFN-γ, IL-12, TNF-α) or Th2 cytokines (IL-4, IL-5, IL-10) results in cure or progression of the disease, respectively [Leishmania IgG) immune response can be observed in different clinical forms of the disease. Our group has recently shown that patients with localized cutaneous leishmaniasis (LCL) display a diminished Th1 response during the early phase of disease, which is reverted after treatment [in vivo [L. major [Leishmaniasis is endemic in several parts of the world, with a global prevalence of over 12 million cases and 1.5–2 million new cases emerging every year [ectively . In humareatment , we were[in vivo . Althoug[in vivo , only feL. major . In thisLeishmania braziliensis and, sporadically, Leishmania amazonensis. During a one year period, 14 patients with LCL (single lesion with less than 4 weeks of duration) were selected and treated (20 mg/kg of Sb (Glucantime) i.v. during 20 days). Of those, only 7 patients returned to draw blood after 3 months of treatment, but all patients cured during follow-up. Seven patients with ML were selected after several rounds of unsuccessful treatment and severe disease progression. Blood samples from 10 patients (at diagnosis, before treatment) with VL were obtained from two different urban areas . Ten healthy urban controls were recruited among students and laboratory staff . Diagnosis was confirmed by Montenegro skin test, serology, direct culture of parasites from lesions [Blood samples (10 ml heparinized tubes, Vacutainer) from 21 patients and 15 healthy controls (mostly patient's relatives) were obtained in an outpatient clinic in the district of Corte de Pedra, (Bahia state, Northeast of Brazil). This rural area has a low socio-economic status and a high incidence of infection with Cu and Zn were quantified by atomic absorption spectrophotometry (Varian 220) using an air/acetylene flame. One ml of plasma was diluted tenfold with 0.05 % Triton X-100, 1 % HNO3, sonicated for 10 min and analyzed in triplicate. All reagents used were analytical grade (Merck). Due precautions were taken to avoid external and internal (hemolysis) trace metal contamination.ex vivo cellular immune response, we used a recently described model using whole blood and live Leishmania promastigotes [in vivo events following a sandfly bite. Whole blood was diluted tenfold in culture medium  and stimulated with L. amazonensis promastigotes (105/mL). Buffy coats from normal blood donors were used to obtain large quantities of cells required for in vitro experiments to examine the effect of exogenous trace metals upon cytokine production. Mononuclear cells were separated by density gradient centrifugation  and cultured in complete culture medium . Cu and Zn concentrations were below the detection limit in RPMI and less than 2 μM in complete culture medium. Supernatants were collected after 72 h of culture and frozen in aliquots for cytokine determination. Lymphoproliferation was quantified by measuring [H]-thymidine incorporation after 120 h of culture. IFN-γ, TGF-β1, TNF-α and IL-5 in plasma or culture supernatants were quantified using commercial ELISA kits (DuoSet, R&Dsystems). All results are expressed as mean ± SEM. Statistical evaluation of data was performed using GraphPad Prism software: Mann-Whitney test for comparing patients and control groups, Spearman rank test for correlation and Wilcoxon signed rank test for comparing in vitro treatments; a p-value <0.05 was considered significant.In order to investigate the influence of trace metals, antibodies and other endogenous plasma components, such as cytokines, on the stigotes , closelyA significant decrease in plasma Zn was observed for both LCL and ML patients, as compared to controls from the same endemic area  in untreated patients only, whereas no significant correlation was observed for Leishmania-or mitogen-induced lymphoproliferation, TGF-β1, TNF-α and IL-5 levels in plasma or culture supernatants (not shown). To verify the hypothesis that increased Cu levels might down-regulate IFN-γ production, we added Cu (10 μM, corresponding to the increase of plasma Cu observed ex vivo in LCL patients) to mitogen-or anti-CD3-stimulated in vitro cultures from healthy controls. As shown in Figure 2 significantly decreased anti-CD3-induced IFN-γ production (40.5 ± 9.3 % inhibition, p < 0.05). Interestingly, the addition of physiological concentrations of Zn (10–30 μM) to ex vivo or in vitro cultures did not revert the apparently inhibitory effect of endogenous or exogenous Cu on IFN-γ secretion. (not shown).To determine if these observations reflect possible changes in the patients' immune status, we quantified humoral and cellular anti-VL Table . In addiups Fig. , confirmin vitro [Leishmania immune response, e.g. by leading to a non-protective Th2/humoral immune response, known to be exacerbated in visceral leishmaniasis. Increased plasma Cu cannot be considered as a mere marker of inflammation, since it was not observed in ML, a chronic inflammatory condition characterized by high production of pro-inflammatory cytokines, such as TNF-α and IFN-γ [Leishmania response.Although plasma Zn was significantly decreased in all three patient groups, plain Zn deficiency was only observed in seven VL patients and one ML patient, being absent in LCL patient and in both control groups. In parallel, plasma Cu in VL patients increased to levels which have been shown to be toxic in vitro . In addind IFN-γ . Absencein vitro findings indicate Zn as a monocyte/macrophage activator [Since our ctivator , the sigctivator . We obsectivator ,15.Leishmania and other intracellular pathogens, such as Listeria and mycobacteria, e.g. by directly interfering with cytokine production as previously shown [It is tempting to speculate that environmental exposure to copper might increase susceptibility to ly shown . Increasly shown . In addily shown . Thus, ily shown  and becaly shown ,19 were Administration of Zn in vivo has been shown to down-regulate increased Cu levels in patients with Wilson disease and to revert its toxicity , suggestin vitro (protective) IFN-γ production, implying that environmentally or genetically determined increases in Cu levels might augment susceptibility to infection with intracellular pathogens. Our data indicate that Cu/Zn imbalance can be a useful marker for immune dysfunction in leishmaniasis and suggest that trace metals are implicated in both humoral and cellular anti-Leishmania immune response, which should inspire future strategies for therapy and immunoprophylaxis of human leishmaniasis.Zn deficiency in visceral and mucosal leishmaniasis indicate possible therapeutic administration of Zn in these severe forms of leishmaniasis. Plasma Cu positively correlates to (non-protective) humoral immune response across patient groups, and reciprocally, increased Cu levels decreased The author(s) declare that they have no competing interests.JVW designed the scientific project, analyzed the data and wrote the paper. GS was responsible for sample processing and data collection. AD selected LCL and ML patients, as well as controls from the endemic area. CHC selected VL patients. AFS did the trace metal analysis. AB and EMC supervised the field work and reviewed the paper. MBN analyzed the data and reviewed the paper.The pre-publication history for this paper can be accessed here:


A 31-year-old man presents with central obesity, hypertension, and abnormal lipids. How would you manage this patient? A 31-year-old white male with no significant past medical history is referred by his workplace to a primary care physician for an elevated blood pressure (BP). He presents to the clinic with no complaints. His mother and grandmother both have diabetes, and his father has hypertension. He has had a 15-pound (lb) weight gain over the last year and has become more sedentary.His BP is 142/90 mm Hg, pulse is 88 beats per minute (bpm), weight is 209 lb, and height is 5′ 11″. On examination he displays moderate central obesity, but otherwise the examination is normal. His fasting cholesterol is 228 mg/dl , low-density lipoprotein (LDL) is 166 mg/dl, high-density lipoprotein (HDL) is 32 mg/dl, triglycerides (TG) are 223 mg/dl (to convert mg/dl of triglycerides to mmol/l, divide by 89), and fasting glucose is 114 mg/dl (to convert mg/dl of glucose to mmol/l, divide by 18).This patient meets the diagnostic criteria for the metabolic syndrome as defined by the National Cholesterol Education Program Adult Treatment Panel III guidelines . Any thrThe patient returns to the clinic two years later. He presents with complaints of increasing frequency of urination and episodes of blurry vision. He has nocturia and has lost 5 lb in the last week. Otherwise, his review of systems is unremarkable. His blood pressure is 146/88 mm Hg, pulse 80 bpm, and weight 216 lb. His fundoscopic examination is normal. He continues to have moderate central obesity. Current medications are a thiazide diuretic, 12.5 mg once daily (QD), started one year prior. A non-fasting blood sugar is 267 mg/dl.There are three criteria for the diagnosis of type 2 diabetes as defined by the American Diabetes Association (ADA), of which any one is sufficient to make the diagnosis see . This paorRandom plasma glucose ≥200 mg/dl (11.1 mmol/l) and symptoms a (6.99 mmol/l) orFasting plasma glucose =126 mg/dla (11.1 mmol/l) in oral glucose tolerance test.Two-hour plasma glucose =200 mg/dlaIn the absence of symptoms, these criteria should be confirmed by repeat testing on a different day.Source: .1c), 8.6%; and urine albumin-to-creatinine ratio, 2.0 mg/mmol . LDL is 176 mg/dl, HDL 32 mg/dl, and TG 292 mg/dl. His electrocardiogram is normal.His additional investigations are as follows: fasting glucose, 215 mg/dl; hemoglobin A1c  QD. With continued elevated systolic BP >130 mm Hg and diastolic BP >80 mm Hg, an angiotensin-converting enzyme inhibitor (ACE-I) is started. An ACE-I at this time is appropriate for BP control and has the additional preventative effects of reducing progression to nephropathy and CVD events ,11. In aThe patient returns to clinic today after spending the last three years overseas and has not seen a physician in two years. He complains of fatigue, occasional blurry vision, awakening three to four times at night to urinate, and diarrhea at least once a week. He says that he has been compliant with his diabetes medications but has gained 15 lb in the last six months. His medications include metformin at 1 g BID, a TZD BID, and an ACE-I QD. His blood sugar is 289 mg/dl (fasting), BP is 130/90 mm Hg, pulse is 88 bpm, and weight is 221 lb. There are no foot sores or ulcers, but he has diminished sensation to monofilament on the plantar surfaces of both feet. The remainder of his examination is unchanged, including normal fundoscopy. His HbA1c is 9.6%, LDL is 143 mg/dl, and spot urine albumin-to-creatinine ratio is 15 mg/mmol. His creatinine and liver enzymes are normal. His pre-meal blood sugars average 210–250 mg/dl.He continues to have an elevated HbA1c, worsening neuropathy, and weight gain, which prompt a more effective treatment strategy. There are several options for pharmacotherapy available to choose from at this point. The patient could begin a third oral agent after maximizing the doses of metformin and TZD, or he could begin insulin injections with or without additional oral agents. Because of the significant cost associated with three oral medications and his need for further glycemic control, insulin would be an appropriate choice at this time. However, he should be advised of the side effect of additional weight gain when beginning insulin therapy.This case presentation illustrates an otherwise healthy appearing patient who is found to have the metabolic syndrome and despite evidence-based management develops type 2 diabetes. This patient likely represents the natural history of type 2 diabetes in most patients. Mild hypertension is often the only presenting sign of metabolic syndrome and prediabetes, allowing an opportunity for prevention of type 2 diabetes.There is an association between metabolic syndrome and the development of CVD and type 2 diabetes . This syMetformin is in the class of biguanides and works by decreasing hepatic glucose output and increasing insulin action in tissues. Metformin has been suggested to help prevent the onset of diabetes but is less effective than diet and lifestyle changes . Other mIntensive therapy in patients with type 2 diabetes results in a decreased risk of microvascular complications; therefore, it is appropriate to use combinations of medications in patients with suboptimal glycemia . The claThe pathophysiology of type 2 diabetes involves, in part, a “relative” deficiency of insulin. Although a state of endogenous hyperinsulinemia occurs, the degree of tissue resistance causes a total decrease in “effective” endogenous insulin. Progression of disease is also attributed to worsening beta-cell dysfunction and decreased release of insulin . InsulinIn summary, diabetes prevention and management is an important goal in practice. The morbidity and mortality from diabetes is a significant burden to health care, emphasizing the need for effective prevention and control of diabetes in improving outcomes.PLoS Medicine EditorsThe management of the patient in this Learning Forum article is in keeping with two national guidelines—those of the United States National Cholesterol Education Program and the ADA. Both peer reviewers pointed out that clinicians in other countries would follow their own national or regional guidelines. For example, the guidelines for the management of type 2 diabetes published by the United Kingdom's National Institute for Clinical Excellence differ in key ways from the ADA guidelines. We as editors debated whether to insist that the authors include guidance from other parts of the world. We decided that as an international journal we should reflect global variations in practice and allow authors to discuss how patients would be optimally managed in their own countries. There is much we can learn from different approaches to clinical practice worldwide.—The www.nice.org.uk/pdf/NICE_full_blood_glucose.pdfNational Institute for Clinical Excellence Clinical Guidelines for Type 2 Diabetes: www.staff.ncl.ac.uk/philip.home/t2dg1999.htmInternational Diabetes Federation (European Region) Desktop Guide to Type 2 Diabetes: The natural history of diabetes suggests that it is a progressive disease, and therapy may need to be frequently changed or augmented over time.The diagnosis of the metabolic syndrome should alert primary care physicians to prescribe intensive lifestyle modifications for prevention of diabetes.Strict BP, lipid, and weight control is just as essential as strict glycemic control in preventing CVD in patients with diabetes.Metformin can reduce the risk of CVD in obese patients with diabetes independent of glycemic control.The decision of combination oral therapy with or without insulin should be individualized to optimize glycemic control and reduce micro- and macrovascular complications.


HJV) in whites is a cause of early age-of-onset iron overload (juvenile hemochromatosis), and of hemochromatosis phenotypes in some young or middle-aged adults. HJV coding region mutations have also been identified recently in African American primary iron overload and control subjects. Primary iron overload unexplained by typical hemochromatosis-associated HFE genotypes is common in white and black adults in Alabama, and HJV I222N and G320V were detected in a white Alabama juvenile hemochromatosis index patient. Thus, we estimated the frequency of the HJV missense mutations I222N and G320V in adult whites and African Americans from Alabama general population convenience samples.Homozygosity or compound heterozygosity for coding region mutations of the hemojuvelin gene  technique. Analysis for HJV I222N was performed in 240 whites and 124 African Americans. Analysis for HJV G320V was performed in 241 whites and 118 African Americans.HJV I222N; she was also heterozygous for HFE C282Y, but had normal serum iron measures and bone marrow iron stores. HJV I222N was not detected in 124 African American subjects. HJV G320V was not detected in 241 white or 118 African American subjects.One of 240 white control subjects was heterozygous for HJV I222N and G320V are probably uncommon causes or modifiers of primary iron overload in adult whites and African Americans in Alabama. Double heterozygosity for HJV I222N and HFE C282Y may not promote increased iron absorption. HJV) in whites is a cause of early age-of-onset iron overload (juvenile hemochromatosis), and of hemochromatosis phenotypes in some young or middle-aged adults [HJV coding region mutations have also been identified recently in African American primary iron overload and control subjects [HJV missense mutations I222N and G320V in Alabama white and African American adults who reported no history of hemochromatosis or iron overload, because primary iron overload unexplained by typical hemochromatosis-associated HFE genotypes is common in whites and blacks in Alabama [HJV I222N and G320V were detected in an Alabama juvenile hemochromatosis index patient [HJV I222N and G320V have been described in white adults with hemochromatosis phenotypes in other geographic areas [Homozygosity or compound heterozygosity for coding region mutations of the hemojuvelin gene . No subject reported a previous diagnosis of hemochromatosis or iron overload. Serum transferrin saturation, serum ferritin concentration, or other indicators of iron metabolism were not measured in most study subjects.HJV I222N and G320V were genotyped by standard PCR-RFLP and agarose electrophoresis techniques. Exon 4 of the HJV locus was amplified using the primers HJEx4AF and HJEx4BR previously described [HJV locus. Reaction conditions were as follows: 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8), 0.1% Triton X-100, 3.2 mM MgSO4, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 1 μM of each primer, 0.2 U/μL of Taq Polymerase. Amplification conditions on a Biometra UNO II thermocycler (LabRepCo/LabRepNet, Horsham, PA) consisted of an initial denaturing at 94°C for five minutes followed by thirty cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds.Genomic DNA was isolated from blood buffy coat as described previously . HJV I22HJV exon 4 nucleotide sequence defining I222N was made utilizing the restriction endonuclease Bcc I (New England Biolabs, Beverly, MA), which recognizes the nucleotide c.665T of I222 as a restriction site, and not the nucleotide c.665A of N222. Similarly, the nucleotide polymorphism site c.959G→T defining the G320V polymorphism of the HJV exon 4 locus was determined by using the restriction endonuclease Ban I (New England Biolabs, Beverly, MA). Ban I recognizes the nucleotide c.959G of G320 as part of the restriction site sequence, but not the nucleotide c.959T of V320. DNA specimens from the parents of a white juvenile hemochromatosis index case were used as positive controls for HJV I222N and G320V [Delineation of c.665T→A of the HJV I222N was performed in 240 whites and 124 African Americans. Analysis for HJV G320V was performed in 241 whites and 118 African Americans. Results are expressed as allele frequencies and 95% confidence intervals (CI). To estimate the CI for frequencies of HJV mutations that were not detected in the present subjects, we computed the allele frequency as the quotient of single minimal hypothetical value (= 0.01) and the number of chromosomes corresponding to the respective groups of subjects. Computations were performed with GB-STAT .The data set consisted of observations on 241 Alabama white and 124 African American adults. Analysis for HJV I222N, but this mutation was not detected in African American subjects. HJV G320V was not detected in either white or African American subjects.These results are displayed in Table HJV I222N were available for review. This 29 year-old woman was also heterozygous for HFE C282Y. She reported that she had had two normal pregnancies and one spontaneous abortion. She has always experienced heavy menstrual flow, but she reported no other significant blood loss. She reported no blood donation. She reported that she eats a variety of meats, vegetables, and other foods; there was no history of supplemental iron ingestion. Her hemoglobin level was 12.8 g/dL, platelets 113,000/mm3, transferrin saturation 21%, and serum ferritin 23 ng/mL; marrow examination performed to evaluate thrombocytopenia revealed normal cellular morphology and normal iron stores.Medical records of the white subject heterozygous for HJV coding region mutations occur in homozygous or compound heterozygous configuration in persons with juvenile or adult-onset hemochromatosis phenotypes [HJV-associated hemochromatosis are collectively uncommon [HJV mutations were also uncommon in control whites studied with dHPLC (denaturing high-performance liquid chromatography) [HJV G320V mutation detectable by PCR-RFLP analysis, suggesting that the frequency of the G320V allele in the Greek population is lower than 0.004 [HJV missense mutations L101P (two heterozygotes) and E302K (one heterozygote) were identified in 333 control subjects, representing an aggregate HJV mutation frequency of 0.0045 (95% CI: 0, 0.0096) [HJV coding region mutations is low in general populations of whites [A variety of enotypes -5,9-11. uncommon ,11,12. HHJV missense mutation was associated with increased severity of iron overload in nine of 310 French patients with hemochromatosis and HFE C282Y homozygosity who were studied with dHPLC [HFE C282Y homozygosity studied with dHPLC, no HJV coding region mutation was detected [HJV coding region mutation frequencies in C282Y homozygotes with a hemochromatosis phenotype in France and the U.S. are similar . The aggregate frequency of HJV coding region mutations was similar in French hemochromatosis patients with HFE C282Y homozygosity and in French control subjects , although the hemochromatosis patients with HJV coding region mutations had more severe iron overload, on the average, than did hemochromatosis patients without HJV coding region mutations [Heterozygosity for a th dHPLC . In 48 wdetected . Thus, autations .HJV I222N and HFE C282Y was not associated with evidence of increased iron absorption in the present case. Some persons with JH have common HFE genotypes, including C282Y heterozygosity, H63D heterozygosity or homozygosity, and S65C heterozygosity [HFE genotypes are infrequently associated with a severe hemochromatosis phenotype [HFE introns and exons in English and French Canadian JH cases did not reveal novel HFE mutations that could likely explain the development of iron overload [HJV mutations were relatively common and were associated with greater severity of iron overload [HJV coding region mutation was detected [HFE H63D [HJV alleles in the clinical expression of iron overload with particular reference to HFE hemochromatosis is warranted [HJV alleles may vary among racial/ethnic groups.Double heterozygosity for zygosity -18. Howehenotype ,19-25. Foverload ,15. In 3detected . The brodetected . AlthougHFE H63D , other pHFE mutations, a common allele of the ferroportin gene (FPN1 Q248H), or heritable types of anemia [HJV I222N and G320V were not detected in the present African American subjects. However, HJV coding region mutations were recently identified in two of 51 African American subjects with iron overload: exon 3, nt 205–207 ins GGA (ins G69) (frequency 0.0098); and exon 4, nt 929 C→G (A310G)  [HJV alleles ins G69 and A310G were also identified in African American control subjects  [HJV coding region mutations are uncommon in African Americans, but may account for some cases of primary iron overload [Primary iron overload in African Americans is phenotypically and genetically heterogeneous . Some pa FPN1 Q24H, or herf anemia ,28, but HJV I222N and G320V are uncommon causes or modifiers of primary iron overload in adult whites and African Americans in Alabama. Double heterozygosity for HJV I222N and HFE C282Y may not promote increased iron absorption.The author(s) declare that they have no competing interests.JCB participated in conceiving the study, provided DNA specimens, performed statistical evaluation, and wrote part of the manuscript. CAR devised the PCR-RFLP assay, provided DNA specimens, participated in data collection, and wrote part of the manuscript. SN and SB performed testing on DNA specimens and participated in data collection. RTA participated in conceiving the study, provided DNA specimens, and wrote part of the manuscript. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Mandrillus leucophaeus) was detected in two mandrills (Mandrillus sphinx) from Amsterdam Zoo. These captivity-born mandrills had never been in contact with drill monkeys, and were unlikely to be hybrids. Their mitochondrial haplotype suggested that they descended from founder animals in Cameroon or northern Gabon, close to the habitat of the drill. SIVdrl has once before been found in a wild-caught mandrill from the same region, indicating that mandrills are naturally infected with a SIVdrl-like virus. This suggests that mandrills are the first primate species to be infected with three strains of SIV: SIVmnd1, SIVmnd2, and SIVdrl.A pol-fragment of simian immunodeficiency virus (SIV) that is highly related to SIVdrl-pol from drill monkeys ( Mandrillus sphinx) are quite exceptional among African monkeys in that they harbour two distinct SIV strains, designated SIVmnd1 and SIVmnd2, with a separate geographic distribution [To date over 30 strains of simian immunodeficiency virus (SIV) have been isolated from African primate species and sequenced . Mandrilribution ,5. Howevribution . In 2003Both EDTA-plasma and PBMC (isolated with the OptiPrep system (Nycomed, Oslo, Norway)) was obtained from the animals for further analysis. Our goal was to investigate whether the monkeys were virus carriers, and which strain of SIV they harboured. To detect both SIVmnd1 and SIVmnd2, we designed two nested primers sets based on published pol gene sequences that amplify an RT fragment of 282 nucleotides (Table M. sphinx [Mandrillus leucophaeus), with a lower sequence identity to SIVmnd2 (± 85% to GenBank acc. no. AF367411), and to SIVmnd1 (<64% to GenBank acc. no. M27470). Although SIVmnd2 and SIVdrl are more closely related to each other than the two SIVmnd strains, SIVdrl has several mismatches with the PCR primers designed for detection of SIVmnd2. This could explain why one seropositive animal was tested as PCR-negative. Therefore, we designed a new primer set that amplifies the same gene fragment based on the SIVdrl sequence  at any time. Drills are rare in European zoos, and only the zoos of Nikolaev (Ukraine) and Saarbruecken (Germany) reported keeping both drills and mandrills in a 1992 survey [Mandrillus cannot hybridise in the wild, as the habitats of the two species do not overlap, but it does so in captivity. The morphologic differences between female drills and mandrills are less obvious than those between males and are mainly noticeable in the colouration of the muzzle and the size of the animal. A single hybrid M. sphinx × M. leucophaeus has been reported from Vienna Zoo, Austria, in a 1992 survey [M. leucophaeus × M. sphinx were described from a Wildlife Rescue Centre in Cameroon [All four mandrills examined here were born in captivity. Male CAS was born in 1983 at the now closed Wassenaar Zoo (The Netherlands), and moved to Artis Zoo in 1986. The female, REB, was born in Budapest Zoo (Hungary), and moved to Artis Zoo when she was 5 years old. Their offspring, RAF and HAB, were both born in Amsterdam. Exposure to drills during their lifetime is unlikely as none of the zoos kept drills  left Africa at least ten years ago , its genome conservation is remarkable: only 2 conserved amino acid differences separate the consensus pol-sequence from the SIVdrl reference sequence Figure . HoweverThe author(s) declare that they have no competing interests.ACvdK designed the study, analysed the sequences, and drafted the manuscript. RvdB carried out the PCR assays and performed the cloning and sequencing. MJH did the medical examinations and collected the blood samples. RAG carried out the SIV antibody assays. ADMEO and BB conceived of the study, and participated in its coordination.


A common assumption about malaria, dengue, and other mosquito-borne infections is that the two main components of the risk of human infection—the rate at which people are bitten (human biting rate) and the proportion of mosquitoes that are infectious—are positively correlated. In fact, these two risk factors are generated by different processes and may be negatively correlated across space and time in heterogeneous environments. Uneven distribution of blood-meal hosts and larval habitat creates a spatial mosaic of demograPhic sources and sinks. Moreover, mosquito populations fluctuate temporally, forced by environmental variables such as rainfall, temperature, and humidity. These sources of spatial and temporal heterogeneity in the distribution of mosquito populations generate variability in the human biting rate, in the proportion of mosquitoes that are infectious, and in the risk of human infection. To understand how heterogeneity affects the epidemiology of mosquito-borne infections, we developed a set of simple models that incorporate heterogeneity in a stepwise fashion. These models predict that the human biting rate is highest shortly after the mosquito densities peak, near breeding sites where adult mosquitoes emerge, and around the edges of areas where humans are aggregated. In contrast, the proportion of mosquitoes that are infectious reflects the age structure of mosquito populations; it peaks where old mosquitoes are found, far from mosquito breeding habitat, and when mosquito population density is declining. Finally, we show that estimates for the average risk of infection that are based on the average entomological inoculation rate are strongly biased in heterogeneous environments. A modeling approach reveals that incorporating the demography and behavior of mosquitoes can substantially change estimates of the risk of infection from diseases such as malaria Understanding the spatiotemporal distribution of risk for mosquito-borne infections is an important step in planning and implementing effective infection control measures . Remote The risk of a mosquito-borne infection is estimated by the entomological inoculation rate (EIR): the number of bites by infectious mosquitoes per person per day . EIR is We develop theory to illustrate simple patterns in the EIR in heterogeneous environments, focusing on EIR's separate components. We follow the a priori approach of Ross, developing simple mathematical models as tools for qualitative and quantitative reasoning . We deveFluctuating mosquito density affects EIR through changes in HBR: transmission increases as mosquito density increases. Following an increase in the rate at which adult mosquitoes emerge, mosquito density and HBR peak .We illustrate this principle for one particular set of parameters that leads to increased mosquito aggregation. The human population is distributed heterogeneously in blocks of low, high, and medium density, approximating a town with a rural population on one side and an intermediate-density population on the other. The distribution of adult mosquitoes is influenced by the distribution of humans A. AggregWhen mosquitoes and humans are distributed unevenly, the distribution of mosquitoes and risk may be dominated either by proximity to larval habitats and gradients away from them or by the tendency of mosquitoes to aggregate around humans. The realized pattern depends on the relative distribution of larval habitat and humans, and whether mosquito aggregation around humans increases HBR. We illustrate one kind of pattern for parameters that lead to increased HBR. In this case, human density increases away from larval habitat. The density of mosquitoes peaks a short distance from the source, and the density of infected mosquitoes peaks slightly further away A. HBR deVariability in EIR across a landscape can lead to systematic bias in the estimation of risk. In In contrast, the relationship between average EIR and average prevalence of infection in humans is biased, such that average prevalence always falls below the true relationship A. The biThe local density of infectious mosquitoes B and theThe patterns illustrated in The tendency of mosquitoes to aggregate at the edges of a town or away from larval habitat depends on mosquito searching behavior and demography. Three important parameters that affect these patterns are the maximum daily flight distance of a mosquito, mosquito longevity, and mosquito searching efficiency. The distribution of a mosquito cohort initially reflects the distribution of larval habitat. As mosquitoes search for hosts, the distribution of the cohort shifts to reflect the distribution of human hosts. These tendencies are also reflected in the static spatial distributions of mosquitoes. The distribution of long-lived mosquitoes with long daily flight distances will tend to reflect the underlying distribution of humans, while the distribution of short-lived mosquitoes with short flight distances will tend to reflect the distribution of larval habitat. Mosquito searching efficiency determines the relative rates of movement through habitats that vary in human density. A strong tendency for mosquitoes to aggregate at the edges of dense human populations occurs when mosquitoes move quickly through areas that are sparsely populated by humans and linger in areas that are heavily populated. In other words, mosquitoes tend to become more aggregated than their hosts, increasing HBR, when mosquito searching is relatively inefficient at low human densities.The distribution of relative risk also changes with the time required for incubation of the infectious agent, with mechanically transmitted agents at one extreme. When all else is equal, HBR is higher in areas in which human density is low, since human population density is in the denominator of HBR. On the other hand, HBR may decline in low-human-density areas because mosquitoes tend to move up a gradient of human population density in search of a blood-meal host. Such migration will tend to lower the average age of mosquitoes in low-human-density patches, especially near the edge of a town. This will tend to lower PIM for infectious agents with a long incubation period. In contrast, PIM for mechanically transmitted infectious agents will not be as strongly affected, so in comparison, the relative risk may be higher at that same edge of town.EIR is generally considered to be the best estimate of the risk of mosquito-borne infections, but EIR varies over space and time. EIR varies spatially because larval habitat and blood-meal hosts are heterogeneously distributed across a landscape. Temporal variability is generally driven by weather, especially rainfall, temperature, and humidity. To compound the problem, heterogeneity in human feeding over short distances can be caused by vector preferences for individual humans based on odor or other cues . HeterogHeterogeneous biting also has important implications for the measurement of EIR. Estimates of EIR may vary substantially over short distances depending on the place and time at which the measurement is made. Depending on the method used, EIR may also vary with the relative attractiveness of the human bait. Our mathematical models have shown that average EIR in heterogeneous environments gives a strongly biased estimate of average risk, even when local estimates of EIR provide a perfect measure of local risk. The bias is unavoidable because the relationship between EIR and the proportion of humans who are infected is nonlinear, which leads to a bias due to Jensen's inequality . A similMathematical models provide a sound approach to understanding risk and planning for control in heterogeneous environments, especially when the models are based on the ecology of the local vector populations and a sound understanding of the entomological parameters relevant for transmission , 2000b. A dominant component of EIR is the density of mosquito vectors relative to human density. Our models show that mosquito densities and the proportion of humans who carry a mosquito-borne infection decline with distance away from larval habitat because of random movement and mosquito mortality. Such patterns have been documented by numerous field studies . The steUnlike the patterns in human biting, the proportion of mosquitoes that are infectious depends on the age structure of the mosquito population. The likelihood of infection in mosquitoes shows a strong association with the age or parity of mosquitoes . The aveMosquito aggregation around dense human populations depends on the details of mosquito searching behavior. Long-lived mosquitoes with long flight distances tend to become more aggregated than their human hosts over intermediate distances. For example, EIR may peak at the edges of a village, as has been documented by one field study . At largThe use of remote sensing and GIS provides a potentially powerful tool for understanding the distribution of mosquito-borne infections at large spatial scales, but dynamics and control of mosquitoes and mosquito-borne infections occur locally. These technologies will be most effective if they are coupled with micro-epidemiological models of malaria, dengue, and other mosquito-borne infections . Such moWe use mathematical models strategically, to illustrate general principles that may apply to many mosquito-borne infections, not to make predictions about the distribution of a particular infectious agent or the incidence of disease. The models we present and analyze are based on the models for malaria infection developed by x denote the proportion of humans who are infected and infectious and H denote the population density of humans. We assume that the human infectious period is exponentially distributed with average duration of infection 1/r. Thus, we are following Ross in developing a model for infection ignoring superinfection, immunity, and clinical disease (Let  disease .ɛ(t) denote the rate adult female mosquitoes emerge from larval habitat; we do not assume that the emergence of adults is explicitly linked to the density of adult mosquitoes. Let M denote the population density of mosquitoes, Z the density of infectious mosquitoes, and z = Z/M the proportion of mosquitoes that are infectious. We assume that the mosquito lifespan is exponentially distributed with a mean lifetime of 1/g d.We extend the Ross model by adding temporal variability in mosquito density. Let n stages of equal duration; the proportion of mosquitoes that are infected and incubating in stage k is denoted k,y and the density of mosquitoes in that stage is kY. We assume the incubation period has mean of 1/q d. The probability of surviving the incubation period is (qn/(qn + g))n (approximately eg/q− for large n), and the duration of the incubation period (for surviving mosquitoes) has a Gamma distribution with mean 1/q and variance 1/(q2n); for the numerical simulations, we use n = 64 . Therefore, when mosquito density changes, HBR changes proportionally. In contrast, EIR is the number of infectious bites per human per day. Thus, it is the product of PIM and HBR (i.e., EIR = zHBR = azM/H). The transmission dynamics of mosquito-borne infections are complex, and it is easy to lose sight of what terms such as EIR and HBR actually mean. HBR is the number of bites received by a human each day. Thus, it is the product of the human feeding rate, i is added to variable names to denote the value in the ith patch. Thus, iH denotes local human population density and ix the local prevalence of infection in humans. Similarly, iM denotes local mosquito population density, and iZ denotes the density of infectious mosquitoes. The density of infected mosquitoes in patch i and incubation stage k is denoted i,kY. This deterministic approach to incorporating space has some limitations ; the emergence of adult female mosquitoes depends predictably on time and location. Following emergence, female mosquitoes spread into surrounding areas seeking blood hosts; they feed, oviposit, and then repeat the cycle. We assume that heterogeneity in larval habitat takes the form of differences in quality of larval development rather than availability of places to oviposit. In other words, we assume that suitable sites for oviposition are distributed homogeneously throughout the habitat, but that patches may vary in the successful development of adults. Some patches may produce no adults. Heterogeneity in the availability of oviposition habitat would affect the distribution of risk because mosquitoes would alternate between finding a place to oviposit and finding a blood meal. If oviposition were not possible in most patches, those that allowed oviposition would become focal points for mosquito aggregation. Thus, these results apply mainly to mosquito species for which heterogeneous availability of oviposition sites is relatively unimportant for the distribution of risk.Larval habitat and human distributions form a template that determines mosquito distributions and the distribution of risk. The emergence rate of adults in the We assume that humans do not move among patches. The density of humans and the productivity of the larval habitat may vary over space. As we change the distribution of humans and larval habitat to explore the effects of spatial heterogeneity, we hold the total emergence rate of adult mosquitoes per human constant; only the distribution of humans and adult mosquito emergence changes.iH) denote the per capita emigration rate of mosquitoes away from patch i regardless of infection status. We assume that Φ(H) is a decreasing function of H; the more humans, the less likely mosquitoes are to leave a patch in search of another blood-meal host. Thus, mosquitoes move more rapidly through patches with low human densities. A parameter, i,j,κ describes the fraction of mosquitoes leaving patch i that fly to patch j, and Σjκi,j = 1. Thus, the rate that mosquitoes move from patch i to patch j is Φ(iH)i,jMiκ.We assume that mosquitoes are more likely to stay in a patch if they encounter a human, and that they are more likely to find humans where humans are more abundant. Let Φ. The human infectious period for this case is 100 d (r = 0.01), roughly consistent with malaria.Our intent is to focus on the effects of temporal and spatial heterogeneity. Consequently, we have used a single set of mosquito life-history parameters and a single duration of infection in humans. The parameters are roughly consistent with iɛ(t) = ig,K while temporal heterogeneity was modeled using the seasonal forcing function iɛ(t) = igK(1 sin(2πt/365)). In a homogeneous landscape, iK is the long-term average density per patch, often called the carrying capacity. Throughout, K was chosen such that the average number of mosquitoes per human across all patches was 2, i.e., ΣiiM/ΣiiH = 2. x = 0.01 and i,kY = iZ = 0. We generated numerical solutions for 4 y and plotted the last three.Constant mosquito populations were modeled using a constant birthrate, i,jκ = 0 unless two patches are adjacent, and we plot the values at equilibrium. We assume that no humans live in the patches at the extreme ends of the transect, and that all of the mosquitoes leaving one of these edges return to the adjacent patch; thus 1,2κ = 17,16κ = 1, a reflective boundary. Otherwise, we assume that mosquitoes move in either direction at random; thus, i,jκ = 0.5 for i = 2…16 and j = i ± 1. Mosquito migration was described by the functionΦ(Hi) = ζei−θH. In ξ = 10 and θ = 4. These correspond to a maximum daily flight distance (i.e., without humans) of about ten patches per day.For gK(P − 2) in patch 1 (K = 2 and P = 17); no adults emerged within other patches. The adult emergence rate for gK in each patch with humans (K = 2 in patches 2–16).Adult mosquito emergence for For Protocol S1(285 KB PDF).Click here for additional data file.


PLoS Biology, volume 2, issue 10:In Regulation of Muscle Fiber Type and Running Endurance by PPARδ.Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, et al.10.1371/journal.pbio.0020294The following competing interest should have been indicated in the above research article. R. M. Evans wishes to acknowledge a consulting relationship with Ligand Pharmaceuticals. Under a licensing agreement with GlaxoSmithKline, Ligand receives milestone payments on the development of GW501516, a PPARδ-specific drug used in this study. For clarification, no materials or support were received from either company, and no agreements were in place concerning the execution or publication of this work.Published January 18, 2005


Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs.Through Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor.Through a gain-of-function approach, we analyze the interaction of Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances. In that context, early studies [mab-21 allele as a dominant enhancer of egl-5, a loss-of-function mutation affecting the nematode homolog of the Drosophila homeotic gene AbdominalB. Subsequent studies [mab-21(+) plays a key role in the formation of the nematode male tail. Homozygous mab-21 mutations act cell-autonomously on the identity of a sensory ray, leading to alterations in anteroposterior identities akin to a homeotic transformation. Furthermore, they act non-cell-autonomously on the binary switch between hypodermal and neuroblast specification. Finally, hypomorphic mab-21 mutants exhibit pleiotropic changes affecting movement, body shape and fecundity, suggesting that mab-21(+) also plays key developmental roles outside the male tail region.The mab-21 gene encodes a 41 kD basic protein with no significant homologies to any other published proteins. Homologs of the mab-21 gene have been identified in many other species, from Drosophila to humans. The first vertebrate counterparts of mab-21 were isolated in amniote genomes: a human homolog, MAB21L1, was cloned during a systematic search for transcripts carrying trinucleotide repeats [Mab21l1 within a chromosomal deletion discovered in a patient with autism and language deficit secondary to an auditory processing abnormality [The  repeats  and potemab-21 genes (Mab21l1, Mab21l2) [MAB21L2 gene, and showed that they encode nuclear proteins [Mab21l1 and Mab21l2 are expressed in largely overlapping territories [Xenopus laevis (Xmab21l2) [Danio rerio [Zmab21l2). Mab21l2 is an early marker of the tectum as well as primitive eye field, optic cup and retina in anamniotes, in agreement with observations previously made in mouse embryos.Our group, in parallel with others, identified two murine Mab21l2) -8 and thproteins  that areritories -7, suggemab21l2) , , while they do not affect body size in double mutants [mab-21 gene transcription by the sma signaling pathway.Some of these important advances point to a critical role for the mab-21 gene family as a whole in early embryogenesis on one hand, and in various aspects of neural development on the other. However, our knowledge on the molecular mechanisms through which mab-21 genes operate remains inadequate at best. Interestingly, by genetic analysis of loss-of-function mutations in nd CET-1 , also knnd CET-1 , and reg mutants . Reportemab-21 – TGF-beta interaction, but stop short of addressing several key questions. What is the molecular counterpart of the genetic interaction described in C. elegans between mab-21 and the sma pathway? Is this interaction conserved in vertebrate development, and what TGF-beta superfamily molecules does it involve? Does it play a relevant role in the establishment of vertebrate dorsoventral (DV) polarity? If so, at what level do MAB-21 and TGF-beta pathways converge into a single regulatory cascade? In the present study, through a gain-of-function strategy, we analyze the molecular interplay between MAB21L2, a vertebrate homolog of C. elegans MAB-21, and TGF-beta superfamily signaling molecules. In particular, we focus on the signaling pathway initiated by BMP4, a ligand structurally related to CET-1/DBL-1, that regulates DV axis formation and numerous key aspects of neural development in chordata.The above genetic studies prove the biological relevance of the BMP4 in Xenopus embryos can lead to the formation ventralized embryos, i.e. embryos devoid of axial structures, whereas BMP4 depletion can lead to dorsalized embryos, i.e. embryos exhibiting an expansion of dorsal mesoderm [Xmab21l2 is restricted to the primitive eye field, optic recess, optic cup and retina (excluding the choroid fissure), to the tectum and dorsal neural tube, and to branchial arches .To determine whether Xmab21l2 and BMP signaling molecules, we analyzed the effects of isolated Xmab21l2 overexpression. Embryos were injected at the one-cell stage with 800 pg in vitro-transcribed Xmab21l2 alone. We analyzed the phenotype of injected tailbud embryos as well as the expression of dorsal markers at gastrulation. A significant percentage of injected embryos (48%, N = 200) featured various degrees of dorsalization/anteriorization (DAI 6–8), with a well formed, sometimes enlarged head, a broad cement gland, a short and/or bent axis  with expanded and/or enhanced Chordin gene expression . Our results are in sufficient agreement with those previously obtained by others [Xmab21l2 overexpression produces neither signs of complete dorsalization, such as Janus twins or radial eyes [Prior to addressing the nature of the interaction between s Figure . We preps Figure , suggesty others , confirmXmab21l2 can compensate the effects of BMP4 overexpression, Xenopus embryos were injected at the one-cell stage with in vitro-transcribed BMP4 mRNA alone (0.6 ng, n = 111), or coinjected with Xmab21l2 mRNA . We scored the formation of dorsal structures in tadpole stage embryos by using the dorsoanterior index [BMP4 overexpression (0.6 ng) . Likewise, whilst complete dorsal structures (DAI = 5) , suggesting that Xmab21l2 antagonizes BMP4 in its ability to ventralize embryonic mesoderm in vivo  Figure . Coinjec) Figure , bottom in situ hybridization, we analyzed the expression of molecular markers of dorsal . Both cell- and embryo lysates were subjected to immunoprecipitation, using an anti flag monoclonal antibody, whereas an anti-myc antibody was utilized for immunodetection in Western blotting. Experiments conducted in transfected P19 cells showed that SMAD1 coprecipitates with MAB21L2, and that the interaction is enhanced by activation of BMP signaling, which is required for nuclear localization of receptor-dependent SMADs  [Mab21l2-cotransfected COS7 cells was compared to the baseline reporter activity scored in cells transfected with the GAL4 DBD alone (G-D) or with a transcriptionally inactive GAL4 DBD fusion protein, (GAL4HOXB3NT, G-N) [Gal4-Mab21l2-transfected cells only, suggesting that MAB21L2 may function as a transcriptional repressor or co-repressor  , either NT, G-N) . Reporter Figure . The samC. elegans mab-21 undergoes inhibitory regulation by TGF-beta superfamily signals [Mab-21 genes have been isolated in several species and have been shown, through genetic and epigenetic loss-of-function experiments, to play key roles in various developmental processes, ranging from gastrulation and neural tube closure to eye and lens formation ,10,12,13 signals , the molXenopus embryo, and in murine embryonic carcinoma cells, indicate that MAB21L2 interacts functionally with SMAD1, a nuclear transducer of BMP2/4/7 signaling. Overexpression of Mab21l2 complements the effects of BMP4 overexpression, in keeping with the epistatic interactions observed in nematodes [pseudocoelomates appear to be strongly conserved in evolution.Our results, obtained in an anamniotic model system, the ematodes . Thus, gAs the nuclear localization of MAB21L2 suggested, our pull-down and immunoprecipitation results indicate that MAB21L2 is a new interactor of the receptor-activated transducer SMAD1. Whilst the interaction between MAB21L2 and SMAD1 appears to be direct, our results do not seem to favor a direct contact between MAB21L2 and SMAD4.Xenopus laevis and mouse embryos. The increasing levels of MAB21L2 protein observed starting at midblastula transition may be required to compensate mesoderm-ventralizing signals triggered by BMP2/4. In the absence of MAB21L2 genes, completion of gastrulation may be hampered, resulting in various abnormalities of dorsoventral polarity [Mab21l2 downstream genes will make it possible to investigate mab-21 – BMP4 interactions from the opposite standpoint, i.e. by looking at the modulatory effect of BMP signaling on the expression of Mab21l2 targets.The interaction of MAB21L2, a transcriptional repressor, with nuclear transducers of BMP signaling offers a possible explanation for the results of loss-of-function experiments conducted by other investigators in polarity ,12. Resupolarity . Our resMab21l2 in early embryogenesis, the gene is clearly expressed at high levels and in a tightly localized fashion in neurulation and morphogenesis as well, likely contributing to neural tube and eye formation. Indeed, because of redundancy in the mab-21 gene family, many of the early effects of mab-21 gene mutation may be masked, uncovering these genes' roles in later stages of development and postnatal life.Although our results are more directly relevant to understanding the functions of Mab21l1 in lens formation has been recently documented in knock-out mice [mab-21 leads to disruption of the retinal anlage, where the gene is strongly expressed in a territory overlapping with the Bmp4 and Xvent2 expression domain. Recent studies have shown that BMP signaling plays a relevant role in guiding morphogenesis along the dorsoventral axis of the chick [Xenopus [In late developmental processes, well beyond the end of gastrulation, MAB-21 proteins may prolong their dynamic interaction with BMP signaling transducers. As mentioned, a cell-autonomous requirement for out mice  that devhe chick  and Xeno[Xenopus  retina. Mab21l1 and Mab21l2 are prominently expressed in the embryonic midbrain, both dorsally and ventrally, and in prosomere 1 [somere 1 ,6. Rolessomere 1 , and, mosomere 1 .Mab21l2 is expressed mostly in dorsal territories. In cerebellar development, it is expressed in the cerebellar plate and, later on, in granule cell neurons [Mab21l2 at several of those sites, and their developmental roles have been at least partially addressed: as an example, granule cell specification can be induced by BMP7 overexpression in the dorsal and ventral rhombencephalon [Mab21l2 is expressed in a dorsal territory, where BMP2/4 signaling plays a key role in the specification of neuronal identities alongside the dorsoventral axis of the neural tube [Throughout the hindbrain and spinal cord,  neurons . BMP sigcephalon . BMP7 iscephalon . Likewisral tube -34.Mash1 maintains competence for neuronal differentiation [Mab21l2 may thus participate in specifying neuronal and non-neuronal cell fates by interacting with BMP signals.Finally, in postmigratory neural crest, the interplay between BMP2/4 and ntiation . In neurmab-21 gene function in vertebrate systems, demonstrating that MAB21L2 interacts functionally with the BMP4 signalling pathway and physically with its best characterized nuclear transducer, SMAD1. Furthermore, we show that MAB21l2 can act as a powerful transcriptional repressor when targeted to an heterologous promoter. Further work is clearly required to determine if MAB21L2 binds DNA, what its binding specificity is, or if it only exerts its effect in a DNA-binding-independent fashion. More broadly, additional studies are required to clarify the role of mab-21 genes in the context of BMP signaling, and their likely function as a molecular switch linking different regulatory pathways in development and disease.Our results provide a biochemical and molecular foundation for future studies of Images were obtained either by traditional photography (slides 1–3) or by electronic scanning of autoradiography films. All images were processed through the Adobe Photoshop 7.0 software or through the Adobe Illustrator 10 software.Xenopus laevis homolog of mouse Mab21L2 was isolated by screening a Xenopus stage 28–30 lambdaZapII library (Courtesy of Richard Harland) using the murine homolog as a probe. The 2.8 Kb clone isolated contained the full length Xmab21l2 cDNA (GenBank AF040992) as well as 5' and 3' regions. The coding region of Xmab21 was excised using NotI and BalI (1.3 Kb) and subcloned into pBluescript for whole mount in situ hybridization, or BamHI-StuI (1.1 Kb) and subcloned into pT7TS for overexpression in embryos and into pCDNA3 for cell transfection.The Xenopus females were primed 1000 U of human chorionic gonadotropin (Profasi HP 5000, Serono) the night before collection. Ovulated eggs were fertilized with testis homogenates and allowed to develop in 0.1X MMR (1X MMR is 0.1 M NaCl, 2.0 mM KCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 5 mM HEPES, pH7.4). Jelly coats were removed in 3.2 mM DTT, 0.2 M Tris pH 8.8. Embryos were staged according to Nieuwkoop and Faber [All animal experimentation was conducted according to the stipulations of the Institutional Animal Care and Use Committee, San Raffaele Scientific Institute. In order to obtain embryos, nd Faber  and fixend Faber  for in sFor histological examination, wt or injected and stained embryos were fixed in MEMFA, embedded in wax, cut into 10 μm sections, dried onto slides, dewaxed with xylene and dehydrated with alcohol. Sections were rehydrated and stained with an orange G solution  and subsequently with an orange G-aniline blue solution , and dehydrated ,38.Whole-mount in situ hybridization was performed on staged embryos as described by . The antXenopus Mab21l2 protein wastested by Western blot on total extracts from X. laevis embryos. The antibody did not work successfully in IHC or IP experiments, suggesting that it fails to recognize the native epitope.A 14aa C-terminal stretch from the mMab21l2 protein was chosen as immunogenic peptide to obtain antisera in rabbits. Peptide was synthesized, lyophilized and coupled to Keyhole limped hemocyanin (KLH). Coupled peptide was used for immunization of two rabbits. Immune and preimmune serum were controlled by ELISA using the same peptide coated at different serial dilutions. Immune serum was then purified by affinity chromatography: briefly, 20 mg peptide was coupled on CNBr-Sepharose-4B resin (Pharmacia), according to the standard procedure suggested. 50 ml antiserum diluted in 1:1 in 1X PBS was applied on the peptide/CNBr-Sepharose-4B over night at 4°C. After washing the resin with 50 ml PBS and 50 ml 3 M NaCl, bound antibodies were eluted using 100 mM Glycine pH 1.8. Amount, specificity and purity of the antiserum was tested by the Bradford assay, ELISA and SDS-PAGE/Coomassie staining. Purified antibody was tested for specificity in western blot on total protein extracts from cos-7 cells transfected with mMab21l2-myc fusion protein or mock vector, and on total brain extract from mouse embryos. One single band of 41 KDa was detected by anti Mab21l2 antibody. The same 41 kD band was detected by WB in lysates from transfected COS-7 cells, and was missing in untransfected cells. Cross-reactivity of anti mouse Mab21l2 antibody with the Protein concentrations in extracts were quantitated through Bradford assays. Extracts were gel-fractionated by denaturing polyacrylamide gel electropohoresis SDS-PAGE. Gels were transfered onto nitrocellulose filters. Even loading was confirmed by Ponceau staining of nitrocellulose filters. Imunostaining was conducted as described .Xmab21-T7TS was linearized with EcoRI and transcribed with the T7 RNA polimerase, N-MycMab21L2-CS2+ was linearized with Asp718 and transcribed with the SP6 RNA polymerase. The Flag-Smad1 was excised from the human construct Flag-Smad1-CMV5 (Courtesy of J. Massagué) and subcloned into the vector pCS2+. The Flag-Smad1-pCS2+ vector was linearized with Asp718 and transcribed with SP6. BMP4 expression constructs were a kind gift of N. Ueno. Capped mRNA was synthesized using the Ambion Message Machine kit according to manufacturer's instructions. Injections were performed in 4% Ficoll in 1X MMR. Embryos were injected animally at the one-cell or two-cell stage into one or both blastomeres. The amount of mRNA injected is given in the text.The vector COS7 cells were maintained in DMEM Medium (Gibco Brl) supplemented with 10% FBS (Euro Clone), 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 É g/ml streptomycin. Calcium phosphate transfection was performed in a 60 mm Petri dish with different amounts of the expression constructs, 5 É g of reporter plasmids, and 100 ng of pRL-TK (Promega) as an internal control. 18 h after transfection the cells were washed twice with PBS1X and the medium was replaced with DMEM Medium supplemented with 10% FBS, 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 É g/ml streptomycin. Cells were harvested 36 h after the transfection, lysed, and assayed for luciferase activity . All luciferase assays were performed in triplicate.Xenopus laevis lysates were obtained in the same way starting from injected embryos harvested by centrifugation.P19 cells were maintained in MEM alpha medium (Gibco Brl) supplemented with 10% FBS (Euro Clone), 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 μg/ml streptomycin. Calcium phosphate transfection was performed in a 100 mm Petri dish with 10 μg of mycMab21l2 expression construct and 10 μg of flagSmad1 expression construct. 24 h after transfection the medium was replaced with MEM Alpha Medum supplemented with 1% FBS 2 mM L-glutamine (Euro Clone), 100 IU/ and 40 ng/ml BMP4 (R&D Systems). Cells were harvested 36 h after the transfection with TEN Solution  and centrifugated for 15 minutes at 5000 RPM. Cell pellets were frozen at -80°C o/n, thawed at RT and resuspended in 5 volumes of Extraction Buffer . The samples were centrifugated at 34000 RPM for 30 minutes at 4°C and the supernatants were harvested. The concentration of the proteins was determined by BCA assay (Pierce). 3; 0.1% triton), once with TSA solution  and once with 50 mM Tris-Cl pH 6.8. The protein complexes were eluted by addition of Sample buffer  followed by boiling for 5 minutes and separated on an SDS-polyacrylamide gel followed by anti-myc (1 μg/ml of clone n°9E10) immunoblotting. The secondary antibody was a goat anti-mouse antibody HRP-conjugated (1:30000 Bio-Rad). The blots were developed with the Supersignal West Pico reagent (Pierce).After preclearing with 50 μl of protein G-sepharose o/n at 4°C, lysates were incubated with 25 μg of M2 anti-Flag monoclonal antibody (Sigma) and 50 μl of protein G-Sepharose o/n at 4°C. The resin was washed twice with Dilution Buffer  was induced by β-D-thiogalactopyranoside (IPTG)  when the optical density (OD600) reached 0.7–0.9. Cells were harvested after 3 hours, resuspended in lysis buffer , and sonicated. The purification protocol was performed with Ni-NTA Agarose (Quiagen) beads and the elution with 250 mM Imidazole (Sigma).The protocol was based on manufacturer's recommendations for Ni-NTA Agarose (Qiagen). His-ZZ fusion protein expression in bacterial cultures (37°C, BL21 120 μg of His-ZZ-Mab21l2 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose beads (Amersham). The beads were recovered by centrifugation and washed ten times with 0.4 M KCl. The beads were incubated 1 h with 250 μg of precleared lysates of P19 treated and untreated with 40 ng/ml of BMP4. The beads were washed 5 times with 0.2 M KCl; the protein complexes were eluted by addition of Sample buffer followed by boiling for 5 minutes, and separated on an SDS-polyacrylamide gel for anti-Smad1 (1:2000 Santa Cruz) immunoblotting. The secondary antibody was a goat anti-rabbit HRP-conjugated antibody (Bio-Rad). The blots were developed with the Supersignal West Pico reagent (Pierce).120 μg of His-ZZ-Smad1 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose resin (Amersham). The beads were recovered by centrifugation and washed ten times with 0.4 M KCl.The beads were incubated 1 h with 250 μg of precleared lysates of COS7 cells transfected with mycMab21l2. The beads were washed 5 times with 0.2 M KCl; the protein complexes were eluted by addition of Sample buffer,boiled for 5 minutes and separeted on a SDS-polyacrylamide gel for anti-myc immunoblotting.2; 1 mM DTT; 0.2% NP-40, 10% glycerol). The beads were recovered by centrifugation and washed with Binding buffer. In-vitro translated, 35S-methionine-labeled Smad1 and Smad4 were prepared with the TNT coupled transcription/translation system (Promega). 45 μl of the TNT reaction were mixed with the His-ZZ-Mab21l2 bound to the IgG-Sepharose beads. The mixture was incubated for 2 hours at 4°C in Binding buffer. The Sepharose-protein complex was washed four times with Wash buffer , eluted by addition of Sample buffer followed by boiling for 5 minutes and separated on SDS-polyacrylamide gels. Gels were fixed and stained with 50% methanol, 10% acetic acid and 0.1% Comassie Blue for 20 minutes. After the incubation with Sodium salicylate 1 M (Fluka) for 20 minutes to improve the radioactive signal the gels were dried and exposed to Kodak X-Omat film o/n at -80°C.120 μg of His-ZZ-Mab21l2 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose beads (Amersham) in Binding buffer (50 mM Tris pH8; 50 mM KCl; 5 mM MgClin situ hybridizations and in vivo overexpression experiments.DB did AB did pull-downs, immunoprecipitations and luciferase assays.RL designed peptides and produced an anti MAB-21 polyclonal antibody.VZ produced constructs for, and carried out DNA-binding assays and luciferase assays. In addition he helped design and evaluate all cellular assays.GGC designed and coordinated the experimental work.All authors read and approved the final manuscript


A 24-y-old woman was admitted to the emergency department having had a generalized seizure (acute loss of consciousness, convulsive movements of her arms and legs, and confusion on regaining consciousness). She was on the sixth day of treatment with 300 mg daily of slow-release bupropion (Zyban SR) as an aid to smoking cessation. She had a past medical history of tonsillectomy and hay fever, for which she was taking budesonide nasal drops (two drops daily, each drop 200 mcg). She was on no other medication. There was no history of head trauma, liver disease, or alcohol withdrawal. Clinical examination, including neurological examination, was normal. The patient's weight was 48 kg. Her blood pressure was 130/80 mm Hg. Electrocardiogram showed a sinus tachycardia at 102 beats per minute. Radiography of the skull and a computed tomography scan of the brain without contrast were both normal. The patient's blood glucose, urea, electrolytes, and liver function tests were all normal. Her serum calcium was 2.01 mmol/l  and her hemoglobin was 116 g/l . The bupropion was discontinued, and the patient recovered without any further seizures or other neurological sequelae. Bupropion recently came onto the Mauritian market as an aid to smoking cessation. This case report is a useful reminder to clinicians of the risks of taking the drug Originally developed as an antidepressant, bupropion has more recently been licensed in many countries as an aid to smoking cessation. It came onto the Mauritian market as a smoking cessation aid in November 2003.www.bnf.org) recommends starting the drug one to two weeks before the target smoking stop date, initially at a dose of 150 mg daily for 6 d and then 150 mg twice daily. The maximum period of treatment is 7–9 wk; treatment should be discontinued if abstinence is not achieved by 7 wk.The British National Formulary .Bupropion is associated with a dose-related risk of seizure. The Medicines Control Agency states that the incidence of seizures is one in 1,000 based on doses up to the maximum recommended daily dose of 300 mg per day . The seiUp to 24 July 2002, in the UK there were 184 reports of seizures suspected as being associated with the use of bupropion [In the case we have presented, the patient had no current or previous history of seizure disorders, bulimia, or anorexia nervosa. She was on the sixth day of treatment with bupropion, taking 300 mg per day in two separate doses. She had no other predisposing risk factors for seizure. In particular she was taking no prescription medications, or over-the-counter medications (such as those containing ephedrine products) that are known to lower the seizure threshold. While systemic steroids can lower the seizure threshold, systemic absorption of the nasal steroid drops the patient was taking is an extremely unlikely cause for her seizure (she was not taking a high dose of high-strength drops). Her weight (48 kg) may have been an important factor, since bupropion reaches higher plasma levels in smaller individuals—indeed clinical trials of the drug have generally excluded patients weighing under 100 lb (about 45 kg) .This case report is a useful reminder to clinicians that bupropion is associated with a dose-dependent risk of seizures.In about half of the reports of seizure associated with bupropion, there was a past history of seizures and/or risk factors for their occurrence.It is extremely important to adequately assess seizure risk before prescribing bupropion.Patients should be made aware of the possible adverse effects of the drug.