Testing life history theory in a contemporary African population Meij, J.J.

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(1)Testing life history theory in a contemporary African population Meij, J.J.. Citation Meij, J. J. (2008, February 21). Testing life history theory in a contemporary African population. Retrieved from https://hdl.handle.net/1887/12615 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/12615. Note: To cite this publication please use the final published version (if applicable)..

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(3) Chapter 6 Chronic diseases in low-income countries: a role for infectious diseases and inflammation. submitted for publication. AHJ van den Biggelaar JJ Meij AJM de Craen J Amankwa M Frölich RGJ Westendorp. 107.

(4) Summary Background. Low-income. countries. are. confronted. with. an. emerging. epidemic of chronic diseases that in general is accounted for by behavioural and biological risk factors related to progressive urbanization. An alternative explanation could be the continuous exposure to infectious diseases and associated host inflammatory responses. Methodology/Principal Findings. Population dynamics and age-related. changes in inflammatory host responses were compared for a population living under adverse conditions in Ghana and an affluent Dutch population. Mortality rates at young age were high in Ghana and low in the Netherlands, a difference that disappeared in old age. In Ghana survival up to old age was mirrored by significant age-related increasing (lipopolysaccharide-induced) pro-inflammatory Tumour Necrosis Factor (TNF)-α responses (1.13 ± 0.05 fold increase for each 10 years, p=0.006) and decreasing anti-inflammatory Interleukine(IL)-10 responses (0.88 ± 0.04 fold decrease for each 10 years, p=0.001). In contrast, TNF-α and IL-10 responses were similar for all age groups in the Dutch population. Conclusions/Significance. Given the parallel between low survival proba-. bilities and increasing inflammatory response with age in Ghana, our data favour the hypothesis that in low-income countries continuous exposure to infections contributes to the burden of chronic inflammatory diseases by selection of subjects with strong pro-inflammatory responses.. 108.

(5) Introduction Chronic diseases are the leading cause of death worldwide [1,2]. In contrast to general expectation over 80% of these deaths occur in low- and middleincome countries. Not only does a higher absolute number of people live in low- and middle-income countries, also age- and sex specific death rates attributable to chronic diseases are higher [2]. Poor availability of preventive and therapeutic interventions as well as an elevated exposure to risk factors in people reaching middle and old age are supposed to underlie the high incidence of chronic diseases in these countries. Progressive urbanization and associated behavioural and biological risk factors such as smoking, high blood pressure, cholesterol, and overweight are increasingly recognised as important. factors. in. the. development. of. cardiovascular-. and. other. non-. communicable diseases [3,4].. An additional risk factor that, to our knowledge, has received little attention in this emerging epidemic of chronic diseases is the continuous exposure to infectious diseases and its associated inflammatory processes in populations living under adverse conditions. Whereas pro-inflammatory responses are produced to facilitate pathogen clearance, counteracting anti-inflammatory responses are required to control inflammation [5-8]. There is increasing evidence, that imbalanced pro- and anti-inflammatory processes contribute to the development of cardiovascular disease. For example, levels of C-reactive protein, a marker of low grade systemic inflammation, have been associated with coronary disease, myocardial ischemia, and infarction [9-11]. Moreover, results. from. population-based. studies. have. demonstrated. that. increased. levels of markers of inflammation such as cytokines, adhesion molecules, and acute-phase reactants, are associated with cardiovascular events [12-14]. In line with earlier analyses on historical cohorts, we therefore hypothesized that. a. continuous. challenge. by. infectious. pathogens. would. drive. host. immune responses towards an inflammatory risk profile that associates with chronic diseases in old age [15,16].. In the present study we assessed population demographic profiles together with age-related changes in inflammatory host responses in two different populations.. One. population. has. lived. under. lifelong. strong. adverse. conditions in a remote area of the Garu-Tempane district in Ghana; the other population was born and raised under affluent conditions in the Netherlands. For. both. populations. the. production. capacity. of. ex. vivo. bacterial. lipopolysaccharide (LPS)-induced levels of the pro-inflammatory cytokine Tumour Necrosis Factor (TNF)-α and anti-inflammatory Interleukin (IL)-10 were studied in a representative group of participants aged 20 through 70 years old. We hypothesized that with increasing age Ghanaian subjects would. 109.

(6) expose an immune response that is skewed towards a phenotype resistant for infectious. diseases. and. at. the. same. time. favours. the. development. of. cardiovascular and other chronic diseases.. Results The 2002 demographic data obtained from all 19.797 subjects in the GaruTempane district of the Upper-East region of Ghana show a pyramid-like distribution of age catagories (figure 1). In 2002, the median age of the population in the Garu-Tempane research area was thirteen years. In contrast, age groups in the general Dutch population are equally distributed up to old age. There is a higher frequency of middle-aged individuals born during the post World War II baby boom. Median age of the Dutch population is 33 years.. In. addition,. age-specific. mortality. rates. were. assessed. for. both. populations (figure 2). Mortality was higher for young and middle-aged people in the Garu-Tempane district compared to the general Dutch popula-. Figure. 1.. Population. distribution. in. the. Garu-Tempane. District,. Ghana. and. the. Netherlands. The top panel shows the population distributions in decades for males and females as assessed in the Garu-Tempane district in Ghana in 2002. The lower panel shows the population distributions for males and females in the general Dutch population in 2003 [28].. 110.

(7) tion whereas it was similar in old age.. The 91 Ghanaian blood donors for whom whole blood culture were available included 49 men and 42 women, with a median age of 46 years (IQR 36 – 54 years). The 87 Dutch subjects included in the cytokine analysis consisted of 47 men and 40 women with a median age of 46 years (IQR 33 – 54 years old). Very few participants produced cytokine levels above the detection limit in the non-stimulated control cultures, and those that did, produced very low levels.. All. producing Ghanaian. 1360. participants cytokine. subjects. responded. levels. far. in. the. exceeding. LPS-induced. cytokine. the. LPS-stimulated detection. production. cultures. by. limit. Among. the. levels. ranged. from. pg/ml to 17565 pg/ml (GM 4677 pg/ml, 95% CI 4092-5271) for TNF-.

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(9) . α and from 659 pg/ml to 10556 pg/ml (GM 3182 pg/ml, 95% CI 2823 –. 3583) for IL-10. For the Dutch subject, LPS-induced production levels of TNF-α ranged from 2055 pg/ml to 24056 pg/ml (GM 7422 pg/ml, 95% CI. . 6605 – 8339) and levels of IL-10 ranged from 489 pg/ml to 8636 pg/ml (GM.

(10) . 3406 pg/ml, 95% CI 2823 – 3719).

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(12). Figure 2. Age-related mortality rates in Garu-Tempane as observed over the period 2002-2005, and in the general Dutch population in 2003.. 111.

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(21) . . . . . . . . . . . . . Figure 3.. Age-related pro-inflammatory and anti-inflammatory responses in. Ghana and the Netherlands. Gender-adjusted TNF-alfa and IL-10 levels are expressed as geometric means with 95%. confidence. intervals. Associations. between. cytokine. levels. and. age. (P for. trends) were derived from linear regression models adjusted for gender, with age as a continuous variable. Unavoidable minor differences in cell culture conditions in Ghana. and. the. Netherlands. may. have. affected. the. absolute. level. of. cytokine. production. erefore, in order to facilitatie in comparing patterns between the two populations, the Y axis for TNF-alfa. and IL-10 respectively are optically similar for. Ghana and The Netherland, and hence do not start at zero.. 112. .

(22) In. the. Ghanaian. population. LPS-induced. production. of. TNF-α. levels. increased significantly with age (1.13 ± 0.05 fold increase for each 10 year increase of age, p = 0.006), whereas production levels of IL-10 decreased significantly (0.88 ± 0.04 fold lower for each 10 year increase of age, p = 0.001) (figure 3). In contrast, LPS-induced production of both TNF-α (0.98 ± 0.04 fold change for each 10 year increase of age, p = 0.715) and IL-10 (0.96 ± 0.03 fold change for each 10 year increase of age, p = 0.213) were similar for all age groups from the Dutch population.. Discussion Among the population of the Garu-Tempane district in the Upper-East region of Ghana, that still exhibits high mortality figures at young age, we found a progressive. imbalance. inflammatory. response. of. innate. with. immune. increasing. responses. age.. No. such. towards. a. pro-. imbalance. with. increasing age was present in a Dutch control population of similar age and sex.. We. postulate. that. in. the. Ghanaian. population. increasing. pro-. inflammatory host responses are driven by a survival mechanisms acting under the continuous burden of infectious diseases. Such survival mechanism is absent in the Dutch population under current affluent conditions.. There are two possible explanations for our observations. First, host immune responses may be modulated by infectious pathogens depending on the age and/or time of exposure of the host. The second explanation is selective survival. of. people. with. a. strong. pro-inflammatory. response.. If. a. large. proportion of a population dies young, subjects who manage to reach middle age will likely represent a sub-group of subjects genetically predisposed with mechanisms that helped them to survive. Given the environmental conditions in an underdeveloped area like the Garu-Tempane district of Ghana, such mechanisms are likely to be associated with resistance to infectious diseases [7,17,18].. Given. the. parallel. between. the. low. survival. probabilities. and. increasing pro-inflammatory responses from younger to older age groups in Ghana, our data favour the second explanation of selection of subjects with a strong pro-inflammatory innate immune response. Using historical data from cohorts born before the 20th century in European countries, it has been shown that increasing longevity and declining mortality in the elderly occurred among the same birth cohorts that experienced a reduction in mortality at younger ages and increasing adult height [15,16]. It was thus hypothesized that both the decline in old-age mortality and the increase in height were promoted by the reduced burden of infections and inflammation, a morbidity phenotype that persists from early age into adult life and is rooted in the adverse external environment. Here, for the first time,. 113.

(23) we provide data from contemporary cohorts that underpin this hypothesis. Systemic inflammation contributes to the development of atherosclerosis [11-14],. whereas. rupture. of. atherosclerotic. plaques. may. activate. pro-. inflammatory mechanisms alike induced by infectious pathogens that in the absence of a well-balanced anti-inflammatory response may reinforce the inflammatory host response. We therefore postulate that under continuous infectious. pressure. the. selection. for. pro-inflammatory. high. responders. contributes to the burden of chronic inflammatory diseases in middle-age people in low-income countries.. Despite the clear relationship with progressive urbanization, chronic diseases are clearly not solely related to a modern affluent lifestyle. A high prevalence of stroke morality has been shown in both poor rural and urban areas in Tanzania [19] and atherosclerotic lesions have been identified on autopsy in mummies of people who died at middle age [20]. This emphasizes that humans have always suffered from chronic diseases, which are therefore more ancient than a phenomenon of modern life. Whereas behavioural and biological risk factors such as smoking, high blood pressure, cholesterol, and body-mass. index. are. undoubtedly. important. risk. factors. for. the. current. epidemic of cardiovascular and other chronic diseases [2], inflammatory host response mechanisms are likely to play an inportant role in the development of these diseases outside the context of urbanization.. Genotypes associated with resistance to infection differ in frequency across different. populations. most. likely. due. to. (historical). selection. pressure. [18,21]. This includes both pro-and anti-inflammatory genotypes [22,23]. Although. selection. for. higher. anti-inflammatory. responses. does. not. immediately fit our observations, there are indications that survivors in lowincome countries may benefit from higher IL-10 production in an attempt to optimize the immune response [5,7,8]. When exposed to affluent conditions these. people. responses. may. strong. even. enough. lose to. their. ability. counterbalance. to. produce. anti-inflammatory. disproportionate. specific. and. non-specific inflammatory processes [24]. It even implies that under the optimistic scenario that infectious diseases will be eradicated, populations that were exposed to evolutionary pressure will remain at a high risk to develop excessive inflammation and chronic diseases. We are aware that our study has shortcomings. First, given the cross-sectional nature of our study, we could not associate the inflammatory immune profiles of the subjects with specific causes of mortality. Second, we have no data on immune responses or genotypes of subjects in the youngest age groups who are at the highest risk of dying of infectious diseases. We are currently. 114.

(24) undertaking a large cohort study in Ghana in which we further address this issue. Third, minor differences in the experimental culture conditions in Ghana and the Netherlands were unavoidable and may have affected the absolute level of cytokine production. This precludes a direct comparison of the cytokine production profiles between the two populations.. With the increasing control of infectious diseases, global mean life expectancy will continue to rise, as is the burden of chronic diseases. A healthy diet, regular physical activity, and avoidance of tobacco use may reduce the incidence of chronic disease in old age. However, the reported increase towards a pro-inflammatory host response with increasing age, together with high frequencies of genotypes associated with pro-inflammatory immune responses, may play a major additional role. Therefore, a further understanding. of. the. activation,. complex. and. interactions. genetics. is. between. essential. to. pathogen. properly. exposure,. anticipate. on. immune. the. global. increase in chronic diseases in old age.. MATERIAL and METHODS Populations The Ghanaian part of our study was conducted in the remote Garu Tempane district. in. the. Upper-East. region. of. Ghana.. This. densely. populated. agricultural area is inhabited by several tribes, mostly Bimoba and Kusasi. The. whole. Ghana. Upper-East. region,. and. especially. the. Garu-Tempane. district, is underdeveloped, with an estimated gross domestic product per head of less than $ 100, whereas for Ghana as a whole this is $ 2150. The region has a semi-Saharan climate with an average temperature of 32 ºC throughout the year and a yearly rain season from June to August. Our research area is situated around the village of Garu and measures approx. 375. 2 km . The vast majority of the people is farmer and the total agricultural. process is done by hand labour. The area is highly endemic for malaria, schistosomiasis, filariasis, and intestinal helminth infections. In recent years, the area has seen several outbreaks of meningitis. The prevalence of HIVinfection in the Garu-Tempane region is less than 1% [25]. Recently, in the area some health clinics have been set up, but these are not fully in service yet. Hospitals and medical services are absent. Vaccination of children was introduced. in. the. early. 1990s,. but. coverage. amongst. children. is. highly. variable. It is estimated that about 50% of the children under the age of 10 years has been vaccinated at least once against either measles, poliomyelitis, or diphteria-tetanus-pertussis. In 2001, we mapped our research area using a GPS system [26]. Since 2002, we have revisited the area annually to assess population changes. In 2002, 19.797 people, of whom 50% was under the age of fourteen years, lived in the research area. In order to study inflammatory. 115.

(25) immune responses, we matched 95 subjects from the Garu Tempane research area on age and sex with 95 Dutch subjects (described below). For all 95 Ghanaian subjects a venous blood samples was drawn in 2004. For four of the 95 Ghanaian subjects whole blood cultures were unsuccessful. None of the participants suffered from febrile illness and all were free from any medication that might have interfered with the immune system.. The Dutch individuals from whom whole blood cultures were available, originated from a series of 95 healthy Caucasians aged 20 to 70 years, all born and raised in the Netherlands. The subjects were enrolled in 2000 as a healthy control group in a study on the association between immune profile and risk of autoimmune diseases [27]. All control subjects were unrelated to the patients with the autoimmune disease. All were checked for health issues using a semi-structured questionnaire and were excluded when they suffered from diseases that might influence cytokine productions (medication and acute infections). For 87 of the 95 participants spare aliquots of supernatants that had been kept frozen since whole blood cultures were performed in 2000, were available for re-measurements.. The Medical Ethical Committee of the Ghana Health Service, as well as the Medical. Ethical. Committee. of. the. Leiden. University. Medical. Center. approved the studies. Witnessed observed informed consent was obtained from all Ghana participants and written informed consent was obtained from the Dutch participants.. Population mortality Age-specific mortality of the Dutch population was obtained from the 2003 Dutch population statistics database from the National Bureau of Statistics (CBS) in the Netherlands [28] For the Ghanaian Garu-Tempane population age-specific mortality was calculated from the number of age-specific deaths we observed during our annual population surveys. in the period 2002 –. 2005. Age specific mortality rates were calculated and are presented as Gompertz Curves.. Whole blood stimulation assay Both in the Ghanaian and the Dutch sample the pro-inflammatory and antiinflammatory cytokine production capacity of the innate immune system was assessed by stimulating ex vivo whole blood samples with lipopolysaccharide (LPS) as described elsewhere [29]. In short, all venous blood samples were drawn in the morning to exclude circadian variation, diluted twofold with RPMI-1640, and within two hours after collection cultured with medium alone. or. with. an. optimal. dose. of. 1. 116. pg/ml. E.coli-derived. LPS. (Difco.

(26) Laboratories, Detroit, Michigan, USA) in 24-well plates at duplicate volumes of. 1. ml. for. 24. hours. in. 370 C. incubators.. Whole. blood. cultures. were. performed at the study sites in Ghana and the Netherlands. Procedures and conditions were kept similar in both settings, except that a CO2 incubator set at 5% was used in the Netherlands, and ambient CO2 levels were induced by a candle jar incubation system in Ghana. In the candle jar incubation system culture. plates. are. placed. in. an. airtight. container. enclosed, and transferred as a whole to a 37. 0. with. a. burning. candle. C incubator once the candle has. faded [30]. The compatibility of both systems was compared in a small experiment in which whole blood assays were performed for the same five staff members at both study sites: LPS-induced levels of TNF-α and IL-10 were comparable (Wilcoxon Rank Test) for the ambient CO2 conditions in Ghana and the incubator set 5% CO2 conditions in the Netherlands (data not shown).. After 24 hours of incubation supernatants were collected and kept at –20° C in Ghana until transported on dry ice to the Netherlands. In the Netherlands all samples, including those of the Dutch population, were stored at –80° C until. cytokine. levels. were. determined. by. ELISA in. one. batch. in. 2005.. Cytokine ELISA for human TNF-α and IL-10 were performed according to manufacturers’ guidelines. (Central. Laboratory. of. the. Blood. Transfusion. Service, Amsterdam, the Netherlands), with detection limits of 4.0 pg/ml and 3.0 pg/ml respectively. Supernatants from the Dutch sample were tested for a possible denaturation of cytokine proteins during their 5-year storage at –80° C. No significant differences between aliquots measured in 2000 and 2005 were found (data not shown).. Statistical analysis Cytokine levels were log-transformed and are described as geometric means with 95% confidence intervals. Since we cannot exclude that the absolute values of cytokine production are influenced by unavoidable differences in the experimental conditions, we refrained from comparing cytokine levels between. the. Ghanaian. and. Dutch. study. groups.. Associations. between. cytokine responses and age were assessed with linear regression analysis with. log-transformed. continuous. cytokine. independent. data. variable.. as. dependent. Back. variable. transformation. of. and the. age. as. a. obtained. regression coefficients results in a multiplicative model, meaning that the coefficient from the linear regression model equals the relative change in cytokine levels per year increase of age. All models were adjusted for gender. Calculations were performed with SPSS version 12.0.. 117.

(27) ABBREVIATIONS CI. Confidence interval. GM. Geometric mean. IL-10. Interleukine-10. IQR. Interquartile range. LPS. Lipopolysaccharide. TNF-α. Tumour Necrosis Factor-α. 118.

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