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Changes in the levels of cytokines, chemokines and malaria-specific antibodies in response to Plasmodium falciparum infection in children living in sympatry in Mali
Stéphanie Boström1,
Pablo Giusti1,
Charles Arama1,2,
Jan-Olov Persson3,
Victor Dara2,
Boubacar Traore2,
Amagana Dolo2,
Ogobara Doumbo2 &
Marita Troye-Blomberg1
The Fulani are known to be less susceptible to Plasmodium falciparum malaria as reflected by lower parasitaemia and fewer clinical symptoms than other sympatric ethnic groups. So far most studies in these groups have been performed on adults, which is why little is known about these responses in children. This study was designed to provide more information on this gap.
Circulating inflammatory factors and antibody levels in children from the Fulani and Dogon ethnic groups were measured. The inflammatory cytokines; interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF) and the chemokines; regulated on activation normal T cell expressed and secreted (RANTES), monokine-induced by IFN-gamma (MIG), monocyte chemotactic protein (MCP)-1 and IFN-gamma-inducible protein (IP)-10 were measured by cytometric bead arrays. The levels of interferon (IFN)-alpha, IFN-gamma and malaria-specific antibodies; immunoglobulin (Ig) G, IgM and IgG subclasses (IgG1-IgG4) were measured by ELISA.
The results revealed that the Fulani children had higher levels of all tested cytokines compared to the Dogon, in particular IFN-gamma, a cytokine known to be involved in parasite clearance. Out of all the tested chemokines, only MCP-1 was increased in the Fulani compared to the Dogon. When dividing the children into infected and uninfected individuals, infected Dogon had significantly lower levels of RANTES compared to their uninfected peers, and significantly higher levels of MIG and IP-10 as well as MCP-1, although the latter did not reach statistical significance. In contrast, such patterns were not seen in the infected Fulani children and their chemokine levels remained unchanged upon infection compared to uninfected counterparts. Furthermore, the Fulani also had higher titres of malaria-specific IgG and IgM as well as IgG1-3 subclasses compared to the Dogon.
Taken together, this study demonstrates, in accordance with previous work, that Fulani children mount a stronger inflammatory and antibody response against P. falciparum parasites compared to the Dogon and that these differences are evident already at an early age. The inflammatory responses in the Fulani were not influenced by an active infection which could explain why less clinical symptoms are seen in this group.
Infection with Plasmodium falciparum remains one of the most common infectious diseases worldwide and is still a major cause of morbidity and mortality in tropical regions, especially in children below the age of five. Immunity to malaria is dependent on both the innate and the adaptive arms (both cell- and antibody mediated) of the immune system, which are required for adequate protection [1].
Inflammatory cytokines play an important role in human immune responses to infection with P. falciparum but also seem to contribute to immunopathology and the severity of the disease if not controlled properly. Available data are consistent with a requirement for an early production of in particular interferon (IFN)-γ to mount resistance against infection [2], and protection from clinical episodes [3]. However, other pro-inflammatory cytokines such as interleukin (IL)-12 and tumour necrosis factor (TNF) have also shown to be essential mediators in this protection [4]. They all appear to be necessary for the inhibition of parasite growth and stimulation of phagocytosis to enhance clearance of parasitized erythrocytes. Plasma levels of TNF and nitric oxide, secreted by activated macrophages and neutrophils, are associated with resolution of fever and parasite clearance [3]. Other cytokines such as IL-1, IL-6, IL-8, IL-10 and IL-12 have been implicated in the pathogenesis of severe malaria cases compared to uncomplicated and matched healthy controls [5, 6].
Chemokines are chemotactic cytokines that play important roles in bridging the innate and the adaptive immune system [7]. They orchestrate the migration of leucocytes and other cells by activating corresponding receptors on responsive cells, thereby inducing chemotaxis of immune cells to sites of infection. Studies have shown that chemokine levels in children with acute P. falciparum infections vary with disease severity [8] and some, such as IP-10 and RANTES, have been associated with mortality during cerebral malaria [9–11].
Even though the innate immune system is essential during P. falciparum infections, the importance of antibody-mediated response against P. falciparum parasites was shown 50 years ago in passive transfer studies, in which immunoglobulin (Ig) G antibodies from immune African adults reduced the level of parasites in Gambian children [12]. However, accumulating evidence suggests a role for the cytophilic antibodies IgG1 and IgG3 to be protective against P. falciparum infection, whereas the role for IgG2 and IgG4 in terms of protection are less clear and has been suggested to interfere with the protective mechanisms seen by the cytophilic antibodies [13–16]. Thus, protection against malaria depends on early, and intense but carefully balanced pro-inflammatory response to control/reduce the parasite numbers in the human host, which in turn shape the following adaptive immune response, and when such is properly induced it will protect against challenge.
In Mali, previous studies have shown different susceptibility to P. falciparum infection between two ethnic groups; the Fulani and the Dogon [17]. Similar findings have been shown in Burkina Faso between Fulani and their sympatric ethnic groups Rimaibé and Mossi [18]. These populations live under similar social, cultural and geographic conditions and are exposed to similar malaria pressure [17, 18]. Despite this, the Fulani are more resistant to P. falciparum infection than the other ethnic groups as reflected by fewer clinical symptoms of malaria and less parasite clones and numbers found in their blood [17, 18]. In addition, the Fulani have also been shown to have higher titres of malaria-specific IgG [15, 16, 19] and IgM [14] antibodies as well as higher total IgG and IgM levels than other sympatric groups [14]. This higher humoral response in the Fulani has been associated with increased splenomegaly compared to neighbouring ethnic groups [20]. The consistently increased spleen rates found in the Fulani compared to the Dogon (whose spleen rates change upon infection) suggest a more acute response in the spleen to the parasites and clinical disease in the Dogon. Thus, the two groups appear to have different responses when coping with malaria infections. Furthermore, the Fulani has been shown to have higher percentages of IL-4 and IFN-γ producing cells [19] and, recently, lower expression of genes distinctive of Treg activity (foxp3 and CTLA-4) has been observed indicative of a stronger inflammatory response [21]. Established genetic malaria resistance factors such as haemoglobin S and C, alpha thalassemia, G6PD and HLA B have been shown to occur at a lower frequency in the Fulani than in their sympatric neighbours [22], suggesting that various hemoglobinopathies is not the reason for Fulani's lower susceptibility to malaria. The Fulani seem to mount a more potent early IFN-γ response against the parasite [23] compared to other sympatric ethnic groups, which indicates differences in immune regulation between these groups. This is emphasized by the findings that Fulani and Dogon children respond differently to P. falciparum infection in terms of antigen-presenting cell (APC) subset activation and toll-like receptor (TLR) stimulations [24]. Nevertheless, the mechanism underlying the lower susceptibility to P. falciparum infection in Fulani as compared to other sympatric ethnic groups is not fully understood. Earlier studies have mostly focused on asymptomatic adults, and little is known about this in children. The focus of this study has, therefore, been to investigate inflammatory- and antibody responses in infected and uninfected children from Dogon and Fulani ethnic groups living in Mali, by measuring selected cytokines, chemokines and IgG subclasses in plasma obtained from these children.
Children between 2 and 10 years of age belonging to either the Fulani or the Dogon ethnic group was included in the study. In total, 40 children from the Dogon and 37 from the Fulani ethnicity were recruited. Healthy children and children diagnosed with asymptomatic malaria were included from both ethnic groups. A thick blood smear was made from each donor and the slides were stained in 3% Giemsa and examined for the presence of P. falciparum parasites. Malaria infection was defined as having a positive thick blood smear with or without any malaria symptoms. Axillary temperature was measured in all children and symptomatic malaria was defined as fever ≥ 37.5°C, or history of fever, plus the presence of any density of parasites in the blood. Among the Dogon, 20 children were undergoing a malaria infection and 20 children were healthy, while in the Fulani, 14 children were having a malaria infection and 23 were healthy. Written informed consent was obtained from both the community and from the children's guardians before inclusion in this study. The study was approved by the institutional review boards of the University of Bamako Mali (N°08_64/FMPOS).
This study was conducted in a rural area of the Dogon valley located approximately 850 km from the capital of Bamako, in Mali. The malaria transmission in the area is meso-endemic with intense transmission during the rainy season that usually extends from June to October [17]. Samples were collected from October to November 2008, from two rural villages (Manteourou and Binédama) where people from the Dogon and Fulani ethnic groups live together in sympatry. The study area and the study population have been described in detail elsewhere [17].
Blood samples were collected from uninfected and infected children of both ethnicities. From each donor, 6 ml of venous blood were collected in heparin tubes (BD Vacutainer® Plasma Tube, 143 USP Units of Sodium Heparin freeze dried) and plasmas were obtained by centrifugation and were kept at -80°C and transported to Stockholm University and analysed for cytokine and antibody levels.
Determination of cytokine levels in plasma samples using ELISA
To quantify levels of IFN-α and IFN-γ in plasma, ELISA kits using pairs of capture and detection antibodies were used according to the manufacturer's recommendation (Mabtech, Stockholm, Sweden). All samples were diluted 1:2 and tested in duplicates. The enzyme-substrate reaction was developed using p-nitrophenyl phosphatase (Sigma, St Louis, MO, USA) and the optical densities were measured at 405 nm in an ELISA plate reader (VmaxTM Kinetic Microplate Reader, Menlo Park, CA, USA). The concentrations were calculated from a standard curve obtained in a sandwich ELISA with eight dilutions of lyophilized native human IFN-α standard (range 3.0-10,000 pg/ml) and recombinant human IFN-γ standard (range 3,000-1.0 pg/ml).
Determination of cytokines and chemokines in plasma samples using cytometric bead array
Levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF, regulated on activation normal T cell expressed and secreted (RANTES), monokine-induced by IFN-γ (MIG), monocytes chemotactic protein (MCP)-1 and IFN-gamma-inducible protein (IP)-10 were measured in plasma using cytometric bead array (CBA) (BD Biosciences, San Diego, CA, USA) according to the manufacturer's recommendation. Briefly, 24 μl of bead populations with discrete fluorescent intensities coated with cytokine capture antibodies, were added to 24 μl of plasma sample and the mixtures were incubated for 90 min in room temperature. At the same time, standards for each cytokine/chemokine (range 0-10.000 pg/ml) were also mixed with cytokine specific capture beads. After incubation, the samples and standards were washed to remove unbound material and 24 μl of phycoerythrin (PE)-conjugated anti-human inflammatory cytokine or chemokine antibodies were added and mixtures were incubated again for 90 min followed by washing an acquisition on a fluorescent activated cell sorter (FACS) Calibur (BD Biosciences). Samples and standards were analysed using FCAP Array software v1.0.1 (BD/Softflow, Pécs, Hungary). To determine levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF samples were incubated with anti-cytokine capture beads for 90 min, washed and incubated with PE-detection reagent for another 90 min and after final washing acquisition was done on FACS. Calibration was performed on the flow cytometer before acquisition using BD FACSComp™ and BD CaliBRITE™. The lower detection limit for the various cytokines and chemokines was 3.6, 7.2, 2.5, 3.3, 3.7, 1.9, 1.0, 2.5, 2.7 and 2.8 pg/ml for IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70, RANTES, MIG, MCP-1 and IP-10 respectively.
Preparation of crude malaria antigen
Plasmodium falciparum parasites of the strain F32 were held in continuous cultures as described elsewhere [25] and kept synchronized by treating the parasites repeatedly with sorbitol. Crude malaria antigen was prepared as previously described by Troye-Blomberg and colleagues [26]. A control antigen was prepared the same way as the crude malaria antigen by sonication of erythrocytes that had been held in cultures and had been treated the same way as the parasite cultures.
Determination of circulating levels of malaria-specific antibodies
Levels of malaria-specific antibodies were quantified using sandwich ELISAs as described by Bolad and colleagues [14] but with some modifications. Briefly, 96-well ELISA plates (Costar, Corning, NY) were coated with 50 μl crude malaria antigen (10 μg/ml) in coating buffer (pH 9.6) over night at 4°C. After blocking for 2 hr with 100 μl of coating buffer containing 0.5% bovine serum albumin, the plates were washed with phosphate buffered saline containing 0.05% Tween 20. Plasma dilutions for the determination of the IgG subclasses (50 μl, 1:20 for IgG2 and IgG4 and 1:400 for IgG1 and IgG3) were added to the plates and incubated for 1 hr at 37°C. For determination of total malaria-specific IgG and IgM antibodies, plasma samples were diluted 1:1000 and 1:500, respectively. To detect IgG2, IgG3 and IgG4, 50 μl biotin-conjugated mouse anti-human monoclonal antibodies (IgG2 diluted 1:3000 (PharMingen, San Diego, CA, USA), IgG3 diluted 1:1000 (Caltag laboratories, Burlingame, CA, USA) and IgG4 diluted 1:2000 (PharMingen), and strep-ALP (Mabtech, Stockholm, Sweden) were used for each subclass. Detection of IgG1 was done using a mouse anti-human monoclonal IgG1 antibody (SkyBio, Bedfordshire, England) and alkaline phosphatase conjugated goat anti-mouse Ig (Dako Denmark A/S, Glostrup, Denmark). To detect malaria-specific IgG and IgM, 50 μl goat-anti-human IgG-ALP (Mabtech, räcker med stad och land första gången företaget nämns sedan bara namnet på företaget) and 50 μl goat-anti-human IgM-ALP (Jackson ImmunoResearch Laboratories, PA, USA) were added to the wells. The assays were developed using p-nitrophenyl phosphatase as a substrate (Sigma, St Louis, MO, USA) and the optical densities were measured at 405 nm in an ELISA plate reader (VmaxTM Kinetic Microplate Reader, Menlo Park, CA, USA). The concentrations of malaria-specific antibodies were calculated from standard curves obtained from sandwich ELISA with six dilutions of myeloma proteins of the IgG1-4 isotypes (Biogenesis, Poole, England) or for total malaria-specific IgG and IgM antibodies, using highly purified IgG or IgM antibodies, respectively (Jackson ImmunoResearch Laboratories, PA, USA).
Differences between the Dogon and Fulani were analysed for statistical significance using Kruskal-Wallis non-parametric test. If differences were seen, a Mann-Whitney U non-parametric test was performed, to distinguish intra- and inter ethnic differences between infected and non-infected Fulani and Dogon. Spearman rank correlation was employed when analyzing possible correlations. Multiple regression was employed to correct for age on antibody titres. Statistical significance was assumed when p < 0.05. The data were analysed using StatView software 5.0.1 and Stata 12.
Study subject characteristics
The results from study subject characteristics have been described elsewhere [24]. There were no differences in age, haemoglobin levels and axillary temperature between the Dogon and Fulani children groups, irrespective of infectious status (Table 1). Likewise, no differences were seen in haemoglobin and axillary temperature between uninfected individuals from both ethnic groups. However, when children were divided into infected and uninfected individuals, infected Dogon children had significantly higher axillary temperature compared to uninfected Dogon children (37.74°C versus 36.43°C, p < 0.0001) and to infected Fulani children (37.74°C versus 36.81°C, p = 0.002). There was no difference between infected and uninfected Fulani children (36.81°C versus 36.71°C, p = 0.97). The infected Fulani children were found to be younger than both the infected Dogon (4.50 versus 6.70, p = 0.01) and the uninfected Fulani children (4.50 versus 6.44, p = 0.007). No difference in age was seen between infected and uninfected Dogon children (6.70 versus 6.35, p = 0.47). The mean parasite density was 24,151 (100-122,000) asexual parasites/μl for the Fulani and 13,692 (575-48,625) asexual parasites/μl for the Dogon (p = 0.17). There were two outliers among the infected Fulani children with very high parasitaemia. When these were excluded from the analysis the mean parasite density in Fulani was 1,618 (100-7,875) asexual parasites/μl and hence being less parasitized compared to Dogon (p = 0.004).
Table 1 Description of the Fulani and the Dogon children in the present study
Increased plasma cytokine levels in Fulani children as compared to Dogon
Blood plasma samples from the Dogon and Fulani children were analysed for levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF, IFN-α and IFN-γ (Figure 1). Detectable levels of each cytokine were divided according to infectious status and/or ethnicity. When comparing the Fulani to the Dogon children regardless of infectious status, higher levels of IL-6 (p = 0.0003; Figure 1A), IL-8 (p = < 0.0001; Figure 1B), IL-12p70 (p = 0.01; Figure 1C), IFN-α (p = 0.003; Figure 1D) and IFN-γ (p = 0.017; Figure 1E) were found in the Fulani. When comparing the uninfected children from both groups with each other, higher levels of IL-6 (p = < 0.0001), IL-8 (p = 0.0002), IL-12p70 (p = 0.001) and IFN-α (p = 0.003) were found in Fulani compared to Dogon children.
Levels of inflammatory cytokines in plasma from Dogon and Fulani children. Blood plasma samples from 77 children were analysed for circulating cytokines. Levels of IL-6 (A), IL-8 (B) and IL-12p70 (C) were measured with cytometric bead array. Levels of IFN-α (D) and IFN-γ (E) were measured with ELISA kits. The children were subdivided in terms of ethnicity and with regard to being infected or not according to slide positivity to malaria; i.e. uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 23) and infected Fulani (n = 14). The boxes represent the values between 25% and 75% quartile and the line indicates the median. The whiskers indicate the 10% and 90% percentiles. Statistical analysis was done by Mann-Whitney U test.
When comparing the levels in the infected children of the two groups, IFN-γ was the only factor out of all tested that showed a significant difference, where the levels were higher in infected Fulani as compared to infected Dogon (p = 0.04).
When the levels were compared within the same ethnic group in relation to P. falciparum infection, it was found that infected Dogon children had significantly higher levels of IL-6 (p = < 0.0001) and IL-12p70 (p = 0.0002) compared to the uninfected Dogon. Likewise, the only factor that showed significant difference between infected and uninfected Fulani was IL-8, which was lower in the infected Fulani (p = 0.048).
In most cases levels of IL-1β and TNF were under the detection limit for the kit and were therefore excluded from analysis.
Chemokine levels differ in infected Dogon and Fulani children
Blood plasma from the Dogon and Fulani children were analysed for levels of RANTES, MIG, MCP-1 and IP-10 (Figure 2). When comparing the Fulani children to the Dogon children, regardless of infectious status, MCP-1 but not RANTES, MIG and IP-10 showed significant difference between the two ethnic groups (p = 0.01; Figure 2C).
Levels of chemokines in plasma from Dogon and Fulani children. Blood plasma samples from 77 children were analysed for circulating chemokines. Levels of RANTES (A), MIG (B), MCP-1 (C) and IP-10 (D) were measured with cytometric bead array. The children were subdivided in terms of ethnicity and with regards to being infected or not according to slide positivity to malaria; i.e. uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 23) and infected Fulani (n = 14). The boxes represent the values between 25% and 75% quartile and the line indicates the median. The whiskers indicate the 10% and 90% percentiles. Statistical analysis was done by Mann-Whitney U test.
When comparing the uninfected children from both groups, the uninfected Dogon children had higher levels of RANTES (p = 0.05) but lower levels of MCP-1 (p = 0.007) and IP-10 (p = 0.05) compared to uninfected Fulani. In contrast, when comparing infected Dogon to infected Fulani children, no such difference was seen for any of the factors tested.
When comparing children from the same ethnic group, there were no differences for any of the factors tested between infected and uninfected Fulani children. In contrast, infected Dogon children had significantly lower plasma levels of RANTES (p = 0.003) and significantly higher plasma levels of both MIG (p = 0.006) and IP-10 (p = 0.001) as compared to uninfected Dogon. The infected Dogon also had increased levels of MCP-1 compared to uninfected Dogon, even though this did not reach statistical significance (p = 0.15).
Malaria-specific antibody titres are higher in Fulani children as compared to Dogon
Levels of malaria-specific total IgG and IgM as well as the IgG subclasses; IgG1, IgG2, IgG3 and IgG4 antibodies were measured in blood plasma samples from Dogon and Fulani children (Figure 3). When analysing the data regardless of infectious status, the Fulani children exhibit higher titres of both malaria-specific IgG and IgM antibodies as compared to the Dogon population (p ≤ 0.0001; Figure 3A + B). When comparing infected and uninfected children within the same ethnic group, infected Dogon children had higher IgM titres as compared to uninfected Dogon (p = 0.05). In concordance with this, higher titres of malaria-specific IgG subclasses in Fulani population as compared to Dogon for IgG1 (p ≤ 0.0001; Figure 4A), IgG2 (p ≤ 0.0001; Figure 4B) and for IgG3 (p ≤ 0.0001; Figure 4C) but not for IgG4 (p = 0.19; Figure 4D) were found. The same differences were also seen when only comparing the uninfected individuals from the two ethnic groups.
Distribution of malaria-specific IgG and IgM antibodies in response to Plasmodium falciparum infection in Fulani and Dogon children. Blood plasma levels from 77 children were analysed for malaria-specific IgG (A) and IgM (B) antibodies. The children were subdivided in terms of ethnicity and with regards to being infected or not according to slide positivity to malaria; i.e. uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 23) and infected Fulani (n = 14). The boxes represent the values between 25% and 75% quartile and the line indicates the median. The whiskers indicate the 10% and 90% percentiles. Statistical analysis was done by Mann-Whitney U test.
Distribution of malaria-specific IgG subclasses in response to Plasmodium falciparum infection in Fulani and Dogon children. Blood plasma samples from 77 children were analysed for malaria-specific IgG subclasses IgG1 (A), IgG2 (B), IgG3 (C) and IgG4 (D). The children were subdivided in terms of ethnicity and with regards to being infected or not according to slide positivity to malaria; i.e. uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 23) and infected Fulani (n = 14). The boxes represent the values between 25% and 75% quartile and the line indicates the median. The whiskers indicate the 10% and 90% percentiles. Statistical analysis was done by Mann-Whitney U test.
When comparing the infected children of both ethnicities, the infected Fulani children had higher titres of malaria-specific IgG1 (p = 0.03) and IgG3 (p = 0.001) as compared to infected Dogon children (Figure 3A + C).
When comparing infected and uninfected children within the ethnic groups, IgG3 was significantly higher in infected Dogon as compared to uninfected individuals (p = 0.034). The reverse pattern was seen in Fulani, in which uninfected Fulani had higher titres as compared to infected individuals (p = 0.045) (Figure 3C). No antibody reactivity for any of the groups was seen for the control antigen (not listed).
Available studies trying to resolve the reason for the Fulani's relative protection against malaria have so far focused on immunological responses mostly in adults and not so much is known about these responses in children. The previously established relative protection for Fulani seen in adults was in this study shown to be evident during early childhood in this ethnic group. Children from the Fulani ethnic group had higher titres of malaria-specific antibodies and pro-inflammatory cytokine levels as well as unchanged chemokine levels upon an infection, as compared to Dogon children, suggesting that these differences are established already early in life.
Cytokines and chemokines are essential mediators during P. falciparum infection and the balance between pro- and anti-inflammatory cytokines may be important for the clinical outcome of malaria [27]. Higher levels of all detectable cytokines were found in the Fulani group when comparing all Fulani children to all Dogon children, irrespective of infectious status. When children were further subdivided and the uninfected children from both ethnic groups were compared, the uninfected Fulani were found to have higher levels of IFN-α, IL-6, IL-8 and IL-12 compared to the uninfected Dogon. This indicates that Fulani children might be more prone to combat P. falciparum infections, possibly due to higher baseline levels of these cytokines.
When comparing the infected children from both ethnic groups, IFN-γ was the only factor out of all tested that was significantly increased in infected Fulani compared to infected Dogon. This observation is in line with previous findings by McCall and colleagues who reported that mononuclear cells from Fulani are able to produce a 10-fold stronger IFN-γ response after stimulation with Plasmodium parasites compared to cells from Dogon [23]. Available studies in both humans and mice have demonstrated the importance of an early IFN-γ response as a crucial determinant in the outcome of the infection and the well-being of the patient [28, 29]. The result suggests that the increased levels of IFN-γ seen in infected Fulani may play a critical role in the difference in susceptibility to malaria in these two ethnic groups.
When comparing the uninfected children from both ethnic groups, the levels of IL-8 were found to be higher in uninfected Fulani compared to uninfected Dogon. However, when comparing differences within the same ethnic group, the levels of IL-8 were significantly lower in infected Fulani compared to their uninfected peers and this difference was not evident between infected and uninfected Dogon. IL-8 is a chemoattractant cytokine known to be involved in recruiting neutrophils to inflammatory sites. Very little information concerning the role for IL-8 in malaria pathogenesis/protection exists. IL-8 has been shown to be elevated in a small group of severe malaria cases in adults [30]. In addition, high expression levels of IL-8 mRNA have also been demonstrated in placental malaria [31]. Thus, the relevance for the lower levels of IL-8 that Fulani children exhibit upon infection is presently unknown and needs to be further investigated.
Further, the only cytokines that showed differences between infected and uninfected Dogon were IL-6 and IL-12p70. The results showed higher levels of IL-6 and IL-12p70 in infected Dogon compared to their uninfected counterparts, and these differences were not seen between infected and uninfected Fulani children. IL-6 is a major mediator of the acute phase response. Increased P. falciparum-induced IL-6 production has previously been associated with increased incidence of clinical episodes [32] and others have suggested IL-6 to be a marker for complicated P. falciparum malaria [33]. The fact that IL-6 increased upon infection in Dogon but not in Fulani, might imply this cytokine to be important in the pathogenesis of malaria. IL-12p70 has a fundamental role in the induction of Th1-associated immunity. Low levels of IL-12p70 have been reported to be associated with severe malaria disease in children [34, 35]. The Fulani children had unchanged levels of IL-12p70 upon infection, while the levels increased in Dogon children. However, there were no correlations between the levels of IL-6 (p = 0.24, Rho = 0.28) and IL-12p70 (p = 0.26, Rho = -0.28) with parasitaemia, which could be due to small sample size in this study population.
The role of chemokines and their distribution in plasma has not been investigated in the Fulani and Dogon ethnic groups. The results from this study show that all chemokines tested were unchanged upon an infection in Fulani children, while this was not the case in Dogon. Infected Dogon children undergoing an infection had lower levels of RANTES compared to uninfected Dogon. Low levels of RANTES have previously been reported to be associated with mortality in children with severe malaria [36], in particular in cerebral malaria [9], and decreased levels have also been reported to be correlated with increased disease severity [8]. One reason for the low levels of RANTES in severe malaria may be due to thrombocytopaenia that is often seen during these conditions, as platelets are a major reservoir of RANTES. The low level of RANTES seen in infected Dogon could thus be a result of destroyed platelets even though this was not tested.
The observed higher levels of MIG, MCP-1 and IP-10 in infected Dogon children compared to uninfected children. This marked up-regulation of chemokines was not evident in the infected Fulani children. Chemokines are involved in attracting different immune cell populations to the site of infections thereby aiding in the inflammatory response. Some of these factors have been implicated in severe malaria disease as well as in inflammatory diseases [37]. High levels of IP-10 have been shown to be associated with increased risk of cerebral malaria associated mortality [10], IP-10 and MIG are required for development of murine cerebral malaria [38] and elevated levels of these factors have also been found in visceral leishmaniasis [39].
Taken together it seems Fulani already have high baseline levels of these chemokines in circulation compared to Dogon since the levels do not change when they become infected, again suggesting that they are more prepared for combating infections. Since chemokines such as MIG, MCP-1 and IP-10 are involved in cell migration, the lower frequencies of these chemokines found in uninfected Dogon might imply that these cells are not able to migrate as well as cells from uninfected Fulani. Interestingly, Arama and colleagues [24] reported that infected Dogon children have higher percentages of certain antigen-presenting cell (APC) subsets in circulation but that they are less activated compared to APC obtained from Fulani children. In contrast, the Fulani children had fewer but more activated APC in the circulation, which suggests that the cells have migrated to the secondary lymphoid organs and are therefore not seen in the circulation.
It has been known since the early 1960s that immunoglobulins (Ig) play an important role in malarial immunity [12]. The result from this study indicates that the Fulani children have a stronger anti-malaria specific antibody response as compared to the Dogon, as revealed by higher titres of malaria-specific IgG and IgM antibodies. This has been reported earlier by others who compared adult Fulani with adults from their neighbouring ethnic groups, living in both Burkina Faso [14] and in Mali [14–16]. Previous studies have shown that antibody titres are correlated with age and parasitaemia [40], therefore the relationship between high parasitaemia and low antibody titres as well as a correlation between age and high antibody titres in these children was investigated. However, such analysis showed that there were no correlations for total IgG in infected Dogon (p = 0.63, Rho = 0.11) as well as for infected Fulani (p = 0.43, Rho = -0.32) between high parasitaemia and antibody titres. In contrast, the correlation between age and antibody titres showed an increase of total IgG antibodies with increasing age in uninfected Dogon (p = 0.005, Rho = 0.64), which was not the case in uninfected Fulani (p = 0.47, Rho = -0.17), suggesting that Fulani children already have high antibody titres irrespective of age in this study population. Similar results were found for IgG1 (p = 0.07, Rho = 0.40) and IgG3 (p = 0.02, Rho = 0.52) between age and antibody titres in the uninfected Dogon, which was not the case for uninfected Fulani (p = 0.57, Rho = -0.14) and (p = 0.23, Rho = 0.25) for IgG1 and IgG3, respectively.
To reassure that the malaria-specific antibodies are specific for the parasite, a control antigen of sonicated erythrocytes were included on all plates. There was no reactivity in any sera tested with sonicated erythrocytes confirming that the antibodies detected against the crude antigen are parasite specific.
Growing evidence supports that not all immunoglobulin subclasses are associated with protection against malaria, but rather it is the cytophilic subclasses; i.e. IgG1 and IgG3, that are important. IgG1 and IgG3 antibodies have shown to be crucial in parasite clearance by binding to the surface of the infected erythrocyte or to mediate opsonization by cooperating with human monocytes [13]. The observed higher titres of malaria-specific antibodies in the Fulani compared to the Dogon for IgG1-IgG3 but not for IgG4 are in concordance with previous studies [14–16]. These differences were still significant when correcting for age (not listed). Regarding IgG3 levels, one interesting observation was made when dividing the children into infected and uninfected individuals. The infected Fulani had lower titres of IgG3 antibodies than uninfected children. This is surprising since IgG3 is an isotype that has previously been suggested to play an important role in the protection against malaria [13]. One reason for the lower levels seen in Fulani could be that these antibodies have formed immune-complexes and thus are no longer detectable in the circulation. However, after correcting for age this difference was not statistically significant between the two groups anymore (not listed). The reason for this is not know but might be due to the different age distributions in the two groups (the infected Fulani children included the youngest children in the study) as well as the small sample size. Further studies are needed to clarify this.
The result showed that the titres of malaria-specific IgG2 and IgG4 antibodies were relatively low in both Fulani and Dogon. These antibodies are thought to inhibit the protective effects mediated by the cytophilic antibodies (IgG1 and IgG3) and could thereby potentially inhibit their neutralizing effect on the parasite. However, the role of IgG2 antibody has shown somewhat conflicting results and recent studies have indicated that this antibody may have a protective role against P. falciparum malaria in individuals carrying a certain Fcγ receptor subtype [15]. Furthermore, it has also been shown that high levels of IgG2 in combination with low levels of IgG4 antibodies are associated with protection against malaria [41]. There are also studies indicating that it is not the relative amount of these malaria-specific antibodies that is important, but rather the balance between the cytophilic and the non-cytophilic antibodies that determines the outcome during an infection [42].
Taken together, the results from this study demonstrate that there are marked differences between Fulani and Dogon immune responses to P. falciparum infection and that these differences are evident at an early age. The Fulani were shown to have higher levels of all tested inflammatory cytokines compared to the Dogon children, especially levels of IFN-γ, a cytokine that is crucial in determining the outcome of an infection. When children were further subdivided into infected and uninfected individuals, infected Dogon children had significantly lower levels of RANTES compared to uninfected Dogon, while significantly higher levels of MIG and IP-10 and also higher levels of MCP-1. Such patterns of differences in chemokine levels between infected and uninfected individuals were not evident in Fulani children whose levels remained unchanged upon an infection with P. falciparum. In addition, the results showed that the Fulani children had higher titres of malaria-specific antibodies compared to the Dogon children. This was true for levels of IgG1 and IgG3 antibodies, which have been shown previously to be involved in protection against malaria. The findings presented here suggests that there are marked differences in the cytokine and chemokine levels as well as in malaria-specific antibody titres between Fulani and Dogon that can be found at childhood age in these ethnic groups. Such difference might be important for the relative protection against malaria seen in the Fulani as compared to the Dogon. However, due to the small sample size in this study a prospective evaluation of a larger cohort of patients is required to confirm the results presented here. Further investigations are needed to elucidate the underlying mechanisms responsible for Fulani's relative resistance against malaria and hopefully, such insights may help in control strategies in malaria-endemic areas.
antigen presenting cell
CBA:
cytometric-bead array
FACS:
fluorescent activated cell sorter
IFN:
interferon
immunolglobulin
IL:
interleukin
IFN-gamma-inducible protein
MCP:
monocyte chemotactic protein
MIG:
monokine-induced by IFN-γ
PE:
phycoeryhtrin
P. falciparum:
RANTES:
regulated on activation normal T cell expressed and secreted
TLR:
toll like receptor
TNF:
tumor necrosis factor.
Perlmann P, Troye-Blomberg M: Malaria and the immune system in humans. Chem Immunol. 2002, 80: 229-242.
Favre N, Ryffel B, Bordmann G, Rudin W: The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice. Parasite Immunol. 1997, 19: 375-383. 10.1046/j.1365-3024.1997.d01-227.x.
Artavanis-Tsakonas K, Riley EM: Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes. J Immunol. 2002, 169: 2956-2963.
Torre D, Speranza F, Giola M, Matteelli A, Tambini R, Biondi G: Role of Th1 and Th2 cytokines in immune response to uncomplicated Plasmodium falciparum malaria. Clin Diagn Lab Immunol. 2002, 9: 348-351.
Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, Kone A, Harley R, Plowe CV, Doumbo OK, Sztein MB: Serum levels of the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun. 2004, 72: 5630-5637. 10.1128/IAI.72.10.5630-5637.2004.
Wroczynska A, Nahorski W, Bakowska A, Pietkiewicz H: Cytokines and clinical manifestations of malaria in adults with severe and uncomplicated disease. Int Marit Health. 2005, 56: 103-114.
Luster AD: The role of chemokines in linking innate and adaptive immunity. Curr Opin Immunol. 2002, 14: 129-135. 10.1016/S0952-7915(01)00308-9.
Ochiel DO, Awandare GA, Keller CC, Hittner JB, Kremsner PG, Weinberg JB, Perkins DJ: Differential regulation of beta-chemokines in children with Plasmodium falciparum malaria. Infect Immun. 2005, 73: 4190-4197. 10.1128/IAI.73.7.4190-4197.2005.
John CC, Opika-Opoka R, Byarugaba J, Idro R, Boivin MJ: Low levels of RANTES are associated with mortality in children with cerebral malaria. J Infect Dis. 2006, 194: 837-845. 10.1086/506623.
Jain V, Armah HB, Tongren JE, Ned RM, Wilson NO, Crawford S, Joel PK, Singh MP, Nagpal AC, Dash AP, Udhayakumar V, Singh N, Stiles JK: Plasma IP-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in India. Malar J. 2008, 7: 83-10.1186/1475-2875-7-83.
Wilson NO, Jain V, Roberts CE, Lucchi N, Joel PK, Singh MP, Nagpal AC, Dash AP, Udhayakumar V, Singh N, Stiles JK: CXCL4 and CXCL10 predict risk of fatal cerebral malaria. Dis Markers. 2011, 30: 39-49.
Cohen S, McGREGOR IA, CARRINGTON S: Gamma-globulin and acquired immunity to human malaria. Nature. 1961, 192: 733-737. 10.1038/192733a0.
Groux H, Gysin J: Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. Res Immunol. 1990, 141: 529-542. 10.1016/0923-2494(90)90021-P.
Bolad A, Farouk SE, Israelsson E, Dolo A, Doumbo OK, Nebie I, Maiga B, Kouriba B, Luoni G, Sirima BS, Modiano D, Berzins K, Troye-Blomberg M: Distinct interethnic differences in immunoglobulin G class/subclass and immunoglobulin M antibody responses to malaria antigens but not in immunoglobulin G responses to nonmalarial antigens in sympatric tribes living in West Africa. Scand J Immunol. 2005, 61: 380-386. 10.1111/j.1365-3083.2005.01587.x.
Israelsson E, Vafa M, Maiga B, Lysen A, Iriemenam NC, Dolo A, Doumbo OK, Troye-Blomberg M, Berzins K: Differences in Fcgamma receptor IIa genotypes and IgG subclass pattern of anti-malarial antibodies between sympatric ethnic groups in Mali. Malar J. 2008, 7: 175-10.1186/1475-2875-7-175.
Vafa M, Israelsson E, Maiga B, Dolo A, Doumbo OK, Troye-Blomberg M: Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali. Acta Trop. 2009, 109: 12-16. 10.1016/j.actatropica.2008.09.001.
Dolo A, Modiano D, Maiga B, Daou M, Dolo G, Guindo H, Ba M, Maiga H, Coulibaly D, Perlman H, Blomberg MT, Toure YT, Coluzzi M, Doumbo O: Difference in susceptibility to malaria between two sympatric ethnic groups in Mali. AmJTrop Med Hyg. 2005, 72: 243-248.
Modiano D, Petrarca V, Sirima BS, Nebie I, Diallo D, Esposito F, Coluzzi M: Different response to Plasmodium falciparum malaria in west African sympatric ethnic groups. Proc Natl Acad Sci USA. 1996, 93: 13206-13211. 10.1073/pnas.93.23.13206.
Farouk SE, Dolo A, Bereczky S, Kouriba B, Maiga B, Farnert A, Perlmann H, Hayano M, Montgomery SM, Doumbo OK, Troye-Blomberg M: Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali. Microbes Infect. 2005, 7: 110-117. 10.1016/j.micinf.2004.09.012.
Bereczky S, Dolo A, Maiga B, Hayano M, Granath F, Montgomery SM, Daou M, Arama C, Troye-Blomberg M, Doumbo OK, Farnert A: Spleen enlargement and genetic diversity of Plasmodium falciparum infection in two ethnic groups with different malaria susceptibility in Mali, West Africa. Trans R Soc Trop Med Hyg. 2006, 100: 248-257. 10.1016/j.trstmh.2005.03.011.
Torcia MG, Santarlasci V, Cosmi L, Clemente A, Maggi L, Mangano VD, Verra F, Bancone G, Nebie I, Sirima BS, Liotta F, Frosali F, Angeli R, Severini C, Sannella AR, Bonini P, Lucibello M, Maggi E, Garaci E, Coluzzi M, Cozzolino F, Annunziato F, Romagnani S, Modiano D: Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria. Proc Natl Acad Sci USA. 2008, 105: 646-651. 10.1073/pnas.0709969105.
Modiano D, Luoni G, Sirima BS, Lanfrancotti A, Petrarca V, Cruciani F, Simpore J, Ciminelli BM, Foglietta E, Grisanti P, Bianco I, Modiano G, Coluzzi M: The lower susceptibility to Plasmodium falciparum malaria of Fulani of Burkina Faso (west Africa) is associated with low frequencies of classic malaria-resistance genes. Trans R Soc Trop Med Hyg. 2001, 95: 149-152. 10.1016/S0035-9203(01)90141-5.
McCall MB, Hopman J, Daou M, Maiga B, Dara V, Ploemen I, Nganou-Makamdop K, Niangaly A, Tolo Y, Arama C, Bousema JT, van der Meer JW, van der Ven AJ, Troye-Blomberg M, Dolo A, Doumbo OK, Sauerwein RW: Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in susceptibility to parasitemia between sympatric Fulani and Dogon in Mali. J Infect Dis. 2010, 201: 142-152. 10.1086/648596.
Arama C, Giusti P, Bostrom S, Dara V, Traore B, Dolo A, Doumbo O, Varani S, Troye-Blomberg M: Interethnic differences in antigen-presenting cell activation and TLR responses in Malian children during Plasmodium falciparum malaria. PLoS One. 2011, 6: e18319-10.1371/journal.pone.0018319.
Jensen JB, Trager W: Plasmodium falciparum in culture: establishment of additional strains. AmJTrop Med Hyg. 1978, 27: 743-746.
Troye-Blomberg M, Perlmann H, Patarroyo ME, Perlmann P: Regulation of the immune response in Plasmodium falciparum malaria. II. Antigen specific proliferative responses in vitro. Clin Exp Immunol. 1983, 53: 345-353.
Clark IA, Budd AC, Alleva LM, Cowden WB: Human malarial disease: a consequence of inflammatory cytokine release. Malar J. 2006, 5: 85-10.1186/1475-2875-5-85.
Luty AJ, Lell B, Schmidt-Ott R, Lehman LG, Luckner D, Greve B, Matousek P, Herbich K, Schmid D, Migot-Nabias F, Deloron P, Nussenzweig RS, Kremsner PG: Interferon-gamma responses are associated with resistance to reinfection with Plasmodium falciparum in young African children. J Infect Dis. 1999, 179: 980-988. 10.1086/314689.
De Souza JB, Williamson KH, Otani T, Playfair JH: Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria. Infect Immun. 1997, 65: 1593-1598.
Friedland JS, Ho M, Remick DG, Bunnag D, White NJ, Griffin GE: Interleukin-8 and Plasmodium falciparum malaria in Thailand. Trans R Soc Trop Med Hyg. 1993, 87: 54-55. 10.1016/0035-9203(93)90417-O.
Abrams ET, Brown H, Chensue SW, Turner GD, Tadesse E, Lema VM, Molyneux ME, Rochford R, Meshnick SR, Rogerson SJ: Host response to malaria during pregnancy: placental monocyte recruitment is associated with elevated beta chemokine expression. J Immunol. 2003, 170: 2759-2764.
Robinson LJ, D'Ombrain MC, Stanisic DI, Taraika J, Bernard N, Richards JS, Beeson JG, Tavul L, Michon P, Mueller I, Schofield L: Cellular tumor necrosis factor, gamma interferon, and interleukin-6 responses as correlates of immunity and risk of clinical Plasmodium falciparum malaria in children from Papua New Guinea. Infect Immun. 2009, 77: 3033-3043. 10.1128/IAI.00211-09.
Kern P, Hemmer CJ, Van Damme J, Gruss HJ, Dietrich M: Elevated tumor necrosis factor alpha and interleukin-6 serum levels as markers for complicated Plasmodium falciparum malaria. Am J Med. 1989, 87: 139-143. 10.1016/S0002-9343(89)80688-6.
Luty AJ, Perkins DJ, Lell B, Schmidt-Ott R, Lehman LG, Luckner D, Greve B, Matousek P, Herbich K, Schmid D, Weinberg JB, Kremsner PG: Low interleukin-12 activity in severe Plasmodium falciparum malaria. Infect Immun. 2000, 68: 3909-3915. 10.1128/IAI.68.7.3909-3915.2000.
Chaiyaroj SC, Rutta AS, Muenthaisong K, Watkins P, Na Ubol M, Looareesuwan S: Reduced levels of transforming growth factor-beta1, interleukin-12 and increased migration inhibitory factor are associated with severe malaria. Acta Trop. 2004, 89: 319-327. 10.1016/j.actatropica.2003.10.010.
Were T, Hittner JB, Ouma C, Otieno RO, Orago AS, Ong'echa JM, Vulule JM, Keller CC, Perkins DJ: Suppression of RANTES in children with Plasmodium falciparum malaria. Haematologica. 2006, 91: 1396-1399.
Gerard C, Rollins BJ: Chemokines and disease. Nat Immunol. 2001, 2: 108-115. 10.1038/84209.
Campanella GS, Tager AM, El Khoury JK, Thomas SY, Abrazinski TA, Manice LA, Colvin RA, Luster AD: Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria. Proc Natl Acad Sci USA. 2008, 105: 4814-4819. 10.1073/pnas.0801544105.
Hailu A, van der Poll T, Berhe N, Kager PA: Elevated plasma levels of interferon (IFN)-gamma, IFN-gamma inducing cytokines, and IFN-gamma inducible CXC chemokines in visceral leishmaniasis. AmJTrop Med Hyg. 2004, 71: 561-567.
Crompton PD, Kayala MA, Traore B, Kayentao K, Ongoiba A, Weiss GE, Molina DM, Burk CR, Waisberg M, Jasinskas A, Tan X, Doumbo S, Doumtabe D, Kone Y, Narum DL, Liang X, Doumbo OK, Miller LH, Doolan DL, Baldi P, Felgner PL, Pierce SK: A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci USA. 2010, 107: 6958-6963. 10.1073/pnas.1001323107.
Aucan C, Traore Y, Tall F, Nacro B, Traore-Leroux T, Fumoux F, Rihet P: High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria. Infect Immun. 2000, 68: 1252-1258. 10.1128/IAI.68.3.1252-1258.2000.
Bouharoun-Tayoun H, Druilhe P: Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity. Infect Immun. 1992, 60: 1473-1481.
To all the people living in Manteourou and Binédama for their kind contributions and willingness to participate in this study.
This work was supported by grants from the Swedish Agency for Research and Development with Developing Countries (Sida/SAREC) as well as grants within the BioMalPar European Network of Excellence (LSHP-CT-2004-503578) and from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement N° 242095." and lastly from the European & Developing Countries Clinical Trials Partnership (EDCTP; Grant CG-TA 05 40204_003).
Department of Immunology, Wenner-Gren Institute, Stockholm University, Svante Arrheniusväg 20C, 10691, Stockholm, Sweden
Stéphanie Boström
, Pablo Giusti
, Charles Arama
& Marita Troye-Blomberg
Malaria Research & Training Centre, Faculty of Medicine, Pharmacology & Dentistry, University of Bamako, Bamako, Mali
Charles Arama
, Victor Dara
, Boubacar Traore
, Amagana Dolo
& Ogobara Doumbo
Division of Mathematical Statistics, Department of Mathematics, Stockholm University, Stockholm, Sweden
Jan-Olov Persson
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Correspondence to Stéphanie Boström.
Authors' contributions
PG, CA, VD, BT, AD, OD and MTB conceived, designed and coordinated the study. PG and CA participated in the sample collection and processing. SB and MTB designed and supervised the immunoassays. SB carried out the immunoassays. SB and JP performed statistical analysis. SB drafted the first version of the manuscript. All authors read and approved the final manuscript.
Pablo Giusti, Charles Arama contributed equally to this work.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Boström, S., Giusti, P., Arama, C. et al. Changes in the levels of cytokines, chemokines and malaria-specific antibodies in response to Plasmodium falciparum infection in children living in sympatry in Mali. Malar J 11, 109 (2012) doi:10.1186/1475-2875-11-109


text:
Q: sum values of an array when keys are not values of another array i have 2 arrays:
$a=Array
(
[25] => 21.00
[33] => 63.00
[12] => 45.00
[14] => 12.00
[17] => 22.00
)
$b=Array
(
[0] => 24
[1] => 25
[3] => 14
)

I want to sum only the values of array $a that their keys do not much the values of array $b.
For instance in this example the output would be 130.
Any idea would be appreciated.Thanks in advance!

A: $a = array(
    25 => 21.00,
    33 => 63.00,
    12 => 45.00,
    14 => 12.00,
    17 => 22.00,
);
$b = array(24, 25, 14);

$result = array_sum(
    array_diff_key(
        $a,
        array_flip($b)
    )
);
var_dump($result);

A: Try this:
$sum = 0;
foreach($a as $arr => $value_a){
   $find = 0;
   foreach($b as $arr_b => $value_b){
       if($arr == $value_b)
           $find = 1;
   }
   if($find == 0)
      $sum += $value_a
}

echo'sum: '.$sum;

A: This is probably one of the most readable ways. What's the purpose of this because there are more efficient ways for sure that only require one loop.
$total = 0;
foreach($a as $k => $v){
    $sum = true;
    foreach($b as $kk => $vv){
        if($vv == $k){
            $sum = false;
        }
    }
    if($sum){
        $total += $v;
    }
}

redpajama_set_name: RedPajamaStackExchange


text:
Q: dev c++. Получить координаты курсора dev c++. Создать проект Windows Application. Получить координаты курсора через каждую 1 секунду и записать все результаты в текстовый файл.

A: Для получения координат воспользуйтесь функцией GetCursorPos
POINT p;
if (GetCursorPos(&p))
{
    //позиция курсора: p.x  p.y
}

Так же вам пригодится ScreenToClient для маппинга координат в win координаты.
if (ScreenToClient(hwnd, &p))
{
    //p.x и p.y относительны вашего окна
}

Что бы периодически получать координаты, воспользуйтесь например таймером:
#include <iostream>
#include <chrono>
#include <thread>
#include <functional>

void timer_start(std::function<void(void)> func, unsigned int interval)
{
    std::thread([func, interval]() {
        while (true)
        {
            func();
            std::this_thread::sleep_for(std::chrono::milliseconds(interval));
        }
    }).detach();
}

void do_something()
{
    std::cout << "I am doing something" << std::endl;
}

int main() {
    timer_start(do_something, 1000);

    while(true);
}

Записать данные в файл можно следующим образом:
#include <iostream>
#include <fstream>
using namespace std;

int main () {
  ofstream myfile;
  myfile.open ("тест.txt");
  myfile << "Данные для записи.\n";
  myfile.close();
  return 0;
}


Listed below are the rates and fees for the cards available through the RebelCard office.
Once a RebelCard leaves our office, we are no longer responsible for the condition of the card. When cared for properly, the RebelCard can last your entire college career. Store your card in a soft holder that allows for easy removal. Avoid storing your card with other cards or in a tight wallet sleeve as repetitive removal from such conditions can wear down the imagery on the card. Do not punch a hole in your card or put your card next to a magnet. Avoid leaving your card in your car or outdoors during the hotter months of the year. A broken card returned to our office can be replaced for a $10 fee.


Getting Involved and Giving Back . . .
Gaston Electrical is proud to play an active role in the communities where we live and work. Through partnerships with local organizations and regional institutions – we strive to better the lives of our neighbors and those in need of enrichment or critical care. We value the relationships we have forged over the years. The hard work and dedication of our leadership and employees have resulted in a measure of success for our company – as such, we are thankful for the opportunity to give back, build new bridges, and create a positive environment for those around us.
Below is a sample of the organizations that we are proud to support and partner with.


So here I am, 8 months, 26 days and a 14-hour time difference later, sitting in Newell while enjoying one of my last veggie omelets and writing my last ever article for the Towerlight as a study abroad student from Australia.
What a marvelous plethora of experiences, knowledge and memories I have accumulated during this time. Aside from my extra possessions, I'm going to need an extra 25kg check in suitcase just to take home the memories!
What have been the highlights, lowlights and in-between lights of my time here? Let me try (in the most superficial way) to summarize.
Favorite foods: Thanksgiving yams with cinnamon butter and toasted marshmallows. And Chipotle – how could I forget Chipotle?
Least favorite food: American chocolate. Ew! It is too oily and sickly sweet compared to the darker, creamier chocolate I am used to in Australia.
Weirdest food thing: The ice obsession. The portion sizes, especially at diners! The 10 kazillion and one soda flavors available on those Coca Cola soda machines at Five Guys.
Favorite places visited: Ocean City boardwalk, walking trails around the Eastern Shore, the Batwing Rollercoaster at Six Flags, New York, New York and the Amazonian exhibit at the Smithsonian Zoo in D.C.
Biggest culture shocks: The size and scale of US sports industries, in particular football. The super trashy aspects of pop culture including reality TV shows like 'Four Weddings' and 'Real Housewives'. The sobering and distressing differences in income and opportunity between the richest and the poorest. The ongoing issues of race and equality in this country.
Most surreal 'Merkun experience: Sometime in February, a squirrel ran across the path in front of me near Glen carrying an ice cream cone between it's teeth. He noticed me gawping and stopped and glared at me indignantly with a look of unreserved entitlement, as if to say "'MERKUH!!" It was hysterical.
Most annoying things: The novelty of being Australian and having an "adorable" accent wore off pretty quick. Sometimes I just want to have a normal conversation without having to repeat myself three times and then explain that no, I do not have a pet kangaroo and yes, everything does try to kill you Australia, why do you think I came to 'Merkuh?
The money – why are all your notes green? It makes much more sense to have a value-correlated colored notes like they do in Australia.
Halloween – the whole lot: Crazy dress-up parties, the Survive Towson Apocalypse, the Towson Percussion and Voice Faculty Concert I was a part of and the amazing atmosphere on campus during Homecoming Week.
The experience of living in dorms with all your friends who became your family over the course of the entire academic year.
To conclude: studying abroad has changed my life. The process of adjusting to and understanding another culture pushes you so far out of your comfort zone that you are forced to change your beliefs and attitudes about the world as you know it.
Too often, I feel this country has a blinkered view on life – if it ain't happening in 'Merkuh, it don't matter.
I want each of you individually to step out of your everyday norms and comforts and look at the world around you to see how much diversity and difference there really is.
You don't even have to leave America to see it; America is one of the largest and most diverse countries in the world, yet many of you have never left Maryland. Heck, you don't even have to leave Towson campus to see it, you just need to engage with the community.
Have you ever been a cultural club that you don't identify with? LASO? ADC? FCAT? How about going to an International Student Association meeting? Have you ever engaged with someone, I mean REALLY engaged with someone who is from a completely different background, economically, culturally and socially?
They could be the person sitting opposite you in Math 101.
Have you ever read a foreign news website or understood a global issue like carbon-dioxide induced climate change or learned about how well-intentioned foreign aid can create poverty traps in developing countries? Have you ever read the Quran, the Tripitaka, the Shruti or the Dao de jing? Do you know what these are?
I challenge you, fellow Towsonites, to step outside your comfort zone and become a world citizen, not just an American one. I hope that my articles have made some small contribution to your understanding of the greater world and perhaps inspired you to learn something new.
Thank you Towson for hosting me. I will never forget this amazing school.
I have been reading your article's all semester and this one was your best by far! I wanted to wish you well on your return to Australia, I will miss reading your quirky Australian jokes! I will be keeping an eye out for you in the year's to come. I'm sure this won't be the last I see of Stef Foster!


We offer two types of payment methods. No matter how you prefer to to pay for your procedure, you will receive the highest quality care.
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We accept all major insurance methods (if applicable) to pay for your procedure. Like a traditional hospital, we simply file the cost to your insurer.
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Thank you so much for taking such nice care of me this morning for my procedure. And I'm extremely appreciative of you sending the pathology to Lab Corp Va, which will save me considerable money. I felt comfortable with you because of your friendly, professional approach.


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redpajama_set_name: RedPajamaGithub


I took this photo in the bathroom of my favorite bookstore, McNally-Jackson Books in SoHo…after I finally found a quarter to get in.
That's wild. They kept it toooo real. ha!
hahaha damn i forgot to put them away didn't i. damn.
Wealth has more to do with access, than excess.


text:
Local police departments
Cable TV and Internet
Klimentovo village
See it on the map Bulgarian properties in Varna Region - Klimentovo
Klimentovo is a village in north-eastern Bulgaria, part of Aksakovo municipality, Varna region. Klimentovo is located 23 km away from the city of Varna, 40 km away from the town of Dobrich and 16 km away from Albena resort. It is situated on Varna-Albena main road.
Its former name used to be Kapaklii. Its present name is to honour St. Kliment Ohridski - a medieval scholar, teacher and the first bishop to preach in Old-Bulgarian language, who was declared a saint after he died.
The population of the village numbers to 252 people. Altitude - 260 m. The relief is various - hills, hollows and Batova river flowing nearby. The climate is temperate-continental, influenced by the proximity of the Black Sea - the winter is mild with plenty of snowfalls and the summer is cool.
The village is known as "the village with a 100 flags", the mayor has started an initiative to put a national flag in front of every house in the village. Until 1942 the border with Romania used to pass 1 km away from Klimentovo. Even today the guests of the village are wondering why are there dormer-windows on the building mayor's office is housed. In the past those were the watch towers of the border guards.
In the village there is a community center, grocery shops, a cafe, a small park, a mayor's office, a big fountain in the center of the village of which flows drinkable spring water, a hotel complex with a restaurant and a park, an Orthodox church consecrated in1864, a doctor's office. In Klimentovo there is also a hotel and a school for dogs, where the pets get shelter, training and qualified medical service.
The infrastructure is very well organized - all streets are asphalted and are in a very good condition, all properties in the village are supplied with water and electricity. A regular public bus-line /No. 33/ is available to the city of Varna, three times a day.
tourism and landmarks
Klimentovo is amongst the first settlements not only in the municipality but in the region, as well to start developing rural tourism. A resourceful family has started to meet in their home foreign tourists from the nearby resorts and offering them honey and mead. In front of the fountain in the center of the village the foreign guests are invited with a special ritual to taste Easter cake and buttermilk. For about half an hour the local authentic folklore group entertains the guests afterwards everyone is invited to try meals, wine and brandy produced by the locals.
A new building is rising in the yard of the church. It will house a colourful ethnographic exposition. The people from the village are bringing out exhibits to represent the lifestyle of their predecessors.
Over 30 properties in the village are purchased by foreigners as six foreign families are permanent residents of Klimentovo.
The locals are engaged predominantly into farming and tourism. The area is ecologically clean, the nature is preserved and very beautiful. The village provides excellent conditions for the development of rural and eco-tourism.
Souce: internet

redpajama_set_name: RedPajamaCommonCrawl


text:
SETTLE, Doris Settle - Obituary
SETTLE, Doris (nee Ball) - It is with much sadness that the family of Doris Settle of Whitby announces her passing on Sunday, March 4th, 2018, at the age of 91 at Lakeridge Health Whitby Hospital surrounded by her loving family.
She was predeceased by her loving and caring husband, Geoffrey (2002).
Proud mother of daughters Lesley and Hilary and son Graham (wife Lawrene).
She was a beloved grandma ("gma") to Adam, Ryanne, Cath, Samantha (husband Richard), Chris (wife Sami), Ben (wife Ashleigh), Natasha, Jake, and great-grandchildren Olivia, Caity, Alfie and Freddie.
Doris was born in Bolton, England and is fondly remembered by brother Eric (wife Florence), sister-in-law Jackie, and nephews Gary, Keith and Andrew.
Doris will be missed by good friend Larry (wife Olga, deceased 2000). Also missed greatly by her furry family, Chloe and Charlie. In 1964 Doris and Geoff decided that England could no longer hold them and so made plans for the Settle family to spread their wings and venture into the wide blue yonder, applying to Australia and Canada.
Whichever country first saw their potential as upstanding citizens would be where the family would settle, no pun intended. With the house sold, a week off school, and an overnight coach trip to London, the family of five, with their little bags on their laps, faced nine hours in a Boeing 707 headed for Toronto, about to realize their dreams.
Doris and Geoff eventually set roots in Oshawa to raise their family. Both enjoyed satisfying careers and travelled often, but struggled to learn German and nearly squandered the children's inheritance during their "occasional" side trips to play the slots in Las Vegas.
In later years, after retirement, they became snowbirds, travelling between Mesa, Arizona and Fergus, Ontario before moving permanently to Whitby to be closer to family. A voracious reader, Doris was a consummate knitter and had a passion for cross stitch.
When asked her age, she proudly exclaimed she was as old as the queen. She will be dearly missed, but the family rests easy knowing that Doris and Geoff are forever reunited.
Visitation will be held at OSHAWA FUNERAL HOME, 847 King Street West (905-721-1234) on Friday, March 9th from 11:00 a.m. until time of Funeral Service in the Chapel at 12:00 noon. Interment at Union Cemetery.
Memorial donations to the Salvation Army would be appreciated.
Online condolences may be made at oshawafuneralhome.com.
OSHAWA FUNERAL HOME - (Dignity Memorial) - DurhamObituaries.com


WITH three Football League goals in 136 games heading into the season, Max Ehmer could never be described as prolific.
Yet the Gillingham defender has found the Midas Touch this term, with four goals already to his name heading into this weekend – even if he is at a loss to explain where this sudden clinical edge has come from.
The campaign started with a bang for Ehmer as he netted in the 3-1 opening-day victory over Southend before grabbing a 90th-minute winner against Shrewsbury before the end of August.
A September strike at home to Coventry followed, while the 24-year-old slid in at the back post to convert Jay Emmanuel-Thomas' flick against Northampton a fortnight ago.
Ehmer's unlikely purple patch has him as the Gills' second-highest scorer in League One this season – behind only Emmanuel-Thomas – although he laughs off any suggestion he is showing the strikers how it is done.
Far more pertinent for the German-born defender is the fact Gillingham's only clean sheet this term came in the Checkatrade Trophy and admits he would happily give up his goalscoring hot streak for a more miserly defence.
"I'm a defender first and foremost and clean sheets are what you get judged on," explained Ehmer. "Anything up the other end like goals and assists are a bonus.
"I'd rather keep clean sheets than score goals and we haven't kept a clean sheet in the league yet.
"I really don't know why I'm suddenly scoring so many. Obviously I scored on the first day of the season and I thought that was going to be my only goal of the year!
"I've already got three more since and now, when I go up for corners, I'm thinking I'm going to score.
Ehmer came through the QPR academy but only ever made one substitute appearance for the R's, instead having a string of loan spells at clubs such as Yeovil, Preston, Stevenage and Carlisle.
He spent six months on loan at Gillingham from November 2014 and made the switch permanent the following summer.
He helped them to ninth in League One last season and the expectation this year was for a concerted promotion push, although it has been a mixed bag in the early going with the club trapped in mid-table following Tuesday's 1-0 loss at Oxford United.
Ehmer acknowledges that the Gills expect better and is confident the set-up at the MEMS Priestfield Stadium will ultimately lead to them ending their 12-year exile from the second tier of English football.
"We're not that happy with where we are in the league and then I always think you can do better as a player – you can't ever say you're doing unbelievably," added Ehmer.
"I'm happy enough with my performances but we're not as high up in the league as we think we should be.
"In terms of our squad and everything about the club, we should definitely be in the top six. We're not far off it in terms of points but we just haven't been consistent enough.
"It's a tough one to address because it's a bit of everything – concentration, consistency and we're giving away stupid goals.
"We're also not scoring enough. Last season we would be winning games 3-2 but this year we're not scoring as many and we're conceding more.
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text:
Q: Try not to wake her by A) talking too loud or B) talking quietly? As I see it both can be correct:

*

*Try not (to wake her by talking too loud).

*(Try not to wake her) by talking quietly.

I'm not sure which one a native speaker would use. Any preferences?

A: Both are equally grammatical, and for what it's worth equally unlikely.
First off, I submit that wake up is more likely to occur than just wake, but that can depend on where in the world the conversation is taking place.
Secondly and more to the point, what's much more likely to be produced than either of your options is a simple "don't wake her up", which to any sufficiently smart interlocutor clearly implies not shouting, or a more explicit "lower your voice(s)" for those who are not quite that bright.

A: To answer your question, a native speaker would prefer the first example, "Try not to wake her by (as a result of) talking too loud." Although native speakers will understand what you mean in the second example, the implication is that you would wake someone as a result of talking quietly, which is implausible. That being said, there are better alternatives. As mentioned by RegDwigHt, "Don't wake her up" expresses the sentiment you want. Unless, you want to specify that she may be woken up by other means, for example, jumping on her.


text:
Biografia
Nacque a Modena il 7 luglio 1917. Dopo aver frequentato un corso di specializzazione presso la Scuola della Marina di La Spezia, nell'agosto 1936, all'età di 19 anni, si arruola volontario nella Regia Marina. Iniziato a frequentare il corso per allievi specialisti di tiro presso la Scuola del Corpo Reali Equipaggi della Marina (CREM) a San Bartolomeo (provincia della Spezia), fu costretto ad interromperlo, e nel 1939, ultimato il servizio militare di leva, fu trattenuto in servizio a causa del precipitare della situazione internazionale. All'atto dell'entrata in guerra del Regno d'Italia, avvenuta il 10 giugno 1940, si imbarcò sul cacciatorpediniere "Francesco Crispi". Promosso sottocapo S.D.T. (specialista direzione tiro) nel 1941, sbarcò dal Francesco Crispi nel febbraio 1942, decorato con una Croce di guerra al valor militare concessa sul "Campo",  prendendo servizio presso il Comando difesa della base navale di La Spezia.
Dopo l'armistizio dell'8 settembre 1943 rifiutò di collaborare con i tedeschi, entrando nelle file della Resistenza, aggregandosi alle formazioni partigiane che combattevano sugli Appennini. Partecipò a molti scontri, fino a che il 29 aprile 1944 non rimase ferito a morte in uno scontro in località Crocette di Saliceta San Giuliano (provincia di Modena). Portato all'ospedale di Modena, dove moribondo viene anche torturato, si spense in quello stesso giorno. Per onorarne la memoria fu decretata la concessione della Medaglia d'oro al valor militare.

Intitolazioni
A Walter Tabacchi sono state intitolate tre vie: a Modena , a Nonantola  ed a Bomporto .

Onorificenze

Note

Annotazioni

Fonti

Bibliografia

Altri progetti

Collegamenti esterni

Militari italiani del XX secolo
Medaglie d'oro al valor militare
Croci al merito di guerra
Croci di guerra al valor militare
Sottufficiali e comuni della Regia Marina

redpajama_set_name: RedPajamaWikipedia


text:
Picassos Guitar Licks
By David Ebony
David Ebony
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Lessons from a Room
A Recent Book Explores What It Means to Write without Meaning
What's a week without a new Picasso show? As the Virginia Museum of Art, Richmond, gets set to open "Masterpieces from the Musée Picasso, Paris," featuring 176 works from Picasso's own collection [Feb. 19–May 15], the Museum of Modern Art in New York unveils "Picasso Guitars: 1912–1914," an exhibition of some 85 sculptures, paintings and works on paper, from the period when the artist was exploring music themes within the context of Cubism [Feb. 13–June 6].
For those of us who find in this artist's work an endless source of drama, "Guitars" is not just another Picasso show. Organized by MoMA's Associate Curator of Painting & Sculpture, Anne Umland, it's a focused gem of an exhibition highlighting two years of feverish creativity on the eve of World War I, a transitional time when Picasso and Braque's Analytical Cubism was morphing into Synthetic Cubism. Picasso would soon delve into all sorts of other -isms.
The show includes some rarely exhibited works from international public and private collections. It offers an intimate view of Picasso's seemingly effortless ability to convey a sense of pulsing rhythm in even the most rigorously abstract compositions. At times, the works suggest erotic overtones using just a few well-placed marks and shapes.
The show revolves around two guitar sculptures: the famous sheet-metal Guitar (1914) that Picasso donated to MoMA a few years before his death in 1973; and an earlier version, Guitar (1912), a wall relief in cardboard, which is the first work visitors encounter at the third floor gallery entrance. Due to its abject or banal material and outlandish construction, the piece sparked considerable controversy when it was reproduced in the 1913 journal Le Soirées de Paris. Picasso's widow, Jacqueline, gifted the piece to the museum soon after the artist died. But it arrived disassembled and, placed in the Study Collection for works to be researched and restored rather than the museum collection proper, it was all but forgotten.
Partially reconstructed for the big 1980 Picasso retrospective at MoMA, the sculpture was missing its lower portion, a paper cutout representing a kind of tabletop base. Thought to be lost, the section was discovered in the museum's archives only in 2005, so this is the first time the work has appeared intact since 1913. Like its better-known counterpart in metal, the sculpture resembles a guitar in some way, but Picasso seems to have turned the instrument inside out as he heightens the sensuality of its forms. He gives substance to the openings a real guitar has, such as the sound hole, which he renders with a phallic-shaped tube. The guitar's contours—already suggestive of a voluptuous female figure—are exaggerated and doubled asymmetrically to represent both the front and back of the instrument. Perhaps contradictorily, it's an improbable yet plausible interpretation of a musical instrument, albeit expressed as an almost topological experiment. Not merely a cerebral exercise, though, this Guitar begs to be strummed in a lovesick serenade.
A theme Picasso associated not only with the music halls of Paris, but especially with his Spanish roots and heritage, the guitar appears early on in his work, as for instance in the celebrated Blue Period painting Old Blind Guitarist (1903–04), which hangs in the Art Institute of Chicago. During the years covered in this show, the guitar apparently inspired endless spatial games in two-dimensional works as well as sculpture. The exhibition contains a number of striking paintings, including Violin Hanging on the Wall (1912), in which Picasso conveys illusionistic depth with the help of heavy impasto, spackle and sand.
Perhaps most exciting of all, a series of papier collé works, including Musical Score and Guitar (1912), from the Centre Pompidou, and Guitar, Sheet Music and Glass, from the McNay Art Museum, feature cutout bits of wallpaper, newspaper and sheet music glued to the surface. In many of these works, a tonal play ensues as a single rectangle of turquoise paper interacts with the almost terracotta hue of the newspaper strips that have ripened with age. Contrasted with the larger, more symphonic paintings and sculptures, these playful compositions suggest chamber pieces or witty songs.
FROM TOP: STILL LIFE WITH GUITAR. 1915. MUSEUM OF MODERN ART. ESTATE OF PABLO PICASSO/ARTISTS RIGHTS SOCIETY. VIOLIN HANGING ON THE WALL (1912–1913). KUNSTMUSEUM BERN, HERMANN AND MARGRIT RUPF FOUNDATION. © 2011 ESTATE OF PABLO PICASSO/ARTISTS RIGHTS SOCIETY.
Anne Umland