Robert W. Sauerwein

Cytokine patterns in patients after major vascular surgery, hemorrhagic shock, and severe blunt trauma. Relation with subsequent adult respiratory distress syndrome and multiple organ failure.

ObjectiveThis study investigates the course of serum cytokine levels in patients with multiple trauma, patients with a ruptured abdominal aortic aneurysm (AAA), and patients undergoing elective AAA repair and the relationship of these cytokines to the development of adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Summary Background DataSevere tissue trauma, hemorrhagic shock, and ischemia-reperfusion injury are pathophysiologic mechanisms that may result in an excessive uncontrolled activation of inflammatory cells and mediators. This inflammatory response is thought to play a key role in the development of (remote) cell and organ dysfunction, which is the basis of ARDS and MOF. MethodsThe study concerns 28 patients with multiple trauma, 20 patients admitted in shock because of a ruptured AAA, and 18 patients undergoing elective AAA repair. Arterial blood was serially sampled from admission (or at the start of elective operation) to day 13 in the intensive care unit, and the serum concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were determined. ResultsTwenty-two patients died, 15 within 48 hours and 7 after several weeks, as a result of ARDS/MOF. At hospital admission and after 6 hours, these nonsurvivors had significantly higher plasma TNF-α and IL-1β levels than did the survivors. At the same measuring points, TNF-α and IL-1β were significantly more elevated in patients with ruptured AAA than in traumatized patients. However, IL-6 was significantly higher in the traumatized patients. In 10 patients, ARDS/MOF developed, and 41 had an uncomplicated course in this respect. Those with ARDS/MOF exhibited significantly different cytokine patterns in the early postinjury phase. TNF-α and IL-1β levels were higher mainly on the first day of admission; IL-6 concentrations were significantly elevated in patients with ARDS/MOF from the second day onward. The latter cytokine showed a good correlation with the daily MOF score during the whole 2-week observation paeriod. ConclusionsIn the early postinjury phase, higher concentrations of these cytokines are associated; not only with an increased mortality rate, but also with an increased risk for subsequent ARDS and MOF. These data therefore support the concept that these syndromes are caused by an overwhelming autodestructive inflammatory response.

Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry

The annotated genomes of organisms define a ‘blueprint’ of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell–cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.

Protection against a Malaria Challenge by Sporozoite Inoculation

BACKGROUND An effective vaccine for malaria is urgently needed. Naturally acquired immunity to malaria develops slowly, and induction of protection in humans can be achieved artificially by the inoculation of radiation-attenuated sporozoites by means of more than 1000 infective mosquito bites. METHODS We exposed 15 healthy volunteers--with 10 assigned to a vaccine group and 5 assigned to a control group--to bites of mosquitoes once a month for 3 months while they were receiving a prophylactic regimen of chloroquine. The vaccine group was exposed to mosquitoes that were infected with Plasmodium falciparum, and the control group was exposed to mosquitoes that were not infected with the malaria parasite. One month after the discontinuation of chloroquine, protection was assessed by homologous challenge with five mosquitoes infected with P. falciparum. We assessed humoral and cellular responses before vaccination and before the challenge to investigate correlates of protection. RESULTS All 10 subjects in the vaccine group were protected against a malaria challenge with the infected mosquitoes. In contrast, patent parasitemia (i.e., parasites found in the blood on microscopical examination) developed in all five control subjects. Adverse events were mainly reported by vaccinees after the first immunization and by control subjects after the challenge; no serious adverse events occurred. In this model, we identified the induction of parasite-specific pluripotent effector memory T cells producing interferon-gamma, tumor necrosis factor alpha, and interleukin-2 as a promising immunologic marker of protection. CONCLUSIONS Protection against a homologous malaria challenge can be induced by the inoculation of intact sporozoites. (ClinicalTrials.gov number, NCT00442377.)

Hitting hotspots: spatial targeting of malaria for control and elimination.

Teun Bousema and colleagues argue that targeting malaria “hotspots” is a highly efficient way to reduce malaria transmission at all levels of transmission intensity.

Plasmodium falciparum Associated with Severe Childhood Malaria Preferentially Expresses PfEMP1 Encoded by Group A var Genes

Parasite-encoded variant surface antigens (VSAs) like the var gene–encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSASM) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSAUM). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSAUM to in vitro–selected sublines expressing VSASM to identify PfEMP1 responsible for the VSASM phenotype. Expression of VSASM was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

A Central Role for P48/45 in Malaria Parasite Male Gamete Fertility

Fertilization and zygote development are obligate features of the malaria parasite life cycle and occur during parasite transmission to mosquitoes. The surface protein PFS48/45 is expressed by male and female gametes of Plasmodium falciparum and PFS48/45 antibodies prevent zygote development and transmission. Here, gene disruption was used to show that Pfs48/45 and the ortholog Pbs48/45 from a rodent malaria parasite P. berghei play a conserved and important role in fertilization. p48/45- parasites had a reduced capacity to produce oocysts in mosquitoes due to greatly reduced zygote formation. Unexpectedly, only male gamete fertility of p48/45- parasites was affected, failing to penetrate otherwise fertile female gametes. P48/45 is shown to be a surface protein of malaria parasites with a demonstrable role in fertilization.

A Role for Apical Membrane Antigen 1 during Invasion of Hepatocytes by Plasmodium falciparum Sporozoites

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.

TLR4 polymorphisms, infectious diseases, and evolutionary pressure during migration of modern humans.

Infectious diseases exert a constant evolutionary pressure on the genetic makeup of our innate immune system. Polymorphisms in Toll-like receptor 4 (TLR4) have been related to susceptibility to Gram-negative infections and septic shock. Here we show that two polymorphisms of TLR4, Asp299Gly and Thr399Ile, have unique distributions in populations from Africa, Asia, and Europe. Genetic and functional studies are compatible with a model in which the nonsynonymous polymorphism Asp299Gly has evolved as a protective allele against malaria, explaining its high prevalence in subSaharan Africa. However, the same allele could have been disadvantageous after migration of modern humans into Eurasia, putatively because of increased susceptibility to severe bacterial infections. In contrast, the Asp299Gly allele, when present in cosegregation with Thr399Ile to form the Asp299Gly/Thr399Ile haplotype, shows selective neutrality. Polymorphisms in TLR4 exemplify how the interaction between our innate immune system and the infectious pressures in particular environments may have shaped the genetic variations and function of our immune system during the out-of-Africa migration of modern humans.

Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study

BACKGROUND We have shown that immunity to infection with Plasmodium falciparum can be induced experimentally in malaria-naive volunteers through immunisation by bites of infected mosquitoes while simultaneously preventing disease with chloroquine prophylaxis. This immunity was associated with parasite-specific production of interferon γ and interleukin 2 by pluripotent effector memory cells in vitro. We aim to explore the persistence of protection and immune responses in the same volunteers. METHODS In an open-label study at the Radboud University Nijmegen Medical Centre (Nijmegen, Netherlands), from November to December, 2009, we rechallenged previously immune volunteers (28 months after immunisation) with the bites of five mosquitoes infected with P falciparum. Newly recruited malaria-naive volunteers served as infection controls. Our primary outcome was the detection of blood-stage parasitaemia by microscopy. We assessed the kinetics of parasitaemia with real-time quantitative PCR (rtPCR) and recorded clinical signs and symptoms. In-vitro production of interferon γ and interleukin 2 by effector memory T cells was studied after stimulation with sporozoites and red blood cells infected with P falciparum. Differences in cellular immune responses between the study groups were assessed with the Mann-Whitney test. This study is registered with ClinicalTrials.gov, number NCT00757887. FINDINGS Four of six immune volunteers were microscopically negative after rechallenge. rtPCR-based detection of blood-stage parasites in these individuals was negative throughout follow-up. Patent parasitaemia was delayed in the remaining two immunised volunteers. In-vitro assays showed the long-term persistence of parasite-specific pluripotent effector memory T-cell responses in protected volunteers. The four protected volunteers reported several mild to moderate adverse events, of which the most commonly reported symptom was headache (one to three episodes per volunteer). The two patients with delayed patency had adverse events similar to those in the control group. INTERPRETATION Artificially induced immunity lasts longer than generally recorded after natural exposure; providing a new avenue of research into the mechanisms of malaria immunity. FUNDING Dioraphte Foundation.

Identification of hot spots of malaria transmission for targeted malaria control.

BACKGROUND Variation in the risk of malaria within populations is a frequently described but poorly understood phenomenon. This heterogeneity creates opportunities for targeted interventions but only if hot spots of malaria transmission can be easily identified. METHODS We determined spatial patterns in malaria transmission in a district in northeastern Tanzania, using malaria incidence data from a cohort study involving infants and household-level mosquito sampling data. The parasite prevalence rates and age-specific seroconversion rates (SCRs) of antibodies against Plasmodium falciparum antigens were determined in samples obtained from people attending health care facilities. RESULTS Five clusters of higher malaria incidence were detected and interpreted as hot spots of transmission. These hot spots partially overlapped with clusters of higher mosquito exposure but could not be satisfactorily predicted by a probability model based on environmental factors. Small-scale local variation in malaria exposure was detected by parasite prevalence rates and SCR estimates for samples of health care facility attendees. SCR estimates were strongly associated with local malaria incidence rates and predicted hot spots of malaria transmission with 95% sensitivity and 85% specificity. CONCLUSIONS Serological markers were able to detect spatial variation in malaria transmission at the microepidemiological level, and they have the potential to form an effective method for spatial targeting of malaria control efforts.