Eleven years of malaria surveillance in a Sudanese village highlights unexpected variation in individual disease susceptibility and outbreak severity.

An analysis is presented of continuous data collected over 11 years based on 1,902,600 person/days of observation on the malaria experience of the people of Daraweesh, a village in eastern Sudan. Malaria transmission is hypo-endemic: the acquisition of clinical immunity with age is not as obvious as in more holo-endemic areas and malaria remained a problem in all age groups throughout the study. However, this population, who are of Fulani origin, showed a distinctly variable level of disease susceptibility. Thirty-two percent of the village never reported malaria symptoms or required malaria treatment while others experienced up to 8 clinical episodes over the 11 years of observation. Malaria incidence was clearly influenced by drought but much less obviously by rainfall. To what extent outbreak patterns are explicable in terms of anopheline factors, and to human immune factors, remains an interesting question for malaria modelling in this, and in other low transmission zones, such as the burgeoning urban areas of modern Africa.

Assessment of the safety of recombinant tissue factor pathway inhibitor in patients with severe sepsis: a multicenter, randomized, placebo-controlled, single-blind, dose escalation study.

OBJECTIVE: To identify a safe and potentially effective recombinant tissue factor pathway inhibitor (rTFPI) dose for further clinical evaluation in patients with severe sepsis. DESIGN: Prospective, randomized, single-blind, placebo-controlled, dose escalation, multicenter, multinational phase II clinical trial. SETTING: Thirty-eight intensive care units in the United States and Europe. PATIENTS: Two hundred and ten subjects with severe sepsis who received standard supportive care and antimicrobial therapy. INTERVENTIONS: Subjects received a continuous intravenous infusion of placebo or rTFPI at 0.025 or 0.05 mg/kg/hr for 4 days (96 hrs). MEASUREMENTS AND MAIN RESULTS: There were no significant imbalances in demographics, severity of illness, or source of infection in patients randomized to placebo or either dose of rTFPI. A 20% relative reduction in 28-day all-cause mortality was observed when all rTFPI-treated patients were compared with all placebo patients. An improvement in pulmonary organ dysfunction score and in a composite intensive care unit score (pulmonary, cardiovascular, and coagulation) were also noted in the rTFPI-treated patients. Logistic regression modeling indicated a substantial treatment by baseline laboratory international normalized ratio (INR) interaction effect when only treatment and INR were in the model (p =.037) and when baseline Acute Physiology and Chronic Health Evaluation (APACHE II) and log10 interleukin 6 were adjusted for (p =.026). This interaction effect indicates that higher baseline INR is associated with a more pronounced beneficial rTFPI effect. There was no increase in mortality in subjects treated with either dose of rTFPI compared with placebo. Biological activity, as detected by a statistically significant reduction in thrombin-antithrombin complexes (TATc), was noted in the all rTFPI-treated patients compared with those receiving placebo. There were no major imbalances across all treatment groups with respect to safety. The frequency of adverse events (AEs) and severe adverse events (SAEs) was similar among the treatment groups, with a slight increase in SAEs and SAEs involving bleeding in the 0.05 mg/kg/hr rTFPI group. The overall incidence of AEs involving bleeding was 28% of patients in the all placebo group and 23% of patients in the all rTFPI-treated group; a slight but statistically insignificant increase in incidence of SAEs involving bleeding was observed in the all rTFPI group (9%) as compared with the all placebo group (6%; p =.39). CONCLUSIONS: Although the trial was not powered to show efficacy, a trend toward reduction in 28-day all-cause mortality was observed in the all rTFPI group compared with all placebo. This study demonstrates that rTFPI doses of 0.025 and 0.05 mg/kg/hr could be safely administered to severe sepsis patients. Additionally, rTFPI demonstrated bioactivity, as shown by reduction in TATc complexes and interleukin-6 levels. These findings warrant further evaluation of rTFPI in an adequately powered, placebo controlled, randomized trial for the treatment of severe sepsis.

Extremely low doses of tissue factor pathway inhibitor decrease mortality in a rabbit model of septic shock.

OBJECTIVE: We sought to determine the lowest dose of recombinant human tissue factor pathway inhibitor (TFPI) that can provide protection from lethality in a rabbit model of septic shock. METHODS: Sepsis was induced in New Zealand white rabbits by intraperitoneal implantation of 7.0 ml of a solution containing hemoglobin (4.8 g/dl), porcine mucin (6 g/dl), and 0.8-1.4 x 10(4) viable Escherichia coli (strain O:18 K+). Gentamicin (5 mg/kg) was administered 4 h following surgery, and this dose was repeated every 12 h for 3 days. Beginning 4 h following the induction of sepsis, animals were treated with a bolus (1 ml) plus a continuous infusion (100 ml over 24) of either TFPI (various doses) or its vehicle. Four different doses of TFPI were studied, and each experiment included a contemporaneous control group. The primary outcome parameter was survival time. Results were analyzed using the Wilcoxen log rank test. RESULTS: The average survival time for rabbits treated with the highest dose of TFPI tested (50 microg/kg bolus and 0.5 microg/kg per minute infusion) was 118 h, as compared to 81 h in vehicle-treated controls). The average survival time for septic rabbits treated with a much lower dose of TFPI (100 ng/kg bolus and 1.0 ng/kg per minute infusion) was 119 h as compared to 57 h in surviving vehicle-treated controls. Treatment with an even lower dose of TFPI (10 ng/kg bolus and 0.1 ng/kg per minute infusion) still produced a marginally significant prolongation of average survival time (80 h) relative to contemporaneously studied controls (47 h). When the dose of TFPI was decreased still further (1.0 ng/kg bolus and 0.01 ng/kg per minute infusion), average survival times were not significantly different between TFPI-treated and vehicle-treated rabbits (77 and 51 h, respectively). CONCLUSIONS: Delayed infusion with remarkably low doses of recombinant human TFPI prolongs survival in a rabbit model of antibiotic-treated Gram-negative bacterial sepsis. In planning human trials of TFPI as an adjuvant treatment for sepsis it may be reasonable to use much lower doses of the agent than were heretofore contemplated.

Tissue factor pathway inhibitor activity in severe sepsis.

OBJECTIVE: To review the preclinical and clinical evidence that provides the therapeutic rationale for recombinant human tissue factor pathway inhibitor (rTFPI) as a novel treatment for human sepsis. DATA SOURCES: A summary of published English-language literature regarding preclinical studies and limited information published about three phase II clinical studies for the evaluation of rTFPI safety in sepsis patients. DATA SUMMARY: Tissue factor pathway inhibitor, the physiologic inhibitor of the tissue factor pathway, interrupts activation of coagulation at multiple steps, including tissue factor VIIa activity, Xa activity, prothrombinase complex, and thrombin generation. Recombinant human TFPI exhibits anticoagulant and anti-inflammatory activities in animal models and humans with sepsis. These activities appear to have an important therapeutic role in protecting the microvasculature from injury and preventing multiple organ failure in sepsis. CONCLUSIONS: Tissue factor pathway inhibitor is a potent inhibitor of clotting in the microvasculature, which is thought to protect organs from injury. Recombinant TFPI improved survival of septic animals in multiple models. Recent phase II results suggest that rTFPI is well tolerated, and they show a trend toward reduction in 28-day all-cause mortality in rTFPI-treated patients; in addition, rTFPI demonstrated significant reduction in thrombin generation. These results suggest that a powered study is indicated to further evaluate rTFPI utility for the adjunctive management of severe sepsis.

Tissue factor pathway inhibitor does not influence inflammatory pathways during human endotoxemia.

Activation of coagulation induces a proinflammatory response in in vitro and animal experiments. Inhibition of the tissue factor-dependent pathway of coagulation inhibits cytokine release and prevents death in gram-negative sepsis models in primates. This study investigated the influence of blocking the coagulation system by tissue factor pathway inhibitor (TFPI) on endotoxin-induced inflammatory responses in healthy humans. Eight men were studied in a double-blind, randomized, placebo-controlled cross-over study. They received a bolus intravenous injection of 4 ng/kg of endotoxin, followed by a 6-h continuous infusion of either TFPI (0.2 mg/kg/h after a bolus of 0.05 mg/kg) or placebo. Endotoxin induced-activation of coagulation was prevented completely by TFPI. In contrast, TFPI did not influence leukocyte activation, chemokine release, endothelial cell activation, or the acute phase response. Thus, complete prevention of coagulation activation by TFPI does not influence activation of inflammatory pathways during human endotoxemia.

The activity of tissue factor pathway inhibitor in experimental models of superantigen-induced shock and polymicrobial intra-abdominal sepsis.

OBJECTIVES: To study recombinant human tissue factor pathway inhibitor (rhTFPI) in a superantigen-induced shock model and in a cecal ligation and puncture (CLP) model of peritonitis in mice. DESIGN: Prospective, randomized, experimental study. SETTING: An experimental animal research laboratory. SUBJECTS: Eighty BALB/c mice for the superantigen model, and 56 BALB/c mice for the CLP model. INTERVENTIONS: In the superantigen-induced shock model, animals received rhTFPI (350 mg/kg) subcutaneously every 12 hrs (n = 30) or saline control (n = 30) for 60 hrs after staphylococcal enterotoxin B (SEB; 10 microg iv) and a sublethal dose of E. coli 0111:B4 lipopolysaccharide (LPS; 75 microg ip). Control groups received SEB alone (n = 10) and LPS alone (n = 10). In the CLP model, rhTFPI or saline was given every 8 hrs for 48 hrs by using a 21-gauge needle (n = 9) or 23-gauge needle (n = 14) for CLP. A sham surgery control group (n = 10) was also included. MEASUREMENTS AND MAIN RESULTS: There was 0% mortality in the SEB and LPS control groups. The mortality rate was 64% in the saline control group that received both SEB and LPS (19 of 30), whereas the rhTFPI- treated animals had a mortality rate of 20% (6 of 30; p < .01). The rhTFPI-treated group had significantly lower interleukin-6 levels (61.8 +/- 41 pg/mL vs. 285 +/- 63 pg/mL; p < .05) than the control group but no differences in tumor necrosis factor-alpha or interferon-gamma levels. In the CLP experiment, rhTFPI-treated animals did not have any survival advantage over the control group after the large-bore (21-gauge) needle puncture. The rhTFPI group had significantly improved 7-day mortality rate after CLP with the small-bore needle (23-gauge; 21.4% [rhTFPI] vs. 71.4% [control], p < .01). Plasma LPS, interleukin-6, interferon-gamma, and tumor necrosis factor-alpha levels were unchanged by rhTFPI treatment, but significantly reduced LPS (p = .006) and IFNgamma (p = .001) levels were found in the peritoneal fluid. CONCLUSIONS: Tissue factor pathway inhibitor significantly improves the mortality rate in models of superantigen-induced shock and polymicrobial intra-abdominal infection, supporting its potential use in clinical trials for septic shock.

Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia.

Inhibition of the tissue factor pathway has been shown to attenuate the activation of coagulation and to prevent death in a gram-negative bacteremia primate model of sepsis. It has been suggested that tissue factor influences inflammatory cascades other than the coagulation system. The authors sought to determine the effects of 2 different doses of recombinant tissue factor pathway inhibitor (TFPI) on endotoxin-induced coagulant, fibrinolytic, and cytokine responses in healthy humans. Two groups, each consisting of 8 healthy men, were studied in a double-blind, randomized, placebo-controlled crossover study. Subjects were studied on 2 different occasions. They received a bolus intravenous injection of 4 ng/kg endotoxin, which was followed by a 6-hour continuous infusion of TFPI or placebo. Eight subjects received 0.05 mg/kg per hour TFPI after a bolus of 0.0125 mg/kg (low-dose group), and 8 subjects received 0.2 mg/kg per hour after a bolus of 0.05 mg/kg (high-dose group). Endotoxin injection induced the activation of coagulation, the activation and subsequent inhibition of fibrinolysis, and the release of proinflammatory and antiinflammatory cytokines. TFPI infusion induced a dose-dependent attenuation of thrombin generation, as measured by plasma F1 + 2 and thrombin-antithrombin complexes, with a complete blockade of coagulation activation after high-dose TFPI. Endotoxin-induced changes in the fibrinolytic system and cytokine levels were not altered by either low-dose or high-dose TFPI. The authors concluded that TFPI effectively and dose-dependently attenuates the endotoxin-induced coagulation activation in humans without influencing the fibrinolytic and cytokine response. (Blood. 2000;95:1124-1129)

Effects of immune complex formation and complement activation on circulating platelets in the primate.

Primate platelets are different from rodent and rabbit platelets in that they do not express receptors for C3a or C5a or immune adherence receptors. This study assessed the effects of immune complex (IC)-induced complement activation on primate platelets in the circulation. Cynomolgus monkeys (CYN, N = 4) immunized to bovine gamma globulin (BGG) were infused with BGG over 5 min to induce acute intravascular IC formation and complement activation. The studies were carried out under normal complement conditions (N = 12), partial complement inhibition (CAB-2 treated, N = 3), or total complement inhibition (CVF treated, N = 1). Under normal complement conditions, BGG infusion increased C3a levels from undetectable to an average of 11.9 +/- 2.6 micrograms/ml. At this time, decreases occurring in both circulating neutrophils (85 +/- 6%) and monocytes (78 +/- 6%) were significantly greater than decreases in circulating platelets (13 +/- 3%, p < 0.001). Partial complement inhibition had an equivocal effect on the BGG-induced changes in circulating leukocytes, while total complement inhibition abrogated these changes. In contrast, platelet changes were unaffected by complement inhibition. We conclude that, compared to circulating leukocytes, circulating platelets are insensitive to intravascular complement activation induced by IC in the nonhuman primate. These results contrast with previous studies in rodents which demonstrate strong effects of IC-induced intravascular complement activation on both circulating neutrophils and platelets.

Activity of secreted Kunitz domain 1 variants of tissue factor pathway inhibitor.

Tissue factor pathway inhibitors (TFPI and TFPI-2) are Kunitz domain-type serine protease inhibitors which inhibit factor VIIa/tissue factor (VIIa/TF) complexes in a factor Xa-dependent manner. The VIIa/TF and Xa inhibitory activity has been localized to the first two Kunitz domains, respectively. Unlike TFPI, TFPI-2 has been reported to exhibit significant Xa-independent VIIa/TF inhibitory activity, perhaps due to an arginine at the P1 residue in the first Kunitz domain of TFPI-2 as opposed to a lysine at the comparable residue in TFPI. Two domain TFPI variants, differing in the first Kunitz domain but containing the second Kunitz domain of TFPI, were constructed and secreted by Saccharomyces cerevisiae in order to test the possibility that a TFPI first Kunitz domain with a P1 lysine to arginine change or a hybrid containing the TFPI-2 first Kunitz domain may represent more potent VIIa/TF inhibitors. When yeast supernatants were analyzed for specific activity in the Xa-dependent inhibition of VIIa/TF, neither variant was as active as the truncated TFPI.

Randomized, blinded, placebo-controlled trial of tissue factor pathway inhibitor in porcine septic shock.

This study tested the hypothesis that tissue factor pathway inhibitor (TFPI) would improve mortality and morbidity evoked by peritonitis-induced bacteremia in pigs. Secondarily, it sought to determine if TFPI treatment would attenuate cardiodynamic abnormalities produced by this septic model. 32 pigs were chronically instrumented with intracardiac transducers to measure left ventricular pressure and diameter, pulmonary and aortic pressures, and cardiac output. At least 5 days after surgery to implant transducers, basal cardiovascular readings and blood samples were obtained. Using a randomized, blinded study design, either purified, reconstituted TFPI (1 mg/kg bolus, 10 mg/kg/min for 48 h), placebo (arginine buffer), or saline was administered to pigs immediately after Escherichia coli 0111.B4 (3.0-11 x 10(9) colony-forming U/kg)-laden fibrin clots were implanted intraperitoneally, producing peritonitis and bacteremia. Pigs did not receive antibiotics or supportive therapy. No significant differences in primary or secondary endpoints were noted between the arginine and saline groups, so these data were combined into a control group (N = 20). 5 of 12 TFPI pigs survived (42%), while 5 of 20 control pigs survived (25%); this difference was not significant (p = .714, Fisher's exact test). TFPI treatment augmented cardiac output in surviving pigs, but did not affect any other cardiovascular performance variable (heart rate, % diameter shortening, or systemic and pulmonary vascular resistance). In controls, peritonitis induced rapid increase in plasma tumor necrosis factor-alpha (428 +/- 771 to 5,933 +/- 559 pg/mL at 2 h) and interleukin-8 (180 +/- 153 to 1,393 +/- 145 pg/mL at 2 h). TFPI treatment significantly attenuated cytokine responses to sepsis, reducing peak tumor necrosis factor-alpha to 2,103 +/- 813 pg/mL and reducing peak interleukin-8 levels to 534 +/- 211 pg/mL at 2 h (p < .05, Tukey test, two-way ANOVA). In conclusion, TFPI treatment attenuated important mediator components of the inflammatory response but did not provide significant survival benefit.

Potential mechanisms for a proinflammatory vascular cytokine response to coagulation activation.

We have previously shown that an anticoagulant could attenuate inflammation in animal models of sepsis with disseminated intravascular coagulation (DIC) and that coagulation activation of human whole blood ex vivo results in a proinflammatory cytokine response. The current studies were performed to better understand mechanisms for the blood cell cytokine response and extend the investigation of such a response to endothelial cells as likely contributors to a vascular inflammatory response. Utilizing cell separation techniques, it was determined that the whole blood IL-8 response to coagulation activation or thrombin, specifically, was mediated by CD14+ monocytes. Moreover, thrombin was observed to stimulate both IL-8 and IL-6 production in cultured mononuclear cells. Analyses of the effects of coagulation activation and thrombin were extended to cultured human endothelial cells, and a similar cytokine response was observed. Thrombin catalytic activity appeared essential, since hirudin reduced thrombin-stimulated proinflammatory cytokine production in cultured monocytes and endothelial cells and prothrombin only weakly mimicked the thrombin response. The endothelial cell IL-8 and IL-6 response to thrombin could be mimicked by the thrombin receptor agonist peptide (TRAP), implicating a functional role of the classic thrombin receptor. Altogether, the results facilitate a better understanding of potential proinflammatory vascular responses to coagulation activation.

Delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbits with gram-negative peritonitis.

To determine whether treatment with recombinant human tissue factor pathway inhibitor (TFPI), an inhibitor of the extrinsic coagulation pathway, can improve survival in a clinically relevant model of gram-negative sepsis, rabbits were given an intraperitoneal inoculation of a suspension containing hemoglobin (40 microg/mL), porcine mucin (150 microg/mL), and viable Escherichia coli O18:K1 (1.0 +/- 0.5 x 10(5) cfu/kg). Treatment with gentamicin (5 mg/kg every 12 h for five doses) was instituted 4 h after induction of peritonitis. At the same time point, rabbits were randomized to receive a 24-h infusion of vehicle or one of three different doses of TFPI. Treatment groups, 7-day survival rates, and significance versus control were as follows: control, 1 of 20; TFPI(LOW DOSE) (0.1 mg/kg, then 1 microg/kg/min), 3 of 12 (P = .14); TFPI(MID DOSE), (0.5 mg/kg, then 5 microg/kg/min), 7 of 12 (P = .002); TFPI(HIGH DOSE) (10 mg/kg, then 10 microg/kg/min), 4 of 13 (P = .04). Thus, delayed treatment with TFPI improves survival in septic rabbits.

Measurement of Plasmodium falciparum growth rates in vivo: a test of malaria vaccines.

Several prototype vaccines against the asexual blood stage of malaria are undergoing preclinical and phase I testing. Although these vaccines have been chosen for their ability to elicit an anti-parasite response, no practical and sensitive clinical trial procedure has been available for measuring their impact on parasite growth. We describe a system that allows parasite growth rates to be measured in volunteers through the incubation period. Two necessary elements of this system are developed: suitable blood-stage Plasmodium falciparum inocula, and a highly sensitive and quantitative assay to measure parasite growth during the incubation period. We infected five nonimmune volunteers with an inoculum as small as 300 parasites and demonstrated that the resultant in vivo asexual parasite growth rates were reproducible at 12-15-fold per cycle. The system allowed the infection to be followed for eight days before treatment without symptoms developing. These findings suggest that it is feasible to directly measure the anti-parasite efficacy of a prototype malaria vaccine in human volunteers without subjecting them to the risk of disease.

Tissue factor pathway inhibitor blocks cellular effects of endotoxin by binding to endotoxin and interfering with transfer to CD14.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type plasma protease inhibitor that inhibits factor Xa and the factor VIIa/tissue factor catalytic complex. It plays an important role in feedback inhibition of the coagulation cascade (Broze, Annu Rev Med 46:103, 1995). TFPI has also been used successfully to prevent lethality and attenuate coagulopathic responses in a baboon model of septic shock (Creasey et al, J Clin Invest 91:2850, 1993; and Carr et al, Circ Shock 44:126, 1995). However, the mechanism of reduced mortality in these animals could not be explained merely by the anticoagulant effect of TFPI, because TFPI-treated animals also had a significantly depressed interleukin-6 response. Moreover, inhibition of coagulopathic responses by other anticoagulants has failed to block the organ damage or lethal effect of endotoxic shock (Coalson et al, Circ Shock 5:423, 1978; Warr et al, Blood 75:1481, 1990; and Taylor et al, Blood 78:364, 1991). We show here that recombinant TFPI can bind to endotoxin in vitro. This binding prevents interaction of endotoxin with both lipopolysaccharide binding protein and CD14, thereby blocking cellular responses.

The proinflammatory cytokine response to coagulation and endotoxin in whole blood.

Acute inflammatory illnesses, including the sepsis syndrome, often include a component of coagulation. A human whole blood culture system was developed so that the relationship between coagulation activation and cytokine responses in the presence or absence of lipopolysaccharide (LPS) could be evaluated. In the absence of LPS stimulation, coagulation activation resulted in a novel pattern of cytokine production. During a 4-hour culture of coagulating blood, significant production of interleukin-8 (IL-8; >2,000 pg/mL) was observed, whereas other proinflammatory cytokines including IL-1 beta, IL-6, or tumor necrosis factor a were undetectable or less than 35 pg/mL. The cytokine profile was distinct from that of fully anticoagulated, LPS-stimulated blood, which showed levels of all the indicated proinflammatory cytokines > or = 2,000 pg/mL over the same time period. Over 24 to 48 hours, the coagulation-induced cytokine response was characterized by marked and sustained IL-8 production, limited IL-6 generation (with kinetics delayed relative to IL-8), and minimal or undetectable tumor necrosis factor alpha levels. The magnitude of the whole blood IL-8 response correlated with the level of coagulation activation as determined by measurement of thrombin-antithrombin III complex formation. The combined stimuli of coagulation activation and LPS challenge induced a synergistic enhancement of IL-8 production but not of IL-6. Coagulation-induced cytokine production and the synergistic production of IL-8 by coagulation and LPS could be attenuated by hirudin or tissue factor pathway inhibitor (TFPI). Studies to elucidate mechanisms implicated (1) the TFPI third Kunitz and carboxy-terminus as important structural components for TFPI regulation of coagulation activation and (2) thrombin as a candidate mediator of the mononuclear cell cytokine response to coagulation activation. In summary, a unique aspect of the crosstalk between the coagulation and cytokine cascades in whole blood is shown with the identification of IL-8 as a key proinflammatory participant.

Recombinant E. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock.

Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.

Maternal inheritance of extrachromosomal DNA in malaria parasites.

Plasmodium falciparum has two extrachromosomal genomes, the mitochondrial 6-kb DNA element and the 35-kb circular DNA. The mitochondrial gene cytochrome b on the 6-kb element has been shown to be inherited uniparentally. In order to ascertain whether the route is maternal or paternal we have examined preparations of male and female gametes of the closely related Plasmodium gallinaceum for the presence of extrachromosomal DNA. DNA from purified preparations of gametes was hybridised to probes for both the 6-kb and 35-kb extrachromosomal genomes. Both probes hybridised to the preparation of Plasmodium gallinaceum female gametes but not to that of the males. We conclude that the extrachromosomal DNAs of malaria parasites are transmitted maternally.