Correction: Dynamics of serological responses to defined recombinant proteins during Schistosoma mansoni infection in mice before and after the treatment with praziquantel.

[This corrects the article DOI: 10.1371/journal.pntd.0008518.].

Detection of Leishmania donovani DNA within Field-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) in Three Cutaneous Leishmaniasis Endemic Foci of Kurunegala District, Sri Lanka.

Leishmaniasis is a parasitic infection transmitted through the bite of female phlebotomine sand flies. Microscopy is the gold standard to detect parasites within the sand flies and for vector incrimination. However, molecular-based detection has become more popular nowadays in the identification of Leishmania parasites since it provides detection and species identification simultaneously with no need of laborious procedures. The entomological surveys were conducted monthly from May to October 2017 using standard entomological techniques. Field-caught sand flies were identified to the species level followed by DNA extraction. The polymerase chain reaction (PCR) was performed using species-specific primers to detect Leishmania donovani parasites. A total of 1,662 sand flies were encountered from the entomological surveys, and the majority of them were Phlebotomus argentipes (n = 1517; 91.27%), while others were Sergentomyia punjabiensis (n = 140; 8.72%). Leishmania donovani parasite DNA was detected only from P. argentipes (2.3%; n = 2). The detection of Leishmania DNA in P. argentipes suggests the possible role of this species as a vector for leishmaniasis in Sri Lanka.

Potential of antibody test using Schistosoma mansoni recombinant serpin and RP26 to detect light-intensity infections in endemic areas.

Schistosomiasis remains a worldwide public health problem, especially in sub-Saharan Africa. The World Health Organization targets the goal for its elimination as a public health problem in the 2030 Neglected Tropical Diseases (NTDs) Roadmap. Concerted action and agile responses to challenges will be necessary to achieve the targets. Better diagnostic tests can accelerate progress towards the elimination by monitoring disease trends and evaluating the effectiveness of interventions; however, current examinations such as Kato-Katz technique are of limited power to detect light-intensity infections. The point-of-care circulating cathodic antigen (POC-CCA) test shows a higher sensitivity compared to the reference standard, Kato-Katz technique, but it still lacks sufficient sensitivity with low infection intensity. In this study, we examined antibody reactions against recombinant protein antigens; Schistosoma mansoni serine protease-inhibitor (SmSerpin) and RP26, by enzyme-linked immunosorbent assay (ELISA) in plasma samples with light-intensity infection. The sensitivity using the cocktail antigen of recombinant SmSerpin and RP26 showed 83.7%. The sensitivity using S. mansoni soluble egg antigen (SmSEA) was 90.8%, but it showed poor specificity (29.7%), while the cocktail antigen presented improved specificity (61.4%). We conclude that antibody detection to the SmSerpin and RP26 protein antigens is effective to detect S. mansoni light-intensity infections. Our study indicates the potential of detecting antibody against recombinant protein antigens to monitor the transmission of schistosomiasis in low endemicity contexts.

Dynamics of serological responses to defined recombinant proteins during Schistosoma mansoni infection in mice before and after the treatment with praziquantel.

To eliminate schistosomiasis, appropriate diagnostic tests are required to monitor its prevalence and transmission, especially in the settings with low endemicity resulting from the consecutive mass drug administration. Antibodies that react with either crude soluble schistosome egg antigens or soluble worm antigen preparations have been used to monitor infection in low-prevalence regions. However, these detection methods cannot discriminate current and past infections and are cross-reactive with other parasites because both antigens contain numerous proteins and glycans from schistosomes, and standard preparations need maintenance of the life cycle of the schistosome. To evaluate the potential utility of nine recombinant Schistosoma mansoni proteins as single defined antigens for serological diagnosis, we monitored the kinetics of antibodies to each antigen during S. mansoni infection in mice before and after the treatment with praziquantel. C57BL/6 mice were infected with 50 cercariae. The levels of immunoglobulin G (IgG) raised against five recombinant antigens (RP26, sm31, sm32, GST, and LAP1) significantly increased as early as 2-4 weeks after infection and rapidly declined by 2 weeks after the treatment, whereas those raised against crude S. mansoni egg antigens or other antigens remained elevated long after the treatment. The IgG1 raised against RP26, sm31, and serpin decreased after the treatment with praziquantel, whereas the IgE raised against serpin declined strikingly after the treatment. This study clarifies the dynamics of the serological responses to recombinant S. mansoni proteins during infection and after the treatment with praziquantel and identifies several candidate antigens with potential utility in the monitoring and surveillance of schistosomiasis toward the elimination of schistosomiasis.

Leishmaniasis recidivans by Leishmania tropica in Central Rift Valley Region in Kenya.

OBJECTIVES: This study sought to determine the endemic Leishmania species, the clinical features of cutaneous leishmaniasis (CL) in the Central Rift Valley in Kenya and to give an account on unresponsiveness to treatment in the region. METHODS: Participants were clinically identified and grouped into untreated, classical and recidivate based on clinical manifestation and clinical data. Leishmaniasis recidivans lesions were scaly hyperemic papules that appeared before the classic lesion had healed or after healing. The demographics and socio-economic data were recorded and lesion scraping samples screened through microscopy and Internal Transcribed Spacer 1-PCR. Leishmania species were identified using Restriction Fragment Length Polymorphism. RESULTS: A total of 52 participants were sampled, of which, 44.2% of the cases were recidivate and L. tropica the only species identified. All patients had been treated using sodium stibogluconate (SSG) which is the recommended first-line drug in Kenya. 60% of the patients experienced prolonged exposure to the drug (>30 days). CONCLUSION: L. tropica is the endemic Leishmania species for CL leading to classical and leishmaniasis recidivans. Treatment of CL in the area is not effective hence, alternative measures/therapy should be considered to cope with the unresponsiveness.

Development of multiplex serological assay for the detection of human African trypanosomiasis.

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance. We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.

Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin.

Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl.

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay.

BACKGROUND: Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. METHODOLOGY/PRINCIPAL FINDINGS: Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman's rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). CONCLUSIONS/SIGNIFICANCE: Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system.

Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection.

The diagnosis of schistosome infection, followed by effective treatment and/or mass drug administration, is crucial to reduce the disease burden. Suitable diagnostic tests and field-applicable tools are required to sustain schistosomiasis control programs. We therefore assessed the potential of tandem repeat (TR) proteins for sero-diagnosis of Schistosoma mansoni infection using an experimental mouse model. TR genes in the genome of S. mansoni were searched in silico and 7 candidates, named SmTR1, 3, 8, 9, 10, 11 and 15, were selected. Total RNA was extracted from S. mansoni adult worms and eggs. Target TR genes were amplified, cloned, and the proteins were expressed in Escherichia coli competent cells. Female BALB/c mice were infected with 100 S. mansoni cercariae and sera were collected each week post-infection for 18 weeks. The levels of IgG antibodies to SmTR antigens were compared to those to soluble egg antigen (SEA) and to soluble worm antigen preparation (SWAP). Sera of infected mice reacted to all the antigens whereas those of naïve mice did not. IgG responses to SmTR1, 3, 9 and 10 were detected at the early stage of infection. Interestingly, antibodies reacting to SmTR3, 9, 10 and 15 dramatically decreased 4 weeks after treatment with praziquantel, while those against SEA and SWAP remained elevated. Our study suggests that TR proteins, especially SmTR10, may be suitable antigens for sero-diagnosis of infection by S. mansoni and are potential markers for monitoring and surveillance of schistosomiasis, including re-infection after treatment with praziquantel.

Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models.

BACKGROUND: A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya. METHODS: We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized. FINDINGS: Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively. INTERPRETATION: A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.

Comparison of sxx commercially-available dna polymerases for direct ccr / Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .

RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .

Comparison of six commercially-available DNA polymerases for direct PCR.

The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

The in vitro interactions and in vivo efficacy of atovaquone and proguanil against Babesia gibsoni infection in dogs.

In vitro interactions between atovaquone (ATV) and proguanil (PG) against Babesia gibsoni and the clinical efficacy of this combination therapy using Malarone(®) which is the antimalarial drug containing ATV and PG were evaluated. This combination showed synergism against uncloned wild-type and ATV-resistant B. gibsoni in vitro examinations using a modified fixed ratio method. Administration of Malarone(®) to experimentally B. gibsoni infected two dogs in chronic stage and three dogs in acute stage resulted in decrease in parasitemia, and clinical improvements were observed. However, all dogs showed relapse of parasitic infection with a single-nucleotide polymorphism in the cytchrome b gene (M121I). Some side effects were confirmed: self-limiting vomiting in two dogs and hyperphosphatasia in another dog. Mild increases in the levels of alanine aminotransferase were confirmed in two dogs. This is the first study to evaluate the interactions in vitro and the clinical efficacy of ATV and PG against canine B. gibsoni infection in dogs.

Encephalitozoon infections in Rodentia and Soricomorpha in Japan.

Encephalitozoon is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts. In this study, we used nested PCR to investigate the presence of Encephalitozoon infection in Rodentia and Soricomorpha in Japan. We attempted to amplify and sequence Encephalitozoon-specific DNA from brain and viscera samples of 180 animals collected between 2008 and 2010. Forty-three samples (23.9%) from the orders Rodentia and Soricomorpha were positive for Encephalitozoon. This study is the first report of Encephalitozoon infection in Rodentia and Soricomorpha in Japan, and our findings suggest that these hosts may play a role in the spread of microsporidian spores in the environment.

Analysis of effects of meteorological factors on dengue incidence in Sri Lanka using time series data.

In tropical and subtropical regions of eastern and South-eastern Asia, dengue fever (DF) and dengue hemorrhagic fever (DHF) outbreaks occur frequently. Previous studies indicate an association between meteorological variables and dengue incidence using time series analyses. The impacts of meteorological changes can affect dengue outbreak. However, difficulties in collecting detailed time series data in developing countries have led to common use of monthly data in most previous studies. In addition, time series analyses are often limited to one area because of the difficulty in collecting meteorological and dengue incidence data in multiple areas. To gain better understanding, we examined the effects of meteorological factors on dengue incidence in three geographically distinct areas (Ratnapura, Colombo, and Anuradhapura) of Sri Lanka by time series analysis of weekly data. The weekly average maximum temperature and total rainfall and the total number of dengue cases from 2005 to 2011 (7 years) were used as time series data in this study. Subsequently, time series analyses were performed on the basis of ordinary least squares regression analysis followed by the vector autoregressive model (VAR). In conclusion, weekly average maximum temperatures and the weekly total rainfall did not significantly affect dengue incidence in three geographically different areas of Sri Lanka. However, the weekly total rainfall slightly influenced dengue incidence in the cities of Colombo and Anuradhapura.

Effect of sex steroids on Babesia microti infection in mice.

Sex-based-differences are known to affect susceptibility to protozoan infections, but their effects on parasitemia and clinical symptoms in Babesia infections remain unclear. We examined the sex-based susceptibility of various mouse strains to Babesia microti Munich strain infection. In all strains, male mice exhibited significantly higher peak parasitemia and more severe anemia than female mice. Testosterone and estradiol-17ß treatment caused an increase in parasitemia and aggravation of anemia. Orchidectomized male mice receiving testosterone exhibited smaller splenic macrophage populations three days after infection, smaller B cell populations 10 days after infection, and reduced splenic tumor necrosis factor-α and interferon-γ mRNA expression than mice that did not receive testosterone. Mice receiving estradiol-17ß did not exhibit immunosuppressive effects. Thus, a weakened and delayed innate immunity response may lead to acquired immunity failure. The results suggested that testosterone directly affects T or B cells, leading to delayed acquired immunity, dramatically increased parasitemia, and severe anemia.

FOXK2 transcription factor is a novel G/T-mismatch DNA binding protein.

DNA mismatch repair is an important mechanism in the prevention of mutations. We reported the existence in the HL60 cell line of a novel G/T-mismatch DNA-binding protein (nGTBP) requiring strict DNA sequences. In this report, we identify the FOXK2 transcription factor as the nGTBP. FOXK2 fragments were obtained as the only clones with specifically binding activity to the G/T-mismatch DNA by screening of a human brain expression library. The recombinant forkhead domain of FOXK2 specifically recognized the G/T-mismatch DNA. The forkhead domain also recognized hypoxanthine/T and G/uracil, derived from the deamination of the exocyclic amino groups of A/T and G/C, respectively. An electrophoretic mobility shift assay (EMSA) analysis using HL60 cell nuclear extract and antibody raised against FOXK2 resulted in the exclusive binding of FOXK2 to G/T-mismatch DNA. Furthermore, FOXK2 bound to G/T-mismatch DNA with higher affinity than 'match' FOXK2 consensus DNA. We therefore propose that FOXK2 is a G/T-mismatch DNA-binding protein and a deaminated DNA-binding protein.

SATB1 makes a complex with p300 and represses gp91(phox) promoter activity.

The expression of gp91(phox), the key component of the phagocyte NADPH oxidase, is regulated by various factors binding to its proximal promoter. Two nuclear matrix attachment region (MAR)-binding proteins, special AT-rich binding protein 1 (SATB1) and CCAAT displacement protein (CDP), have been reported as rare examples of gp91(phox) gene repressors. However, their individual roles and interactions with other factors in the promoter have not been elucidated in detail. We have focused on these two repressive proteins recognizing the bp -115 to bp -106 segment of the gene and obtained the following results: 1. SATB1 makes a complex, mainly with p300, regardless of the presence of DNA. 2. SATB1/p300 complex binding to the 5' upstream AT-rich region in the bp -115 to bp -106 segment represses the gp91(phox) promoter activity, and the repressed activity is partially released by CDP binding to the CCAAT element directly downstream of the AT-rich region. Our findings imply a novel role for p300 in SATB1-associated global transcription regulation.

A novel GT-mismatch binding protein that recognizes strict DNA sequences with high affinity.

Mismatched or damaged base pairs in DNA are mutagenic and both eukaryotes and prokaryotes have a series of repair systems that decrease a spontaneous mutation rate. All exocyclic amino groups of cytosine(C), adenine(A), and guanine(G) contribute to hydrogen bonds for base pairing. High temperature and oxidative stresses increase the deamination of these bases and methylated C. These deaminated sites would be initially recognized by components of DNA repair system. We discovered a novel G/thymine(T)-mismatch binding protein (nGTBP) that bound, with high affinity, to a minimal 14-mer DNA heteroduplex with a strict 5'-TRT GNB-3' sequence (R for purine, N for any bases, and B for "not A," namely for C, G, or T ). This italicized G position mismatched with T could be replaced by hypoxanthine, the deaminated A. The nGTBP, however, barely recognized DNA duplexes individually containing 8-oxo-G, thymine glycol, and 5-methylcytosine.

PU.1 is dominant and HAF-1 supplementary for activation of the gp91(phox) promoter in human monocytic PLB-985 cells.

Gp91(phox) is a key component of the phagocyte NADPH oxidase. Mutations of its promoter found in patients with chronic granulomatous disease cause deficient binding of PU.1 and HAF-1. Because the two factors bind to the same site (Pu box) of the promoter, we attempted to clarify their relative in vivo contributions to activation of the gp91(phox) promoter in monocytically differentiated PLB-985 cells using a dual luciferase reporter assay and a gel shift competition assay. We found that the activity of a series of single-point-mutated promoters increases or decreases according to an increase or decrease, respectively, in the affinity of the promoters to PU.1 but not to HAF-1. Two of 7 mutants showing weak binding affinity to PU.1 exhibited moderate promoter activity and normal binding affinity for HAF-1. These results suggest PU.1 is the dominant activator and HAF-1 is supplementary. The increased promoter activity of single-, double-, and triple-point-mutated constructs with sequences closer to that of the Ets-binding element correlates with their binding affinity to PU.1 but not to HAF-1, supporting that PU.1 is a more efficient activator than HAF-1. In contrast to co-expressed wild-type PU.1, dominant-negative PU.1 significantly inhibited the activity of a PU.1-optimised gp91(phox) promoter construct. Therefore, we conclude that PU.1 and HAF-1 binding to the Pu box is dominant and supplementary, respectively, for activation of the gp91(phox) promoter in human monocytic cells.