Plasmodium falciparum Drug Resistance Genes pfmdr1 and pfcrt In Vivo Co-Expression During Artemether-Lumefantrine Therapy.

Background: Artemisinin-based combination therapies (ACTs) are the global mainstay treatment of uncomplicated Plasmodium falciparum infections. PfMDR1 and PfCRT are two transmembrane transporters, associated with sensitivity to several antimalarials, found in the parasite food vacuole. Herein, we explore if their relatedness extends to overlapping patterns of gene transcriptional activity before and during ACT administration. Methods: In a clinical trial performed in Tanzania, we explored the pfmdr1 and pfcrt transcription levels from 48 patients with uncomplicated P. falciparum malaria infections who underwent treatment with artemether-lumefantrine (AL). Samples analyzed were collected before treatment initiation and during the first 24 h of treatment. The frequency of PfMDR1 N86Y and PfCRT K76T was determined through PCR-RFLP or direct amplicon sequencing. Gene expression was analyzed by real-time quantitative PCR. Results: A wide range of pre-treatment expression levels was observed for both genes, approximately 10-fold for pfcrt and 50-fold for pfmdr1. In addition, a significant positive correlation demonstrates pfmdr1 and pfcrt co-expression. After AL treatment initiation, pfmdr1 and pfcrt maintained the positive co-expression correlation, with mild downregulation throughout the 24 h post-treatment. Additionally, a trend was observed for PfMDR1 N86 alleles and higher expression before treatment initiation. Conclusion: pfmdr1 and pfcrt showed significant co-expression patterns in vivo, which were generally maintained during ACT treatment. This observation points to relevant related roles in the normal parasite physiology, which seem essential to be maintained when the parasite is exposed to drug stress. In addition, keeping the simultaneous expression of both transporters might be advantageous for responding to the drug action.

Plasmodium falciparum K13 expression associated with parasite clearance during artemisinin-based combination therapy.

BACKGROUND: Delayed parasite clearance and, consequently, reduced efficacy of artemisinin-based combination therapies have been linked with Plasmodium falciparum K13 gene SNPs in Southeast Asia. In Africa, significantly prolonged clearance has not yet been observed and the presently restricted variation in parasite clearance cannot be explained by K13 polymorphisms. OBJECTIVES: Our aim was to study the in vivo pfK13 transcriptional response in patients treated with artemether-lumefantrine and explore whether the pfk13 transcripts can explain the patients' parasite clearance outcomes. PATIENTS AND METHODS: A total of 47 Tanzanian children with microscopically confirmed uncomplicated P. falciparum malaria were hospitalized and received artemether-lumefantrine treatment (clinical trial ID: NCT00336375). RNA was extracted from venous blood samples collected before treatment initiation and at five more timepoints after treatment. cDNA was synthesized and pfk13 transcripts measured by real-time PCR. RESULTS: A wide range of pfk13 transcript variation was observed throughout all timepoints after artemether-lumefantrine treatment. Taking parasite clearance data together with the pfk13 transcripts profile, we observed a negative correlation inferring that pfk13 down-regulation is associated with longer parasite clearance time. CONCLUSIONS: The findings suggest that a reduced PfK13 transcriptional response may represent a first step towards artemisinin tolerance/resistance.

From high to low malaria transmission in Zanzibar-challenges and opportunities to achieve elimination.

BACKGROUND: Substantial global progress in the control of malaria in recent years has led to increased commitment to its potential elimination. Whether this is possible in high transmission areas of sub-Saharan Africa remains unclear. Zanzibar represents a unique case study of such attempt, where modern tools and strategies for malaria treatment and vector control have been deployed since 2003. METHODS: We have studied temporal trends of comprehensive malariometric indices in two districts with over 100,000 inhabitants each. The analyses included triangulation of data from annual community-based cross-sectional surveys, health management information systems, vital registry and entomological sentinel surveys. RESULTS: The interventions, with sustained high-community uptake, were temporally associated with a major malaria decline, most pronounced between 2004 and 2007 and followed by a sustained state of low transmission. In 2015, the Plasmodium falciparum community prevalence of 0.43% (95% CI 0.23-0.73) by microscopy or rapid diagnostic test represented 96% reduction compared with that in 2003. The P. falciparum and P. malariae prevalence by PCR was 1.8% (95% CI 1.3-2.3), and the annual P. falciparum incidence was estimated to 8 infections including 2.8 clinical episodes per 1000 inhabitants. The total parasite load decreased over 1000-fold (99.9%) between 2003 and 2015. The incidence of symptomatic malaria at health facilities decreased by 94% with a trend towards relatively higher incidence in age groups > 5 years, a more pronounced seasonality and with reported travel history to/from Tanzania mainland as a higher risk factor. All-cause mortality among children < 5 years decreased by 72% between 2002 and 2007 mainly following the introduction of artemisinin-based combination therapies whereas the main reduction in malaria incidence followed upon the vector control interventions from 2006. Human biting rates decreased by 98% with a major shift towards outdoor biting by Anopheles arabiensis. CONCLUSIONS: Zanzibar provides new evidence of the feasibility of reaching uniquely significant and sustainable malaria reduction (pre-elimination) in a previously high endemic region in sub-Saharan Africa. The data highlight constraints of optimistic prognostic modelling studies. New challenges, mainly with outdoor transmission, a large asymptomatic parasite reservoir and imported infections, require novel tools and reoriented strategies to prevent a rebound effect and achieve elimination.

Glycaemic control and excess risk of ischaemic and haemorrhagic stroke in patients with type 1 diabetes: a cohort study of 33 453 patients.

OBJECTIVE: To estimate the excess risk of stroke in relation to glycaemic control in patients with type 1 diabetes. METHODS: In this prospective, matched cohort study, we identified patients with type 1 diabetes, aged ≥18 years, who were registered in the Swedish National Diabetes Register from 1998-2011 and five control subjects for each case from the general population, matched for age, sex and county of residence. The risks of all strokes, ischaemic stroke and haemorrhagic stroke were estimated using Cox hazard regression. RESULTS: Of 33 453 type 1 diabetes patients [mean age, 35.5 (SD 14.4) years; mean follow-up, 7.9 (SD 4.3) years; and mean diabetes duration, 20.2 years (SD 14.6)], 762 (2.3%) were diagnosed with stroke compared with 1122 (0.7%) of 159 924 control subjects [mean follow-up, 8.2 (SD 4.3) years]. The overall multiple-adjusted hazard ratios (HRs) for type 1 diabetes patients versus control subjects were 3.29 (95% CI: 2.96-3.66) and 2.49 (95% CI: 1.96-3.16) for ischaemic and haemorrhagic stroke, respectively. The risk of ischaemic and haemorrhagic stroke incrementally increased with increasing HbA1c; the risk of ischaemic stroke was significantly increased with HbA1c within target [≤6.9% (≤52 mmol mol-1 )] [multiple-adjusted HR 1.89 (95% CI: 1.44-2.47)]. For HbA1c ≥9.7% (≥83 mmol mol-1 ), there was a markedly increased risk of both ischaemic and haemorrhagic stroke, with multiple-adjusted HRs of 7.94 (95% CI: 6.29-10.03) and 8.17 (95% CI 5.00-13.35), respectively. CONCLUSIONS: Individuals with type 1 diabetes have an increased risk of ischaemic and haemorrhagic stroke, increasing markedly with poor glycaemic control.

Mutation analysis of Swedish haemophilia B families - high frequency of unique mutations.

INTRODUCTION: Haemophilia B is caused by a heterogeneous spectrum of mutations. Mutation characterization is important in genetic counselling, prenatal diagnosis and to predict risk of inhibitor development. AIMS: To study the mutation spectrum, frequency of unique recurrent mutations, genotype-phenotype association and inhibitor development in a population-based study of the complete Swedish haemophilia B population. METHODS: The study included, facilitated by centralized DNA diagnostics, the complete registered Swedish haemophilia B population (113 families: 47 severe, 22 moderate and 44 mild), each represented by a single patient. Mutation characterization was performed by conventional sequencing of all exons and haplotyping by genotyping of single nucleotide variants and microsatellites. RESULTS: A mutation was found in every family: eight had large deletions, three had small deletions (<10 base pair) and 102 had single base pair substitutions (69 missense, 26 nonsense, four splice site and three promoter). Ten novel mutations were found and were predicted to be deleterious. Sixteen mutations (one total gene deletion, 14 substitutions and one acceptor splice site) were present in more than one family. Of the single nucleotide mutations (37/102), 36% arose at CpG sites. Haplotyping of families with identical mutations and present analyses showed that the frequency of unique mutations was at least 65%. Inhibitors developed in 9/47 (19%) patients with severe haemophilia B. CONCLUSION: The spectrum of haemophilia B mutations reveals at least 65% of the families carry a unique mutation, but with more inhibitor patients than reported internationally, probably as a result of many 'null' mutations.

Origin of mutation in sporadic cases of severe haemophilia A in Sweden.

UNLABELLED: Many newly diagnosed Swedish severe haemophilia A (HA) patients are sporadic cases. Some genotypically non-carrier mothers have gone on to have two descendants with the same mutation, presumably because of mosaicism. AIMS: To define the origin of mutation in sporadic cases of HA, reveal possible sex-specific differences in mutagenesis and identify potential mosaics among non-carrier mothers. METHOD: Sanger sequencing characterized the mutations and microsatellite haplotyping determined the origin of the X chromosome carrying the mutation in 3 generations of 45 families with sporadic severe HA. Droplet digital polymerase chain reaction (ddPCR) was used in five cases to reveal that mosaicism mutations are not found on conventional DNA sequencing. RESULTS: In 23 out of 45 families, the mother carried the mutation and in 5 out of 28 families, the grandmother was also a carrier. The X chromosome was of grandpaternal origin in 17 out of 23 cases. One of five tested mothers was a mosaic with a mutation frequency of 7%. CONCLUSION: In 40 out of 45 families, the sporadic case resulted from a mutation in the last two generations. In 82% (23/28), the carrier mothers had a de novo mutation where the X chromosome was of paternal origin in 74% (17/23). ddPCR is a potentially powerful and promising analysis for mosaicism in HA.

Prenatal diagnosis of haemophilia in Sweden now more commonly used for psychological preparation than termination of pregnancy.

The aims of the study were to define the frequency, outcome and reasons for prenatal diagnosis (PND) in Sweden during a 30-year period in order to study trends and changes. The study population, from the Swedish nationwide registry of PND of haemophilia, consisted of 54 women, compromising >95% of all, who underwent PND (n = 90) of haemophilia during 1977-2013. PND was performed by amniocentesis (n = 10), chorionic villus sampling (n = 64) or by analysis of foetal blood (n = 16). A total of 27/90 foetuses were found to have haemophilia. Sixteen went to termination and the remaining 11 were born during the end of the study period (2000-2013). Three of 90 pregnancies were terminated due to findings other than haemophilia and 3/90 PNDs led to miscarriage. In the 30 families with known haemophilia, PNDs (n = 55) were used in 27/55 cases for 'psychological preparation' and in 23/55 cases with the aim to terminate the pregnancy. A subgroup of women (n = 17) who consecutively underwent PND in the years 1997-2010 were further interviewed. For 11/17, being a carrier had a negative effect on the decision to become pregnant, and in 11 cases PND had influenced their decision to conceive. Our study show that PND of haemophilia is stable over time but increasingly used during the last decade as a psychological preparation for having a child with haemophilia as compared to earlier where more terminations of pregnancies were conducted.

Origin of Swedish hemophilia B mutations.

BACKGROUND: More than 1100 mutations that cause hemophilia B (HB) have been identified. At the same time, specific F9 mutations are present at high frequencies in certain populations, which raise questions about the origin of HB mutations. OBJECTIVES: To describe the mutation spectrum of all HB families in Sweden and investigate if mutations appearing in several families are due to independent recurrent mutations (RMs) or to a common mutation event (i.e. are identical by descent (IBD)). PATIENTS/METHODS: The registered Swedish HB population consists of patients from 86 families. Mutations were identified by resequencing and identical haplotypes were defined using 74 markers and a control population of 285 individuals. The ages of IBD mutations were estimated using ESTIAGE. RESULTS: Out of 77 presumably unrelated patients with substitution mutations, 47 patients (61%) had mutations in common with other patients. Haplotyping of the 47 patients showed that 24 patients had IBD mutations (51%) with estimated ages of between two and 23 generations. A majority of these patients had mild disease. Eight of the 15 mutations observed in more than one family were C>T transitions in CpG sites and all eight were RMs. CONCLUSIONS: The association of IBD mutations with a mild phenotype is similar to what has been previously observed in hemophilia A. Noteworthy features of the mutations that are common to more than one family are the equal proportions of patients with RM and IBD mutations and the correlation between the occurrence of RMs and C>T transitions at CpG sites.

Inoculation with arbuscular mycorrhizae does not improve 137Cs uptake in crops grown in the Chernobyl region.

Methods for cleaning up radioactive contaminated soils are urgently needed. In this study we investigated whether the use of arbuscular mycorrhizal (AM) fungi can improve (137)Cs uptake by crops. Barley, cucumber, perennial ryegrass, and sunflower were inoculated with AM fungi and grown in low-level radionuclide contaminated soils in a field experiment 70 km southwest of Chernobyl, Ukraine, during two successive years (2009-2010). Roots of barley, cucumber and sunflower plants were slightly or moderately infected with AM fungus and root infection frequency was negatively or non-correlated with (137)Cs uptake by plants. Roots of ryegrass were moderately infected with AM fungus and infection frequency was moderately correlated with (137)Cs uptake by ryegrass. The application of AM fungi to soil in situ did not enhance radionuclide plant uptake or biomass. The responsiveness of host plants and AM fungus combination to (137)Cs uptake varied depending on the soil, although mycorrhization of soil in the field was conditional and did not facilitate the uptake of radiocesium. The total amount of (137)Cs uptake by plants growing on inoculated soil was equal to amounts in plant cultivated on non-inoculated soil. Thus, the use of AM fungi in situ for bioremediation of soil contaminated with a low concentration of (137)Cs could not be recommended.

Effect of arbuscular mycorrhizal (AM) fungi on 137Cs uptake by plants grown on different soils.

The potential use of mycorrhiza as a bioremediation agent for soils contaminated by radiocesium was evaluated in a greenhouse experiment. The uptake of (137)Cs by cucumber, perennial ryegrass, and sunflower after inoculation with a commercial arbuscular mycorrhizal (AM) product in soils contaminated with (137)Cs was investigated, with non-mycorrhizal quinoa included as a "reference" plant. The effect of cucumber and ryegrass inoculation with AM fungi on (137)Cs uptake was inconsistent. The effect of AM fungi was most pronounced in sunflower: both plant biomass and (137)Cs uptake increased on loamy sand and loamy soils. The total (137)Cs activity accumulated within AM host sunflower on loamy sand and loamy soils was 2.4 and 3.2-fold higher than in non-inoculated plants. Although the enhanced uptake of (137)Cs by quinoa plants on loamy soil inoculated by the AM fungi was observed, the infection of the fungi to the plants was not confirmed.

Leachability and metal-binding capacity in ageing landfill material.

In order to study the stability of landfilled heavy metals, landfill material from a combined household and industrial waste landfill was aerated for 14 months to simulate the natural ageing processes as air slowly begins to penetrate the landfill mass. During aeration, the pH of the landfill material decreased from around 8.6 to 8.1 and the carbon content also decreased. In order to investigate the possible fate of metals in ageing landfills, a four-stage sequential extraction technique was applied. The ability of the materials to bind metal ions by electrostatic attractions and to form stronger complexes was studied separately. The amount of exchangeable cations, the capacity to bind metal ions by electrostatic attraction and the capacity of the landfill material to complex copper ions were increased by the aeration process. However, results from the sequential analysis showed an increased solubility of sulphur and some metals (Cd, Co, Cu, Ni and Zn). Equilibrium speciation models (Medusa) indicated that the organic matter deposit had a significant capacity to bind metal ions provided that pH was sufficiently high. However, as carbonates are consumed over time, the risk for metal mobility increases. Therefore, the landfills can become an environmental risk, depending on variations in the solubility of metal ions due to changes in pH, redox status and the availability of organic material.

Swedish recovered wood waste: linking regulation and contamination.

In Sweden, large amounts of wood waste are generated annually from construction and demolition activities, but also from other discarded products such as packaging and furniture. A large share of this waste is today recovered and used for heat production. However, previous research has found that recovered wood waste (RWW) contains hazardous substances, which has significant implications for the environmental performance of recycling. Improved sorting is often suggested as a proper strategy to decrease such implications. In this study, we aim to analyse the impacts of waste regulation on the contamination of RWW. The occurrence of industrial preservative-treated wood, which contains several hazardous substances, was used as an indicator for contamination. First the management of RWW during 1995-2004 was studied through interviews with involved actors. We then determined the occurrence of industrial preservative-treated wood in RWW for that time period for each supplier (actor). From the results, it can be concluded that a substantially less contaminated RWW today relies on extensive source separation. The good news is that some actors, despite several obstacles for such upstream efforts, have already today proved capable of achieving relatively efficient separation. In most cases, however, the existing waste regulation has not succeeded in establishing strong enough incentives for less contaminated waste in general, nor for extensive source separation in particular. One important factor for this outcome is that the current market forces encourage involved actors to practice weak quality requirements and to rely on end-of-pipe solutions, rather than put pressure for improvements on upstream actors. Another important reason is that there is a lack of communication and oversight of existing waste regulations. Without such steering mechanisms, the inherent pressure from regulations becomes neutralized.

Sources of heavy metal contamination in Swedish wood waste used for combustion.

In this paper, wood waste (RWW) recovered for heat production in Sweden was studied. Previous research has concluded that RWW contains elevated amounts of heavy metals, causing environmental problems during waste management. This study extends previous work on RWW by analysing which pollution sources cause this contamination. Using existing data on the metal contents in various materials, and the amounts of these materials in RWW, the share of the elevated amounts of metals in RWW that these materials explain was quantified. Six different materials occurring in RWW were studied and the results show that they explain from 70% to 100% of the amounts of arsenic, chromium, lead, copper and zinc in RWW. The most important materials contributing to contamination of RWW are surface-treated wood, industrial preservative-treated wood, plastic and galvanised fastening systems. These findings enable the development and evaluation of strategies aiming to decrease pollution and resource loss from handling RWW. It is argued that source separation and measures taken further downstream from the generation site, such as treatment, need to be combined to substantially decrease the amount of heavy metals in RWW.

Metal and organic matter contents in a combined household and industrial landfill.

A combined household/industrial landfill in a humid and cold temperate climate was characterised with respect to its chemical composition. Cores taken at three randomly chosen sites on the landfill and at different depths at each site were analysed. Carbon, nitrogen and pH were measured by standard laboratory methods. The chemical elements analysed included metals and the non-metals B, P and S. pH ranged between 8.0 and 8.5. The total carbon content was in the interval 4.5-26.9% and the total nitrogen content in the interval 0.05-0.48%. The C/N ratio was high, indicating that there was not enough nitrogen available to ensure the stabilisation of carbon. The metal contents varied substantially. The water and carbon contents were related to each other as well as to the metal content, which increased with the content of water. Based on the results obtained regarding the chemical composition of the landfill, it is evident that the landfill consists of two layers. This indicates that the landfill body might have different levels of chemical development, due to water content, and different long-term leachability in the future.

Cervical muscle afferents play a dominant role over vestibular afferents during bilateral vibration of neck muscles.

A previous study showed that vibratory stimulation of neck muscles in humans induced short-latency electromyographic (EMG) activation of lower leg muscles, producing postural reactions at the feet. These findings indicated that cervical proprioception contributes to stabilization of stance through rapidly integrated pathways. However, as vibration may excite both proprioceptive and vestibular afferents, and because of the proximity of neck muscles to the vestibular apparatus, neck muscle vibration could also have activated the vestibular system thereby contributing to the effect observed. To investigate any possible contribution of vestibular stimulation, vibratory stimuli were applied bilaterally and separately to the splenius muscles of the neck and the planum mastoideum overlying the vestibular organs. Ten normal subjects, with eyes closed, were exposed to vibratory stimulation of two different amplitudes and frequencies. Responses were assessed by EMG activity recorded from tibialis anterior and gastrocnemius muscles of both legs and by changes in center of pressure as measured by a force platform. Results indicated that vibration induced reproducible EMG and postural responses in the anteroposterior direction, particularly on cessation of vibration. EMG and postural responses were considerably lower and less consistent with mastoid vibration compared with neck muscles vibration. Previous reports suggest that vibratory stimulation could propagate to the vestibular organs and generate a vestibular-induced postural activation. However, our findings indicate that cervical muscles afferents play a dominant role over vestibular afferents when vibration is directed towards the neck muscles.

The Nordic Reference Interval Project 2000: recommended reference intervals for 25 common biochemical properties.

Each of 102 Nordic routine clinical biochemistry laboratories collected blood samples from at least 25 healthy reference individuals evenly distributed for gender and age, and analysed 25 of the most commonly requested serum/plasma components from each reference individual. A reference material (control) consisting of a fresh frozen liquid pool of serum with values traceable to reference methods (used as the project "calibrator" for non-enzymes to correct reference values) was analysed together with other serum pool controls in the same series as the project samples. Analytical data, method data and data describing the reference individuals were submitted to a central database for evaluation and calculation of reference intervals intended for common use in the Nordic countries. In parallel to the main project, measurements of commonly requested haematology properties on EDTA samples were also carried out, mainly by laboratories in Finland and Sweden. Aliquots from reference samples were submitted to storage in a central bio-bank for future establishment of reference intervals for other properties. The 25 components were, in alphabetical order: alanine transaminase, albumin, alkaline phosphatase, amylase, amylase pancreatic, aspartate transaminase, bilirubins, calcium, carbamide, cholesterol, creatine kinase, creatininium, gamma-glutamyltransferase, glucose, HDL-cholesterol, iron, iron binding capacity, lactate dehydrogenase, magnesium, phosphate, potassium, protein, sodium, triglyceride and urate.

Reference individuals, blood collection, treatment of samples and descriptive data from the questionnaire in the Nordic Reference Interval Project 2000.

The rules for recruitment of reference individuals, inclusion and preparation of individuals, blood collection, treatment of samples (and control materials) and analysis at the 102 medical laboratories attending the Nordic Reference Interval Project (NORIP) are given as well as the rules for central exclusion of reference individuals. The individuals (18-91-year-olds) should be evenly distributed on age and gender groups. The 3002 reference individuals who contributed at least one reference value to the finally suggested reference intervals were characterized using the information in the questionnaire. Gender, age and country are the main entries in the tables. Other variables in the cross-tables or figure are height, weight, body mass index, ethnic origin, heredity for diabetes, chronic disease, oestrogens or oral contraceptives, other medication, hard physical activity, previous blood donations, smoking habits, use of alcohol, hours since last meal and time of blood collection (hour, day of week, month, year). The Danes had the highest alcohol consumption and the Icelanders had the highest body mass index. The information in this article may interest potential users of the Nordic Reference Interval Project bio-bank and database (NOBIDA) in which serum, Li-heparin plasma and EDTA buffy coat from the mentioned individuals are stored below -80 degrees C.

A multicentre study of reference intervals for haemoglobin, basic blood cell counts and erythrocyte indices in the adult population of the Nordic countries.

Eight haematological quantities were measured in EDTA anticoagulated venous blood specimens collected from 1826 healthy male and female individuals between 18 and 90 years of age in the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). The samples, collected between November 1999 and November 2001 as part of the Nordic Reference Interval Project (NORIP), were analysed on 12 different types of modern automated haematology instruments currently in use among the 60 laboratories participating in the study. Non-parametric reference intervals (between 2.5 and 97.5 percentiles) have been calculated for B-Haemoglobin (females 117-153 g/L, males 134-170 g/L), B-Erythrocytes (females 3.94-5.16 x 10(12)/L, males 4.25-5.71 x 10(12)/L), B-EVF (females 0.348-0.459, males 0.395-0.500), B-MCV (82-98 fL), Erc-MCH (27.1-33.3 pg), Erc-MCHC (317-357 g/L), B-Trc (females 165-387 x 10(9)/L, males 145 x 348 x 10(9)/L) and B-Lkc (3.5-8.8 x 10(9)/L). Partitioning of data according to age and gender was done according to a standardized procedure. For most variables the calculated reference intervals corresponded well with older and less well-defined reference intervals. The mean concentration of B-Haemoglobin increased by 0.08 g/L per year of age in women, and decreased by 0.1 g/L per year of age in men. B-Haemoglobin increased with body mass index in both men and women. Smoking increased the mean of B-Lkc by 1.1 x 10(9)/L and regular use of alcohol increased the mean of B-MCV by 0.8 fL. The influence of these factors was small overall and did not promote specific reference intervals.

A pTalpha-negative subpopulation of CD25+ TN thymocytes revealed by a transgenic marker.

We have recently generated 5'lambda5-huTAC mice, which express the human CD25 (huTAC) gene under the control of the 5'-flanking region of the mouse lambda5-gene. The huTAC-transgene was expressed in pre-B cells but neither in mature B cells nor in T cells of these mice. In this report we demonstrate that the transgene is also transiently expressed by adult CD25+ CD3-CD4-CD8- (triple negative, TN) thymocytes and in fetal thymocytes. The huTAC+, in contrast to the huTAC- subpopulation of the CD44+CD25+ TN cells, was unexpectedly found not to express the pTalpha-gene. Still the huTAC+CD44+CD25+ TN cells reconstituted the development of both alphabeta and gammadelta lineage cells equally efficiently as the pTalpha-expressing huTAC- fraction, demonstrating that this pTalpha-negative subpopulation contained precursors for both T-cell lineages. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated that also in normal mice only a fraction of CD44+CD25+ and CD44-CD25+ TN cells expressed this gene. Taken together, these data indicate that huTAC transgene expression revealed a truly pTalpha-negative fraction of the CD44+CD25+ TN cells. The observation that not all precursors in the CD25+ TN population express the pTalpha-gene has important implications for the understanding of early T-cell development and T-cell lineage commitment.