Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.     

Early helper T-cell dysfunction in simian immunodeficiency virus but not in human immunodeficiency virus type-2-infected macaques

Both naive and vaccinated macaques acquired a virus-specific proliferative helper T-cell reactivity in response to infection with the nonpathogenic human immunodeficiency virus type 2 (HIV-2). In contrast, macaques infected with the pathogenic simian immunodeficiency virus of the macaque strain (SIV_mac) did not develop a helper T-cell response. Furthermore, a vaccine-induced preexisting T-cell reactivity was abrogated after SIV_mac infection in vaccine failures. These differences may reflect the different pathogenicity of the two closely related viruses.     

Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

In vitro rearing ofExorista larvarum on tissue culture-based diets

Exorista (=Tachina) larvarum (L.) (Diptera, Tachinidae), a polyphagous parasitoid that attacksLymantria dispar L. andHyphantria cunea (Drury), was rearedin vitro from egg to adult on four tissue culture media-based diets (TMM-FH, SCHNEIDER'S, EX-CELL 400, and SF-900). The kind of tissue culture media in the diets did not influence the adult yield (34 to 55%) and puparium weight (26–27 mg). Adult yield and the puparium weight ofE. larvarum developed on TNM-FH and SCHNEIDER'S-based diets containing different amounts ofGalleria mellonella pupal extract (PE) (0, 1.25, 2.5 and 5%), were lower on diets without PE. In diets without PE development times from oviposition to adult emergence, were shorter on TNM-FH (19 days) than on SCHNEIDER'S-based diet (25–26 days). The adults that developed on artificial diets were able to parasitize the factitious hostG. mellonella and produce viable progeny. The results demonstrate thatE. larvarum is the most promising parasitoid ever studied forin vitro mass production.     

Changes in total and free tryptophan levels in serum following acute morphine administration in arthritic rats

In ‘arthritic’ rats a decrease in total tryptophan and an increase in free tryptophan levels was observed in serum after morphine administration (10 mg kg, s.c.). These changes were maximum within 15 and 30 min after injection.A decrease in total and an increase in free tryptophan levels in serum were observed 30 min after naloxone administration (1 mg/kg, i.m.).An increase in tryptophan and 5-hydroxyindoleacetic acid levels was also observed in the brain after morphine and naloxone.These observations suggest that the rise in 5-hydroxytryptamine synthesis provoked by morphine may be partly related to an increase in the availability of tryptophan from blood. However, the analgesia induced by the opiate appears unlikely to be directly related to this effect.     

Utilisation illégale d’androgènes

Drug abuse for synthetic anabolic androgenic steroids in order to ameliorate sports results is illegal since the law of june 89 in France. No exception whatsoever, therapeutical purpose(s) included, is accepted. Means for controlling such abuse are reviewed briefly here together with data from our research on epitestosterone modifications following physical exercise and testosterone undecanoate controled administration in 15 nor In France the national body responsible for doping analysis in sports is the «Laboratoire National de Dépistage du Dopage» (LNDD). In fact, the control of drug abuse in sports requires laboratory means enabling the detection of banned substances with unlimited certainty. Briefly, untimed urine samples are analyzed for such purpose by gas (liquid or high pressure) chromatography coupled to mass spectrometry (GC/MS) which is the only technique accepted by the medical commission of I.O. C. and relevant bodies world-wide. However, since testosterone itself can now be used as a mean of steroid abuse in man, detection has to solve new problems arising from such manipulation. After considering various approches in order to prove the offense, such as the isotopic ratio for testosterone and different urinary metabolites, and indirect technique, based on the ratio of testosterone to epitestosterone glucuronides, has proved valuable but is now questionned. Epitestosterone is the 17α epimere of testosterone. It is not readily known to clinicians as it has no androgenic potency [5] and does not bind to the specific plasma protein TeBG (26) or androgen receptor [5, 26]. Epitestosterone was first isolated from human urines, simultaneously, by Brooks [6] and Korenman [18]. Wilson and Lipsett [30] among others demonstrated, in man, that this steroid originated both from adrenals and testis, results confirmed recently by Dehennin [8]. Dray et al. and studied its production and circadian rythm in man pointing out that epitestosterone is found in the sulphate fraction of plasma steroids and in the glucuronide fraction of urinary androgen metabolites [12, 20]. Epitestosterone is recovered as such in urines. It is not metabolized [12, 28]. However the pathway for epitestosterone biosynthesis is still uncertain today. The best (for it’s the only one!) hypothesis at present being that of Weustein et al. [30] who reported that epitestosterone could be synthesyzed in man from Δ5 androstène- 3 β, 17α-diol through a possible non enzymic modification of Δ5 androstène-3β, 17β- diol. Donike et al. [10] showed, in man, that the mean GT/GEPIT ratio in urines was 1,5±0,9 (±SD) using epitestosterone as a marker for endogenous androgens. This added index has being implemented, as an official test for androgen abuse’s detection in sports, since the Los Angeles Olympic Games in 1982. All other parameters being normal the definition of a positive androgen doped case is based on GT/GEPIT values over 6. This value of 6 was obtained by adding 6 SD to the mean obtained in man. However, is the use of this parameter justified and 100% safe? Some have questioned its used arguing that epitestosterone, not a well known substance, can not be reliable and could lead to false positive results. We summarize here the results of the French Research Network to which we participated. Mathian et al. [22] showed that epitestosterone production follows testosterone production whatever the age of the subject. The ratio GT/GEPIT doesn’t vary according to age, even over puberty, it remains at 1,40±0,86 from Tanner Stade II to Tanner Stade V. It doesn’t vary significantly after exercise or with fatigue. We also report our study of 15 young men (18–45 year old) over a year. Extensive blood (T, Δ₄, DHT, DHA, SDHA, E2, TeBG, FSH, LH) and urinary parameters (GT, DHT, GEPIT, ADIOL, BDIOL) were measured before, during and after a 21 days course of testosterone undecanoate (TU). Whatever the technique used (GC/MS or RIA) results are identical. We confirmed that GT/GEPIT was very stable for each individual and could be considered as a personnal marker. After TU, at the dosage of 40 to 80 mg/day, GT/GEPIT increased significantly in all instances (athletes and sedentary subjects alike), but not permanently. This change resulted from an increase in testosterone excretion wheras epitestosterone remained non statistically changed. However at the dosage used no permanent modification was found and most of the time GT/GEPIT returned to basal values rapidly. The analysis of the results of our study, according to the limit set by the I.O.C. at 6 for GT/GEPIT, pointed out a lot of false negative (over 50%). Values for GT excretion rate corrected with the creatinine content in the same urinary sample (GT/mg creatinine) have therefore been considered together with GT/GEPIT values. In our opinion, a more suitable and reliable index is thus obtained. The setting of a new limit at 3 for GT/GEPIT (Mean ± 3 SD) together with values under 75 ng/mg creatinine for GT is analyzed. It is also stressed that only a medical commission (aware of the significance of epitestosterone) can interpret the results obtained by analytical chemistry. This is the case in France.     

Expression and Activation of Caspase-6 in Human Fetal and Adult Tissues

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.     

Wild Anopheles funestus Mosquito Genotypes Are Permissive for Infection with the Rodent Malaria Parasite,Plasmodium berghei

Background

Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.

Methods

An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.

Results

P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18–20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.

Conclusions

The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus.