Variation in murid Plasmodium desequestration and its modulation by stress and pentoxifylline.

Changes in the parasitaemia and the characteristics of parasitic infection for three species of rodent Plasmodium (P. chabaudi chabaudi, P. vinckei petteri and P. yoelii yoelii) were investigated under conditions of stress and after treatment with pentoxifylline (POF), a drug that increases red blood cell deformability and causes peripheral vasodilatation. The results indicated that under stress, late parasite stages became less abundant in the tail blood of mice. These changes might be the consequence of parasite sequestration. Attempts to assess sequestration intensity were made by measuring the release rate (RR) of late stages for 10,000 red blood cells. The RR is given by the product of the parasitaemia (P) by the percentage of old trophozoites (OT) and schizonts (S) in the peripheral blood: RR = P(%OT + %S) . With all three species, RR decreased considerably within 5 min following the manipulation of the mice. Injections of POF had the opposite effect. POF had a protective effect against infection by P.v. petteri, causing a delay of 48 h in the development of infection and a higher survival rate in treated mice.

Liver and plasma concentrations in paf-acether and its precursors after partial hepatectomy.

Liver and plasma concentrations in paf-acether (paf) and related phosphocholines, i.e., lysopaf and the ether lipid 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (AAGPC) were studied in rats following two-third hepatectomy. We report a rapid increase in hepatic content of the 3 phospholipids at early steps of the regeneration process, when hepatocytes are switching from G0 to G1 (time 2-6 h). Later on, throughout G1 and at the G1-S transition, these concentrations decreased progressively. They were back to sham-operated or intact control levels at 50 h. In the plasma of hepatectomized animals, no comparable changes were detected. However, an increase in both circulating paf and lipoprotein-bound paf concentrations was measured during the regenerating response. This report is, to our knowledge, the first one on paf level variations following 2/3 hepatectomy. In rats, partial resection of the liver was shown to initiate rapid and complex cascades of biochemical changes involving growth factors, neurotransmitters and interleukins among others. Our data are in good agreement with reported increases in both total phospholipid content and synthesis of phosphatidylcholine, a paf precursor, in the regenerating liver. At present, the possible functional significance of high paf concentrations measured over the 'priming' stage of the induced proliferative wave is suggested as a working hypothesis. However, on the one hand, the observed paf response is noteworthy in view of its cytokine-related action, i.e., stimulation of IL-6 production by different cell types (endothelial, macrophagic). On the other hand, it could represent an in vivo confirmation of previously reported in vitro paf effects inducing c-fos and c-jun expression, two members of the so-called 'cellular immediate-early gene' family.

Liver cells can spontaneously resume proliferation in long-term quiescent primary cultures.

We report here data on the spontaneous resumption of proliferation in long-term primary cultures and we show that the proliferating areas are neoplastic. Normal rat hepatocytes were explanted in serum-supplemented Ham F12 medium and maintained over 8 months without transfer. The cells remained quiescent for the first 10 weeks and they were not tumorigenic when injected into nude mice. Later, without any modification of the culture conditions or transfer, progressive changes spontaneously occurred. Foci of dividing cells were detected, some displaying gamma-glutamyl-transpeptidase (gamma-GT) activity and F-actin fragmentation. These proliferating foci overcame the quiescent population. When injected into nude mice, the 15-week-old primary cultures were highly tumorigenic, with a 3-6 week latency for tumour formation. Furthermore, a cell line was derived from a primary culture started with a liver carcinogen promoter (biliverdin-enriched medium). This cell line proliferated rapidly and differed from a liver epithelial line, also established from our primary cultures, in its 1 karyotype (hyperploidy and translocation on chromosome 3), 2 requirement for arginine to proliferate, 3 gamma-GT positive reaction correlated to changes in actin fibre pattern, 4 sensitivity to protease inhibitors (i.e. alpha2 macroglobulin, PMSF) and 5 tumorigenicity. Long-term primary cultures and the karyotypically defined cell line are useful tools for further studies on in vitro genetic deviations.

Detection on liver tissue sections of S-phase markers in synchronized cycling rat hepatocytes by SIMS microscopy.

The aim of this study was to localise two ionic S-phase markers in tissue sections using SIMS microscopy: aluminium as a potential endogenous marker and bromine as an exogenous marker after in vivo injection of bromodeoxyuridine (BrdU). This study was performed in an experimental model of hyperplastic proliferation after partial hepatectomy in rat. Aluminium was never detected in nuclei which were positive or negative for tritiated thymidine uptake, as determined by autoradiography in tissue prepared by cryotechniques. In contrast, bromine of BrdU was found in hepatocyte nuclei. However, there was a discrepancy between SIMS bromine images and BrdU immunohistochemistry detection which appears more sensitive. This is probably due to problems of stereology intrinsic to the correlation method which requires serial sections for this multi-instrumental approach.

In vivo effect of the tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro, on the G1-S transition of rat hepatocytes.

The synthetic molecule N-Acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), corresponding to the low molecular weight inhibitory factor preventing in vivo haematopoietic stem cell (CFU-S) entry into DNA synthesis, was tested in two heterologous systems in vivo: adult regenerating rat liver and 10-day-old rat hepatocytes synchronized by an irritating trigger. In both systems, it was shown that doses of 2-8 micrograms kg-1 of tetrapeptide inhibited 50-70% of the hepatocyte G1-S transitions.

[Increase in nuclear aluminum concentration during hepatic regeneration: study by analytical ionic microscopy]. / Augmentation de la concentration d'aluminium nucléaire pendant la régénération hépatique: étude par microscopie ionique analytique.

20 hrs. after partial hepatectomy, a significant increase in the aluminum concentration of liver cell nuclei has been measured by analytical ion microscopy. These results indicate a possible role of aluminium in an in vivo induced proliferative response.

In vitro effect of a glycopeptide acting in vivo on hepatocyte cell cycle.

A factor specifically inhibiting the hepatocyte cell cycle in vivo was found to block the G1-S transition of liver cells in vitro. It proved to be non-toxic in our culture conditions, as judged by the reversibility of the effect on cell proliferation. It was not active on DNA synthesis in fibroblastic cell lines (3T3).

Liver DNA synthesis after phenytoin injection in adult NMRI mice.

Stimulation of hepatocyte DNA synthesis was observed in adult mice, as measured by the number of parenchymal cells in S phase, at two different times after a single i.p. injection of diphenylhydantoin (DPH): 18 and 25 hr. No such stimulation was obtained for Kupffer or endothelial cells.

Plasma levels of growth hormone, corticosterone and insulin, during induced hepatocyte synchronization in young rats.

A wave of synchronous hepatocytes entering the cell cycle can be obtained in vivo after a subcutaneous injection (e.g. of casein) in rats at around Post-natal Day 10, when plasma growth hormone (GH) levels reach a low plateau (40 +/- 2 ng/ml) and liver cell proliferation rate is high. The present work reports the following changes in plasma hormone concentrations after synchronization of 20% of the hepatocyte population: (1) during the G1 phase (i.e. 6-12 hr after the mitogenic trigger), plasma GH concentration has dropped further (25 +/- 1.5 ng/ml). It was back to 90% of control levels during the S phase, mitosis and the following response including a transitory decrease in labelling index below control values. Injected together with the irritating mitotic trigger, a single dose of rat GH reduced the cell synchronization and post-synchronization effects by 50%. (2) Plasma corticosterone levels varied inversely to those of GH, increasing to twice the control values during G1 and were back to physiological levels when synchronized hepatocytes entered the S phase. (3) Variations in insulin levels were similar to that of corticosterone, with narrower ranges and reduced amplitudes. Our data suggest a possible correlation between the observed variations in plasma hormone levels and the induced synchronous hepatocyte response.

Phenytoin administration to pregnant mice: a mutagenic action?

Phenytoin orally administered to pregnant NMRI mice results in limb haemorrhages, necrosis, and in most cases in limb amputations in the offspring. By mating these offspring without any treatment, the same type of abnormalities appeared throughout seven successive generation. In the second and third generation, other malformation, such as haemorrhages of the eyelids, and genitourinary defects were observed. These findings raise the question of the mutagenic role of phenytoin. Phenytoin is a drug of considerable medical use, but very little is known about the various possible intermediate steps involved in its action.

Interference of sex-related factors in the response of liver cells to experimental mitotic stimuli.

Stimulation of liver cell multiplication was obtained under two different experimental conditions. (1) A single injection of casein solution resulted in (a) an identical synchronized mitotic wave response in 10-day old male and female rats and (b) a significantly lower response in adult male rats compared to females, a difference which was reduced by castration of males at birth but essentially maintained if animals were operated when 10 days old. (2) Partial hepatectomy shortly after puberty resulted in active hepatocyte multiplication occurring 3 hr earlier in females were ovariectomized at birth and significantly reduced when they were spayed at a later age. Hepatocytes of castrated females entered actively into S phase 2 hr later than the sham-operated controls. Unilateral ovariectomy on the other hand indicated that during compensatory and/or hypercompensatory activity of the single ovary there was a maximum difference between the male and female rate of [3H]thymidine uptake in liver nuclei 20 hr after hepatectomy. A further kinetic study (t = 25, 30,40, 65, 90 hr) indicated no significant sex-related difference in the number of S phases per 10,000 cells. The DNA content of regenerating versus control livers was comparable in both sexes at t = 22 and 90 hr but higher in females at t = 40 and 65 hr. A possible early postnatal interference of certain hormonal mechanisms in the receptivity to mitotic stimuli is postulated and discussed.

Differentiation of ovarian and testicular interstitial cells during embryonic and post-embryonic development in mice. Ultrastructural observations.

Different steps in mouse ovarian and testicular development have been studied in order to compare the time sequences during the in vivo differentiation of steroidogenic cell populations growing in contact with male and female gonocytes. These time sequences indicated a basic common developmental pattern: early signs of steroid synthesis in the male gonad, but late entering into meiotic prophase of XY germ cells; early meiosis but late steroidogenic activity in the ovary. In both male and female interstitial tissues, signs of involution were found following a period of exponential development.

Inhibition of rat hepatocyte multiplication by serum and liver factors: physiological development and experimental induction.

Inhibition of the G1-S transition in synchronized baby rat hepatocytes was obtained by a subcutaneous injection of adult rat liver cytosol. This inhibitory activity was observed only with liver cytosol and not with kidney or spleen cytosol. The liver cell was a relatively specific target: no modifications were recorded in the kidney or submaxillary gland and inconsistant variations were found with tongue epithelium. The activity was associated with a non-dialysable factor. Physiological investigations support the opinion that the liver factor is the origin of the serum factor which had previously been described. Both factors were absent during the first three weeks of life. They appeared together during the 4th week in correlation with a decreasing rate of liver cell multiplication and then reached progressively their definitive adult levels. After 2/3 hepatectomy in adults, the liver cytosol retained its inhibitory activity, but the serum factor was reversibly neutralized by an antagonistic factor. Both inhibitory factors could be prematurely induced in baby rats and appeared transiently during the period of low mitotic activity following a wave of synchronized liver cells generated by an irritating stress. This inhibitory system is characteristic of the last developmental stages of the liver when growth decelerates before reaching a steady state. It seems to reduce the multiplication of hepatocytes by decreasing their sensitivity to stimuli initiating cell replication.

[Metabolism of pregnenolone 16-3H and progesterone 4-14C by 18 day fetal mouse gonads in organotypic culture. Effect of the gonadotropic hormone LH]. / Métabolisme de la prégnènolone 16-3H et de la progestérone 4-14C par les gonades foetales de souris de 18 jours en culture organotypique. Influence de l'hormone gonadotrope LH

Fetal mice testes convert pregnenolone-16-3H and progesterone-4-14C to testosterone in organ culture. The 3H/14C ratio in progesterone and testosterone fractions isolated from culture media suggests the importance of the delta5-3 beta hydroxysteroid pathway in our experimental conditions. LH decreases radioactive testosterone production and increases the activity of the kelta4-3-ketosteroid pathway.