Neural stem cell separation from the embryonic avian olfactory epithelium by sedimentation field-flow fractionation.

The aim of the present study was to isolate neural stem cells from a complex tissue: the avian olfactory epithelium; by using sedimentation field flow fractionation (SdFFF). By using "Hyperlayer" elution mode, fraction collection and cell characterization methods, results shows that SdFFF could be a useful cell sorter to isolate an enriched, viable and sterile immature neural cell fraction from which the reconstitution of a complete epithelium was possible. In culture, SdFFF eluted cells first led to a "pseudoplacodal" epithelioid cell type from which derived "floating cells". These cells were then able to generate neurosphere-like structures which were composed of cell having many features of immature cells: undifferentiated, self-renewable and multipotentiality. Such a population might be used as a model to improve our understanding of the mechanisms of olfactory neoneurogenesis.

Light chain inclusion permits terminal B cell differentiation and does not necessarily result in autoreactivity.

Mice in which the Jkappa cluster was replaced with a VkappaJkappa rearranged gene were studied. More than 90% of B cells from homozygous mutant mice expressed the transgenic kappa chain but showed a slightly reduced level of kappa transcripts compared with WT B lymphocytes. Light chain inclusion was apparent in 10% of B cells from these mice and raised 25% in hemizygous mice with a still lower expression of the knockin kappa chain. Beyond the rules of clonal selection, peripheral B cells developed in such animals, with included cells being activated and differentiating into class-switched or antibody-secreting cells. The high amount of included mature B cells was associated with an increase of hybrid kappa/lambda immunoglobulins but not with the increased prevalence of autoantibodies. Altogether, these data suggest that light chain exclusion prevalent in normal B cells mostly results from ordered rearrangements and stochastic mechanisms but is neither tightly ensured by a stringent cell selection process nor absolutely required for normal B cell function.

Differential responses of olfactory neurons to axotomy at embryonic and postnatal stages.

In the nervous system, apoptosis is a major process during embryonic and postnatal stages. In chick, experimental cell death can be obtained by axotomy. We have compared the responses of olfactory neurons to axotomy at embryonic stage E17 and postnatal stages.Forty-three chicken embryos and 32 young chickens less than 3 weeks old were used. We combined optic microscopy, electronic microscopy, terminal (TdT)-mediated dUTP-nick end labeling (TUNEL) method and gel electrophoresis of genomic DNA to analyze cell death. Cells in synthetic phase were labeled by bromodeoxyuridine injected i.p. and detected by immunohistochemistry. Apoptotic index and 5-bromo-2'-deoxyuridine (BrdU) labeling index were calculated for each stage. The Wilcoxon test was used for statistical analysis. A P value <0.05 was considered significant. Within 3 days following axotomy in E17 chicken embryos, there was no significant increase of apoptosis on the lesion side analyzed 3, 6, 12, 18, 24, 48 or 56 h later. A slight increase of bromodeoxyuridine incorporation appeared at 3 h, was weakly significant at 24 h (P=0.045) and the return to basal rate took place at 48 h. In postnatal stages, an apoptotic wave appeared 12 h after axotomy, reached a maximum at 24 h, and then decreased between 48 h and 72 h. A significant elevation of bromodeoxyuridine incorporation occurred on 48 h with a 24 h delay after the peak of apoptotic death. This differential response to axotomy in embryos and postnatal chickens might be due to a less complete maturation and higher plasticity of embryonic olfactory neurons corresponding to different requirement for survival and differentiation factors. Thus the embryonic or immature neurons would depend more on local epithelial environment and mature or postnatal neurons would require target-derived survival factors and die from apoptosis after their shortage resulting from axotomy.

Insulin-like growth factor I induced survival of axotomized olfactory neurons in the chick.

Insulin-like growth factor 1 (IGF1) receptor is expressed in avian olfactory neurons and IGF1 in the bulb. To explore the function of IGF1 in olfactory system in the chick, we infused IGF1 at the lesion site 0 and 12 h after olfactory axotomy. The animals were killed 1-3 days later. TdT mediated dUTP nick end labeling method and bromodeoxyuridine incorporation allowed the evaluation of programmed cell death and mitotic activity respectively in the olfactory epithelia of IGF1 treated or untreated lesioned animals and controls. IGF1 treatment suppressed the apoptotic wave, stimulated mitosis which peaked within 24 h (instead of 48 h), to return promptly to normal, and transiently maintained the number of calmodulin related kinase II expressing neurons at normal levels. It reveals a long lasting effect of IGF1 on the survival of lesioned olfactory neurons and transient effects on maintenance of differentiation and mitosis stimulation.

Cyclo-oxygenase 2 tissue distribution and developmental pattern of expression in the chicken.

1. Cyclo-oxygenase 2 (COX-2) is implicated in multiple physiopathological processes. We have studied its physiological expression during chicken embryogenesis. 2. An original procedure was set to prepare a COX-2 probe from red blood cells. In situ hybridization, reverse transcription-polymerase chain reaction and northern blots, were performed on chick embryos from embryonic day (E) 3 to postnatal day 15. 3. In the mesonephros, the signal detected in mesonephric tubules presented a slow increase from E5 to E9, a plateau up to E12 and then a decrease, while the signal increased in the metanephros and then decreased after hatching. Transient expression of COX-2 mRNA in endothelial cells of the infundibulum of the aorta was also detected between E12 and E17. 4. This enzyme may have important roles in kidney morphogenesis during early embryonic stages and in tubular functions during development and in adult life. In the cardiovascular system, its vasodilatory effect could modulate the vasoconstrictor effect of the systolic pressure between E12 and E17 and contribute to a normal morphogenesis of the arterial tree and heart.

An exploratory pilot study of acupuncture on the quality of life and reproductive hormone secretion in menopausal women.

The majority of menopausal women suffer from climacteric symptoms. The purpose of this study was to assess the effects of acupuncture on the quality of life and reproductive hormones secretion in menopausal women. Eleven (11) menopausal women with climacteric symptoms entered this prospective study. The Menopause Specific Quality of life Questionnaire was filled out by the patients before the first acupuncture session, after the last one (5 weeks later), and 3 months after the last acupuncture session. Reproductive hormones including follicular-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, and prolactin were measured before and after treatment. Acupuncture significantly improved menopausal vasomotor symptoms (p = 0.001 and p = 0.003 for the end of treatment and 3 months later, respectively) and physical symptoms (p = 0.014 at the end of treatment and p = 0.046 3 months later). It did not change psychosocial or sexual symptoms, nor did it change the measured reproductive hormones. In conclusion, acupuncture is shown to be effective in relieving vasomotor and physical disturbances of menopausal women with effects lasting at least up to 3 months after termination of the treatment. Acupuncture may be a useful treatment alternative for women who are unable or do not want to receive hormone replacement therapy. A prospective study with larger sample sizes will be needed to define the role of acupuncture in the management of menopausal symptoms.

Intrauterine insemination: evaluation of the results according to the woman's age, sperm quality, total sperm count per insemination and life table analysis.

We report on 332 infertile couples who underwent 1115 cycles of intrauterine insemination (IUI) with washed husband's semen. The indication for IUI was an abnormal post-coital test due to either a male or cervical infertility factor. The mean number of IUI cycles per patients was 3.4, the overall pregnancy rate 18.7%, and the pregnancy rate per cycle 5.6%. The cumulative pregnancy rate calculated by life table analysis showed that 16.0% of pregnancies occurred in the first three treatment cycles, while the cumulative pregnancy rate was 26.9% by the sixth cycle. The outcome of the therapy was adversely affected if the woman's age was > 39 years and/or total motile sperm count per insemination was < 1 x 10(6). No pregnancy occurred in women older than 44 years or in cases with a total motile sperm count before semen preparation of < 1 x 10(6).