Confocal microscopy of hair.

Confocal microscopy is an excellent method for studying the localization of fluorescent stains. Used in this way, superior 3D images can be obtained from multiple optical sections with very shallow depth of field. The main advantage of this technique is that the sample is not damaged. We have taken serial confocal sections of hair and via specific image enhancement routines have obtained high-quality 3D images enabling the visualization of cuticle scale and its pattern of distribution. This has been done on various types of hair: bleached, permed and in certain pathological conditions. This first step will allow us to characterize the hair surface in terms of its roughness, and the distribution and form of cuticular scale, parameters that have potential in the assessment of dermocosmetic efficacy.

Genetic difference in the proliferative response to T mitogens between Hi/PHA and Lo/PHA lymphocytes is independent of accessory cell function.

The role of the macrophage as accessory cell in the proliferative response of lymphocytes to phytohemagglutinin (PHA) was studied in two lines of mice genetically selected for high and low responsiveness to T mitogens. Adherent cell depletion of lymph node cells abrogated the low (Lo)/PHA response, but only partially inhibited the high (Hi)/PHA response. Addition of peritoneal cells provided either by Hi/PHA or by Lo/PHA mice equally restored Hi/PHA responsiveness but had only a slight reconstituting effect on the inhibited Lo/PHA response. Equivalent enhancement or suppression of proliferation of untreated lymph node cells was obtained by the addition of increasing percentages of each of the two peritoneal cell populations. However, the maximum level of the Lo/PHA response never reached that of Hi/PHA cells. These data indicate that the bidirectional selective breeding has not modified the potentialities of the macrophages as accessory cells but has resulted in an impaired response of Lo/PHA lymphocytes to the signals delivered either by accessory cells or by T mitogens.

Selection of BW 5147 subclones devoid of non-specific suppressive activity for use in cell hybridization.

Culture supernatants of BW 5147 cells widely used for T-cell hybridization often manifest non-MHC-restricted, non-antigen-specific regulatory activities on the mixed lymphocyte reaction (MLR) of mouse cells. This report demonstrates that, whereas supernatants of BW 5147 cells grown at low concentrations (2 X 10(5)/ml) enhanced MLR, high cell concentration (2 X 10(6)/ml) supernatants markedly inhibited this reaction. BW 5147 cell-free extracts significantly inhibited MLR and in vitro antibody production (PFC), as well as the mitogenic response to lipopolysaccharide E. coli (LPS) of mouse spleen cells, but did not affect the response to an optimal dose of phytohaemagglutinin (PHA). Both supernatant and cell-free extract inhibitory activities were located in 60,000 MW fraction. Inhibitory material of low MW (less than 12,000) was also found in high cell concentration supernatants. A similar suppressive activity was exerted by cell-free extracts of P3 X 63 NS cells used for B-cell hybridization. The suppressive activity seemed to stem from some kind of interaction between BW 5147 cells and the fetal calf serum (FCS) of the culture medium. Supernatants from subclones of BW 5147 cells obtained in selected batches of FCS and maintained in the same serum, even at high cell concentrations, did not affect MLR, whereas the supernatants from the same subclones maintained in other batches definitely suppressed this reaction. Thus, provided that culture conditions are chosen carefully, subclones of BW 5147 devoid of effect on in vitro immune reactions can be obtained.

Effects of T mitogens on in vivo antibody production to a T-dependent antigen in lines of mice genetically selected for high or low in vitro responsiveness to PHA.

The influence of genes which regulate the in vitro T-cell proliferative response to T mitogen upon in vivo antibody production to a T-dependent antigen was studied in two lines of mice genetically selected for a high or a low in vitro lymphocyte response to phytohaemagglutinin (PHA). Kinetics of agglutinin production to increasing doses of sheep erythrocytes was similar in the two lines, except for the titres of mercaptoethanol-resistant antibodies, which were slightly lower in the low-responder line. Treatment with mitogen prior to immunization modified the antibody response in the two lines differently. This finding would indicate that the genes which regulate in vitro stimulation of T cells by PHA also control in vivo activation of T-cell subsets involved in immunoresponsiveness.

In vitro viability of lymphoid cells from lines of mice genetically selected for high or low responsiveness to phytohemagglutinin.

The kinetics of viability of lymph node and spleen cells of mice genetically selected for "high" or "low" in vitro lymphocyte responsiveness to PHA were studied in PHA or PPD-stimulated short-term cultures. Lo/PHA cells were found to be less viable than Hi/PHA cells in unstimulated control cultures. PHA improved the viability of Lo/PHA cells while inducing proliferation of Hi/PHA cells with the appearance of more and larger lymphoblasts in the latter. PPD only improved the viability of spleen cell cultures, more so for the Hi/PHA line. The interline difference in thymidine uptake was smaller after PPD than after PHA stimulation. Modifications of culture conditions designed to decrease the interline difference in cell viability lessened but did not abolish the separation between the two lines for the PHA response as measured by thymidine uptake.

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin.

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.

Comparative blood level of dihydroergotoxine (DHET) after administration of two oral forms (solution & tablet): a preliminary study.

In this preliminary study, using a radioimmunoassay, we demonstrate that the DHET blood levels observed after administration of an oral solution to human subjects, were different from those obtained after administration of a slow-release table. After administration of the oral solution, the DHET blood levels rose quickly, reached a peak between 1 and 2 hours and the decrease rapidly. On the contrary, after ingestion of a slow-release tablet of DHET, the plasma levels took 6 hours to reach the maximum due to the slow release of the drug from this dosage form. In both cases, the areas under the curves were very similar but the relative bioavailability of DHET in these two forms is very different if one considers the two components of availability extent and rate. The equality of the areas under the curves indicated that the extent of DHET available was the same, but time-course of the plasma levels showed that the rate at which DHET became available was significantly slower. Therefore the tablet form has given the desired "slow-release" availability for which it was designed.

[Genetic regulation of T-lymphocyte responsiveness to PHA is independent of culture conditions (author's transl)]. / La régulation génétique de la réponse des lymphocytes T a la PHA est indépendante des conditions de culture.

A maximal interline separation has been obtained after 10 consecutive generations of selective breeding for the character "quantitative in vitro response of lymph node lymphocytes to the mitogenic effect of phytohaemagglutinin". At the selection limit the difference between high and low responder lines was about 20-fold. A similar interline separation has been demonstrated for the T-mitogen effect of concanavalin A. The identical response to PPD (purified protein derivative of tuberculin), a B mitogen, proved that the genetic selection has only modified the potentialities of T lymphocytes. During the selective breeding, responsiveness to PHA stimulation has been always measured under identical culture conditions. To demonstrate that the interline difference in responsiveness was due essentially to genetic factors independent of environmental effects, a systematic study of various culture conditions has been undertaken. The optimal stimulation was found after two days of culture for high line cells and after three days for low line cells. The difference between maximal responses was only slightly lower than that obtained after a two-day culture as used for the selection test. Increase in cell concentrations produced higher thymidine incorporation. In the two lines, a linear correlation was established between the cell concentration and the response produced. The maximal response given by the highest number of low line lymphocytes was equivalent to that given by a number, 11-fold smaller, of high line cells. Within certain limits, changes in the amount of tritiated thymidine added to the culture did not affect the interline separation. With a thymidine of high specific activity, a sub-evaluation of uptake by high line cells decreased the interline difference. Results in mixed culture of lymph node cells from high and low lines indicated that the low response was not due to the release of inhibiting factors or to the presence of suppressive cells in low responder mice. In conclusion, separation of these two lines was due to genetic factors acting independently of the cell culture conditions.

The capacity of histocompatibility antigens solubilized in hypertonic salt solution to induce allograft tolerance in rats.

Treatment of Fisher rats (AgB 1,26,28) with a soluble extract of histocompatibility antigens (SAE) prepared from the liver of donor August rats (AgB 5, 28, 31) associated with a few injections of anti-lymphocyte serum (ALS) provoked a specific prolongation of the median survival time of skin grafts from 27.6 +/- 11.4 days in ALS-treated controls to 55.1 +/- 8.8 days (p less than 0.01). The SAE was obtained from liver homogenates by hypertonic KCl (3M) extraction. Further purification by chromatography in DEAE-cellulose column resulted in the separation of fractions possessing a specific inhibitory activity on a Fisher anti-August cytotoxic serum that was almost 100 times higher than that of the initial SAE preparation. Analysis of the state of unresponsiveness induced by SAE and ALS showed that most of the unresponsive animals had in their serum blocking factors. On the other hand, in vitro study of proliferative and cytotoxic components of cell-mediated immunity by mixed lymphocyte reaction and cell-mediated lympholysis, respectively, showed that the proliferative reactivity remained unimpaired whereas the cytotoxic reactivity was clearly inhibited in the tested animals. These results suggest that a central tolerance to histocompatibility antigens (equivalent to those coded by the K and D end in the mouse) could have been induced in the experimental animals whereas reactivity to Ia region antigens was not affected.

Genetic selection of mice for quantitative responsiveness of lymphocytes to phytohemagglutinin.

A two-way selection was performed in mice according to the quantitative in vitro response of lymph node lymphocytes to the mitogenic activity of phytohemagglutinin (PHA). The foundation population was composed of outbred mice produced by reciprocal mating of equal numbers of mice from four different colonies. The selective breeding was carried out by mating of mice at each generation giving the best or the lowest response, respectively. The progressive interline separation produced by 6 generations of selective breeding demonstrates that responsiveness to PHA is submitted to polygenic regulation. The heritability of the character investigated is 0.28 +/- 0.08. The interline separation is also found with another T mitogen, concanavalin A (Con A). In spleen cells PHA and Con A produce a similar interline difference. In contrast, the purified protein derivative of tuberculin (PPD) stimulated both lines equally, and E. coli lipopolysaccharide gave only a slightly higher response in high line. This finding implies that our selection based upon response to PHA did not influence B cell function.

[Genetic selection of mice for quantitative responsiveness of lymphocytes to phytohemagglutinin]. / Sélection génétique de souris d'après l'intensité de la transformation blastique des lymphocytes induite par la phytohémagglutinine

A two-way selection was performed in mice according to the quantitative response of small lymphocytes to the mitogenic activity of phytohaemagglutinin (PHA). The response of inguinal lymph node cells of each mouse to an optimal dose of PHA was measured by 3H-thymidine incorporation using a micro-plate method. Starting from four outbred mouse strains we mated on the one hand mice getting the best response and on the other hand mice getting the poorest response. A progressive separation of the two lines was observed. At the 7th generation a 3-fold difference was found between the two lines. A similar interline difference was observed when concanavalin A (ConA) was used as mitogen. The separation of the two lines was also evident when spleen cells or thymus cells were cultured with PHA or ConA.

[Study of anti-macrophage serum and of its effect on antibody formation]. / Etude du sérum anti-macrophage et de son action sur la production des anticorps

A specific anti-macrophage serum (AMS) has been obtained by immunizing rabbits with mouse peritoneal exudate cells and after repeated absorption with mouse erythrocytes, thymocytes and splenic lymphocytes. A comparative study of the cytotoxic activities of this serum on macrophages, thymocytes and lymph node cells, before and after absorption, showed that macrophage posses antigens which are common to both thymic and lymph node lymphocytes, and other antigens which are in common with lymph node lymphocytes but absent on thymocytes, as well as antigens being particular to macrophages. The absorbed sera had a definite cytotoxic effect on macrophages in vitro. This effect was observed when complement was added immediately or 4 hours after incubation with serum. It was lost after 24 hours. An inhibitory effects of AMS on phagocytosis in vivo has also been observed, which disappeared after one day. Antibody production in vivo was only moderately affected by a pretreatment with AMS. However, addition of AMS to spleen cell cultures stimulated with sheep red blood cells inhibited the appearance of hemolytic plaque forming cells.