Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions.

Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.

A new CD43 monoclonal antibody induces homotypic aggregation of human leucocytes through a CD11a/CD18-dependent and -independent mechanism.

We describe a monoclonal antibody (mAb), designated 1.C1, that causes rapid and vigorous aggregation among normal leucocytes and among T and myeloid/monocytic cell lines. As shown by competitive binding and sequential immunoprecipitation experiments, the antigen recognized by mAb 1.C1 is a 115,000 MW sialoglycoprotein, that corresponds to the human CD43 antigen, also known as leukosialin or sialophorin. The aggregation process starts within minutes and reaches maximum level 6-18 hr after addition of the antibody. It is dependent on active cell metabolism (inhibited at low temperatures and by a mixture of the metabolic poisons azide and 2-deoxy-D-glucose), a fluid plasma membrane (inhibited by pretreatment of the cells with paraformaldehyde) and an intact cytoskeleton (inhibited by cytochalasin B). Two reference CD43 antibodies (MEM-59 and DF-T1), both binding the same or closely related sialic acid-dependent epitope as mAb 1.C1, are also capable of inducing cell clump formation. CD11a/CD18 mAb block the 1.C1-induced adhesion of resting peripheral blood leucocytes, but not of haematopoietic cell line cells. In addition, mAb 1.C1 induces homotypic aggregation of K-562 cells, which do not express members of the beta 2 integrin subfamily on their surface. These data suggest that triggering of the CD43 antigen promotes homotypic cell adhesion that is mediated by both CD11a/CD18-dependent and -independent pathways.

Inflammatory properties of recombinant tumor necrosis factor in rabbit skin in vivo.

We have investigated the ability of recombinant TNF (mouse and human) to produce acute inflammatory lesions in an established experimental model of inflammation. Upon intradermal injection in rabbit skin, TNF, in amounts as low as 3 x 10(-14) mol/site, was found to be very potent at inducing local neutrophil accumulation and neutrophil-dependent oedema formation, thereby fulfilling two important criteria to be considered as an inflammatory mediator. Our findings further indicate that the pro-inflammatory properties of TNF are probably more related to its immediate stimulatory effects on neutrophils rather than to its slow (protein biosynthesis-dependent effects on endothelial cells. Our data thus show that very low amounts of mouse and human recombinant TNF can initiate an acute inflammatory reaction in vivo in rabbit skin and that TNF is able to evoke two of the four cardinal signs of inflammation.

Different pro-inflammatory profiles of interleukin 1 (IL 1) and tumor necrosis factor (TNF) in an in vivo model of inflammation.

The pro-inflammatory properties of IL 1 and TNF were investigated in an in vivo model of inflammation. IL 1 induced PMN leukocyte accumulation that was slow in onset, reaching a peak rate at 3-4 h and that was inhibitable by Actinomycin D and Cycloheximide. PMN leukocyte emigration was not associated with any significant plasma leakage. In contrast, TNF induced PMN leukocyte accumulation and oedema formation, that were rapid in onset and very short of duration (t1/2 6-10 min). TNF-induced plasma leakage was PMN leukocyte-, but not protein biosynthesis-dependent. The differences in time course and biological profile suggest that IL 1 and TNF exert their pro-inflammatory effects in vivo via different mechanisms.

Mab. 198: a monoclonal antibody recognizing the complement type 3 receptor (CR3) in the rabbit.

A mouse monoclonal (Mab.198, IgG1) was generated that recognizes an epitope expressed on rabbit peripheral phagocytes and tissue macrophages. The membrane antigen recognized by Mab.198 consisted of two polypeptide chains of 165,000 and 95,000 molecular weight (MW). This Mab efficiently inhibited complement receptor-mediated granulocyte functions such as phagocytosis of complement-opsonized particulate antigens and zymosan-induced chemiluminescent responses. Fc receptor-mediated phagocyte functions were, on the contrary, barely affected by Mab.198. The cellular distribution, molecular structure and functional characteristics of the membrane antigen defined by Mab.198 suggested that this antibody recognizes the complement type 3 receptor (CR3). We found that Mab.M1/70, specific for mouse CR3 (i.e. CD 11 or Mac-1 antigen), also binds on rabbit phagocytes. However, this Mab recognizes a different CR3 epitope than Mab.198 as shown by cross-blocking experiments. This anti-rabbit CR3 Mab will be useful in the characterization of rabbit complement receptors and their involvement in immune reactions.

A Brodie's abscess caused by Salmonella give.

This report describes a typical Brodie's abscess caused by a Salmonella subspecies in an otherwise healthy individual. The rarity of the causative agent of this infection is stressed.

Rabbit leukocyte surface antigens defined by monoclonal antibodies.

Several monoclonal antibodies (mAb) against rabbit leukocytes were characterized in binding and functional studies. mAb 1.24 stains thymocytes, bone marrow cells, peripheral T and B cells and blood monocytes. T cells express more 1.24 antigen than B cells. In the absence of added complement (C), mAb 1.24 inhibits alloantigen-, concanavalin A (Con A)-, and phytohemagglutinin (PHA)-, but not pokeweed mitogen (PWM)- or anti-immunoglobulin (Ig)-induced cell proliferation. It also strongly blocks anti-sheep erythrocyte plaque-forming cell responses. A second mAb, designated 4.B9, binds to 20% of thymocytes and to most, if not all, peripheral T cells and in vitro-activated T cell blasts. A third one, 10.B3, is reactive with the nearly entire thymocyte and a major peripheral T cell population. Two-color membrane immunofluorescence reveals the presence of a small population of peripheral blood leukocytes which bear surface Ig and are weakly stained by mAb 4.B9 and 10.B3. Without C, both 4.B9 and 10.B3 inhibit Con A- and PHA-induced mitogenesis, but have no effect on PWM-, antigen-, or alloantigen-induced cell proliferation. Depletion of 4.B9+ cells by panning or complement lysis completely abrogates proliferative responsiveness to antigen and alloantigen, significantly reduces responsiveness to the T cell mitogens Con A and PHA, but enhances that to the B cell mitogen anti-Ig. A fourth mAb, 12.C7, binds to 60% of thymocytes and to 10-30% of peripheral T lymphocytes at high-level fluorescence. T cell blasts obtained in mixed leukocyte reactions are partially stained by mAb 12.C7, while those obtained after Con A or PHA activation are not. In addition, mAb 12.C7 is completely unreactive with B cells or monocytes. Without complement, it does not seem to interfere with any of the in vitro functions tested. All antigens studied here do not appear to be expressed in nonleukon tissues, as they do not bind to erythrocytes and are absent from brain, heart, liver and kidney as shown by quantitative absorption analysis.

Effects of anti-Ig reagents on T cell functions I. Activation of rabbit T cells by anti-allotype antibodies.

Rabbit lymphocytes were analyzed by flow microfluorometry, using anti-T cell and anti-Ig reagents. Rabbit T cells and cells expressing surface Ig (B cells) appeared to belong to distinct subpopulations which could be separated on the basis of their selective adherence to nylon wool columns or to anti-Ig-coated dishes. Using flow microfluorometry, no evidence was obtained for the expression of a allotypes (VH framework) on T cells. Separated lymphocyte populations were functionally characterized using an in vitro proliferation assay. B and T cells from rabbit spleen or peripheral blood responded in a differential fashion to B and T cell-specific mitogens and to anti-Ig antibodies. Although such T cells did not respond upon stimulation with anti-Ig antibodies alone, significant proliferation could be induced by simultaneous addition of anti-Ig and T cell growth factor. In addition, activated T cells, derived from lymph nodes of immunized rabbits, generated a proliferative response upon stimulation with anti-Ig reagents alone. The above-mentioned effects on T cells could be obtained using heterologous anti-Ig antibodies or isologous anti-allotype antibodies, directed either against a allotypes (VH framework) or against b allotypes (kappa light chain). Antibodies against the Fc portion of rabbit Ig or against irrelevant allotypic specificities were ineffective in triggering T cells. Fab fragments from anti-allotype antibodies were equally stimulatory for T cells as compared to intact IgG, indicating that cross-linking of Ig-like molecules is not a necessary requirement for anti-Ig-induced T cell activation.

Production of IgG-specific auto-anti-allotypes in b4.2-suppressed rabbits.

Rabbits were completely suppressed for kappa chain allotype b4.2, and autoantibodies against b4.1 or b4.2 could be raised in 2 out of 3 animals. The animal immunized with b4.1 produced anti-b4 and anti-Ms3, two activities which have never as yet been found together in one antiserum. Both autoantisera lacked the capacity to bind b4.2-IgM, whereas they precipitated 4.2-IgG very well. In one animal, anti-b4 IgM activity appeared after the sixth immunization, at the age of 14 months. Allotype suppression was maintained in both rabbits till the end of their lives whereas all control animals recovered within 6 months. The autoantisera which were specific for b4 IgG could not induce suppression in vivo. However, specific inhibition of b4 IgG secretion was observed in vitro.

Antigen-induced proliferation assay for rabbit T lymphocytes. I. Characteristics of the response.

An in vitro assay which measures specific antigen-induced proliferation of primed rabbit lymph node and peripheral blood cells is described. This response was found to be mediated by T cells, since it could be obtained with nylon-wool passed cells and cells which do not adhere to anti-Ig-coated plastic plates. The proliferative response was found to be highly antigen-specific and restricted to the draining lymph node if assessed up to 15 days post-priming. Purified T cells required antigen-pulsed accessory cells to proliferate. The proliferative response of peripheral blood lymphocytes was found to be low unless the lymphocytes were fractionated on insolubilized histamine. The histamine-adherent cells could suppress the above peripheral blood response, implying a certain regulatory role of suppressive cells in the periphery. The ease, reproducibility and specificity of the assay provides a simple method to evaluate the characteristics of a T-cell response in the rabbit.

Histamine-binding suppressor T cells in rabbit peripheral blood.

Peripheral blood leukocytes (PBL) from primed rabbits were able to suppress the in vitro anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) response of autologous spleen cells. A population containing the suppressor cells could be isolated from PBL by cell fractionation on columns of insolubilized histamine. In contrast to spleen cells, PBL generatd a weak secondary anti-SRBC response in vitro. A strong response was obtained with PBL freed from histamine-binding (H+) cells. The addition of these H+ cells to cultures of H-PBL caused strong suppression. The H+ suppressor cell was further characterized as a radioresistant T cell. Low-dose irradiation of H- cells resulted in a supplementary enhanced PFC response suggesting that PBL also contain a radiosensitive regulator cell which is not histamine binding.

A comparison of the electrophoretic haemoglobin patterns of the vertebrates.

A comparative study of the haemoglobins of 648 animals distributed over 300 species of vertebrates and belonging to different classes has been made by horizontal starch gel electrophoresis is tris EDTA-borate pH 8.6. Multiple haemoglobins were found within the different classes. The percentage of multiple haemoglobins varies from class to class. The frequency-distribution of the mobility values of haemoglobins is rather specific for the different classes and in certain instances for the different orders.

The total protein content in the blood serum of vertebrates.

The total protein content in the blood serum of 416 species and subspecies of vertebrates is determined by the microbiuret method. Generally speaking the total protein content of the serum shows an increase with a higher evolutionary level. The difference between the mammals and the other groups is particularly large. This increase is probably attributable to a number of factors, for instance adaptation to living on land, development of the circulatory system, the development of adaptive immunity and the forming of specific transport proteins. A connection between albumin content and level of development can also be noticed.

[First stages of development of the mesodermic leaf in Polypterus senegalus Cuvier]. / Les premiers stades de développement du feuillet mésodermique chez Polypterus senegalus Cuvier

Investigations into the origin of the excretory system cells in Polypterus have shown a few interesting items about the first stages of mesoderm development in these fishes. After the gastrulation the mesoderm is not completely separated from the endoderm. The appearance of cavities in the mesoderm is quite limited and it even is but ephemeral in the superior part (in the epimeres). Lateral extensions of the mesomeric zones of the six most anterior metameres (especially metameres II to V) form the anlage of the pronephric duct. The latter will continue its way in a posterior and inferior direction without any participation of other mesoderm material. The elongation of the undifferentiated material that had been concentrated around the blastoporus will be responsible for the formation of the major part of the body, and mesoderm will play an important role in it. An enterocelic relation between mesoderm and endoderm in the anal region, as Kerr had conjected to see it, is due to the existence of evaginations of endoderm but gives no support to the enterocelic theory.

[Development of the gonads and gonaducts in the polypterids (Pisces)]. / Considerations sur le développement des gonades et des gonoductes chez les polyptères (Pisces)

No study of the development of the gonads and gonoducts in Polypterus has ever been made in a more or less complete way. The present study does not fill up this gap, but repairs several lacks of knowledge, by putting clear a few items that may contribute to a better understanding of this development. In larvae between 5,5 mm and 9,6 mm the primary gonocytes appear under the pronephric ducts in the anterior region of the opisthonephros. A genital crest is foreshadowed there; one of its characteristics is a cubial epithelium. The latter does not have any relation with peritoneal channels and it does not contribute to the formation of gonocytes. In the following stages the gonocytes get very numerous because of cellular multiplication. They cause a local transformation of the genital crest into a genital fold; when the number of gonocytes has increased very much, a part of the fold gets transformated into a gonad. At no stage is there any intrusion of medullary tissue into this gonad anlage.

[Origin of the coelomic cavities and the heart in the polypterids (Pisces)]. / Sur l'origine des cavités coelomiques et du coeur chez les polyptères (Pisces)

In polypterus the mesodermal cavities appear quite late during embryonic life. They are generally small and they only get somewhat more voluminous in the anterior mesomeres (where they establish the pronephrie chambers) and in the ventral anterior mesoderm (where they become an embryonic pericardial cavity). The anlage of the heart appears in the anterior part of the tissue that is situated between the paired mesodermal cavities of these stages. It assumes some unawaited dispositions that are truly confusing in the case of a superficial inspection. It is only during larval life that a coelomic cavity appears all along the truncal part of the mesoderm. In the beginning this is a pericardio-peritoneal cavity. But because of the coalescence between several membranes an anterior cavity gets isolated and this one is the pericardial cavity of the adult specimens; this cavity is much more limited than its homonymic counterpart of the embryonic stages.

[Development of the cloaca and its ulterior transformations in the polypterids (Pisces)]. / Le développement du cloaque et ses transformations ultérieures chez les polyptères (Pisces)

Generally spoken cloacae have been but little studied. In Polypterus, a brachiopterygian fish, a cloaca only exists during parts of the embryonal and larval life and it gets replaced by structures consisting of an anus and an urinary sinus. In the beginning of embryonal life the posterior opening of the body seems to be nothing else than the original blastoporus. The wall of this opening acquires evaginations; the anlagen of the excretory ducts come in contact with them, establishing in that way a structure, that merits the name of cloaca. During the elongation of the post-vitellin body, the latter gets shifted from the level of metamere XII to that of metamere XXX. Another migration of the cloaca occurs when the digestive system acquires its development. Finally the cloaca may be found under the 47th and 48th muscular segments (at least in the species Polypterus senegalus senegalus Cuvier). During these stages the excretory ducts have build up an urinary sinus that gets separated from the gut. In adults no communication exists any more between the gut and the urinary sinus; the latter is a very destinct organ that may have an impair or pair aspect according to its being filled up. Because of the establishing of a communication with the genital ducts the sinus becomes an uro-genital sinus in the adult.