In vitro growth of the nasal septal cartilage of the rat in a serum-free culture medium. [3H]-thymidine incorporation studies

To evaluate the capacity for growth of cartilage in vitro, the cartilaginous nasal septum was cultured in a serum-free medium in short- and long-term incubation periods. The proliferative activity was assessed by relating counts per minute (cpm) obtained in the liquid scintillater after [3H]-thymidine incorporation with the DNA content of the tissue under investigation. Cell proliferation was interpreted as being synonymous with the DNA synthesizing activity or the cpm/microgram DNA ratio. Short-term incubation (24 h) tended to show the same proliferative activity which we have previously found in vivo and as autotransplants, showing that the presence of serum seems to be of little importance under these conditions. Long-term incubation in a serum-free medium tended to show a gradual loss of proliferative activity, emphasizing the need for either serum or other growth-promoting factors as necessary for continued normal proliferation of cartilage in vitro.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Regulation of cultured rat vaginal epithelial cells by 17 beta-estradiol and progesterone

We have previously described a technique to obtain short-term cultures of epithelial cells from Wistar rat vaginae. In order to improve the efficiency and life span of these cultures, in the present study we have cultured the vaginal cells with lethally irradiated 3T3 cell feeder layers. Under this condition, cells can grow for several weeks while retaining epithelial characteristics and can eventually be subcultured. The proliferative effect of the ovarian hormones in these cultures was studied using two different approaches, [Methyl-3H]Thymidine (3HTdr) incorporation and increase in cell number. Both assays indicated a proliferative effect of 17 beta-estradiol and progesterone at physiological concentrations. This proliferative effect was also shown in feeder layer-free cultures, ruling out an indirect effect through the mesodermal cells. The capacity of the hormones to modify terminal differentiation in the culture was also studied, using colony stratification as an indicator of differentiation. Progesterone and fetal calf serum had an inhibitory effect on terminal differentiation, whereas 17 beta-estradiol induced a stimulatory action. This culture model allowed us to show a direct effect of the ovarian hormones on vaginal cells in vitro and seems to be a useful model to study hormone-cell interactions in vitro.     

Functional differentiation in the genetic control of murine T lymphocyte responses to human fibrinopeptide B

The ability to generate proliferative and helper T lymphocyte responses in mice was compared by using the 14 amino acid peptide, human fibrinopeptide B (hFPB). Lymph node or peritoneal exudate T cells from mice immunized with hFPB were assessed for in vitro proliferation to soluble hFPB as determined by the uptake of 3H-thymidine. The T cell proliferative response to hFPB was found to be under MHC-linked Ir gene control; mice possessing the H-2a,k haplotypes were responders, whereas H-2b,d,q,s mice were nonresponders. The influence of non-H-2 genes on these responses was not investigated, so exclusive regulation by H-2 is provisional. The absence of a detectable lymph node and peritoneal exudate T cell proliferative response persisted in H-2b,d,q,s mice after immunization and boosting with several doses of hFPB. In addition, the capacity to produce a T cell proliferative response was inherited in an autosomal dominant manner and gene(s) controlling responsiveness to hFPB mapped to the I-A subregion of the H-2 complex. To measure peptide-specific helper T cell activity, an in vitro microculture assay in which hFPB-primed lymph node T cells and normal spleen B cells and macrophages were used was developed measuring anti-fluorescein isothiocyanate (FITC) IgM and IgG plaque-forming cell (PFC) responses after culture with FITC-conjugated peptide. Immunization of B10.BR, C57BL/10, B10.D2, and B6AF mice with hFPB primed for significant helper T cell activity as assessed by the ability to augment a primary in vitro IgM response to FITC. The normal B cell IgM responses were completely dependent on hFPB-primed T cells and required that hapten (FITC) and carrier (peptide) be linked. In addition, immunization with FITC-conjugated peptide elicited positive in vivo PFC responses to FITC in B10.BR and C57BL/10 mice, indicating similar genetic control of helper activity in both the intact animals and the in vitro microcultures. Thus, B10.BR mice show both T help and T proliferative responses to hFPB, whereas C57BL/10 mice show only T help and no T proliferative responses. In contrast to B10.BR mice, C3H and CBA mice immunized with hFPB were completely unresponsive when assayed for helper T cell activity in vitro despite their ability to generate positive lymph node T cell proliferative responses. These results indicate responsiveness to hFPB by T helper and proliferating cells is different and is under separate genetic control.     

Induction of splenic granulopoiesis in vitro.

Murine spleen cells were cultured in vitro to study the induction of committed granulopoietic stem cell (CFU-C) proliferation and maturation. Marbrook-type diffusion cultures were established with and without the addition of colony-stimulating activity (CSA) and harvested at intervals up to 14 days for viable and differential cell counts, [3H]TdR autoradiography, and quantitation of CFU-C by the agar plate method. Without CSA there was poor cell viability and little proliferative capacity. In CSA-stimulated cultures there was a prominent rise in viable cell counts and [3H]TdR labeling indices rose from a mean of 2% at 0 time to 47% after 5 days in vitro. CFU-C increased by 70-fold in these cultures. Peak numbers of CFU-C, immature cells, and [3H]TdR-labeled cells occurred at about 7 days. Thereafter, mature granulocytes and macrophages predominated in culture. Because the liquid spleen cell culture system begins in a resting state and undergoes a wave of proliferative activity in response to CSA, it can provide a useful model system for studying phenomena associated with stem cell activation and differentiation in vitro.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

Proliferative response of peripheral blood lymphocytes to mitogens in the owl monkey Aotus nancymae

The new world primate Aotus sp. has been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and P. vivax. The present study examined the in vitro proliferative response of peripheral blood mononuclear cells (PBMCs) isolated from Aotus monkeys, utilizing a wide range of mitogens. Results presented herein demonstrate that the in vitro proliferative response of PBMCs from the Aotus sp. is quite variable from monkey to monkey for each of the mitogens assessed. PBMCs from the Aotus monkey exhibited a delayed kinetic proliferative response and, particularly, a different sensitivity to proliferation in response to various concentrations of Phytohemagglutinin-P and favin lectins, the phorbol ester Phorbol myristate acetate and the calcium ionophore ionomycin. Altogether, our findings are consistent with the conclusion that the in vitro proliferative response of PBMCs from the Aotus differ in their activation requirements compared with PBMCs from humans.     

Defect of CD2- and CD3-mediated activation pathways in T cells of atopic patients: role of interleukin 2.

In the present work we analyzed the proliferative response of T lymphocytes from 11 atopic patients stimulated in vitro via either the CD2 or the CD3 pathway of cell activation. In both cases we found a significant decrease of thymidine incorporation in cell DNA in comparison with T cells from normal donors. The mechanism of this impaired proliferative response was analyzed. Atopic patients' T cells were found to secrete low quantities of interleukin 2 (IL2) and to express low amounts of Tac antigen, measured as both a percentage of Tac-positive cells and a mean fluorescence intensity of Tac antigen per cell. Addition of recombinant IL2 to cultures completely restored both cell proliferative response and Tac antigen expression. This effect was specific of IL2 since addition of IL1 or IL4 did not significantly affect T cell proliferative response. We conclude that atopic patients' T lymphocytes have a defect in both CD2 and CD3 pathways of cell activation relying on impairment of IL2 production, without involving IL2 responsiveness or other lymphokine defects.     

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.     

Chemically modified antigen-presenting cells induce T lymphocyte allospecific hyporesponsiveness.

We have investigated the interaction between murine T lymphocytes and allogeneic APC in an in vitro proliferative mixed leukocyte reaction. Our results demonstrate that freshly isolated potentially alloreactive murine splenic T lymphocytes, in primary culture, can be induced to develop a state of allospecific proliferative hyporesponsiveness in vitro by exposure to 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-modified allogeneic APC, a method similar to that previously used to induce nonresponsiveness in murine Ag-specific self-MHC-restricted T lymphocyte clones. This hyporesponsiveness was: specific for the allohaplotype of inducing APC, maintained for 96 h in vitro, not due to cellular inhibitory mechanisms, and associated with reduced ability to secrete IL-2 but not IL-3. Induction of this hyporesponsiveness was not due to altered expression of class II MHC gene products on the APC but was associated with markedly reduced T lymphocyte-APC adhesive interactions despite the lack of a detectable immunophenotypic change in lymphocyte function-associated Ag 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) expression on the modified APC. Therefore, we propose that TCR occupancy in the absence of normal T lymphocyte-APC adhesive clustering may induce T lymphocyte tolerance.     

Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases

The prevention of cancer through dietary intervention is currently receiving considerable attention. Several epidemiological studies substantiate that green tea has a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer. This preventive potential of green tea against cancer is attributed to the biologically active flavonoids called catechins. Epigallocatechin 3-o-gallate, the major catechin found in green tea, mediates diverse physiological and pharmacological actions in bringing about the regression of the tumors and also lowers the risk of nonmalignant cardiovascular proliferative diseases. Much of the current research is being focused on how these catechins specifically bring about the regression of the experimentally induced tumors both in vitro and in vivo. These catechins exert diverse physiological effects against proliferative diseases by several mechanisms, most of which are not completely characterized. This review summarizes the mechanisms by which these catechins play an essential role in regulating the process of carcinogenesis, with a special emphasis on how these catechins antagonize the growth factor-induced proliferative disorders.     

Role of CD2 in activation and cytotoxic function of CD8/Leu-7-positive T cells

CD3/CD8-positive, Leu-7-positive cells comprise about 3 to 5% of PBL in normal individuals, but the proportion of these cells is increased in patients with a variety of diseases including chronic viral infection, Crohn's disease, and AIDS. To study further the function of these cells, the proliferative and cytotoxic responses of highly purified CD8/Leu-7-positive cells were studied in vitro. These cells had low proliferative responses when exposed to PHA or mitogenic anti-CD3 mAb compared to CD8/Leu-7-negative cells, and their proliferative responses were significantly lower after addition of IL-2 or autologous adherent cells. However, the proliferative responses of both Leu-7-positive and Leu-7-negative CD8 cells were similar when stimulated with PHA, Ionomycin, or anti-CD3 in combination with phorbol ester. In addition, CD8/Leu-7-positive cells demonstrated high proliferative responses when exposed to a combination of both PHA and SRBC, and these responses could be inhibited by prior addition of non-stimulating anti-CD2.1 mAb. CD8/Leu-7-positive cells, but not CD8/Leu-7-negative cells, mediated lectin- and anti-CD3-induced cytotoxicity against K562 target cells. Cytotoxicity was in part dependent on the CD2 Ag because it was inhibited by anti-CD2.1 mAb. Finally, when small CD8-positive T cells having low cytotoxic potential were activated with PHA plus SRBC, but not PHA alone, there was significant enhancement of their cytotoxic function. Thus, the CD2 receptor may be an important activation pathway for cytotoxic cells.     

Immunosuppression of T lymphocyte function by fractionated serum from tumor-bearing mice.

Sera from mice with transplanted 3-methylcholantrene-induced tumors have been shown previously to inhibit the function of normal lymphoid cells. When chromatographed on Sephadex G-150, the fraction eluting with immunoglobulin has been shown to inhibit the proliferative response of normal spleen cells to concanavalin A and to inhibit the in vitro antibody response to a T-dependent antigen, but has a lesser effect on the antibody response to a T-independent antigen. This paper deals with studies on the mode of action of the serum factor. The immunoglobulin containing fraction of serum from tumor-bearing mice inhibited the in vitro generation of both allogeneic and syngeneic cytotoxic lymphocytes. Time course studies demonstrate that the serum fraction inhibits the generation of antibody-producing and cytotoxic lymphocytes if added during the first 2 days of a 5-day culture. Serum fractions added after day 2 had no effect on the in vitro response. The serum factor appears to inhibit the generation of specific T cell function during the proliferative stage of development but has no effect on the differentiation stage which leads to either antibody-producing cells or cytotoxic lymphocytes.     

Discrimination of two fibroblast progenitor populations in early explant cultures of hamster gingiva

Summary Numerous metabolic studies have demonstrated heterogeneity of fibroblast populations in culture, yet little is known about the structure of fibroblast populations in adult tissues in vivo. To determine if populations of both cycling and non-cycling cells are present in gingiva, hamsters were labelled with [³H]-thymidine to label cycling cells in vivo, and explanted biopsies were subsequently incubated with bromodeoxyuridine to label cycling cells in vitro. Cycling cells were identified by combined immunohistochemistry and radioautography. Fibroblasts were recognized by the presence of vimentin and the absence of keratin as determined by immuno-fluorescence. The largest proportion of cells were double-labelled with [³H]-thymidine and bromodeoxyuridine (43.8%) indicating the presence of actively cycling populations that maintained their proliferative status upon explantation. Cultures also exhibited a second population of cells labelled only with bromodeoxyuridine (38.7%) that did not cycle in vivo, but retained the capacity for proliferation in vitro. However, limiting dilution analysis of single-cell suspensions revealed only a single class of progenitors capable of forming large colonies in vitro. Approximately 1 in 190 plated cells was capable of colony-formation, indicating that, upon explantation, a subset of the cycling cells in vitro exhibits extensive proliferative capacity. There was also a small population of cells unlabeled with either [³H]-thymidine or bromodeoxyuridine (9.4%) that appeared to be terminally differentiated. Different substrates, including glass and thin films of gelatin and collagen, did not significantly alter the fraction of cells labelled with [³H]-thymidine. These data demonstrate the existence of 2 separate progenitor-cell populations with different capacities for proliferation in vivo and in vitro.     

Effect of 5'-deoxy-5'-S-isobutyladenosine on hematopoietic stem cells

Unlike known cytostatics, 5'-deoxy-5'-S-isobutyladenosine (SIBA) does not inhibit proliferative activity of hemopoietic stem cells of intact bone marrow. On the contrary, it raises CFUs number of 15-28% in vitro and in vivo. SIBA inhibits by 40% the 3',5'-cAMP-induced increase in CFUs proliferation.     

Cell kinetics of gliomas by serial stereotactic biopsy

The potential proliferative activity of glial tumors has been investigate by serial stereotactic biopsies and by "in vitro" 3H-thymidine incorporation procedure (labeling index, LI). The methodology of this combined approach and the preliminary results in 33 patients are reported. Cell kinetic data have been matched with the histological classification (W.H.O.).     

In vitro proliferation and in vivo malignancy of cell lines simultaneously derived from a chemically-induced heterogeneous rat mammary tumor

Summary Identification of clones in primary tumors responsible for proliferation, invasion, and metastasis was carried out. Four different aneuploid established cell lines derived from a ductal infiltrating mammary rat tumor induced by 7,12-dimethylbenz[a]anthracene were studied for proliferative and growth features in vitro and for tumorigenic and metastatic potential in vivo in nude mice. Clones, named RM1, RM2, RM3, and RM4, were characterized by different proliferative activity. Clone RM1 showed the highest proliferative activity by both tritiated thymidine incorporation and S-phase flow cytometry, followed by clone RM4. Conversely, clones RM2 and RM3 showed a lower proliferation rate. Growth-promoting activity, tested on 3T3 Swiss cells, was high in all clones, although RM1 showed significantly lower growth factors—releasing activity. Nude mice tumorigenesis demonstrated a strong tumor induction of line RM1 (100% of the mice after 47±7 d) and a slightly lower tumor induction of line RM4 (70% of the mice after 69±9 d). Line RM3 showed tumor induction in 40% of the mice after 186±16 d. Lines RM2 showed no tumor induction. Metastasis occurred in mice treated with line RM1 only. Therefore, tumorigenesis and metastasis correlate with proliferation but not with the release of growth factors. In conclusion, flow cytometry monitoring of clones from heterogeneous primary tumors proved to be a suitable model for the study of in vivo malignancy and in vitro proliferation.     

Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax.

The cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequence of the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen-specific in vitro proliferative response to the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative response when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, within the major surface antigen of P. vivax sporozoites, of epitopes capable to induce proliferation of human PBMC.     

Nicotine Promotes Late Endothelial Progenitor Cells Functional Activity in a PI 3-Kinase-Dependent Manner

Recent studies have shown that endothelial progenitor cells (EPCs) participated in angiogenic effects of nicotine and nicotine dose dependently increased the functional activity of early EPCs. The effects of nicotine on late EPCs remain to be determined. Therefore, we investigated whether nicotine had influences on the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood and characterized. Late EPCs of 3–5 passages were treated for 32 h with either vehicle or nicotine. The proliferative, migratory, and in vitro vasculogenesis activities of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in matrigel, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Nicotine enhanced proliferative, migratory, adhesive, and in vitro vasculogenesis capacities of late EPCs. These effects were significantly reduced in the presence of phosphatidylinositol (PI) 3-kinase inhibitor.     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. II. Induction of helper activity demonstrated by TNP-haptenated ABA-peptide-Ficoll

In an effort to study T cell functions in Lewis rats immunized with ABA-N-acetyl-L-tyrosine (ABA-tyr), we developed an antigen that provides a sensitive assay of ABA-specific helper function that is read as an increase in TNP-specific plaque-forming cells (PFC). This antigen has ABA coupled to AECM-Ficoll by virtue of a tripeptide (tyr-ala-ala) spacer and TNP coupled to the AECM side chains. At subimmunogenic doses, this antigen induced 400 anti-TNP PFC/10(6) spleen cells in ABA-tyr-immunized rats. As many as 8000 PFC/10(6) spleen cells were induced with larger doses of antigen (200 micrograms). By contrast, only 490 PFC/10(6) spleen cells could be induced with 1 mg of the conventional doubly haptenated protein carriers such as ABA-BSA-TNP. Both direct and indirect PFC were induced by this antigen in primed rats. The use of this antigen and passive transfer techniques to study ABA-specific helper activity revealed some differences from ABA-specific delayed-type hypersensitivity (DTH) and in vitro proliferation, which were studied previously. Cells responsible for helper activity appeared sooner after immunization and were found most prominently in peritoneal exudates but also significantly in spleen where the cells responsible for DTH or in vitro proliferative responses were never found. By contrast, helper cells were not seen in lymph nodes, where some proliferative activity could be found. Of these three ABA-specific T cell functions, helper activity was least easily suppressed by the previously used regimens of ABA-tyr in incomplete freunds adjuvant (IFA). Moreover, helper activity appears after injection of ABA-tyr in IFA, a method that has never in our hands yielded detectable DTH or in vitro proliferative responses. Despite these differences, phenotyping with monoclonal antibodies indicated that cells responsible for helper and proliferative activities were both W3/25+ and OX8-.