A regulatory role of activated T-lymphocytes on human megakaryocytopoiesis in vitro

Cellular interactions responsible for regulating in vitro megakaryocytopoiesis were studied using a microagar culture system which permits the simultaneous proliferation of human megakaryocytic progenitor cells (CFU-M) and T-lymphocytic colonies (CFU-TL). The proliferation of these colony types depends mainly on two factors: phytohaemagglutinin (PHA) and erythropoietin (EP). The direct addition of increasing PHA concentrations to the liquid overlayer resulted in a parallel increase of CFU-M and CFU-TL. If the T-lymphocytes were removed by an E-rosetting technique a marked diminution of CFU-M and CFU-TL numbers was observed. However, monocyte depletion resulted in a marked augmentation of CFU-M proliferation compared to unfractionated mononuclear cells. In order to confirm that the reduction of CFU-M proliferation observed after T-depletion was primarily mediated by the absence of T-lymphocytes, we have co-cultured different concentrations of previously removed autologous T-lymphocytes with a constant number of T-depleted bone marrow cells. A parallel increase of CFU-TL and CFU-M was found if 0.75 - 7.5 X 10(4) T-lymphocytes were added to the culture. In conclusion, our results indicate that activated T-lymphocytes augment proliferation of human bone marrow CFU-M and that monocytes are less important for the growth of megakaryocytic colonies.     

Rosette forming B- and T-lymphocytes in allergic contact eczema (author's transl)

The number of rosette forming lymphocytes with mouse and sheep red blood cells was examined in the time course of more localized (n = 16) and generalized (n = 12) allergic contact eczema. 1. In 25 healthy probands the mean number of B and T lymphocytes were 10 +/- 2.1% and 51 +/- 4.9%, respectively. 2. In generalized eczema a short significant increase of the number of both lymphocyte populations was observed. At the first day of examination 12 +/- 1.6% (p less than or equal to 1%) B and 59.1 +/- 5% (p less than or equal to 0.1%) T lymphocytes were counted. At the second day only the number of T lymphocytes (55 +/- 4.8%; p less than or equal to 5%) was increased, at the third day the number of T lymphocytes had normalized, too. 3. In the more localized eczema no changes in the number of lymphocytes could be found.     

Evidence for in vitro senescence of T-lymphocytes cultured from normal human peripheral blood

Long-term T-cell cultures from 15 donors of various ages were established using T-cell growth factor (TCGF). The donors were divided in equal numbers into three age groups, under 40, 40 to 80 and over 80 years of age. The cell cultures showed a growth pattern similar to the Hayflick Phenomenon observed in the cultured human fibroblasts with a limited total number of doublings. Cell cultures from younger groups grew longer with higher total number of doublings. The total number of doublings averaged 16.5, 12.2 and 6.5 for the cultured cells obtained from donors under 40, 40 to 80 and over 80 years of age respectively.     

Effect of splenectomy on B- and T-lymphocytes and a benzpyrene-induced murine sarcoma.

The investigation of 400 adult mice had the objective to find out 1. The influence of splenectomy on genesis and growth of a benzpyrene-induced sarcoma and 2. The behavior of B- and T-lymphocytes under the influence of splenectomy. Results: 1. A significantly lower number of tumors developed in splenectomized animals (28.5%) as compared to controls (49.5%). 2. A significant decrease of B-lymphocytes and an increase in T cells after splenectomy were found in the peripheral blood. 3. The examination of the tumor marginal zone showed a decreased number of B-lymphocytes and an increase of T-lymphocytes, while the total count of round cells remained unchanged. These results are discussed and compared with results of other authors.     

Effect of generalized graft-versus-host-reaction on B- and T-lymphocytes and a benzpyrene-induced murine sarcoma.

The investigations of 400 adult hybrids from A/Jax fem. BALB/c male were aimed at answering the following questions: 1. What is the influence of a generalized graft-versus-host-reaction (GVHR) on the induction and growth of benzpyren-induced sarcoma? 2. What is the behavior of B- and T-lymphocytes during GVHR, and during induction and growth of benzpyren-induced sarcoma? 3. Which are the deductions to be drawn with regard to the effect of B- and T-lymphocytes during tumorigenesis and tumor growth? Results: 1. The number of tumors was significantly greater in the GVHR animals (74.5%) than in the controls (49.5%). 2. A significant decrease of T-lymphocytes and increase of B-lymphocytes during GVHR were found in the peripheral blood. 3. The examination of the tumor marginal zone showed a marked decrease of the total round cell count and the T cells, while the count of B-lymphocytes remained uninfluenced. These results are discussed and compared with results of other authors.     

Characterization and in vivo distribution of influenza-virus-specific T-lymphocytes in the murine respiratory tract

Cloned populations of antigen-specific, murine T-lymphocytes, maintained in long-term continuous culture, have been reported to exhibit functional activity upon adoptive transfer. Few data are currently available on the in vivo distribution of these homogeneous, functional cell populations. Such information is important to understanding the mechanisms by which T-lymphocytes exert their effector activity. We examined 2 cloned populations of influenza virus-specific T-lymphocytes. One clone was a Lyt-2+, L3T4-, class I MHC-restricted, cytolytic T-lymphocyte subset. The other clone was a Lyt-2-, L3T4+, Class II MHC-restricted clone of the helper/amplifier T-cell subset. Both clones promote recovery from lethal pulmonary influenza infection upon adoptive transfer. Using cell populations labeled with [3H]thymidine, we examined the distribution of these cells in tissue sections from various organs. These cloned cell populations were preferentially retained in the lungs of uninfected and influenza-virus-infected animals. This retention is independent of the cell's viral antigenic specificity, but may be dependent on the phenotype of the clone. Once retained in the lungs, these cells migrated across the bronchial lamina propria and entered the epithelium and pulmonary lumen. The significance of these observations for in vivo T-lymphocyte functions is discussed.     

Tetracyclines inhibit human synovial collagenase in vivo and in vitro

To determine if tetracyclines can inhibit human synovial collagenase from rheumatoid tissue, paired synovial tissue (or synovial fluid) was collected from 7 patients before and after oral administration of minocycline (100 mg BID) for 10 days. With each patient serving as his own control, the postminocycline collagenase activities fell an average of 67% from pretreatment values. Qualitative SDS-PAGE revealed decreased loss of alpha collagen components and reduced formation of alpha A digestion fragments. Addition of minocycline or a chemically modified tetracycline to synovial culture media in vitro profoundly inhibited collagenase activity. Further study of this action of tetracyclines could serve as a probe of the role of collagenase in rheumatoid arthritis and lead to development of agents capable of modifying the tissue destructive actions of collagenase.     

Effects of cholecystokinin-octapeptide on the human gallbladder both in vivo and in vitro

To determine the sites and mechanisms of action of cholecystokinin-octapeptide (CCK-OP) on the human gallbladder, effects of atropine sulfate on CCK-OP-evoked contractions were studied in both in vivo and in vitro experiments. In vivo studies performed by means of real time ultrasonography in six healthy volunteers showed remarkable contractions of the gallbladder after intramuscular injection of CCK-OP (0.07 microgram/kg), which was nearly abolished by premedication of atropine sulfate (0.015 mg/kg). Atropine sulfate (10(-6) M) slightly but significantly reduced CCK-OP (10(-11) M-3 X 10(-7) M) induced contractions and the dose-response curve for CCK-OP was shifted to the right of the muscle strips of the human gallbladders. It is suggested that CCK-OP acts mainly on cholinergic neurons in vivo. On the contrary, the most sensitive sites of action of CCK-OP might be smooth muscles rather than cholinergic neurons in vitro.     

Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones

Gonadotropins are synthesized and released in different molecular forms. In this article, we present evidence that the glycosylation variants of human pituitary FSH exhibit differential and divergent effects at the target cell level and that less sialylated, short-lived variants may exert significant effects in in vivo conditions. Less acidic/sialylated glycoforms (elution pH value 6.60-4.60 as disclosed by high resolution chromatofocusing of anterior glycoprotein extracts), induced higher cAMP release, estrogen production and tissue-type plasminogen activator (tPA) enzyme activity as well as cytochrome P450 aromatase and tPA mRNA expression in cultured rat granulosa cells than the more acidic analogs (pH<4.76). By contrast, the more acidic/sialylated glycoforms induced higher alpha-inhibin subunit mRNA expression than their less acidic counterparts. In cumulus enclosed oocytes isolated from mice ovaries, addition of less acidic isoforms induced resumption of meiosis more efficiently than the more acidic analogs. Interestingly, the least acidic isoform (pH>7.10) behave as a strong antagonist of several FSH-mediated effects. Assessment of the in vivo effects of the isoforms on granulosa cell proliferation in follicles from immature rats, revealed that short-lived isoforms were equally or even more efficient than their more acidic counterparts in maintaining granulosa cell proliferation when administered immediately after hypophysectomy. These results show that the naturally occurring human FSH isoforms may exhibit differential or even unique effects at the target cell level and that factors other than the metabolic clearance rate of the molecule (including receptor-binding affinity and capability of the ligand to activate its receptor and trigger intracellular signaling) also play an important role in determining the net in vivo effects of a particular FSH variant.     

Effects of loperamide on the human hypothalamo-pituitary-adrenal axis in vivo and in vitro.

Loperamide, an opiate agonist of high specificity for mu-receptors, was recently reported to suppress ACTH and cortisol levels in normal subjects, but not in patients with proven ACTH-dependent Cushing's disease. However, there is little information on the site of action of loperamide in the hypothalamo-pituitary-adrenal axis of man. We investigated the effect of loperamide on pituitary hormone secretion in vivo and in vitro. In seven normal subjects, basal ACTH plasma levels were significantly suppressed 3 h after loperamide administration (16 mg, orally) from 5 +/- 1 to 2 +/- 0 pmol/L (P less than 0.0001). After the combined pituitary stimulation test (100 micrograms human CRH, 100 micrograms GnRH, 100 micrograms GH-releasing hormone, and 200 micrograms TRH), the ACTH peak (maximum increase at 30 min) was significantly blunted by loperamide from 9 +/- 1 to 4 +/- 1 pmol/L (P less than 0.001) and the area under the curve of ACTH from 0-120 min was reduced from 35 +/- 5 to 23 +/- 4 pmol/L.2 h (P less than 0.05). In the insulin-hypoglycemia test (0.15 IU/kg BW), neither the ACTH peak nor the area under the curve of ACTH was affected by loperamide. In six patients with Cushing's disease and one patient with secondary adrenal insufficiency due to hypothalamic failure, neither basal ACTH and cortisol levels nor CRH-stimulated levels were influenced by loperamide. In four cultured human corticotropic adenomas, loperamide was not able to reduce basal and CRH-induced ACTH secretion. In summary, loperamide is able to reduce basal and CRH-induced ACTH and cortisol levels in normal subjects, but not in patients with Cushing's disease or secondary adrenal failure of hypothalamic origin. Loperamide has no significant effect on insulin-hypoglycemia-induced ACTH and cortisol levels and, therefore, no effect on stress-induced elevation of cortisol levels. Loperamide might act at a suprapituitary site in man in vivo, but, nevertheless, a pituitary site cannot be excluded.     

Cellular aging and senescence characteristics of human T-lymphocytes

CD28−, CD57+ and KLRG1+ are cell surface markers that have been used to describe senescent T-lymphocytes in humans. However, the relationship among these phenotypes during aging, and their relationship with the concept of in vitro cellular aging have not been well established. Using five-colour flow cytometry, we analyzed peripheral blood T-lymphocytes for their expression of CD28, CD57 and KLRG1 in 11 young (Y) and 11 old (O) apparently healthy human subjects. The proportions of CD28− and CD57+ cells were significantly higher among the T-cell populations of O compared to Y subjects; the proportion of KLRG1+ cells was significantly higher only among CD8+ cells. Populations that were more frequent in the elderly participants were characterised as CD28+ CD57+, CD28− CD57+ or CD28− CD57−. The expression of p16 and p21, considered as markers for in vitro senescence, was higher in CD28+ CD57+ cells than in other subpopulations in both age groups. The expression of p21 was age-related, which was not the case for p16. Thus, although both p16 and p21 are involved in T-cell senescence, they appear to behave differently. CMV infection and shifts in subpopulations are unlikely as explanations of the observed differences. Their higher levels of p16 and p21 expression, coupled with their higher prevalence in the elderly participants make CD28+ CD57+ cells the subpopulation of T-cells most closely corresponding to the concept of senescent cells.     

Estimation of B- and T-lymphocytes in lymphoid tissue by means of photometry and 51Cr labelled marker erythrocytes.

Some new techniques for estimating B- and T-lymphocytes in lymphoid tissue are introduced. The density of marker erythrocytes on closed chamber incubated sections was estimated by the light transmission in relation to the number of the respective lymphocyte subpopulation, measured on suspensions of eluted lymphocytes. Using 51Cr labelled marker erythrocytes, the number of adsorbed marker erythrocytes per lymphocyte could be estimated from the gamma-irradiation of the section. The number of lymphocytes in the section was determined by the amount of protein in the section compared with the protein amount of a tissue specimen of known wet weight and known lymphocyte content. Furthermore, the number of adsorbed marker erythrocytes per rosette in suspensions of eluted lymphocytes was estimated by means of radioactively labelled marker erythrocytes.     

Origin of T-lymphocytes in human mixed hematopoietic colonies

The presence of T-lymphocytes in mixed hematopoietic colonies (CFU-MIX) has been reported by some investigators. Though depletion before culturing was performed, residual T cells might be responsible for the observed phenomenon. Using nondepleted marrow or bone marrow depleted to about 2%, T-lymphocytes could be detected in mixed colonies. However, reduction of the T-lymphocytes to less than 0.7% by using a modified E-rosette technique or a cocktail of anti-T-cell monoclonal antibodies (WT1, WT32, WT82) in the presence of baby rabbit complement, resulted in mixed colonies free of T-lymphocytes. After addition of 1.75% T-lymphocytes to this T-cell-depleted bone marrow, T-lymphocytes could be detected in most mixed colonies, but not after the addition of the same percentage of irradiated T-lymphocytes. The presence of T cells in mixed colonies was determined by an adapted immunofluorescence technique (WT32 plus GAM-FITC). The results indicate that mononuclear cells with T-lymphocyte antigens are not the offspring of mixed hematopoietic colony-forming progenitors, but of a low number of T-lymphocytes contaminating the bone marrow after insufficient T-cell depletion.     

New inhibitors of human renin tested in vitro and in vivo in the anaesthetized baboon

A new inhibitor of human renin (H. 189) is described. It is a decapeptide analogue of human renin substrate with the amino acid, statine, substituted for leucine in the scissile bond. Its inhibitory potency as shown by IC50 is 1.0 X 10(-8) M with human plasma renin and 1.5 X 10(-8) M with baboon plasma renin. It is less effective with dog and rat renin, but its inhibitory potency with human renin is similar to that of another inhibitor of ours (H. 142) having a reduced isostere in the scissile bond. H. 189 has some inhibitory effect on cathepsin D (IC50 6.5 X 10(-5) M) but H. 142 has no discernible effect. Pepstatin, on the other hand, was highly effective against cathepsin D (IC50 1.2 X 10(-8) M). H. 142 and H. 189 were infused intravenously at 10 mg/kg/h in four anaesthetized salt-deplete baboons (Papio hamadryas). The activity of renin in plasma decreased markedly as did the circulating concentration of its products, angiotensin I and angiotensin II.     

Effects of prolactin on cloned human T-lymphocytes

To evaluate the possible role of prolactin (PRL) in T-lymphocytes, we monitored gene induction in one cytotoxic T-lymphocyte (CTL) clone derived from a patient with hemochromatosis and in several T-helper clones generated from a normal donor and a patient with multiple sclerosis. The CTL clone expressed conventional PRL receptor (PRLR), and PRL induced the expression of suppressor of cytokine signaling-3 (SOCS-3) and increased the expression of SOCS-2 and cytokine-inducible src homology-2-containing protein (CIS, another member of the SOCS family). As is the case in granulocytes, expression of a conventional receptor for PRL could not be shown by polymerase chain reaction analysis on three helper clones. In addition, as in granulocytes, PRL modulated the expression of genes such as the interferon-regulatory factor-1, inducible nitric oxide synthase, CIS, and SOCS-2. These effects were also elicited with ovine PRL and could be prevented by anti-PRL antibodies. Thus, the use of clones allowed the detection of direct effects of PRL on T-cells, even when these have few or no detectable PRLR, confirming that human T-lymphocytes are targets for PRL.