Blocking 17β‐hydroxysteroid dehydrogenase type 1 in endometrial cancer: a potential novel endocrine therapeutic approach

The enzyme type 1 17β‐hydroxysteroid dehydrogenase (17β‐HSD‐1), responsible for generating active 17β‐estradiol (E2) from low‐active estrone (E1), is overexpressed in endometrial cancer (EC), thus implicating an increased intra‐tissue generation of E2 in this estrogen‐dependent condition. In this study, we explored the possibility of inhibiting 17β‐HSD‐1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo, and ex vivo models. We generated EC cell lines derived from the well‐differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of 17β‐HSD‐1 similar to human tissues. In these cells, HPLC analysis showed that 17β‐HSD‐1 activity could be blocked by a specific 17β‐HSD‐1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17β‐HSD‐1 inhibition. In vivo, tumors grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by 17β‐HSD‐1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17β‐HSD‐1‐negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17β‐HSD‐1 enzyme activity (ex vivo in tumor tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the 17β‐HSD‐1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumor, we next demonstrated the mRNA expression of the potential drug target, 17β‐HSD‐1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17β‐HSD‐1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17β‐HSD‐1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A study of the relationship of metabolic MR parameters to estrogen dependence in breast cancer xenografts

¹H MRS, ³¹P MRS and diffusion‐weighted MRI (DW‐MRI) were applied to study the metabolic changes associated with estrogen dependence in estrogen receptor (ER)‐positive BT‐474 and triple‐negative HCC1806 breast cancer xenografts supplemented with or without 17β‐estradiol (E₂) at a dose of 0.18 or 0.72 mg/pellet. Furthermore, the effect of estrogen withdrawal on the metabolism of BT‐474 and HCC1806 breast cancer xenografts was studied on day 0, day 2 and day 10. Increasing the dose of E₂ resulted in a rapid growth and increases in the lactate level and phosphomonoester/β‐nucleoside triphosphate (PME/βNTP), phosphocreatine/inorganic phosphate (PCr/Pi) and βNTP/Pi ratios in BT‐474 breast cancer xenografts; however, no significant changes were found in HCC1806 breast cancer xenografts. Estrogen withdrawal resulted in a marked decrease in lactate level and PME/βNTP ratio and an observed increase in βNTP/Pi, PCr/Pi and apparent diffusion coefficient (ADC) values of BT‐474 breast cancer xenografts on day 10. These data suggest that the lactate level and PME/βNTP, PCr/Pi and βNTP/Pi ratios of ER‐positive tumors are closely related to ER dependence. Copyright © 2015 John Wiley & Sons, Ltd.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

Effect of estradiol and degree of cell differentiation on human melanoma susceptibility to killing by monoclonal antibodies

An estradiol non-responsive melanotic (E^?M⁺ShA) was derived from an estradiol-responsive melanotic (E⁺M⁺ShA), and an estradiol-responsive amelanotic (E⁺M^?SR) was derived from estradiol non-responsive amelanotic (E^?M^?SR) cell lines by deprivation and supplementation of the culture medium with estradiol, respectively, E⁺M⁺ShA cells became E^?M⁺ShA after 5 weeks, i.e. 2 subcultures, of culture in the absence of estrogen, and E^?M^?SR cells became E⁺M^?SR after 7 weeks, i.e. 3 subcultures, of culture in the presence of 10^?7 M 17β-estradiol. Spleen lymphocytes from mice pre-immunized with E⁺M⁺ShA, E^?M⁺ShA, E^?M^?SR and E⁺M^?SR have been fused with murine non-producer myeloma cells to obtain mouse mouse hybridoma cultures that synthesize monoclonal antibodies against human malignant melanoma. The complement-dependent cytotoxic activities of these antibodies linked responsiveness to estradiol with melanotropin biosynthesis.     

Sequential appearance of γ/δ- and α/β-bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenes

To search for a potential role of T cell antigen receptor (TcR) γ/β-bearing cells in host-defense against Listeria monocytogenes, we analyzed the sequential appearance of γ/δ and α/β T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 × 10³) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM⁺ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3⁺ T cells in addition to a large number of macrophages. Of the CD3⁺ cells, the proportion of CD4^?CD8^? cells, most of which expressed no TcR α/β, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3⁺CD4^?CD8^?TcR α/β^? cells, high level of TcR γ/δ chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4⁺CD8^? and CD4^?CD8⁺ cells which expressed TcR α/β chain on their surface. These results suggest that the γ/δ T cells precede the α/β T cells in appearance during listerial infection. The γ/β T cells may be involved at the first line of the host-defense against Listeria.     

Effect of Selective Estrogen Receptor Modulator/Raloxifene Analogue on Proliferation and Collagen Metabolism of Tendon Fibroblast

The selective estrogen receptor modulator raloxifene is therapeutically beneficial for postmenopausal connective tissue degradation, such as osteoporosis, vascular sclerosis, and dermal degradation; however, the effects of raloxifene on postmenopausal tendon metabolism have not been clarified. In this study, we investigated the effects of raloxifene analogue (LY117018) on cell proliferation and collagen metabolism using cultured rat Achilles tendon fibroblasts. 17β-Estradiol (E₂; 10^?11–10^?9 M) and LY117018 (10^?9–10^?7 M) had no significant effects on tendon fibroblast proliferation, based on a BrdU (5-bromo-2′-deoxyuridine) incorporation assay (24 hr) and a WST-8 colorimetric assay (2 or 6 days). Neither E₂ nor LY117018 significantly altered the expression of type I collagen, which is a main component of the tendon extracellular matrix (ECM), whereas both E₂ and LY117018 significantly increased the expression of matrix metalloproteinase (MMP)-13, which is responsible for tendon collagen degradation in rat. Also, both E₂ and LY117018 increased the expression of type III collagen and elastin, which are minor components of tendon ECM, but are considered to govern the elastic properties of tendons. These changes in collagen and MMP induced by either E₂ or LY117018 were attenuated by the estrogen receptor alpha blocker ICI 182,780. The results of this study suggest that postmenopausal estrogen deficiency might downregulate tendon collagen turnover and decrease tendon elasticity. Further, raloxifene treatment might restore these changes to premenopausal levels.     

Exploration of the wound healing effect of topical administration of nicotine in combination with collagen scaffold in a rabbit model

Nicotine has been reported to prolong the wound healing; however, we showed that the topical application of 10^?4 M nicotine promoted murine wound healing. The objective of this study was to explore the wound healing effects of nicotine in combination with collagen scaffold using skin defects in rabbit. Three full-thickness skin defects 8 mm in diameter were made on the rabbit auricle. Artificial dermis was applied to the defects, and 10 μl of nicotine solution (10^?5, 10^?4, and10^?3 M), bFGF solution (0.5 μg/10 μl), and both bFGF and 10^?4 M nicotine solutions were injected into the artificial dermis once daily for 7 days. Rabbits were sacrificed on day 10, 15, or 20, and the wound healing process was evaluated. bFGF was superior in the formation of the dermis-like tissue and capillaries. In nicotine groups, the epithelial length and the dermis-like tissue formations in the 10^?4 M group were superior, in contrast, those were inhibited in the 10^?3 M group. The synergistic effect of bFGF and 10^?4 M nicotine was not confirmed. This study suggests that the topical application of 10^?4 M nicotine promoted wound healing in rabbit, but the effect was not apparent compared with murine models.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Steroid Sex Hormones and Macrophage Function: Regulation of Chemiluminescence and Phagocytosis

PROBLEM: Female sex hormones modulate a variety of humoral and cell-mediated immunologic functions. In this study, the effects of estrogen, progesterone, and testosterone on the chemiluminescence (CL) response and phagocytic ability of male rat peritoneal macrophages (Mφ) were examined. METHOD: In Mφ pretreated with 10^?2 ng/ml of 17β-estradiol (E₂) for 20 hours, the CL generated in response to phorbol myristate acetate (PMA), 1,2-dioctanoyl-rac-glycerol (C8:0), or opsonized zymosan (OZ) was significantly increased by 135%, 140%, and 136% of control values, respectively. In addition, Mφ treated with 10^?5 ng/ml or 10 ng/ml of E₂ exhibited a significantly greater PMA-or OZ-stimulated CL response than did untreated controls. RESULTS: At 10^?2 ng/ml, progesterone enhanced and testosterone reduced the CL response, but these changes were not statistically significant. In time course studies, the PMA-stimulated CL response of Mφ treated with 10^?2 ng/ml of E₂ or progesterone for 5 h was significantly less than that of the untreated group. In the presence of endotoxin (12 pg/ml), the CL response in Mφ treated with E₂ or testosterone was significantly depressed as compared to untreated controls. Phagocytosis of opsonized sheep erythrocytes also was significantly enhanced (140% to 190% of control) when Mφ were pretreated with 10^?12 M to 10^?8 M of either E₂ or progesterone. CONCLUSIONS: These findings suggest that, at physiological concentrations, E₂ is capable of modulating both CL generation and phagocytic uptake by Mφ in a manner not shared by other steroid hormones.     

Inhibition of ACTH-induced differentiation of cortical cells and their mitochondria by corticosterone in tissue culture of fetal rat adrenals

Tissue cultures of fetal rat adrenals were used to study the effects of corticosterone on the ACTH-induced ultrastructural differentiation of cortical cells and their mitochondria. Corticosterone in dosages of 0.2, 2.0, 5.0, 10, and 20 μg/ml (corresponding to concentrations of 6 × 10^?7, 6 × 10^?6, 1.5 × 10^?5, 3 × 10^?5, and 6 × 10^?5 molar) was added alone or together with 100 mU/ml of ACTH to the culture medium, daily from the sixteenth day of cultivation up to and including the twenty-first day. Corticosterone alone induced no ultrastructural changes in cortical cells. Corticosterone in concentrations of 6 × 10^?7 to 3 × 10^?5 M given with ACTH induced hypertrophy of Golgi apparatus. Corticosterone in concentrations of 6 × 10^?5 M inhibited the ACTH-induced differentiation of cortical cells. However, the nuclear chromatin increased and Golgi apparatus was strikingly hypertrophied. Mitochondria often aggregated adjacent to the nuclear envelope but their ultrastructure remained undifferentiated with tubular or tubulovesicular cristae. Ribosomes appeared as single particles. A marked increase of smooth surfaced endoplasmic reticulum was noted also in cortical cells treated with 6 × 10^?5 M of corticosterone. The present observations suggest that corticosterone acts as an intracellular inhibitor in cortical cells. It appears to inhibit cytoplasmic protein synthesis at the ribosomal level and prevents synthesis of cytoplasmic mitochondrial protein synthesis stimulating factor and the latter, in turn, inhibits the activation of mitochondrial protein synthesis. A new model is presented to explain the regulation of growth and secretion in the adrenal cortex.     

Increased number of cytotoxic T cells within CD4+8− T cells in β2-microglobulin,major histocompatibility complex class I-deficient mice

Targeted disruption of β₂-microgobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4^?8⁺ T cells. Despite this, β₂M^?/? mice reject skin grafts and cope with most viral infections tested. We asked whether CD4⁺8^? cytotoxic T cells could play a role in compensating for the defect in CD4^?8⁺ cytotoxic T cell function. We found that the cytotoxic activity against class II⁺ targets is significantly higher among CD4⁺8^? T cells of β₂M^?/? than among those of β₂M^+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4⁺8^?, class II-specific T cells is at least fivefold higher in β₂M ^?/?than in β₂M^+/+ mice. These results suggest that CD4+8^? cytotoxic T cells could play a major role in carrying out cytotoxic function in β₂M^?/? mice.     

Quantitative pharmacological characterization of β-receptors and two types of α-receptors mediating sympathomimetic smooth muscle response in the human Fallopian tube at various cyclic stages

The dissociation constants for adrenoceptor-antagonist complexes (K_B) were determined in vitro in circular and longitudinal smooth musculature from the ampullary and isthmic regions of the human Fallopian tube. High extracellular potassium concentrations were used to eliminate the spontaneous contractile activity. Neuronal and extraneuronal amine uptake mechanisms were blocked. The parallel shift of the log dose-response curves was secured in Arunlakshana-Schild plots. K_B for the β-receptor, mediating sympathomimetic relaxation, were determined during α-receptor blockade: the values for propranolol were the same (approximately 10^--⁶ M) in all preparations and at all cyclic stages, as determined from plasma estradiol and progesterone levels. K_B for the complex between the a-receptor (mediating contraction) and phentolamine were determined during preceptor blockade. The values were the same in all types of smooth musculature, but varied with cyclic stage: they were around 7× 10^--⁸ M when plasma estradiol and progesterone were both minimum, and around 2× 10^--⁷ M when these steroid levels were moderate to high, suggesting that the properties of the contractile receptors of the human Fallopian tube are modified during the menstrual cycle.     

Radioautographic Analysis of Surface Markers on Decidual Cells Shared by Cells of the Lymphomyeloid Tissues

ABSTRACT: Stromal type decidual cells recovered from the murine decidua by a mild collagenase dispersion procedure contain immunoregulatory cells whose ultimate precursors may originate from the bone marrow. To explore the familial relationship of these cells with other cells of the immune system, a battery of cell surface markers recognized on lymphomyeloid cells were examined and quantitated at the morphological level on typical stromal type decidual cells of the dispersed CBA mouse decidua at 8–14 days of syngeneic pregnancy, using a sensitive radioautographic technique. Cells were either labeled directly by exposure to ¹²⁵I-labeled monoclonal antibodies against Thy-1, Mac-1, or Lyt antigens or indirectly by a sequential exposure to monoclonal anti-I-A^k (Ia. 17) or monospecific anti-I-J^k antibodies and ¹²⁵I-labeled Protein A. Decidual cells were found to be Thy-1± (13–73% positive, the incidence rising with advancing gestation in the decidua basalis), Mac-1 ± (present on 6–11% on day 8 and 17–32% on day 12), I-A^?, 1-J^?, and Lyt^?. Macrophages within the decidua were Thy-1^?, Lyt^?, Mac-1 ⁺, and I-A± (present on 5–61% of cells, the incidence rising with advancing gestation). These surface properties of decidual cells along with the presence of FcR, an absence of C₃R, and the presence of a unique marker Dec-1 reported by us earlier, distinguish them from most other stromal type immunoregulatory cells such as Langerhans cells (I-A⁺, FcR⁺, C₃R⁺, Mac-1^?, Thy-1^?), follicular dendritic cells (I-A⁺, FcR⁺, Thy-1^?, Mac-1^?, Dec-1^?), and dendritic reticular cells (I-A⁺, FcR^?, C₃R^?, Thy-1^?, Lyt^?, Mac-1). However, a substantial subpopulation of decidual cells share a few properties (1-A^?,Thy-1⁺) with another marrow derived stromal type cell in the epidermis termed as epidermal dendritic cells that remain to be investigated for other surface markers such as Mac-1, Dec-1, and FcR.     

Progenies of fetal thymocytes are the major source of CD4−CD8+ αα intestinal intraepithelial lymphocytes early in ontogeny

Present literature supports the view of an extrathymic origin for the subset of intestinal intraepithelial lymphocytes (IEL) that express the CD4^?CD8⁺ αα phenotype. This subset would include virtually all T cell receptor (TCR) γδ IEL and a portion of TCR αβ IEL. However, these reports do not exclude the possibility that some CD4^?CD8⁺ αα IEL are actually thymically derived. To clarify this issue, we examined the IEL day 3 neonatally thymectomized (NTX) mice. NTX resulted in as much as 80 % reduction in total TCR γδ IEL and in a nearly complete elimination of TCR αβ CD4^?CD8⁺ αα IEL early in ontogeny (3-to 5-week-old mice). The thymus dependency of TCR γδ IEL and TCR αβ CD4^?CD8⁺ IEL was less prominent in older mice (7- to 10-week-old mice), as the total number of these IEL increased in NTX mice, but still remained severalfold less than that in euthymic mice. Furthermore, we demonstrate, by grafting the fetal thymus of CBF1 (H-2^b/d) mice under the kidney capsule of congenitally nude athymic mice of BALB/c background (H-2^d), that a substantial number of TCR γδ IEL and TCR αβ CD4^?CD8⁺ αα IEL can be thymically derived (H-2^b+). In contrast, but consistent with our NTX data, grafting of adult thymi into nude mice generated virtually no TCR γδ IEL and relatively less TCR αβ CD4^?CD8⁺ αα IEL than did the grafting of fetal thymi. These results suggest that the thymus is the major source of TCR γδ and TCR αβ CD4^?CD8⁺ αα IEL early in ontogeny, but that the extrathymic pathway is probably the major source of these IEL later in ontogeny. A reassessment of the theory that most CD4^?CD8⁺ IEL are extrathymically derived is needed.     

An active CD8α/pMHCI interaction is required for CD8 single positive thymocyte differentiation

Recognition of viral antigenic peptides bound to major histocompatibility complex class I molecules (MHCI) by TCR is critical for initiating the responses of CD8⁺ T cells that ultimately lead to elimination of virus‐infected cells. This antigen recognition is enhanced by the CD8 coreceptor through its interaction with the peptide‐MHCI complexes (pMHCI). Mouse CD8αβ can form two different complexes with pMHCI via either the CD8α‐ or CD8β‐dominated interaction. To understand the functional significance of these complexes in vivo, we generated Tg mice carrying a variant CD8αβ (CD8α^m3β) capable of forming only the CD8β‐dominated CD8αβ/pMHCI complex. These mice show sub‐optimal thymic differentiation with reduced populations of CD8⁺ single‐positive thymocytes. Tg CD8⁺ T cells exhibit a compromised developmental capacity when competing with CD8⁺ T cells from B6 mice in mixed bone marrow chimera experiments. However, once these CD8⁺ T cells have emigrated to the peripheral lymphoid organs, they exhibit normal effector function against viral infection. Our observations indicate that, in addition to the CD8 activity conferred by CD8β‐dominated CD8αβ/pMHCI complexes, full thymocyte differentiation requires additional coreceptor activities conferred by CD8αα and/or CD8αβ with CD8α‐dominated CD8/pMHCI complexes.     

Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V

Kharrat M, Trabelsi S, Chaabouni M, Maazoul F, Kraoua L, Ben Jemaa L, Gandoura N, Barsaoui S, Morel Y, M’rad R, Chaabouni H. Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V. Steroid 11β‐hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β‐hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β‐hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population.     

Development of γδ T cells in the adult murine thymus

The development of T cells belonging to the γδ lineage is not well understood. We have analyzed the cells in the adult murine thymus which express the γδ TcR on the surface in order to learn more about this process. Our data demonstrate a number of clear subpopulations of γδ expressing cells in the thymus based on the expression of Thy-1 and HSA (heat-stable antigen). Only one of these subpopulations, the one expressing both Thy-1 and HSA, contains dividing cells or has a significant rate of turnover. Together with the fact that emigrant γδ cells are HSA⁺Thy-1⁺, this suggests that this thymic subpopulation is the sole, or major, source of exported cells. However, the turnover of cells from this population is 5 × 10⁴ - 10 × 10⁴ cells per day, while previous estimates of the rate of export of γδ cells are in the order of 10⁴ cells per day. Furthermore the Vγ profile of recent γδ⁺ emigrants differs from that of the thymic HSA⁺Thy-1⁺ cells. This raises the possibility that only a selected subpopulation of the thymic γδ⁺HSA⁺Thy-l⁺ population is exported, and that some γδ cells may die in situ in the thymus. The function of the other γδ thymic subpopulations, which are turning over very slowly or not at all, (i.e. the HAS^?Thy-l^? and HAS^?Thy-l⁺ subpopulations) remains unclear.     

β-adrenergic stimulation of cellular K+ uptake in rat distal colon

We recently demonstrated that the ratio between colonic K⁺ absorptive and K⁺ secretive pathways was higher in infant than in adult rats. To test the hypothesis that hormones selectively affect these pathways during ontogeny we examined the effect of adrenergic agonists on cellular K⁺ uptake in distal colon from infant (10-day-old) and adult (50-day-old) rats. Here we describe that adrenaline (10^?5 M ) increased total and ouabain-insensitive ⁸⁶Rb uptake in both age groups, but it did not affect ouabain-sensitive ⁸⁶Rb uptake. This stimulation was more pronounced in adult than in infant rats. The effect of adrenaline was mediated via β-adrenergic receptors. Incubation in vitro with β-agonist, isoproterenol, stimulated SCH-28080-sensitive, i.e. H⁺,K⁺-ATPase-dependent, ⁸⁶Rb uptake in adult but not in infant rats. The threshold dose of β-agonist was at 10^?7 M , and the maximal activation was observed at 10^?5 M . In vivo inhibition of β-adrenergic system with propranolol caused a significant decrease in H⁺,K⁺-ATPase-dependent ⁸⁶Rb uptake in infant but not in adult colon. In conclusion, this study suggests that the higher colonic K⁺ absorption in infant rats may be as a result of a selective β-adrenergic up-regulation leading to stimulation of the apical H⁺,K⁺-ATPase.