Prenatal and childhood exposures are associated with thymulin concentrations in young adolescent children in rural Nepal.

The thymus undergoes a critical period of growth and development early in gestation and, by mid-gestation, immature thymocytes are subject to positive and negative selection. Exposure to undernutrition during these periods may permanently affect phenotype. We measured thymulin concentrations, as a proxy for thymic size and function, in children (n = 290; aged 9-13 years) born to participants in a cluster-randomized trial of maternal vitamin A or ß-carotene supplementation in rural Nepal (1994-1997). The geometric mean (95% confidence interval) thymulin concentration was 1.37 ng/ml (1.27, 1.47). A multivariate model of early-life exposures revealed a positive association with gestational age at delivery (ß = 0.02; P = 0.05) and higher concentrations among children born to ß-carotene-supplemented mothers (ß = 0.19; P < 0.05). At ∼9-12 years of age, thymulin was positively associated with all anthropometric measures, with height retained in our multivariate model (ß = 0.02; P < 0.001). There was significant seasonal variation: concentrations tended to be lower pre-monsoon (ß = -0.13; P = 0.15), during the monsoon (ß = -0.22; P = 0.04), and pre-harvest (ß = -0.34; P = 0.01), relative to the post-harvest season. All early-life associations, except supplementation, were mediated in part by nutritional status at follow-up. Our findings underscore the known sensitivity of the thymus to nutrition, including potentially lasting effects of early nutritional exposures. The relevance of these findings to later disease risk remains to be explored, particularly given the role of thymulin in the neuroendocrine regulation of inflammation.

DNA nanoparticle-mediated thymulin gene therapy prevents airway remodeling in experimental allergic asthma.

Thymulin has been shown to present anti-inflammatory and anti-fibrotic properties in experimental lung diseases. We hypothesized that a biologically active thymulin analog gene, methionine serum thymus factor, delivered by highly compacted DNA nanoparticles may prevent lung inflammation and remodeling in a mouse model of allergic asthma. The DNA nanoparticles are composed of a single molecule of plasmid DNA compacted with block copolymers of poly-L-lysine and polyethylene glycol (CK30PEG), which have been found safe in a human phase I/II clinical trial. Thymulin plasmids were detected in the lungs of ovalbumin-challenged asthmatic mice up to 27days after administration of DNA nanoparticles carrying thymulin plasmids. A single dose of DNA nanoparticles carrying thymulin plasmids prevented lung inflammation, collagen deposition and smooth muscle hypertrophy in the lungs of a murine model of ovalbumin-challenged allergic asthma, leading to improved lung mechanics. In the present model of chronic allergic asthma, highly compacted DNA nanoparticles using thymulin analog gene modulated the inflammatory and remodeling processes improving lung mechanics.

Cocaine reduces thymic endocrine function: another mechanism for accelerated HIV disease progression.

Thymulin is a thymic peptide important for the maturation and differentiation of immature thymocytes, which have been found to be depressed in patients with low-level CD4(+) cell recovery despite viral control. Substance use is associated with faster progression of HIV disease, which has been ascribed to poor adherence to antiretroviral medication. Recent findings of an association between cocaine use and decline in CD4(+) cell counts independent of antiretroviral adherence indicate alternative mechanisms for disease progression. We evaluated the relationship between thymulin activity, CD4(+) and CD8(+) cell counts and the CD4(+)/CD8(+) ratio, and the covariate effects of substance use cross-sectionally in 80 HIV(+) active substance users and over 12 months in 40 participants. Thymulin activity was analyzed in plasma using a modification of the sheep rosette bioassay. Thymulin activity was negatively associated with cocaine use (ß = -0.908,95% CI: -1.704, -0.112; p = 0.026). Compared to those who do not use cocaine, cocaine users were 37% less likely to have detectable thymulin activity (RR = 0.634, 95% CI: 0.406, 0.989 p = 0.045) and were 75 times more likely to show a decrease in thymulin activity (OR = 74.7, 95% CI: 1.59, 3519.74; p = 0.028) over time. CD4(+) cell count was positively associated with thymulin activity (ß = 0.127, 95% CI: 0.048,0.205; p = 0.002), detectable thymulin activity was 2.32 times more likely in those with a CD4 cell count ≥200 cells/µl (RR = 2.324, 95% CI: 1.196, 4.513, p = 0.013), and those with an increase in CD4 cell counts were more likely to show an increase in thymulin activity (OR = 1.02, 95% CI: 1.00, 1.034; p = 0.041) over time. Thymulin activity is predictive of HIV disease progression and is depressed in cocaine users independent of antiretroviral treatment (ART) and HIV viral load. Understanding the mechanisms for accelerated HIV disease progression provides opportunities to find alternative strategies to counteract immunosuppression.

Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs.

Thymulin is a well-characterized thymic hormone that exists as a nonapeptide coupled to equimolar amounts of Zn2+. Thymulin is known to have multiple biological roles, including T cell differentiation, immune regulation, and analgesic functions. It has been shown that thymulin is produced by the reticulo-epithelial cells of the thymus, and it circulates in the blood from the moment of birth, maintain its serum level until puberty diminishing thereafter in life. To study the localization of this hormone, we prepared polyclonal and monoclonal antibodies against the commercial peptide and utilized immunocytochemical techniques for visualization. The results indicate that thymulin stains the thymic reticular cells, the outer layers of Hassall's corpuscles and a large round cellular type, which is keratin-negative and does not show affinity for the common leukocyte antigen (CD-45). In mice, this thymulin-positive cell remains in the thymus throughout life and even appears in relatively increased numbers in old involuted thymi. It also appears in thymus-dependent areas of the spleen and lymph nodes, demonstrating that at least one of the thymus cells containing this peptide can be found in peripheral lymphoid tissue.

Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs

Thymulin is a well-characterized thymic hormone that exists as a nonapeptide coupled to equimolar amounts of Zn2+. Thymulin is known to have multiple biological roles, including T cell differentiation, immune regulation, and analgesic functions. It has been shown that thymulin is produced by the reticulo-epithelial cells of the thymus, and it circulates in the blood from the moment of birth, maintain its serum level until puberty diminishing thereafter in life. To study the localization of this hormone, we prepared polyclonal and monoclonal antibodies against the commercial peptide and utilized immunocytochemical techniques for visualization. The results indicate that thymulin stains the thymic reticular cells, the outer layers of Hassall's corpuscles and a large round cellular type, which is keratin-negative and does not show affinity for the common leukocyte antigen (CD-45). In mice, this thymulin-positive cell remains in the thymus throughout life and even appears in relatively increased numbers in old involuted thymi. It also appears in thymus-dependent areas of the spleen and lymph nodes, demonstrating that at least one of the thymus cells containing this peptide can be found in peripheral lymphoid tissue.

Morphometric assessment of the impact of serum thymulin immunoneutralization on pituitary cell populations in peripubertal mice.

Thymulin is a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymus-pituitary communication, particularly during early life. The aim of the present study was to evaluate the impact of serum thymulin suppression from birth to peripuberty on the morphology of different pituitary cell populations in prepubertal C57Bl/6 mice. Animals were submitted to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (age 32 days). From their 1st day of life, the animals were submitted to a protocol of intraperitoneal injections of rabbit anti-thymulin serum (alpha-FTS) and normal rabbit serum (NRS) in the controls. On their 33rd day of life, the animals were killed and their pituitaries were immediately dissected, fixed and immunostained using the EnVision system with primary antibodies against growth hormone, thyrotropin, corticotropin, gonadotropins and prolactin. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density = Sigma cell area/reference area (RA); cell density (CD) = number of cells/RA, and cell size (expressed in microm2). Serum thymulin was measured by a rosette bioassay while pituitary hormones were assayed by radioimmunoassay. Serum prolactin, luteinizing hormone, follicle-stimulating hormone, growth hormone and thyroid-stimulating hormone were significantly lower in the alpha-FTS animals of either sex compared with the corresponding NRS counterparts. The somatotrope, lactotrope and corticotrope populations showed a significant decrease in CD, while cell hypertrophy was observed in some of the pituitary cell populations of the alpha-FTS group compared to the NRS group. In the alpha-FTS group, there were sex differences in the morphometric changes observed. Our results suggest that serum thymulin plays a significant role during early life in the postnatal maturation of endocrine cells of the mouse anterior pituitary gland.

Gene therapy for long-term restoration of circulating thymulin in thymectomized mice and rats.

Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.

[Effect of zinc on thymulin level in mice].

OBJECTIVE: In order to investigate the roles of the different levels of zinc nutrition status on the thymulin of the mice. METHODS: The experiment included two periods: zinc depleted period and repletion period. In the depleted period, mice were divided into 3 dose groups: zinc deficient group (ZD, 5.2 mg/kg), zinc pair-fed group (PZ) and normal zinc group(NZ,25.6 mg/kg). In the repletion period, mice were divided into 3 dose groups: ZD group, DZ-NZ group and PZ-NZ group. RESULTS: The results showed that a signification decrease of thymulin level was observed as a result of zinc deficiency and was increased rapidly follow zinc supplement, while the plasma zinc was kept the same level. CONCLUSION: The thymulin is a sensitive biomarker for assess the zinc status.

[Reduced thymulin production during occupational exposure to lead]. / Ridotta produzione di timulina in corso di esposizione professionale a piombo.

Thymulin is a thymic hormone that being activated by binding a zinc ion promotes differentiation and several functions of T lymphocytes. It has been demonstrated only in experimental animals that metallic lead (Pb) is able to cause adverse effects on thymocyte number and function. The objective of this study is to evaluate the plasmatic level of active thymulin of 58 male workers being exposed for more than one year to low lead doses with respect to 59 male never exposed workers. All these were subjected to anamnesis collection, medical examination and determination of blood lead (PbB), plasmatic lead (PbPl), plasmatic thymulin, urinary lead (PbU) and urinary zinc (ZnU) levels. The mean plasma concentration of active thymulin was significantly lower in lead exposed than in non exposed workers. Active thymulin was also significantly and negatively correlated to PbB, PbPl and PbU level and resulted to be significantly and negatively influenced by PbB. Lead exposed workers had slightly higher zinc concentration in urine than non exposed workers, increasing ZnU levels by class of PbB. It is the first time that a toxic effect of lead on plasmatic active thymulin levels is demonstrated in humans, particularly in occupationally exposed workers. This study opens perspectives for further research that would both confirm the results and verify the mechanisms of action of lead on thymulin either direct or indirect and the possible role of zinc.

Effect of epithalamin on circadian relationship between the endocrine function of the thymus and melatonin-producing function of the pineal gland in elderly people.

The effect of epithalamin on circadian rhythms of thymic serum factor titers and melatonin concentrations in the blood of elderly people was studied. Course treatment with epithalamin modulated the rhythm of the thymic endocrine function. The increase in the titer of thymic serum factor at night coincided with the increase in blood melatonin concentration and shift of its acrophase to 3.00, which was characteristic of young people. In elderly people with preserved nocturnal peak of the thymic serum factor titer before therapy the nocturnal (3.00) concentration of melatonin was high and did not change after drug therapy. The correlation between melatonin concentration and titer of thymic serum factor increased after epithalamin treatment.

Effects of bioactive factors of the pineal gland on thymus function and cell composition of the bone marrow and spleen in mice of different age.

The effects of factors from the pineal gland on the titer of thymic serum factor in the supernatant of 3-h thymus stroma cultures, number of stromal precursor fibroblasts and CD4+ cells in the bone marrow, and CD8+ cells in the spleens of adult and old CBA mice were studied in vitro. Epithalamin, Epithalon, and melatonin appreciably increased the titer of thymic serum factor in the supernatant of thymus stroma cultures from mice of different age and increased the percentage of CD4+ cells in the bone marrow suspension from old animals in vitro. The percentage of CD8+ lymphocytes decreased after incubation of splenic cells from old mice with melatonin. The percentage of bone marrow fibroblast precursor cells from adult and old mice did not appreciably change after incubation with the preparations.

[Capillary zone electrophoresis for determination of thymulin in human plasma].

AIM: To establish a capillary zone electrophoresis (CZE) method for determination of thymulin in human blood plasma. Thymulin is a nonapeptide produced by the thymic epithelium. METHODS: The uncoated capillary used was 75 microns ID x 57 cm (50 cm effective length). The experimental conditions were as follows: the running buffer was phosphate-borate buffer containing 50 mmol.L-1 Na2B4O7-10 mmol.L-1 Na2HPO4 (pH 9.10); running voltage was 12 kV; operated temperature was 25 degrees C; running time was 13 minutes, detective wavelength was 200 nm. RESULTS: The linear range of the thymulin standard curve was 1-10 micrograms.mL-1, gamma = 0.9990, the average recovery was 70.97% with RSD of 3.32%, the RSD of reproducibility was 3.05%. CONCLUSION: The method of CZE for determination of thymulin in human plasma is simple and reliable.

Evidence for a PTH-independent humoral mechanism in post-transplant hypophosphatemia and phosphaturia.

BACKGROUND: Patients undergoing successful kidney transplantation often manifest overt hypophosphatemia associated with exaggerated phosphaturia during the early post-transplant period (2 weeks to 3 months). The mechanism for this phenomenon has not been fully elucidated. We tested the hypothesis that a circulating serum factor [non-parathyroid hormone (non-PTH)], which operates during chronic renal failure (CRF) to maintain phosphate (Pi) homeostasis, can increase fractional excretion of Pi (FE(PO4)) in normal functioning kidney grafts during the early post-transplant period, thereby causing phosphaturia and hypophosphatemia. METHODS: Five groups of patients were studied: control subjects (group 1, N = 16), "early" (2 weeks to 1 month) post-transplant patients (group 2, N = 22), "late" (9 to 12 months) post-transplant patients (group 3, N = 14), patients with advanced CRF (glomerular filtration rate = 30 to 40 mL/min; group 4, N = 8), and patients who suffered from end-stage renal failure and were treated by chronic hemodialysis (group 5, N = 14). Group 2 manifested significant hypophosphatemia and phosphaturia when compared with groups 1 and 3 (Pi = 0.9 +/- 0.003 mg/dL, FE(PO4) = 68+/- 5%, P < 0.0005 vs. groups 1 and 3). Sera were taken from each of the five subject groups and applied to the proximal tubular opossum kidney (OK) cells. The activity of Na/Pi-type 4 (that is, OK-specific type II transporter) was evaluated by measuring Na(+)-dependent (32)Pi flux. The expression of Na/Pi type II mRNA and the abundance of Na/Pi protein were determined by Northern and Western blot assays, respectively. RESULTS: When compared with sera from groups 1 and 3, 10% sera taken from groups 2, 4, and 5 (incubated overnight with OK cells) inhibited (32)Pi flux by 25 to 30% (P < 0.0003). Both Na/Pi mRNA and the expression of Na/Pi protein were markedly augmented under the same conditions (P < 0.05 groups 2, 4, and 5 vs. groups 1 and 3). Time-course analysis revealed that the up-regulation of Na/Pi protein by sera from groups 2, 4, and 5 was observed as early as four hours of incubation, whereas augmented abundance of Na/Pi mRNA was only seen after eight hours of incubation. The addition of PTH (1-34) to sera from groups 2, 4, and 5 abolished the augmented expression of NaPi protein. We labeled OK cell surface membrane proteins with N-hydroxysuccinimide bound to biotin (NHS-SS-biotin). Biotinylated transporters incubated with the different sera were precipitated by strepavidin and identified by Western blot analysis. Cells incubated in sera from group 2 showed increased membrane bound transporter when compared with control sera, whereas the intracellular pool of the transporter was comparable between the two groups. CONCLUSION: A non-PTH circulating serum factor (possibly phosphatonin) that increases FE(PO4) during CRF is also responsible for phosphaturia and hypophosphatemia in the early period following successful kidney transplantation. The putative factor inactivates Na/Pi activity along with inhibition of the transporter trafficking from the cell membrane into the cytosol.

Immunological evaluation of patients with beta-thalassemia major.

Abnormalities in the immune system and zinc homeostasis in patients with beta-thalassemia major (TM) have been reported. Since zinc ion is essential for the efficiency of the immune system and is required to induce biological activity to thymulin (Zn-FTS), a biochemically defined thymic hormone, we investigated the plasma levels of zinc and both active thymulin (Zn-FTS) and total zinc saturable thymulin (Zn-FTS+FTS) in 18 patients with TM aged between 2 and 31 years and 22 normal controls of the same age. Inhibitory molecules anti-thymulin and the distribution of lymphocyte subsets were also analyzed. Patients with TM presented significantly lowered plasma zinc and thymulin levels when compared to normal subjects. The significant enhancement of the active form of the hormone after zinc addition in vitro suggests that low thymulin values found in TM are due not to a thymic failure in synthesizing and secreting the thymic hormone, but a defect in zinc saturation of the hormone. An impairment of cell subset distribution was also demonstrated. This study shows that zinc and thymulin deficiency contribute to the complex mechanisms underlying immune dysfunction in TM.

[Studies on the determination of serum thymic factor by capillary zone electrophoresis].

The determination of serum thymic factor (FTS) by capillary zone electrophoresis was described. The optimum ionic concentration and the pH value of buffer system was examined through orthogonal analysis. The optimum conditions found to be 0.05 mol/L sodium tetraborate, 0.01 mol/L phosphate buffer at pH 8.70, working voltage at 12 kV and wavelength at 200 nm. The recoveries for FTS standards added to the bovine serum albumin and serum samples were measured. The values of the relative standard deviation(RSD), recovery and detection limit were 7.62%, 80.28% and 40 ng/ml respectively. The development of this method provide another way for studying the relations of zinc and thymic factor and investigating the effect of zinc on immune function.

Reconstitución inmunológica en la infección pediátrica por VIH-1 / Immunological reconstitution in HIV-infected children

El objetivo era poner a punto una técnica de cuantificación en tiempo real del número de TRECs. Cuando se produce el reordenamiento de los genes que dan lugar al receptor del linfocito T (TCR), se produce la deleción de unos fragmentos denominados círculos de reordenamiento del TCR (TRECs) Cque son excelentes marcadores de las células T formadas recientemente en el timo. Mediante la cuantificación, por PCR en tiempo real, de estos marcadores se puede estudiar la producción de nuevas células T y, por tanto, la funcionalidad del timo. Dado que el timo conserva toda su funcionalidad en el niño, se realizó un estudio en 8 niños infectados verticalmente por el VIII-1 con el objetivo de estudiar el efecto de la infección por este virus en la producción de células T nuevas y analizar si la disminución de la carga viral por efecto de los tratamientos antirretrovirales conducía a una recuperación en la función del timo que permitiese la reconstitución inmunológica. Los resultados muestran que los niños infectados por el VIH-1 presentan una menor producción de células T nuevas que los niños no infectados, y que la disminución drástica de la carga viral por efecto del tratamiento conduce a una recuperación en la producción de estas células. Además, se ha comprobado que el aumento en el porcentaje de linfocitos CD4, que se produce tras el tratamiento, en los niños no se debe a una redistribución o expansión periférica, sino que responde a una mayor producción de células T nuevas por parte del timo. Estos datos sugieren claramente que la disminución de la carga viral en niños que conservan la funcionalidad del timo conduce a la reconstitución de la población de linfocitos T CD4. El trabajo en este laboratorio está subvencionado por ayudas del Fondos de Investigación Sanitaria (FIS-00/0207), Bristol-Myers Company, Fundación para la Investigación y Prevención del SIDA (FIPSE 3008/99), El Programa Nacional de Salud (SAF 99-0022) y La Comunidad de Madrid. R. Correa dispone de una BEFI del FIS 99/9176 (AU)

Different age-related effects of thymectomy in myasthenia gravis: role of thymoma, zinc, thymulin, IL-2 and IL-6.

Different age-related immune pathogenetic mechanisms in myasthenia gravis (MG) have been suggested because of restoration after thymectomy (Tx) of altered zinc, thymulin (TH) and T-cell subsets exclusively in early-onset patients (younger <50 years), not in late-onset patients (older >50 years). In this context interleukin-2 (IL-2), interleukin-6 (IL-6) and thymoma are crucial because both involved in MG pathogenesis and correlated with acetylcholine receptors (AchRs) Ab production. Moreover, IL-2 and IL-6 are zinc-dependent, are altered in aging and related with zinc and TH age-dependent declines. Moreover, zinc is relevant for immune efficiency. In order to confirm these different age-related pathogenetic mechanisms further, the role of thymoma, zinc, TH, IL-2 and IL-6 is studied in MG patients with generalized MG with and without thymoma before and 1 month and 1 year after Tx. The high IL-2, IL-6, zinc, and AChR Ab levels observed before Tx are significantly correlated each other in younger MG patients (<50 years) independently by thymoma and in older MG patients (>50 years) with thymoma. No correlations exist in older MG patients without thymoma. Thymulin is not correlated with other parameters considered to be both in younger and older MG patients independently by the thymoma. Thymectomy restores zinc; immune parameters and AChR Ab are exclusively in the younger group, not in the older one. These findings suggest that IL-2 and IL-6, via zinc, rather than TH, may be involved in different age-related pathogenetic mechanisms mainly in early-onset MG. By contrast, thymoma may be involved in MG etiology in late-onset representing, as such, a useful discriminant tool for MG etiology between early and late-onset MG patients. Because autoimmune phenomena may rise in aging, a parallelism with altered immune functions during aging is discussed.

Primary thymic endocrine failure in HIV-1-infected children.

Thymulin, an essential hormone for the T lymphocyte differentiation process and function, was evaluated to asses thymic endocrine function in a cohort of 17 HIV-1-infected children aged between 2 months and 14 years, 18 seroreverted subjects and 47 normal controls. The rosette inhibition assay by Dardenne and Bach (1975) is the only method available to evaluate the biologically active form of this hormone (thymulin or Zn-facteur thymique sérique, Zn-FTS), as immunoassays cannot discriminate between thymulin and the inactive form of the hormone not containing Zn (FTS). HIV-1 patients presented undetectable or significantly lowered plasma levels of thymulin. Plasma zinc levels were significantly reduced in patients although inactive, zinc-unbound thymulin molecules were not demonstrated. The investigation of inhibitory anti-thymulin molecules performed in all patients was negative. Thymulin titers did not correlate with CD4+ lymphocyte count at the different disease stages. This study suggests that a primary thymic endocrine deficiency is present in HIV children. The critical importance of these results in assessing disease progression and a potential therapeutic approach are discussed.

Neuropeptides exert direct effects on rat thymic epithelial cells in culture.

To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, 1 mM acetylcholine, and 1 microM isoproterenol (beta-adrenergic agonist) had no effect. Proliferation (incorporation of tritiated thymidine) was increased by CGRP (P = 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P = 0.002), 5-HT (P = 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effects of neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells.

Immuno-electron microscopy of the thymic epithelial microenvironment.

Normal T cell development depends upon interactions between progenitor cells and the thymic microenvironment. Monoclonal antibodies (Mabs) have been used to define subtypes of thymic epithelium by light microscopy (clusters of thymic epithelial staining [CTES]). We have now used a range of these Mabs together with gold-coupled reagents in immuno-electron microscopy to study the fine cellular distribution of the molecules to which the antibodies bind. Anti-cytokeratin antibodies were used to identify all thymic epithelial cells, while the distribution of MHC class II molecules was revealed with Mabs to shared nonpolymorphic determinants. MR6, a CTES III Mab, shows strong surface labelling of cortical epithelial cells and thymic nurse cells and very weak surface staining of thymocytes, medullary macrophages, and interdigitating cells. Mab 8.18 (CTES V) also labels a cell surface molecule; this is present on Hassall's corpuscles and associated medullary epithelial cells. The molecules detected by Mabs MR6 and 8.18 are therefore located in a position where they are available to interact with external cellular and soluble signals within the thymus. In contrast, Mabs MR10 and MR19 (CTES II) recognise intracellular molecules within subcapsular, perivascular, and medullary epithelium. A similar distribution was seen with Mab 4beta, directed against the thymic hormone thymulin, although, in addition to the expected intracellular epithelial staining, large lymphoblasts in the subcapsular zone showed surface positivity, indicating the presence of thymulin bound to surface receptors on these early lymphoid cells. As expected, MHC class II molecules were expressed on some medullary and essentially all cortical epithelial cells. However, although most subcapsular epithelium was class II-negative, some cells did express these MHC molecules on their apical surface and on the surface of their cytoplasmic extensions into the cortex. Interestingly, some cortical epithelial cells surrounding capillaries were positive for both MR6 (CTES III) and for MR10, MR19, and 4beta (CTES II). Double-labelling experiments, using MR6 and MR19 simultaneously, revealed a double-positive perivascular epithelial cell population in the thymic cortex. The possibility that these cells represent a thymic epithelial progenitor population is discussed.