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.

Age-related changes of thymalin content in human epidermis.

Immunomorphological analysis revealed the presence of thymalin in human epidermis and in fetal reticuloepithelium. These structures are developed from the common embryonic primordium ectoderm. In embryos and adult humans thymalin is present only in young epidermal cells, which undergo age-related involution. By the age of 70 years, the layer of thymalin-containing cells looks thinned and discontinuous. The content of thymalin, a thymic factor, decreases with age.

Altered subcellular localization of suppressin, a novel inhibitor of cell-cycle entry, is an independent prognostic factor in colorectal adenocarcinomas.

PURPOSE: Suppressin (SPN), a novel inhibitor of the entry into the cell cycle, has properties of a tumor suppressor gene; however, its role in the development and progression of a human malignancy is not studied. Therefore, we evaluated the status of spn and its prognostic value in human colorectal adenocarcinoma (CRC). EXPERIMENTAL DESIGN: Inhibition of cell proliferation by exogenous/extracellular SPN was assessed by [(3)H]thymidine incorporation. The genetic status of spn in two colon cancer cell lines (LS180 and WiDr) and in a human CRC was determined using direct cDNA sequencing techniques. Phenotypic expression of SPN was evaluated in 105 CRC archival tissues using immunohistochemical methods. Univariate Kaplan-Meier and multivariate Cox proportional hazards models were used to determine the prognostic significance of SPN expression. RESULTS: Exogenous SPN inhibited the proliferation of the LS180 cell line, which also has a mutation in one allele of the spn gene. The spn gene was also mutated in the primary CRC. Expression of SPN was primarily cytoplasmic in nonmucinous CRCs and nuclear in mucinous CRCs. However, the evaluation of 85 nonmucinous CRCs demonstrated that nuclear localization of SPN, nuclear accumulation of p53, and nodal status were independent prognostic indicators with hazard ratios of 2.34, 2.33, and 3.04, respectively. Nuclear localization of SPN plus nuclear accumulation of p53 formed a stronger prognostic indicator (hazard ratios = 5.45) than local nodal status. CONCLUSIONS: This is the first report of genetic alterations in the spn gene in a human malignancy and suggests that genetic alterations in spn and the resulting immunohistochemical phenotypes based on SPN subcellular localization in CRCs may be useful in determining prognosis of patients with subtypes of CRC.

Review of thymic hormones in cancer diagnosis and treatment.

The thymus is an endocrine organ. A unified, physiological concept of humoral regulations of the immune response has emerged in the last three decades. The thymus is the major site of production of immunocompetent T lymphocytes from their hematopoietic stem cells. This complex process required direct cell to cell, receptor based interactions, as well as in situ paracrine information via the numerous cytokines and thymic hormones produced by the cells of thymic microenvironment. Thymic hormones induce in situ T-cell marker differentiation, expression and functions. These polypeptide hormones have also been shown by means of immunocytochemistry to localize in the reticulo-epithelial (RE) cells of the thymic cellular microenvironment. Due to the great complexity of the intrathymic maturation sequence of T lymphocytes and the diverse immunophenotypically unique subpopulations of T lymphocytes, it is quite unlikely that a single thymic humoral factor could control all of the molecular steps and cell populations involved. It is much more likely that an extremely rich and diverse, but genetically determined, milieu is present within the thymus, and that thus the control of intrathymic T lymphocyte maturation and the functional maturation of T cells involves the orchestral interaction of various thymic-specific factors and other molecules during the differentiation process. Thymosin fraction 5 and its constituent peptides influence several properties of lymphocytes including cyclic nucleotide levels, migration inhibitory factor production, T-dependent antibody production, as well as the expression of various cell surface maturation/differentiation markers. Recently, derivatives of thymic hormones, mostly of thymosins, have been detected as products of neoplastically transformed cells and employed in the early diagnosis of neoplasms. In clinical trials, thymic hormones strengthen the effects of immunomodulators in immunodeficiencies, autoimmune diseases, and neoplastic malignancies. Combined chemo-immunotherapeutical anti-cancer treatment seems to be more efficacious than chemotherapy alone, and the significant hematopoietic toxicity associated with most chemotherapeutical clinical trials can be reduced significantly by the addition of immunotherapy.

Characterization of the stimulatory actions of thymic factor(s) on basal and gonadotropin-induced steroidogenesis in cultured rat granulosa cells.

Recently, there have been numerous reports that demonstrate the importance of the thymus gland in reproductive physiology. Previously, we have reported that thymic factors (TFs) which are present in thymic cell culture-conditioned medium (TCM) could stimulate basal progesterone and estradiol production from cultured rat granulosa cells. The current study attempts to characterize the stimulatory actions of TFs on both basal and FSH induced steroidogenesis. Thymic epithelial cells from immature female rats were isolated and used for production of TCM. Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TFs stimulated both basal and FSH-induced progesterone secretions 80 and 17 times, respectively, as compared to the control media. The effects of TFs on basal and FSH-induced 20 alpha-hydroxyprogesterone secretion were comparable to those on progesterone production (40x and 10x, respectively). In addition, TCM stimulated basal and FSH-induced estradiol secretion approximately 4 and 2.5 times, respectively, compared to control. Stimulation of aromatase enzyme activity followed a similar trend as estradiol secretion, and TCM stimulated basal and FSH-stimulated aromatase enzyme activity approximately 15 and 3 times, respectively compared to control. Thus, these results indicate that the observed increases in progesterone and estradiol secretions in TCM-treated rat granulosa cells are likely to be due to elevated activities of specific steroidogenic enzymes. Measurements of total cell protein and DNA synthesis indicate that enhanced steroidogenesis in TCM-treated cells is not due to increased cell growth and/or proliferation. Rather, the enhanced steroidogenesis is probably due to an increased steroid biosynthetic capability of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that appearance of thymulin in plasma follows lymphoid chimerism and precedes development of immunity in patients with lethal combined immunodeficiency transplanted with T cell-depleted haploidentical marrow.

Thymulin, a peptide secreted by human thymic epithelial cells, circulates in peripheral blood. Levels of plasma thymulin (FTS-Zn) activity were analyzed in 21 patients with lethal combined immunodeficiency disorders who were treated with transplantation of HLA-haplotype-mismatched parental bone marrow depleted of T cells by differential agglutination with soybean agglutinin and E-rosetting (SBA-E-BMT). Among these 21 infants, 15 were patients with severe combined immunodeficiency (SCID) and 6 had combined immunodeficiency (CID) with Omenn's syndrome or CID with T cell predominance (CIDTP). In contrast to normal infants who possess high levels of plasma thymulin activity, 20 of the 21 patients demonstrated undetectable or low plasma thymulin levels for their age at admission prior to transplantation. Following SBA-E-BMT, however, thymulin became detectable in the plasma of 17 of 18 evaluable patients and reached normal or near-normal levels between 21 and 125 days posttransplant. In patients in whom the timing of engraftment could be established by emergence of donor lymphocytes, thymulin appeared in the plasma at approximately the same time as lymphoid chimerism was detected, and in all patients who were engrafted and immunologically reconstituted, the increment in thymulin levels preceded development of immune functions. These studies support the concept that normal marrow-derived cells in the graft can provide a stimulus necessary for induction of thymic epithelial secretory function in patients with thymic dysplasia. Further, immunologic reconstitution in these patients was not seen following SBA-E-BMT unless and until recovery of thymus function had been observed.

Changes in thymocytes due to aging--a study of the effects of thymic hormone on adenine nucleotides and thymocyte DNA.

The number of lymphocytes (especially T-lymphocytes) in peripheral blood decreases with aging. The decline is most evident in the thymus. In this study, total AN in the thymocytes of aged mice clearly decreases from that of young mice. The quantity of thymic hormones secreted by thymic cells decreases with the organism's thymus involution with age. After demonstrating the decrease in total AN (primarily ATP) levels within the thymocytes of aged mice, we attempted to reattain the levels of younger thymocytes by the administration of various thymic hormones. Examination of DNA in the thymocytes of 4 and 58-week-old mice, using flowcytometry, showed no differences in content. However, it is significant that aged mouse thymocytes's energy level can be returned to those of young mice with the application of Th-5.

Biochemical characterization of thymic hormones in thymoma tissues.

Thymic hormones induce T-cell markers and functions. These polypeptide hormones have also been shown by means of immunocytochemistry to localize in thymic epithelial cells. Employing biochemical isolation procedures, we have studied the concentration of two thymic hormones, prothymosin alpha and thymosin beta 4, in the thymus of three thymoma patients. After a brief boiling step, thymic tissue obtained from each patient was individually homogenized and centrifuged. The supernatant was then fractionated by gel filtration on Sephadex G-100 and further purified by reversed-phase high performance liquid chromatography (HPLC). Purified components were characterized by amino acid analysis and HPLC tryptic peptide mapping. Our results revealed that the extract from benign thymoma had both prothymosin alpha and thymosin beta 4, similar to normal human thymus. However, the thymus from a patient with invasive malignant thymoma contained only thymosin beta 4, but no prothymosin alpha. In the extract from an undifferentiated carcinoma, neither prothymosin alpha nor thymosin beta 4 could be detected. These results disclose the possible correlation of thymic hormones and the type and differentiation stage of thymomas. The inability of malignant thymic tumors to produce normal amounts of thymic hormones may contribute to their etiology. It is suggested that information on the thymic hormone content might add a new parameter to pathological diagnosis in thymic tumors.

A new radioimmunoassay for the thymic peptide thymulin, and its application for measuring thymulin in blood samples.

A new, specific and sensitive radioimmunoassay, using a polyclonal antiserum raised in rabbits, is described for quantitating plasma thymulin. As little as 300 fg thymulin can be measured in one assay tube. The method has been used to measure thymulin in human blood (umbilical vessel blood, 2191 +/- 123 fg/ml; children and adults up to the age of 20 years, 1499 +/- 119 fg/ml; and adults between 21-65 years, 371 +/- 18 fg/ml). There is a highly significant decrease within these three groups (P less than 0.001 by one way analysis of variance). Also plasma thymulin levels were determined in rats (601 +/- 127 fg/ml) and in pooled plasma samples from mice (638 +/- 56 fg/ml). No thymulin was detected in plasma obtained from nude rats, nude mice and thymectomised mice. These results show that the radioimmunoassay described here is a useful quantitative tool for measuring plasma thymulin that will have applications in basic, applied and clinical research.

Human epithelial thymic tumours: heterogeneity in immunostaining of epithelial cell markers and thymic hormones.

Different hormones (thymulin, thymosin alpha 1, vasopressin), antigenic markers of cortical and subcapsular/medullary thymic areas and tumour associated antigens were studied on paraffin or frozen section and cultures of human epithelial thymic tumours ('thymomas'). Thymulin, thymosin alpha 1 and for the first time vasopressin are found in most tumours. The epithelial cells of five 'thymomas' had markers of both cortical (TE3) and subcapsular/medullary thymic regions (A2B5 and/or TE4 and/or anti-p19). Leu-7, a marker of subcapsular epithelial cells was positive only in two tumours. The histological classification into cortical and medullary tumours does not correspond to our immunofluorescence results. The presence of these markers does not support the theory of different embryologic origin of the cortical and subcapsular/medullary epithelial cells. Transferrin receptors were detected on only some epithelial cells of thymic 'carcinomas'. Adenocarcinoma related antigen and carcino embryonic antigen only stained a few epithelial cells of all the tumours. There is no expected correlation between the presence of epidermal growth factor receptors on cell membranes and the number of proliferative cells stained by the anti-Ki67 antibodies. Immunostainings were heterogeneous according to the epithelial thymic tumours, independent of histological classification and not yet useful for prognosis.

Influence of iron-deficiency anemia on selected thymus functions in mice: thymulin biological activity, T-cell subsets, and thymocyte proliferation.

To define the effects of iron deficiency on thymulin biological activity, T-cell subsets, and thymocyte proliferation, C57BL/6 female mice at weaning were fed an iron-deficient diet (10 mg Fe/kg diet), an iron-sufficient diet (50 mg Fe/kg diet), or restricted amounts of the iron-sufficient diet (the pair-fed group) for 40 d. Iron deficiency did not reduce the concentration of either serum or intracytoplasmic thymulin. Although T-cell subsets in the thymus were not altered, both the cortical and medullar regions were depleted of thymocytes. In the spleen iron deficiency (but not underfeeding) significantly reduced the percentage of L3T4+ cells, of Lyt-2+ cells, and thus of the overall T-cell population. However, it did not affect the ratio of L3T4+ to Lyt-2+ T cells. Thymocyte proliferation was significantly reduced at the concanavalin A (Con A) dose (10 mg/L) that produced maximal stimulation in control and pair-fed mice but not at low (7.5 mg/L) or high (15 mg/L) Con A concentrations. We conclude that the impairment in immune functions associated with iron deficiency is not due to an impairment in thymic endocrine function but rather to decreased immunocompetent lymphocytes.

[Increase of circulating levels of thymulin in hyperprolactinemia and acromegaly]. / Augmentation des taux circulants de thymuline au cours de l'hyperprolactinémie et de l'acromégalie.

The production of thymulin by the thymic epithelium is under complex control involving the endocrine system. Experimental models have suggested that prolactin (PRL) and growth hormone (GH) participate in this regulation but this has not been documented in humans. Using a bioassay we measured circulating thymulin levels in patients with hyperprolactinemia (n = 21), acromegaly (n = 15), or both (n = 6). Thymulin was elevated in these three groups of patients compared with normal subjects or with patients with pituitary disease but no excess in PRL or GH. Contrasting with observations in control groups, thymulin did not decrease as a function of age in patients. No correlation between thymulin and PRL or GH levels was observed while thymulin and insulin-like growth factor 1 levels were correlated. A new radioimmunoassay used in some patients for thymulin determination yielded similar results. Overall these data demonstrate that PRL and GH are involved in the hormonal control of thymulin production by the thymic epithelium in the human.

[The pathogenesis of congenital thymus hyperplasia in children with immune defects]. / Zur Pathogenese der angeborenen Thymushyperplasie (ATH) bei Kindern mit Immundefekten.

Complex clinical and morphological studies were conducted into conditions of the thymus as well as of the lymphatic and neuro-endocrine systems in stillbirths and children up to five years of age. Thymic hormones in blood and thymic tissue were determined, as well. CTH, in most of these cases, was found to reflect dysfunction of the hypothalamic-hypophyseal system which eventually resulted in development of polyglandular endocrinopathy and congenital immune deficiency, primarily in the T-system. CTH has proved quite often to be associated with congenital malformations.

Male BXSB mice develop a thymic hormonal dysfunction with presence of intraepithelial crystalline inclusions.

The thymus is morphologically abnormal in male BXSB mice with cortical involution densification of the epithelium and the presence of intraepithelial crystals. The thymic endocrine function in BXSB mice was appraised using a biological assay to measure the level of the zinc-dependent thymic hormone, thymulin, and an indirect immunofluorescence technique to evaluate the number of cells synthesizing the hormone within the thymus. Unlike the dramatically accelerated age-linked decline of thymulin production reported in females of other autoimmune strains (measured as early as 6 weeks of age), only male BXSB were affected, as compared to normal strains. The number of hormone-producing cells was significantly reduced in male BXSB thymuses, in parallel with this hormonal decrease. Thymulin inhibitory molecules were detected in male BXSB sera, as early as 8 weeks of age, as evaluated by their capacity to absorb in vitro and in vivo the biological activity of the hormone. These inhibitors are thymus dependent since they disappear after adult thymectomy. They are low MW molecules (less than 10 kDa), as previously found in normal aging mice, rather than autoantibodies, as evidenced in two autoimmune strains (B/W and db/db mice). These findings demonstrate that male BXSB mice develop thymic abnormalities very similar to those observed in other autoimmune strains. The presence of intrathymic crystals and of low MW inhibitors suggests the role of abnormal storage and the excretion of thymulin. This thymic dysfunction may play a role in the maintenance of B cell hyperactivity previously shown in BXSB males.

Establishment of the specific radioimmunoassay for serum thymic factor (STF).

Serum thymic factor is a humoral factor involved in the differentiation of T cells. In the present study, a radioimmunoassay system for STF was established using a specific antiserum and an iodinated synthetic STF as a tracer. Serum levels of STF-like immunoreactivities were around 30 pg/ml in human, but in contrast to previous reports serum STF levels did not show age-dependent decreases. STF-like immunoreactivities of the thymic gland were lower than those of the liver and kidney in rats. The livers of human and pig contained high STF-like immunoreactivities. The rat thymic gland extract showed three peaks of STF-like immunoreactivity by gel filtration of Sephadex G-25 which corresponded to the eluted position of authentic STF, a larger molecular size, and a smaller molecular size of STF, respectively, while the rat liver extract showed two peaks corresponding to the void volume fraction and to the position of authentic STF, respectively. When this void volume fraction was digested by trypsin, three peaks which corresponded to authentic STF, a larger molecular size, and a smaller molecular size of STF were observed. Present studies suggest that serum thymic factor is produced as a larger STF molecule in the liver and kidney.

The distribution and structure of FTS immunoreactive cells in the thymus of the mouse.

The intrathymic distributions of facteur thymique sérique (FTS)-containing cells and Ia-expressing cells were examined by a double immunofluorescence technique in C57BL/6 mice of various ages. In the thymic medulla of all the mice examined, there were both FTS-immunoreactive cells and Ia-immunoreactive cells. The former cells, epithelial in nature, extended elongate cytoplasmic processes, while the latter were connected with each other by their spiny processes. The FTS-immunoreactive epithelial cells were all Ia-negative. The FTS-immunoreactive cells and Ia-immunoreactive cells were located in close proximity to each other. The cortex of the adult mice contained no FTS-immunoreactive epithelial cells. The cortex of the newborn and old mice, however, contained scattered FTS-immunoreactive epithelial cells with processes. FTS-immunoreactive cells in the cortex of the old mice possessed Ia-immunoreactive partner cells, while those in the cortex of newborn mice did not. In the marginal zone of the medulla of old mice, there were several cavities bounded by a partly ciliated epithelium and containing lymphocytes. The epithelial cells lining the cavities showed intense immunofluorescence for FTS. In addition to the epithelial cells, a certain population of round cells with thin cytoplasm lacking in discernible processes immunofluoresced for FTS. Located both in the medulla and in the cortex, these round cells were postulated to be thymocytes, the target cells of FTS, binding FTS on their cell surface.

Thymulin (facteur thymique serique) and zinc contents of the thymus glands of malnourished children.

Protein-energy malnutrition (PEM) leads to an immune deficiency, which is now well documented. Some investigators have suggested that the associated zinc deficiency is important in thymic involution and changes in cellular immunity. To evaluate the respective roles of nutritional deficiency, infection, and zinc in the alteration of thymic function, we measured the amounts of thymulin (facteur thymic serique, or FTS) and of Zn in the thymus glands of 58 Senegalese children who died in various stages of malnutrition. In the severe forms (marasmus, kwashiorkor, and marasmic kwashiorkor) the thymus was tiny and contained very little thymulin. The Zn content of the thymus was high whatever the nutritional state of the subject and was related significantly only to the presence of infections. In Senegalese children thymic atrophy and depleted thymulin content are associated with severe PEM but not systemic infection or depleted thymic Zn content.