[Analysis of intestinal microbial diversity in Leopoldamys edwardsi based on illumina sequencing technique].

To explore the composition and diversity of the intestinal microflora of Leopoldamys edwardsi in Hainan Island. In November 2019, DNA was extracted from fecal samples of 25 adult Leopoldamys edwardsi (14 males and 11 females) in Hainan Island at the Joint Laboratory of tropical infectious diseases of Hainan Medical College and Hong Kong University. Based on the IonS5TMXL sequencing platform, single-end sequencing (Single-End) was used to construct a small fragment library for single-end sequencing. Based on Reads shear filtration and OTUs clustering. The species annotation and abundance analysis of OTUs were carried out by using mothur method and SSUrRNA database, and further conducted α diversity and ß diversity analysis. A total of 1481842 high quality sequences, belonging to 14 Phyla, 85 families and 186 Genera, were obtained from 25 intestinal excrement samples of Leopoldamys edwardsi. At the level of phyla classification, the main core biota of the Leopoldamys edwardsi contained Firmicutes (46.04%),Bacteroidetes (25.34%), Proteobacteria (17.09%), Tenericutes (7.38%) and Actinobacteria (1.67%), these five phyla account for 97.52% of all phyla. The ratio of Helicobacter which occupied the largest proportion at the genus level was 12.44%, followed by Lactobacillus (11.39%), Clostridium (6.19%),Mycoplasma (4.23%) and Flavonifractor (3.52%). High throughput sequencing analysis showed that the intestinal flora of Leopoldamys edwardsi in Hainan Island was complex and diverse, which had the significance of further research.

Thymosin enhances the production of IL-1 alpha by human peripheral blood monocytes.

Incubation of human peripheral blood monocytes (PBM) with a partially purified thymic preparation, thymosin fraction (TF5), results in a dose dependent production of an interleukin-1 (IL-1)-like factor. The biological activity of this factor can be blocked by anti-IL-1 alpha, but not by anti-IL-1 beta which neutralizes bacterial induced IL-1 activity. Studies with further purified TF5 fractions show that this activity is not due to the well-characterized peptide, thymosin alpha 1 (T alpha 1), but rather a new thymosin peptide(s) isolated from a more basic fraction. Intraperitoneal injection of TF5 also induces the expression of a membrane-bound IL-1 (mIL-1) on mouse peritoneal cells. This study provides the first evidence that TF5 can influence macrophage activity directly by enhancing IL-1 production. This observation may help explain the mechanism by which TF5 modulates immune responses. These results also point to a more selective role for thymic hormones, growth factors and cytokines which may trigger macrophages to secrete different forms of IL-1, which can then regulate either immune and/or inflammatory processes.

Studies on the effect of methotrexate on macrophage function.

Time course studies in rat adjuvant arthritis have shown that resident peritoneal macrophages synthesize large amounts of interleukin-1 (IL-1) on Day 10-11 postadjuvant immunization. Expression of Ia antigen on these macrophages increased rapidly between Day 15 to Day 20 postadjuvant treatment and remained at these high levels on Day 25. If these adjuvant immunized rats were treated with low doses of methotrexate, resident peritoneal macrophages appeared to synthesize lower amounts of IL-1 and their Ia expression was decreased. Methotrexate, in vitro, has no effect on lipopolysaccharide induced IL-1 synthesis by resident peritoneal macrophages.

Effect of estradiol on interleukin 1 synthesis by macrophages.

The effect of estradiol on interleukin 1 (IL-1) synthesis/secretion by rat peritoneal macrophages was investigated. Peritoneal adherent cells (PAC) from adult female rats secreted greater amounts of IL-1 spontaneously than those from age-matched male rats or prepubescent female rats. Ovariectomy led to reduced synthesis of IL-1 by PAC but estradiol replacement therapy of such rats effectively increased IL-1 synthesis. IL-1 secretion was also stimulated when PAC from male rats was incubated with estradiol. A combination of estradiol and LPS in vitro enhanced secretion of IL-1 by PAC even more than estradiol alone. These data provide new evidence suggesting that estradiol may play an important role in regulating synthesis of IL-1 by macrophages.

Acquisition of paralytic activity by inducer cells.

Clones of inducer cells activated by antigen and class II major histocompatibility complex (MHC) gene products synthesize large amounts of several distinct mRNA species not detected in other activated cell types, including antigen-activated suppressor or killer cell clones. Although inducer cells continued to synthesize and secrete peptides at about the same rate for 5 days after activation by adherent cells and antigen, they expressed a new set of mRNAs approximately 3 days after activation. We therefore tested the functional activity of purified Ly1 cells at different days after activation by adherent cells and antigen. We wished to find out (a) whether there was a qualitative or quantitative change in the level of inducer activity, and if so, (b) whether this functional change was an intrinsic property of Ly1 cells or, rather, was dependent on signals from the cellular environment, in particular from adherent antigen-presenting cells. We incubated purified Ly1 cells and splenic adherent cells for 1-7 days in vitro, and tested inducer activity in cultures containing highly purified B cells and sheep erythrocytes (SRBC). Anti-SRBC plaqueforming cells were counted 5 days later. We found that inducer cells that have been incubated with adherent cells in culture for more than 72 h (a) did not induce B cells to produce antibody, and (b) prevented virgin but not immune B cells from receiving T-helper signals. We term this latter phenomenon "paralysis." Acquisition of the ability to paralyze virgin B cells required an I-E gene-regulated interaction between inducer cells and in vitro-activated adherent cells. This interaction did not require antigen and was associated with transition from Ly1:Qa1- to Ly1:Qa1+ cell-surface phenotype. Taken together these findings indicate that (a) interactions between inducer cells and adherent antigen-presenting cells result first in classical inducer ("helper") activity and later in expression of paralytic activity and (b) sequential expression of these inducer activities depends on two distinct signals, supplied by resting and activated adherent cells, respectively. The signal supplied by autologous activated adherent cells is regulated by I-E gene products and is independent of corecognition of foreign proteins. The physiologic significance of this paralytic inducer activity in the prevention of potentially harmful immune reactions to foreign microbial agents is discussed.

Role of Ly-1:Qa1- and Ly-1:Qa1+ inducer T cells in activation of Ly-23 effectors of suppression of antibody production in mice.

Ly-2+ effectors of T cell-mediated suppression require inducing signals from antigen and a helper cell bearing the Ly-1+:Qa1+ surface phenotype. In this report, we have further examined the helper cell requirements for suppressor cell induction of antibody production in mice. By using the T cell subset education procedure in vitro, we have activated T cells to sheep red blood cells (SRBC) antigens and then purified Ly-2 cells before testing for suppressor activity in assay cultures of defined T and B cell subsets. We have confirmed our previous observations that Ly-1+:Qa1+ cells are required for activation of T suppressors, but have found that under the appropriate conditions, there is not a strict requirement for the Ly-123 subset of T cells. Furthermore, if Ly-23 cells are stimulated in the presence of Ly-1+:Qa1- T cells, effective suppressors can be obtained only if a source of Ly-1:Qa1+ inducers is added to the assay culture. If Ly-23 cells are activated by antigen in the absence of Ly-1 cells, subsequent exposure to the Ly-1+:Qa1+ subset under the conditions tested here is not sufficient to activate suppressors. These results show that effectors of suppression, like B cells and cytotoxic T lymphocytes, may respond to two helper cells.

Definition of two pathways for generation of suppressor T-cell activity.

Antigen-stimulated Ly1 cells induce T cells from nonimmune donors to develop potent feedback suppressive activity. Suppression is mediated by Ly23 suppressor T (Ts) cells, which are generated from either Ly23 or Ly123 precursors. Ts activity generated from Ly23 precursors requires a strong inducer signal and is rapidly expressed but short lived. In contrast, Ts activity from Ly123 precursors is relatively long lived and is efficiently generated by relatively low levels of inducer signals. Induction of both Ly123 and Ly23 precursors to become Ts cells requires that both cells share genes linked to the Ig-H locus.

Effects of 6-hydroxydopamine upon primary and secondary thymus dependent immune responses.

Adult male mice were treated with various doses of 6-hydroxydopamine in order to assess the effects of this drug upon thymic dependent immunity. A consistent decrease in primary antibody titers to sheep erythrocytes was observed following treatment with this drug. Serum levels of thymosin alpha 1 were increased by day three after 6-OHDA with a return to normal by day five. Thymocyte terminal deoxynucleotidyl transferase changes were biphasic with an initial decrease after 6-OHDA followed by an increase. Changes in mitogen responsiveness were observed but were not consistently reproducible. Involvement of both catecholamines and corticosteroids in bringing about these observed changes was discussed.

Modulation of terminal deoxynucleotidyl transferase activity by thymosin.

Thymosin fraction 5, a family of acidic polypeptides isolated from bovine thymus, contains several hormonal-like factors which have been shown to influence the maturation, differentiation and functions of T-cells. Some of these peptides have been chemically defined. Two of them, thymosin alpha 1 (M.W. 3108) and thymosin beta 4 (M.W. 4982) have been sequenced. In murine systems, terminal deoxynucleotidyl transferase (TdT) has been shown to be T-cell specific and to be present primarily in the cortisone sensitive immature T-cell populations. The daily injection of thymosin fraction 5 and two of its components, thymosin beta 3 and beta 4, significantly increases TdT activity in immune suppressed mice as compared to control groups. This study indicates that thymosin can act on prothymocytes and influence the early stages of T-cell differentiation. In an in vivo system, thymosin fraction 5 and the purified peptide, thymosin alpha 1, have high activities in decreasing TdT in normal murine thymocytes after a 22-h incubation. This effect suggests that thymosin can also act on thymocytes and regulate the later biochemical processes during T-cell differentiation.

Complete amino acid sequence of bovine thymosin beta 4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations.

The amino acid sequence of thymosin beta 4, a polypeptide isolated from calf thymus, was determined. Thymosin beta 4 is composed of 43 amino acid residues and has a molecular weight of 4982 and an isoelectric point of 5.1. The NH2 terminus of the peptide is blocked by an acetyl group. This molecule induces expression of terminal deoxynucleotidyl transferase (DNA nucleotidylexotransferase, EC 2.7.7.31) in transferase-negative murine thymocytes in vivo and in citro. Thus, it appears that thymosin beta 4 acts on lymphoid stem cells and may control the early stages of the maturation process of thymus-dependent lymphocytes. This peptide is one of several present in thymosin fraction 5 that participates in the regulation, differentiation, and function of thymus-dependent thymocytes.

Current status of thymosin research: evidence for the existence of a family of thymic factors that control T-cell maturation.

Thymosin fraction 5 contains several distinct hormonal-like factors which are effective in partially or fully inducing and maintaining immune function. Several of the peptide components of fraction 5 have been purified, sequenced and studied in assay systems designed to measure T-cell differentiation and function. These studied indicate that a number of the purified peptides act on different subpopulations of T-cells (see Figure 1). Thymosin beta 3 and beta 4 peptides act on terminal deoxynucleotidyl transferase (TdT) negative precursor T-cells to induce TdT positive cells. Thymosin alpha 1 induces the formation of functional helper cells and conversion of Lyt- cells to Lyt 1+, 2+, 3+ cells. Thymosin alpha 7 induces the formation of functional suppressor T-cells and also converts Lyt- cells to Lyt 1+, 2+, 3+ cells. These studies have provided further evidence that the thymus secretes a family of distinct peptides which act at various sites of the maturation sequence of T-cells to induce and maintain immune function. Phase I and Phase II clinical studied with thymosin in the treatment of primary immunodeficiency diseases, autoimmune diseases, and cancer point to a major role of the endocrine thymus in the maintenance of immune balance and in the treatment of diseases characterized by thymic malfunction. It is becoming increasingly clear that immunological maturation is a process involving a complex number of steps and that a single factor initiating a single cellular event might not be reflected in any meaningful immune reconstitution unless it is the only peptide lacking. Given the complexity of the maturation sequence of T-cells and the increasing numbers of T-cell subpopulations that are being identified, it would be surprising if a single thymic factor could control all of the steps and populations involved. Rather, it would appear that the control of T-cell maturation and function involves a complex number of thymic-specific factors and other molecules that rigidly control the intermediary steps in the differentiation process.

Overview of thymosin activity.

Our current research program centers around the biologic and chemical characterization of the family of polypeptides present in thymosin fraction 5. A system of nomenclature has been developed and the peptides are being systematically isolated and chemically characterized. Thymosin fraction 5 and its component parts influence a variety of lymphocyte properties including cyclic nucleotide levels, migration inhibitory factor production, T-dependent antibody production, and expression of certain surface markers. Thymosin is being used in clinical trials to investigate its effects on immunodeficiency diseases, malignant diseases, and autoimmune diseases.