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.

Islet endocrine-cell behavior from birth onward in mice with the nonobese diabetic genetic background.

BACKGROUND: Glucagon-producing alpha cells play a crucial role during the perinatal period. Because of their peri-islet localization near the early dendritic and macrophage cell infiltration, we thought it pertinent to investigate alpha cells in greater depth in nonobese diabetic (NOD) mice, a well-recognized spontaneous model for human type I diabetes. MATERIALS AND METHODS: We determined alpha-cell distribution (glucagon immunohistochemistry and image analysis) and activity (real-time reverse transcriptase polymerase chain reaction [RT-PCR] and glucagon radioimmunoassay [RIA]), in relationship to glycemia in NOD and lymphocyte-deficient NODscid mice as compared to control mice (C57BL/6) from birth onward. RESULTS: NOD and NODscid mice, particularly at 1 day of age, had twice as many very small islets (<2,000 pixels) as C57BL/6 mice. During the postnatal period, the percentages of glucagon-positive areas in islets less than 2000 pixels were higher in NOD mice than C57BL/6; only a trend was found in NODscid. Pancreatic mRNA expression and glucagon content decreased in all strains at weaning. However, before weaning, pancreatic and blood glucagon levels were significantly lower in NOD and NODscid compared to C57BL/6 mice. Low basal nonfasting glycemia was observed in all strains before weaning with some strain differences: glycemia was significantly lower in NOD than C57BL/6, and higher in NODscid than NOD and C57BL/6. CONCLUSION: These data suggest that, before weaning, NOD and, to some extent NODscid pancreata contain more immature islets (as reflected by their small size and high percentages of glucagon-positive areas, concomitant with lower glucagon storage and basal secretion) than C57BL/6 pancreata.

Pancreatic hormone and glutamic acid decarboxylase expression in the mouse thymus: a real-time PCR study.

We devised a real-time RT-PCR method for the quantification of preproinsulin 1 and 2, proglucagon, prosomatostatin, and GAD 65 and 67 mRNAs in the thymus, using specific primers and internal probes. Corresponding standard cRNA synthesis and normalization to 18S ribosomal RNA allowed direct quantification. Then, during the first month of life, the expression of each substance of interest was measured in the thymus of NOD mice (a spontaneous model of type 1 diabetes), C57BL/6, BALB/c and lymphocyte-deficient mice (NODscid, NODrag, BALB/cscid and C57BL/6rag). In all mouse thymuses, preproinsulin 1 and GAD 65 were undetectable, preproinsulin 2 and proglucagon showed low expression, whereas that of GAD 67 and somatostatin were high. In 7-day-old mice, GAD 67 and prosomatostatin thymic expressions were lower in NOD than in C57BL/6, and at the same age, the scid mutation but not the rag mutation induced higher expression of all investigated genes compared to control mice. In conclusion, our data allowed the quantification of the expression of pancreatic factors in the mouse thymus. Investigations are underway to quantify, at the cellular level, i.e., in thymic dendritic/macrophage cells, the RNA expression of potential autoantigens, such as preproinsulin 2 and GAD 67.

Phase I clinical trial with IL-2-transfected xenogeneic cells administered in subcutaneous metastatic tumours: clinical and immunological findings.

Various studies have emphasized an immunodepression state observed at the tumour site. To reverse this defect and based upon animal studies, we initiated a phase I clinical trial of gene therapy in which various doses of xenogeneic monkey fibroblasts (Vero cells) genetically engineered to produce human IL-2 were administered intratumorally in 8 patients with metastatic solid tumours. No severe adverse effect was observed in the 8 patients analysed during this clinical trial even in the highest dose (5 yen 107 cells) group. This absence of toxicity seems to be associated with rapid elimination of Vero-IL-2 cells from the organism. Indeed, exogenous IL-2 mRNA could no longer be detected in the peripheral whole blood 48 hours after Vero-IL-2 cell administration. In addition, we did not find any expression of exogenous IL-2 mRNA in post-therapeutic lesions removed 29 days after the start of therapy. A major finding of this trial concerns the two histological responses of two treated subcutaneous nodules not associated with an apparent clinical response. The relationship between local treatment and tumour regression was supported by replacement of tumour cells by inflammatory cells in regressing lesions and marked induction of T and natural killer cell derived cytokines (IL-2, IL-4, IFNg ...) in post-therapeutic lesions analysed 28 days after the start of Vero-IL-2 administration. Gene therapy using xenogeneic cells as vehicle may therefore present certain advantages over other vectors, such as its complete absence of toxicity. Furthermore, the in vivo biological effect of immunostimulatory genes, i.e IL-2-, may be potentiated by the xenogeneic rejection reaction.

CD11c+ eosinophils in the murine thymus: developmental regulation and recruitment upon MHC class I-restricted thymocyte deletion.

Eosinophils are bone marrow-derived cells released into the circulation during hypersensitivity reactions and parasitic infections. Under normal conditions most eosinophils are tissue bound, where their physiologic role is unclear. During in situ analysis of the thymic microenvironment for CD11c+ dendritic cell subpopulations (APC critical in the process of thymic negative selection) a discrete population of CD11b/CD11c double-positive cells concentrated in the cortico-medullary region of young mice was detected. Thymic CD11c+ cells were isolated, and the CD11b+ subpopulation (CD44high, class IIlow, CD11cint) was identified as mature eosinophils based on: scatter characteristics, major basic protein mRNA expression, and eosinophilic granules. They are hypodense, release high levels of superoxide anion, and express CD25, CD69, and mRNA for IL-4 and IL-13, but not GM-CSF or IL-5, suggesting a distinct state of activation. Thymic eosinophils are preferentially recruited during the neonatal period; absolute numbers increased 10-fold between 7-14 days to reach parity with dendritic cells before diminishing. In a model of acute negative selection, eosinophil numbers were increased 2-fold 6 h after cognate peptide injection into MHC class I-restricted female H-Y TCR transgenic mice. In both peptide-treated female and negatively selecting male H-Y TCR mice, clusters of apoptotic bodies were associated with eosinophils throughout the thymus. Our data demonstrate a temporal and spatial association between eosinophil recruitment and class I-restricted selection in the thymus, suggesting an immunomodulatory role for eosinophils under nonpathological conditions.

Studies on the gonadotropin-releasing activity of thymulin: changes with age.

We assessed the ability of thymulin, a zinc-dependent nonapeptide produced by the thymic epithelial cells, to influence the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from dispersed anterior pituitary (AP) cells from young, adult, and senescent female rats. Perifusion of young and senescent AP cells with thymulin doses of 10(-6) to 10(-5) M gave a significant stimulatory response for LH but not FSH. Gonadotropin release was always lower in the senescent cells. AP cells from both age groups incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both gonadotropins, with a maximal stimulation at 10(-7) M. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with the secretagogue gonadotropin-releasing hormone (GnRH) revealed a synergistic effect on LH release and an additive effect on the release of FSH. The calcium chelator EGTA blocked the gonadotropin-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF, and forskolin significantly increased the thymulin-stimulated release of gonadotropins. The inositol phosphate enhancer LiCl potentiated the action of thymulin on gonadotropins. It is concluded that the gonadotropin-releasing activity documented here for thymulin is an age- and receptor-dependent effect mediated in part by calcium, cAMP, and inositol phosphates.

Thymic expression of the pancreatic endocrine hormones.

The thymus plays a central role in the selection of T lymphocytes that are tolerant to 'self' antigens and responsive to foreign pathogens. We and others have reported the expression of the pancreatic endocrine hormones, preproinsulin, proglucagon, prosomatostatin and propancreatic polypeptide in the human and mouse thymus. While mRNA expression is very low there is evidence for the presence of the translated product. In addition, we have investigated the cell types responsible for expression. In the thymus, hormone expression is enriched in the antigen-presenting cell population. Interestingly, while proglucagon, prosomatostatin and propancreatic polypeptide appear to be expressed in a macrophage population, preproinsulin expression was restricted to dendritic cells which are more potent antigen-presenting cells. The functional significance of the endogenous expression of insulin in the thymus has been indirectly investigated using transgenic models in which the transgene is introduced by the rat insulin promoter. The data suggest that thymic expression of the transgene is critical in the induction of T-cell tolerance to the transgene in the periphery. Taken together, the evidence suggests that the low-level pancreatic hormone expression in the thymus may be involved in central tolerance to proteins of restricted expression.

Growth hormone-releasing activity of thymulin on pituitary somatotropes is age dependent.

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by growth hormone (GH), among others. We assessed the ability of thymulin to influence the release of GH from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and GH release was measured by RIA. Perifusion of young and senescent AP cells with thymulin doses, ranging from 10(-8) to 10(-5) M, gave a logarithmic dose-response pattern of GH. Supernatants from TEC lines also showed GH secretagogue activity. The GH release was always lower in the senescent cells. AP cells incubated with 10(-8)-10(-3) M thymulin showed a time- and dose-dependent response, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with GHRH revealed a semiadditive release of GH in young and middle-aged AP cells and an additive effect in senescent cells. In middle-aged AP cells, the synthetic GH secretagogue GHRP-6 showed a synergistic effect with thymulin on GH release. The calcium chelator EGTA, but not the calcium ionophore A23187, blocked the GH-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin significantly increased the thymulin-stimulated release of GH while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl potentiated the action of thymulin on GH release. The data suggest that the GH-releasing activity of thymulin is receptor-mediated and involves calcium, cAMP and inositol phosphates. In addition, senescence appears to impair somatotrope responsiveness to thymulin.

Pancreatic hormone expression in the murine thymus: localization in dendritic cells and macrophages.

The expression of preproinsulin (ppIns), proglucagon, prosomatostatin, and propancreatic polypeptide was investigated in thymic extracts, thymic cells, and thymic cell lines from C57BL/6 mice by RT-PCR. The expression of pancreatic hormones was similar in thymic extracts taken from neonatal and 2-, 4-, and 8-week-old animals, but was decreased in 20-week-old animals. Pancreatic hormone expression was not observed in mouse liver, salivary gland, or spleen. Analysis of thymic cell populations revealed a 10- to 20-fold enrichment in expression of all hormones in low buoyant density cells. No expression was detected in high buoyant density cells (predominantly thymocytes) or in thymic epithelial cell lines, primary cultures of epithelial cells, or peripheral macrophages. In addition, immunoreactive insulin, measured by specific RIA, was detectable in the low buoyant density population, but not in high buoyant density cells. The enriched cell population was depleted of contaminating lymphocytes and sorted based on reactivity to the cell surface markers F4/80 (macrophage) or N418 (dendritic cells). Cells gated for N418 demonstrated expression for ppIns, but not the other pancreatic hormones. Conversely, expression for proglucagon, prosomatostatin, and propancreatic polypeptide, but not ppIns, was detected in F4/80-gated cells. Our data indicate that pancreatic endocrine hormones are differentially expressed by dendritic cells and macrophages in a normal mice.

Ontogeny of glutamic acid decarboxylase gene expression in the mouse pancreas.

Using two RT-PCR quantitative assays, we measured the pancreatic expression of the two isoforms of glutamic acid decarboxylase in the foetus, newborn and 14-, 21- and 35-day-old male and female NOD and C57BL/6 mice. In the C57BL/6 mouse, GAD 67 pancreatic expression is stable; in NOD mice, GAD 67 expression is similar to that found in control mice, except at 5 weeks of age, when pancreatic GAD 67 expression is about 2.5 times higher than in C57BL/6 mice. The pancreatic expression of GAD 65 is under the detection limit of the assay until 5 weeks of age. The overexpression of GAD 67 characterized in pancreas from 5-week-old NOD mice could be the result of beta cell hyperactivity, previously reported in this mouse strain.

Glutamic acid decarboxylase (GAD 67) gene expression in the pancreas and brain of the nonobese diabetic mouse.

A quantitative PCR was developed to measure GAD 67 mRNA levels in pancreas from NOD mice. Two nonautoimmune mouse strains were used as controls. An internal standard, rat GAD 67 mRNA, was prepared to quantify the reaction. In NOD mice GAD 67 expression in the pancreas was in the order of 2 x 10(6) molecules/microgram of total RNA; this expression was five times less in the two control mouse strains, whatever the age. The overexpression of GAD 67 characterized in the NOD mouse could be restricted to the pancreas since similar analysis performed in mouse brain did not reveal a large difference between the three mouse strains studied.

Gene expression of pancreatic glutamic acid decarboxylase in the nonobese diabetic mouse.

The 65 kDa isoform of glutamic acid decarboxylase (GAD 65) is an autoantigen implicated in type I diabetes. We have developed a quantitative PCR method to measure GAD 65 mRNA in the pancreas of nonobese diabetic (NOD) mice. Two other nonautoimmune mouse strains were used as controls. In all mice, pancreatic GAD 65 expression declined with age. In 5-week-old NOD mice, a clear difference was detected between males and females. This sexual dimorphism may explain the absence of tolerance to GAD 65 in the NOD female which could contribute to autoimmune destruction of beta cells in the pancreas and subsequent development of diabetes.

Interleukin-1 effect on glycemia in the non-obese diabetic mouse at the pre-diabetic stage.

Cytokines, particularly interleukin 1 (IL-1) and tumor necrosis factor, are known to induce hypoglycemia in normal rodents or different experimental models of type II diabetes. We investigated, at the pre-diabetic stage, the effect of short-term administration of murine recombinant interleukin-1 alpha (mrIL-1 alpha) on the levels of glucose, insulin and corticosterone in the non-obese diabetic (NOD) mouse, a spontaneous model of type I diabetes. Two-month-old, pre-diabetic NOD mice of both sexes were insensitive to mrIL-1 alpha (12.5 and 50 micrograms/kg) 2 h after administration, the time at which the maximal decrease (around 50%) was observed in the C57BL/6 mouse strain. Kinetic studies however showed that mrIL-1 alpha lowered glycemia in both sexes of NOD mice, but the effect was limited and delayed. In the NOD and C57BL/6 strains, mrIL-1 alpha had no influence on insulin levels in females, but significantly increased them in males (P < 0.0001). Castration of NOD males abrogated the stimulatory effect of mrIL-1 alpha on insulin secretion. Corticosterone secretion was stimulated by mrIL-1 alpha in both sexes of NOD and C57BL/6 mice, and this effect was faster and greater in NOD females than in C57BL/6 females. The incomplete hypoglycemic response to mrIL-1 alpha in females may be attributed to the anti-insulin effect of glucocorticoids, an effect which can be demonstrated when mrIL-1 alpha is administered to adrenalectomized animals or when mrIL-1 alpha is administered together with the glucocorticoid antagonist RU38486. In NOD males, in contrast, glucocorticoids did not play a major role in the limited hypoglycemic response to mrIL-1 alpha, since RU38486 and adrenalectomy were not able to unmask a hypoglycemic effect. Moreover, NOD mice of both sexes were less sensitive than C57BL/6 mice to the hypoglycemic effect of insulin (2.5 U/kg), which suggests some degree of insulin-resistance in NOD mice. With regard to the effect of IL-1 on NOD mouse glycemia, therefore, these results suggest that glucocorticoids and/or androgens, according to the animal's sex, may induce a state of insulin-resistance.

Prevention of autoimmune diabetes in nonobese diabetic female mice by treatment with recombinant glutamic acid decarboxylase (GAD 65).

The nonobese diabetic (NOD) mouse spontaneously develops insulin-dependent diabetes (IDDM or type I diabetes), resulting from T-lymphocyte-mediated destruction of pancreatic beta cells. This autoimmune phenomenon includes mononuclear cell infiltration of the islets of Langerhans (insulitis) and the presence of circulating autoantibodies. The specificity of the autoantibodies and of the autoreactive T cells was investigated and several autoantigens were proposed, in particular glutamic acid decarboxylase (GAD). This enzyme exists in two forms (GAD 65 and GAD 67) encoded by two independent genes. To explain the role of GAD in type I diabetes, we prepared recombinant rat GAD 65 as fusion protein, produced in an Escherichia coli expression system, and we treated NOD female mice from 4 to 7 weeks of age by repeated intraperitoneal injections of 5 micrograms fusion protein (3 injections per week); control groups received the fusion partner, maltose binding protein (MBP) or dissolving agent (NaCl 0.9%). We investigated two parameters, the degree of insulitis 5 weeks after the last injection and the overall incidence of the disease. Histological examination of the pancreata from GAD-treated mice revealed a significant reduction in the severity of insulitis compared with the two control groups. Furthermore, we observed that the time of onset and the frequency of diabetes in NOD females injected with GAD fusion protein differed significantly from the control groups receiving MBP or NaCl (P < 0.0001). These results show that a 3-week treatment of NOD female mice starting at 4 weeks of age protects them from diabetes, again emphasizing the crucial role of GAD as autoantigen in type I diabetes.

PCR analysis of interleukin-1 receptor gene in the nonobese diabetic mouse.

Insulin-dependent diabetes mellitus (IDDM) is characterized by a progressive autoimmune destruction of pancreatic beta cells. Many data suggest that interleukin 1 (IL-1) plays a fundamental role in the pathogenesis of the disease. In the nonobese diabetic (NOD) mouse, a spontaneous model of IDDM, it was put forward that the disease is linked to a susceptibility locus, called idd5, which contains the IL-1 receptor (IL-1R) gene. The polymerase chain reaction (PCR) was used to characterize the IL-1R gene in our NOD mouse colony and in two mouse strains taken as controls. Using primers to amplify the IL-1R gene between bp-106 and +378, a 580 bp fragment was obtained from C57BL/6 DNA but not from DBA/2 and NOD DNA. However, amplification of the IL-1R gene region between bp +1 and +378 in the three strains yielded amplicons 480 bp long. The specificity of the amplification was confirmed by restriction analysis. Our results suggest, depending on the strain, the presence of one or two introns: one (480 bp) in the 5'-untranslated region and the other (100 bp) in the region coding for amino acids between 69 and 126, and an exon-intron organization of the mouse IL-1R gene different than that described in the human genome.

Quantitative analysis of glutamate decarboxylase (GAD 65) gene expression in NOD mouse pancreas.

Glutamate decarboxylase (GAD), especially the GAD 65 isoform, is a major autoantigen in autoimmune diabetes. To determine the role of GAD 65 in the pathogenesis of the disease, we developed a quantitative PCR method allowing to establish the absolute number of GAD 65 mRNA molecules expressed in pancreas of male and female non-obese diabetic (NOD) mice at 5 weeks of age, in comparison of the 2 non autoimmune mouse strains. It appeared that pancreatic expression of GAD was similar in the 3 strains (around 30,000 molecules/micrograms total RNA) in males. However, in the NOD mouse, sexual dimorphism was observed with low GAD 65 expression in the female known to show higher incidence of the disease than the male. This finding could contribute to the absence of GAD 65 tolerance in the female and suggests an hormonal control of GAD 65 gene expression.

Interactions between zinc and thymulin.

Thymulin (formerly called "Facteur Thymique Sérique or FTS) is a metallopeptidic hormone selectively produced by thymic epithelial cells (TEC) and known to induce intra and extra-thymic T cell differentiation. It was initially isolated from porcine serum and shown to be present in calf thymus extract. Its amino-acid sequence was determined (

Evidence for antigen driven selection in two monoclonal auto-antibodies derived from nonobese diabetic mice.

The nonobese diabetic (NOD) mouse is a model of human type I diabetes. This diabetes is due to massive infiltration of the pancreatic beta cell of islets by autoreactive T cells (insulitis) followed by the destruction of insulin-producing cells. Circulating autoantibodies are also detected, notably against glutamic acid decarboxylase, peripherin and insulin. Two monoclonal autoantibodies directed against insulin and peripherin were obtained by fusing NOD spleen and myeloma cells. We report here the nucleotide sequence of the genes encoding for the V regions of these two antibodies. Somatic mutations were identified by comparing the light chain nucleotide sequence of one of these autoantibodies with its germline counterpart precursor established from NOD mice after PCR gene amplification. The other one displays N additions on both sides of the D region. These results strongly suggest that both autoantibodies have undergone diversification, either N additions or somatic mutations, and therefore present structural features of antibodies derived from animals immunized against exogenous antigens.

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.