Roquin--a multifunctional regulator of immune homeostasis.

Roquin-1 (Rc3h1) is an E3 ubiquitin ligase originally discovered in a mutational screen for genetic factors contributory to systemic lupus erythematosus-like symptoms in mice. A single base-pair mutation in the Rc3h1 gene resulted in the manifestation of autoantibody production and sustained immunological inflammation characterized by excessive T follicular helper cell activation and formation of germinal centers. Subsequent studies have uncovered a multifactorial process by which Roquin-1 contributes to the maintenance of immune homeostasis. Through its interactions with partner proteins, Roquin-1 targets mRNAs for decay with inducible costimulator being a primary target. In this review, we discuss newly discovered functions of Roquin-1 in the immune system and inflammation, and in disease manifestation, and discuss avenues of further research. A model is presented for the role of Roquin in health and disease.

Hearts from DCD donors display acceptable biventricular function after heart transplantation in pigs.

Cardiac transplantation is in decline, in contrast to other solid organs where the number of solid organ transplants from donors after circulatory death (DCD) is increasing. Hearts from DCD donors are not currently utilized due to concerns that they may suffer irreversible cardiac injury with resultant poor graft function. Using a large animal model, we tested the hypothesis that hearts from DCD donors would be suitable for transplantation. Donor pigs were subjected to hypoxic cardiac arrest (DCD) followed by 15 min of warm ischemia and resuscitation on cardiopulmonary bypass, or brainstem death (BSD) via intracerebral balloon inflation. Cardiac function was assessed through load-independent measures and magnetic resonance imaging and spectroscopy. After resuscitation, DCD hearts had near normal contractility, although stroke volume was reduced, comparable to BSD hearts. DCD hearts had a significant decline in phosphocreatine and increase in inorganic phosphate during the hypoxic period, with a return to baseline levels after reperfusion. After transplantation, cardiac function was comparable between BSD and DCD groups. Therefore, in a large animal model, the DCD heart maintains viability and recovers function similar to that of the BSD heart and may be suitable for clinical transplantation. Further study is warranted on optimal reperfusion strategies.

Ontogeny of the Thy-1-, Lyt-2+ murine intestinal intraepithelial lymphocyte. Characterization of a unique population of thymus-independent cytotoxic effector cells in the intestinal mucosa.

Murine intestinal intraepithelial lymphocytes (IEL) from unprimed thymus-bearing and athymic nude mice were characterized according to the expression of murine lymphocyte antigenic markers and cytotoxic activity. The majority of IEL from thymus-bearing mice were Lyt-2+, L3T4- lymphocytes, over half of which did not express Thy-1 surface antigens. Nude IEL and spleen cells were void of Thy-1+ cells; however, Lyt-2 antigens were expressed on a significant proportion of IEL, but not splenic lymphocytes. Overall, 40-70% of IEL from either thymus-bearing or athymic mice expressed the cytotoxic activation antigen, CT-1, and the J11d lymphocyte marker, both of which were associated with a population of Thy-1-, Lyt-2+ cytotoxic IEL. These data are taken to mean that the intestinal epithelium is a site uniquely enriched for activated cytotoxic cells, a significant proportion of which originate as non-thymus-derived lymphocytes with acquired lytic activity.

Peripheral engraftment of fetal intestine into athymic mice sponsors T cell development: direct evidence for thymopoietic function of murine small intestine.

Adult athymic, lethally irradiated, F1-->parent bone marrow-reconstituted (AT x BM) mice were engrafted bilaterally with day 16-18 fetal intestine or fetal thymus into the kidney capsule and were studied for evidence of peripheral T cell repopulation of 1-12 wk postengraftment. Throughout that time period, both types of grafts were macroscopically and histologically characteristic of differentiated thymus or intestine tissues, respectively. Beginning at week 2 postengraftment, clusters of lymphocytes were present within intestine grafts, particularly in subepithelial regions and in areas below villus crypts. As determined by immunofluorescence staining and flow cytometric analyses, lymphocytes from spleen and lymph nodes of sham-engrafted mice (AT x BM-SHAM) were essentially void of T cells, whereas in AT x BM thymus-engrafted (AT x BM-THG) mice, which served as a positive control for T cell repopulation, normal levels of T cells were present in spleen and lymph nodes by week 3 postengraftment, and at times thereafter. Most striking, however, was the finding that T cell repopulation of the spleen and lymph nodes occurred in AT x BM fetal intestine-engrafted (AT x BM-FIG) mice beginning 3 wk postengraftment. Based on H-2 expression, peripheral T cells in AT x BM-FIG mice were of donor bone marrow origin, and consisted of CD3+, T cell receptor (TCR)-alpha/beta+ T cells with both CD4+8- and CD4-8+ subsets. Peripheral T cells in AT x BM-FIG mice were functionally mature, as demonstrated by their capacity to proliferate after stimulation of CD3 epsilon. Moreover, alloreactive cytotoxic T lymphocytes were generated in primary in vitro cultures of spleen cells from AT x BM-FIG and AT x BM-THG mice, though not in spleen cell cultures from AT x BM-SHAM mice. Histologic studies of engrafted tissues 3-4 wk postengraftment demonstrated that thymus leukemia (Tl) antigens were expressed on epithelial surfaces of intestine grafts, and that both TCR-alpha/beta+ and TCR-gamma/delta+ lymphocytes were present in intestine grafts. Collectively, these findings indicate that the murine small intestine has the capacity to initiate and regulate T cell development from bone marrow precursors, thus providing a mechanism by which extrathymic development of intestine lymphocytes occur.

Nonspecific recruitment of cytotoxic effector cells in the intestinal mucosa of antigen-primed mice.

We have examined cytotoxic responses of lymphocytes derived from the gut epithelium of mice primed systemically and enterically with alloantigens. Both gut intraepithelial (IEL) and splenic lymphocytes from alloantigen-primed mice were found to contain antigen-specific cytotoxic T cell activity. However, after priming, gut IEL also developed high levels of natural killer and spontaneous cytotoxic cell activities. We suggest that this nonspecific activation of additional cytotoxic effector populations during an antigen-specific response is an important host immune defense within the intestinal mucosa.

Cytotoxic T lymphocytes produce immune interferon in response to antigen or mitogen.

Interferon (IFN) was found to be secreted by cloned lines of murine cytotoxic T lymphocytes in response to mitogenic or antigen specific stimulation. The IFN produced was shown to be of the immune type based on its sensitivity to pH 2.0 and on the ability of an antiserum to immune IFN to neutralize the antiviral activity.

CD43 is a murine T cell costimulatory receptor that functions independently of CD28.

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.

Bone marrow cells produce a novel TSHbeta splice variant that is upregulated in the thyroid following systemic virus infection.

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5' rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHbeta splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHbeta splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHbeta construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHbeta construct. The splice variant TSHbeta protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHbeta splice variant, but not the native form of TSHbeta, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHbeta, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.

Thymus-neuroendocrine interactions in extrathymic T cell development.

Studies of the development of murine intestinal intraepithelial lymphocytes (IELs) have yielded markedly different results depending on the experimental system used. In athymic radiation chimeras, IELs consist of all subsets found in euthymic mice; adult mice that were athymic at birth have only IELs that are positive for T cell receptor gamma delta and CD8 alpha alpha. These differences are resolved by the finding that administration of the neuropeptide thyrotropin-releasing hormone to adult mice thymectomized as neonates leads to the development of all IEL T cells. Thus, a neuroendocrine signal initiated by the thymus during fetal or neonatal life appears to be required for subsequent extrathymic maturation of gut alpha beta T cells.

Local hormone networks and intestinal T cell homeostasis.

Neuroendocrine hormones of the hypothalamus-pituitary-thyroid axis can exert positive or negative immunoregulatory effects on intestinal lymphocytes. Small intestine epithelial cells were found to express receptors for thyrotropin-releasing hormone (TRH) and to be a primary source of intestine-derived thyroid-stimulating hormone (TSH). The gene for the TSH receptor (TSH-R) was expressed in intestinal T cells but not in epithelial cells, which suggested a hormone-mediated link between lymphoid and nonhematopoietic components of the intestine. Because mice with congenitally mutant TSH-R (hyt/hyt mice) have a selectively impaired intestinal T cell repertoire, TSH may be a key immunoregulatory mediator in the intestine.

Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-alpha and interleukin-6 secretion, and CCL25 gene expression, in mouse primary gingival cell lines: interleukin-6-driven activation of CCL2.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection is strongly associated with periodontitis. Although P. gingivalis is known to elicit a strong inflammatory response, details of that remain fragmentary. To understand the local response to P. gingivalis, primary cell lines derived from mouse gingival tissues were exposed to P. gingivalis or Escherichia coli lipopolysaccharide, and the production of interleukin-6 and tumor necrosis factor-alpha was measured. CCL25 gene expression was measured by real-time polymerase chain reaction. Cells stimulated with combinations of interleukin-6, soluble interleukin-6 receptor and/or soluble gp130 were assayed for CCL2 and tumor necrosis factor-alpha secretion. MATERIAL AND METHODS: Primary cell lines were generated from mouse gingival tissues. Enzyme-linked immunosorbent assays were used to determine cytokine levels, and real-time polymerase chain reaction was used to quantify CCL25 gene expression. RESULTS: Exposure to P. gingivalis lipopolysaccharide but not to E. coli lipopolysaccharide resulted in significantly elevated levels of both interleukin-6 and tumor necrosis factor-alpha, and stimulation with P. gingivalis lipopolysaccharide also upregulated CCL25 gene expression. In one of three experiments, interleukin-6 induced CCL2 secretion, whereas interleukin-6 plus soluble interleukin-6 receptor induced CCL2 secretion in all three experiments, suggesting that both direct interleukin-6 signaling and interleukin-6 trans-signaling may be involved. However, because soluble gp130 did not inhibit trans-signaling, and because direct stimulation of gingival cells with soluble gp130 resulted in CCL2 secretion, the possibility exists that soluble gp130 forms binary complexes with soluble interleukin-6 receptor that promote direct interleukin-6 stimulation. CONCLUSION: These findings define a pathway in which exposure of gingival cells to P. gingivalis induces the release of interleukin-6 and tumor necrosis factor-alpha; interleukin-6, in turn, induces CCL2 secretion.

Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.

Hemodynamic effects of epinephrine, bicarbonate and calcium in the early postnatal period in a lamb model of single-ventricle physiology created in utero.

OBJECTIVES: A reproducible fetal animal model of single-ventricle physiology was created to examine the effects of pharmacologic agents commonly used in the perinatal and perioperative intensive care management of patients with a single ventricle. BACKGROUND: Single-ventricle physiology is characterized by parallel pulmonary and systemic circulations, with effective blood flow to each determined by the relative resistances in the pulmonary and systemic vascular beds. Perinatal and perioperative management of these patients is largely based on empiric observations and differs considerably between institutions and is further complicated by the transitional physiology of the newborn. The lack of animal models of single-ventricle physiology has hindered the understanding of this problem. METHODS: A 10-mm, Damus-Kaye-Stansel-type aortopulmonary anastomosis was created in 10 fetal sheep at 140 +/- 1.2 days of gestation. The main pulmonary artery was ligated distally, and pulmonary blood flow (Qp) was provided through a 5-mm aortopulmonary shunt. Eight lambs were delivered at term and placed on cardiopulmonary bypass (30 min) 48 to 72 h after birth. Pharmacologic interventions (0.1 microgram/kg body weight per min of epinephrine, 2 mEq/kg of sodium bicarbonate and 10 mg/kg of calcium chloride) were performed before and after bypass, and hemodynamic responses were observed. The response to the epinephrine bolus was determined only in the postbypass study. RESULTS: Both before and after bypass, epinephrine infusion and calcium and bicarbonate administration increased Qp and systemic blood flow (Qs) (total cardiac output) but produced only small changes in the Qp/Qs ratio (-0.5% to -7.3% change). With the epinephrine bolus, Qp increased enormously, and the Qp/Qs ratio increased by 584% (p < 0.001). CONCLUSIONS: In neonatal lambs with single-ventricle physiology created in utero, epinephrine infusion and calcium and bicarbonate administration increased total cardiac output without significantly compromising the Qp/Qs ratio. However, epinephrine bolus seems to be hemodynamically detrimental in circumstances of single-ventricle physiology and should be used with caution and probably in relatively lower doses in the resuscitation of patients with single-ventricle physiology. Further investigation of the dose-dependent effects and the effects of prolonged administration of common pharmacologic agents will enable better management of patients with single-ventricle physiology.

Immune function of thyroid stimulating hormone and receptor.

Thyroid stimulating hormone (TSH) is a central component of the hypothalamus-pituitary-thyroid axis. Although TSH is known for its important biological effects as a neuroendocrine used to regulate thyroid hormone activity and subsequent metabolic functions, TSH also has been shown to be produced and used by cells ofthe mammalian immune system. Moreover, recent findings have linked the use of TSH by cells of the immune system in humans and mice to a group of monocytic cells and lymphocytes--primarily dendritic cells, macrophages, and subset of naïve peripheral T cells. Other studies have demonstrated the capacity of dendritic cells and monocytes to produce biologically active TSH, thereby pointing to a process of paracrine or autocrine TSH-mediated communication during the earliest stages of an immune response to antigen. In this article, these and other features of TSH immune-endocrine interactions are discussed in the context of an intrinsic TSH immunological pathway. Additionally, a hypothesis is proposed in which TSH produced by cells of the immune system during acute antigen exposure plays a dual role, consisting on the one hand of TSH communication during antigen-driven immune activation while concomitantly serving to regulate physiological homeostasis by modulating and adjusting thyroid hormone activity.

T cell receptor delta gene repertoire and diversity of intestinal intraepithelial lymphocytes in athymic mice.

T cell receptor (TCR) delta gene rearrangements in intestinal intraepithelial lymphocytes (IEL) were studied in athymic radiation chimeras using polymerase chain reaction (PCR) and sequence analysis of DNAs spanning the variable (V), diversity (D), and junctional (J) genes. In both thymus-bearing and athymic mice, IEL delta gene rearrangements occurred for V delta 3, V delta 4, V delta 5 and V delta 6. V-D-J junctional-site sequence analyses of cloned DNAs from rearranged IEL delta genes in athymic mice revealed a predominance of in-frame rearrangements; junctional diversity consisting of nucleotide removal from V, D and/or J genes; N segment nucleotide insertions; and high overall gene diversity. Evaluation of PCR-amplified cDNAs made from IEL RNA indicated that all four rearranged V delta genes were expressed in IEL from athymic mice. The high diversity observed at the gene level also was present in amino acid sequences encoded by the V-D-J region of IEL delta genes in athymic mice. These data demonstrate that there is extensive diversity of rearranged delta genes in IEL which develop extrathymically, and suggest that the delta chain of IEL TCR-gamma delta+ T cells has the potential for interactions with polymorphic structures.

Primary structure and features of the genome of the Lactobacillus gasseri temperate bacteriophage (phi)adh.

The complete DNA sequence of the Lactobacillus (Lb.) gasseri temperate phage (phi)adh was determined. The linear and double-stranded genome consists of 43.785bp with a G+C content of 35. 3% and 3' protruding cohesive ends of 12nt. Sixty-two possible ORFs were identified. On the basis of homology comparisons, some of them could be assigned to possible functions, such as a helicase, a nucleic acid polymerase and a protease. In a non-coding area of the (phi)adh genome, structural features of a potential replication origin were detected. After subcloning, this region was functional as a replicon in Lb. gasseri and Lactococcus lactis. N-terminal aa sequencing and electron microscopic analysis of intact and defective phage particles enabled the identification of two capsid protein genes. One of their products, the major head protein, seems to be processed on the posttranslational level.

The arbZ gene from Lactobacillus delbrueckii subsp. lactis confers to Escherichia coli the ability to utilize the beta-glucoside arbutin.

From a genomic library of the industrially used strain Lactobacillus delbrueckii subsp. lactis DSM7290, a gene designated arbZ (869bp; encoding a 33.5kDa protein) was isolated by screening E. coli transformants for the ability to utilize the beta-glucoside arbutin. Out of 9000 transformants nine were able to ferment arbutin, whereas no utilization of the beta-glucosides salicin, esculin or cellobiose could be detected. Overexpression of arbZ using the T7-polymerase-T7-promoter-system resulted in the formation of insoluble, catalytically inactive protein aggregates (inclusion bodies). Accordingly, overexpression was not accompanied by an increase in ArbZ activity. Induction of arbZ controlled by the lac promoter under conditions that reduce protein aggregation resulted in a 12-fold increase in arbutin hydrolyzing activity of intact cells and a 13-fold increase in phospho-beta-glycosidase activity in cell-free extracts of the respective transformants. Nucleotide sequence analysis revealed a second gene upstream of arbZ that was designated arbX (830bp). ArbX (32.6kDa) shared similarity with several glycosyltransferases involved in the biosynthesis of lipopolysaccharides in Gram-negative bacteria. In Lb. delbrueckii subsp. lactis DSM7290 two transcripts, one covering arbX together with arbZ and one covering arbZ alone were detected by Northern blot analysis.

Structure of a genome region of the Lactobacillus gasseri temperate phage phiadh covering a repressor gene and cognate promoters.

By sequencing the DNA regions which flank the intG gene encoding integrase of the temperate Lactobacillus (Lb.) gasseri bacteriophage phiadh, a continuous sequence of 6590 bp was established. It encompasses five newly identified ORFs, of which four are located upstream, and one (orfC) downstream of intG. Proteins corresponding to the expected products of the intG upstream coding regions, orfA (33 kDa), orf2 (14 kDa), rad (12.1 kDa), and tec (7.9 kDa), were identified by in vitro expression of subcloned DNA fragments. Rad shares homology with transcription regulators, including SinR of Bacillus species and the repressor of phage phi105. The gporf2 is similar to predicted products of topologically equivalent coding regions of the Lactococcus lactis phage TP901-1 and the B. subtilis phage phi105. Promoters for the divergently oriented rad and tec genes were mapped within the 435-bp region between them and specify overlapping transcripts with extended 5'-untranslated sequences. As shown with lacZ fusions, Rad repressed transcription from the tec and rad promoters 20- and 5-fold, respectively. In Lb. gasseri, weak expression of cloned rad ws sufficient to mediate immunity towards phiadh.

A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity.

Because murine intestine intraepithelial lymphocytes (IEL) are dispersed throughout the intestine epithelium, it is important that IEL extraction procedures result in lymphocyte preparations of sufficient purity for use in in vitro and in vivo experimental systems. Here, we describe an improved technique for isolating murine IEL consisting of a single 30 min extraction followed by multiple nylon wool filtrations and centrifugation through Percoll. This procedure yields a preparation of IEL with high overall recovery and purity yet takes only 2-2.5 h. Evaluation of individual steps in the extraction process indicated that nylon wool filtration, in particular multiple filtrations, and Percoll fractionation both were important for achieving highly-enriched IEL populations by removal of enterocytes and cellular debris, and demonstrated that multiple nylon wool filtration improved the overall IEL recovery. This procedure has several advantages for studies of murine IEL in that the resultant IEL population is ideal for phenotypic, functional, or molecular analyses. Moreover, this technique is effective for isolating IEL on a single-animal basis, thereby permitting analyses of IEL from individual mice rather than as pooled IEL obtained from several animals.