An alternative B cell development program

Two major types of B cells, the antibody-producing cells of the immune system, are classically distinguished in the spleen: marginal zone (MZ) and follicular (FO). In addition, FO B cells are subdivided into FO I and FO II cells, based on the amount of surface IgM. MZ B cells, which surround the splenic follicles, rapidly produce IgM in response to blood-borne pathogens without T cell help, while T cell-dependent production of high affinity, isotype-switched antibodies is ascribed to FO I cells. The significance of FO II cells and the mechanism underlying B cell fate choices are unclear. We showed that FO II cells express more Sca1 than FO I cells and originate from a distinct B cell development program, marked by high expression of Sca1. MZ B cells can derive from the “canonical” Sca1lo pathways, as well as from the Sca1hi program, although the Sca1hi program shows a stronger MZ bias than the Sca1lo program, and extensive phenotypic plasticity exists between MZ and FO II, but not between MZ and FO I cells. The Sca1hi program is induced by hematopoietic stress and generates B cells with an Igλ-enriched repertoire. In aged mice, the canonical B cell development pathway is impaired, while the Sca1hi program is increased. Furthermore, we showed that a population of unknown function, defined as Lin-c-kit+Sca1+ (LSK-), contains early lymphoid precursors, with primarily B cell potential in vivo. Our data suggest that LSK- cells may represent a distinct precursor for the Sca1hi program in the bone marrow.

Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis

BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. METHODS: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. RESULTS: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. CONCLUSIONS: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a >/=50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.

IMMUNE MODULATION OF THE MYCOBACTERIUM TUBERCULOSIS GRANULOMATOUS RESPONSE

Tuberculosis (TB) remains a major public health burden. The immunocompetant host responds to Mycobacterium tuberculosis (MTB) infection by the formation of granulomas, which initially prevent uncontrolled bacterial proliferation and dissemination. However, increasing evidence suggests that granuloma formation promotes persistence of the organism by physically separating infected cells from effector lymphocytes and by inducing a state of non-replicating persistence in the bacilli, making them resistant to the action of antibiotics. Additionally, immune-mediated tissue destruction likely facilitates disease transmission. The granulomatous response is in part due to mycobacterial glycolipid antigens. Therefore, studies were first undertaken to determine the innate mechanisms of mycobacterial cord factor trehalose-6’6-dimycolate (TDM) on granuloma formation. Investigations using knock-out mice suggest that TNF-a is involved in the initiation of the granulomatous response, complement factor C5a generates granuloma cohesiveness, and IL-6 is necessary for maintenance of an established granulomatous responses. Studies were next performed to determine the ability of lactoferrin to modulate the immune response and pathology to mycobacterial cord factor. Lactoferrin is an iron-binding glycoprotein with immunomodulatory properties that decrease tissue damage and promote Th1 responses. Mice challenged with TDM and treated with lactoferrin had decreased size and numbers of granulomas at the peak of the granulomatous response, accompanied by increased IL-10 and TGF-b production. Finally, the ability of lactoferrin to serve as a novel therapeutic for the treatment of TB was performed by aerosol challenging mice with MTB and treating them with lactoferrin added to the drinking water. Mice given tap water had lung log10 CFUs of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7) and mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Total lung inflammation in lactoferrin treated mice was significantly decreased, with fewer areas of macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. The lungs of lactoferrin treated mice had increased CD4+ IFN-g+ cells and IL-17 producing cells on ELISpot analysis. It is hypothesized that lactoferrin decreases bacterial burden during MTB infection by early induction of Th1 responses.

Human placental lactogen enhancer: Tissue specificity and binding with specific proteins

Human placental lactogen (hPL) is a 22,000 dalton protein hormone produced in the placenta. The physiological actions of hPL are not well understood but its major activity is to regulate both maternal and fetal metabolism. hPL stimulates maternal lipolysis increasing free fatty acids in the maternal blood, allowing their use as an energy source by the mother, and sparing glucose for the fetus. It may also act as a growth promoting hormone for the fetus. hPL is produced in increasing amounts as pregnancy progresses. At term, hPL accounts for 10% of protein and 5% of total RNA in the placenta. This high level of hPL production is tissue-specific, as hPL is only produced in the placenta by syncytiotrophoblast cells.^ The objective of this work was to understand the mechanism by which such high levels of hPL are produced in a tissue-specific manner. A transcriptional enhancer found 2.2 kb 3$\sp\prime$ to one of the hPL genes (hPL$\sb3$) may explain the regulation of hPL expression. Transient transfection experiments using the hPL-producing human choriocarcinoma cell line JEG-3 localized the hPL enhancer to a 138 bp core element. This 138 bp sequence was found to be tissue specific in its actions as it did not promote transcription in heterologous cell lines. Gel mobility shift assays showed the hPL enhancer interacts specifically with nuclear proteins unique to hPL-producing cells. Within the 138 bp enhancer a 22 bp region was shown to be protected from DNase I digestion due to binding of proteins derived from placental nuclear extracts. Proteins binding this region of the enhancer may be instrumental in the tissue specific activity of the hPL enhancer. ^

Expression and regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis

Factors involved in regulating tissue specific gene expression play a major role in cell differentiation. In order to further understand the differentiation events occurring during hematopoiesis, a myeloid specific gene was characterized, the expression pattern during hematopoiesis was analyzed, and the mechanisms governing its regulation were assessed. Previously, our laboratory isolated an anonymous cDNA clone, pD-D1, which displayed preferential expression in myeloid cells. From nucleotide sequencing of overlapping cDNA clones I determined that the D-D1 message encodes a hematopoietic proteoglycan core protein (HpPG). The expression pattern of the gene was assessed by in situ hybridization of bone marrow and peripheral blood samples. The gene was shown to be expressed, at variable levels, in all leukocytes analyzed, including cells from every stage of neutrophil development. In an attempt to ascertain the differentiation time point in which the HpPG gene is initially expressed, more immature populations of leukemic myeloblasts were assessed by northern blot analysis. Though the initial point of expression was not obtained, an up-regulatory event was discovered corresponding to a time point in which granule genesis occurs. This finding is consistent with prior observations of extensive packaging of proteoglycans into the secretory granules of granule producing hematopoietic cells. The HpPG gene was also found to be expressed at low levels in all stages of lymphocyte development analyzed, suggesting that the HpPG gene is initially expressed before the decision for myeloid-lymphoid differentiation. To assess the mechanism for the up-regulatory event, a K562 in vitro megakaryocytic differentiation system was used. Nuclear run-off analyses in this system demonstrated the up-regulation to be under transcriptional control. In addition, the HpPG gene was found to be down regulated during macrophage differentiation of HL60 cells and was also shown to be transcriptionally controlled. These results indicate that there are multiple points of transcriptional regulation of the HpPG gene during differentiation. Furthermore, the factors regulating the gene at these time points are likely to play an important role in the differentiation of granule producing cells and macrophages. ^

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application.

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

Postnatal persistent infection with classical Swine Fever virus and its immunological implications.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

HIV-1 Tat protein mimicry of chemokines

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24–51) encompassing the “chemokine-like” region of Tat induced a rapid and transient Ca2+ influx in monocytes and macrophages, analogous to β-chemokines. Both monocyte migration and Ca2+ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of β-chemokines from the β-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24–51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic β-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.

Long-term suppression of Leydig cell steroidogenesis prevents Leydig cell aging

Male aging is accompanied by reduced testosterone production by the Leydig cells, the testosterone-producing cells of the testis. The mechanism by which this occurs is unknown. Based on the observations that reactive oxygen is capable of damaging components of the steroidogenic pathway and that reactive oxygen is produced during steroidogenesis itself, we hypothesized that long-term suppression of steroidogenesis might inhibit or prevent age-related deficits in Leydig cell testosterone production. To test this, we administered contraceptive doses of testosterone to groups of young (3 months old) and middle-aged (13 months old) Brown Norway rats via Silastic implants to suppress endogenous Leydig cell testosterone production. After 8 months, the implants were removed, which rapidly (days) restores the ability of the previously suppressed Leydig cells to produce testosterone. Two months after removing the implants, when the rats of the two groups were 13 and 23 months of age, respectively, the Leydig cells in both cases were found to produce testosterone at the high levels of young Leydig cells, whereas significantly lower levels were produced by the 23-month-old controls. Thus, by placing the Leydig cells in a state of steroidogenic “hibernation,” the reductions in Leydig cell testosterone production that invariably accompany aging did not occur. If hormonal contraception in the human functions the same way, the adverse consequences of reduced testosterone in later life (osteoporosis, reduced muscle mass, reduced libido, mood swings, etc.) might be delayed or prevented.

Development of an in vitro reconstitution assay for glucose transporter 4 translocation

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca2+-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5′-[γ-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein–protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.