High-fat diet induces systemic B-cell repertoire changes associated with insulin resistance.

The development of obesity-associated insulin resistance is associated with B-lymphocyte accumulation in visceral adipose tissue (VAT) and is prevented by B-cell ablation. To characterize potentially pathogenic B-cell repertoires in this disorder, we performed high-throughput immunoglobulin (Ig) sequencing from multiple tissues of mice fed high-fat diet (HFD) and regular diet (RD). HFD significantly changed the biochemical properties of Ig heavy-chain complementarity-determining region-3 (CDRH3) sequences, selecting for IgA antibodies with shorter and more hydrophobic CDRH3 in multiple tissues. A set of convergent antibodies of highly similar sequences found in the VAT of HFD mice but not RD mice showed significant somatic mutation, suggesting a response shared between mice to a common antigen or antigens. These findings indicate that a simple high-fat dietary intervention has a major impact on mouse B-cell repertoires, particularly in adipose tissues.

A Genetic Animal Model of Alcoholism for Screening Medications to Treat Addiction.

The purpose of this review is to present up-to-date pharmacological, genetic, and behavioral findings from the alcohol-preferring P rat and summarize similar past work. Behaviorally, the focus will be on how the P rat meets criteria put forth for a valid animal model of alcoholism with a highlight on its use as an animal model of polysubstance abuse, including alcohol, nicotine, and psychostimulants. Pharmacologically and genetically, the focus will be on the neurotransmitter and neuropeptide systems that have received the most attention: cholinergic, dopaminergic, GABAergic, glutamatergic, serotonergic, noradrenergic, corticotrophin releasing hormone, opioid, and neuropeptide Y. Herein, we sought to place the P rat's behavioral and neurochemical phenotypes, and to some extent its genotype, in the context of the clinical literature. After reviewing the findings thus far, this chapter discusses future directions for expanding the use of this genetic animal model of alcoholism to identify molecular targets for treating drug addiction in general.

Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation.

Thirty-eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34(+) hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti-thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34(+) cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long-term graft survival in all patients.

Requirement for interactions of natural killer T cells and myeloid-derived suppressor cells for transplantation tolerance.

The goal of the study was to elucidate the cellular and molecular mechanisms by which a clinically applicable immune tolerance regimen of combined bone marrow and heart transplants in mice results in mixed chimerism and graft acceptance. The conditioning regimen of lymphoid irradiation and anti-T cell antibodies changed the balance of cells in the lymphoid tissues to create a tolerogenic microenvironment favoring the increase of natural killer T (NKT) cells, CD4+ CD25+ regulatory T cells and Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs), over conventional T cells (Tcons). The depletion of MDSCs abrogated chimerism and tolerance, and add back of these purified cells was restorative. The conditioning regimen activated the MDSCs as judged by the increased expression of arginase-1, IL-4Rα and programmed death ligand 1, and the activated cells gained the capacity to suppress the proliferation of Tcons to alloantigens in the mixed leukocyte reaction. MDSC activation was dependent on the presence of host invariant NKT cells. The conditioning regimen polarized the host invariant NKT cells toward IL-4 secretion, and MDSC activation was dependent on IL-4. In conclusion, there was a requirement for MDSCs for chimerism and tolerance, and their suppressive function was dependent on their interactions with NKT cells and IL-4.

Tolerance and withdrawal of immunosuppressive drugs in patients given kidney and hematopoietic cell transplants.

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

B lymphocytes as emerging mediators of insulin resistance.

Obesity is associated with chronic inflammation of various tissues including visceral adipose tissue (VAT), which contributes to insulin resistance. T cells and macrophages infiltrate VAT in obesity and orchestrate this inflammation. Recently, we made the surprising discovery that B cells are important contributors to this process. Thus, some B cells and the antibodies they produce can promote VAT-associated and systemic inflammation, leading to insulin resistance. This report will focus on the properties of these B cells, and how they contribute to insulin resistance through T-cell modulation and production of pathogenic autoantibodies. Understanding the mechanisms by which B cells contribute to insulin resistance should lead to new antibody-based diagnostics and B-cell modulating therapeutics to manage this increasingly prevalent disease.

In vivo wireless ethanol vapor detection in the Wistar rat.

Traditional alcohol studies measure blood alcohol concentration to elucidate the biomedical factors that contribute to alcohol abuse and alcoholism. These measurements require large and expensive equipment, are labor intensive, and are disruptive to the subject. To alleviate these problems, we have developed an implantable, wireless biosensor that is capable of measuring alcohol levels for up to six weeks. Ethanol levels were measured in vivo in the interstitial fluid of a Wistar rat after administering 1 g/kg and 2 g/kg ethanol by intraperitoneal (IP) injection. The data were transmitted wirelessly using a biosensor selective for alcohol detection. A low-power piezoresistive microcantilever sensor array was used with a polymer coating suitable for measuring ethanol concentrations at 100% humidity over several hours. A hydrophobic, vapor permeable nanopore membrane was used to screen liquid and ions while allowing vapor to pass to the sensor from the subcutaneous interstitial fluid.

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae).

BACKGROUND AND AIMS: Previous studies have suggested that velamen characteristics are useful as taxonomic markers in Orchidaceae. Members of tribe Cranichideae have been assigned to two velamen types constructed based on combinations of characters such as the presence of secondary cell-wall thickenings and pores. However, such characters have not been analysed on an individual basis in explicit cladistic analyses. METHODS: The micromorphology of roots of 26 species of Cranichideae was examined through scanning electron microscopy and light microscopy, scoring the variation and distribution of four characters: number of velamen cell layers, velamen cell-wall thickenings, presence and type of tilosomes, and supraendodermal spaces. The last three characters were analysed cladistically in combination with DNA sequence data of plastid trnK/matK and nuclear ribosomal internal transcribed spacer (ITS) regions and optimized on the resulting phylogenetic tree. KEY RESULTS: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae, whereas tilosomes, documented here for the first time in Cranichideae, provide an unambiguous synapomorphy for subtribe Spiranthinae. Supraendodermal spaces occur mostly in species dwelling in seasonally dry habitats and appear to have evolved three times. CONCLUSIONS: Three of the four structural characters assessed are phylogenetically informative, marking monophyletic groups recovered in the combined molecular-morphological analysis. This study highlights the need for conducting character-based structural studies to overcome analytical shortcomings of the typological approach.

The role of 5-HT3 receptors in drug abuse and as a target for pharmacotherapy.

Alcohol and drug abuse continue to be a major public health problem in the United States and other industrialized nations. Extensive preclinical research indicates the mesolimbic dopamine (DA) pathway and associated regions mediate the rewarding and reinforcing effects of drugs of abuse and natural rewards, such as food and sex. The serotonergic (5-HT) system, in concert with others neurotransmitter systems, plays a key role in modulating neuronal systems within the mesolimbic pathway. A substantial portion of this modulation is mediated by activity at the 5-HT3 receptor. The 5-HT3 receptor is unique among the 5-HT receptors in that it directly gates an ion channel inducing rapid depolarization that, in turn, causes the release of neurotransmitters and/or peptides. Preclinical findings indicate that antagonism of the 5-HT3 receptor in the ventral tegmental area, nucleus accumbens or amygdala reduces alcohol self-administration and/or alcohol-associated effects. Less is known about the effects of 5-HT3 receptor activity on the self-administration of other drugs of abuse or their associated effects. Clinical findings parallel the preclinical findings such that antagonism of the 5-HT3 receptor reduces alcohol consumption and some of its subjective effects. This review provides an overview of the structure, function, and pharmacology of 5-HT3 receptors, the role of these receptors in regulating DA neurotransmission in mesolimbic brain areas, and discusses data from animal and human studies implicating 5-HT3 receptors as targets for the development of new pharmacological agents to treat addictions.

Neuroadaptive changes in the mesoaccumbens dopamine system after chronic nicotine self-administration: a microdialysis study.

There is little evidence to date to indicate if mesoaccumbens dopamine function at the neurochemical level is altered during early abstinence from chronic i.v. nicotine self-administration. Thus, a quantitative microdialysis (no-net-flux) approach was used to measure basal extracellular concentrations and extraction fractions of dopamine in the nucleus accumbens (ACB) of rats that self-administered nicotine i.v. for 25 days, as well as in rats serving as yoked comparison groups (yoked nicotine and yoked saline). After 24-48 h of the final self-administration session, there was a significant reduction in basal extracellular dopamine levels in the ACB of the self-administration group compared with the yoked saline group (1.35+/-0.15 nM versus 3.70+/-0.28 nM). The basal extracellular dopamine levels in the yoked nicotine group (1.46+/-0.20 nM) were not significantly different compared with the nicotine self-administration group. The in vivo extraction fraction of dopamine, an indirect measure of dopamine uptake, was significantly increased in the nicotine self-administration (86%) and yoked nicotine (91%) groups compared with the yoked saline group (77%). In addition, a marked reduction in the elevation of extracellular dopamine levels in the ACB occurred after a nicotine challenge as measured by conventional microdialysis in the self-administration (112% of basal) and yoked nicotine (121% of basal) groups as compared with a yoked saline (154% of basal) group. The reduced basal ACB dopamine levels in the nicotine groups during early abstinence appears to be due to increased clearance, suggesting increased dopamine uptake is occurring as a result of the chronic nicotine treatment. The reduced elevation of extracellular dopamine levels in the ACB upon nicotine challenge suggests a functional desensitization or downregulation phenomenon involving acetylcholine receptors (nicotinic nAChRs). Overall, these results provide clear evidence for a neuroadaptive change that alters dopamine transmission in the ACB during abstinence from chronic i.v. nicotine exposure.

Ethanol drinking and deprivation alter dopaminergic and serotonergic function in the nucleus accumbens of alcohol-preferring rats.

The alcohol deprivation effect is a temporary increase in the intake of, or preference for, ethanol after a period of deprivation that may result from persistent changes in key limbic regions thought to regulate alcohol drinking, such as the nucleus accumbens. The present study tested the hypothesis that chronic alcohol drinking under continuous 24-h free-choice conditions alters dopamine and serotonin neurotransmission in the nucleus accumbens and that these alterations persist in the absence of alcohol. Using the no-net-flux microdialysis method, the steady-state extracellular concentration (point of no-net-flux) for dopamine was approximately 25% higher in the adult female alcohol-preferring P rats given prior access to 10% ethanol, even after 2 weeks of ethanol abstinence, compared with the P rats gives access only to water. However, the extracellular concentration of serotonin was approximately 35% lower in animals given 8 weeks of continuous access to ethanol compared with water controls and animals deprived of ethanol for 2 weeks. The effect of local perfusion with 100 microM sulpiride (D(2) receptor antagonist) and 35 microM 1-(m-chlorophenyl)-biguanide (5-hydroxytryptamine(3) receptor agonist) on dopamine overflow were reduced approximately 33% in both groups of ethanol-exposed P rats compared with water controls. Free-choice alcohol drinking by P rats alters dopamine and serotonin neurotransmission in the nucleus accumbens, and many of these effects persist for at least 2 weeks in the absence of ethanol, suggesting that these underlying persistent changes may be in part responsible for increased ethanol drinking observed in the alcohol-deprivation effect.

Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy.

Many tumor-associated Ags represent tissue differentiation Ags that are poorly immunogenic. Their weak immunogenicity may be due to immune tolerance to self-Ags. Prostatic acid phosphatase (PAP) is just such an Ag that is expressed by both normal and malignant prostate tissue. We have previously demonstrated that PAP can be immunogenic in a rodent model. However, generation of prostate-specific autoimmunity was seen only when a xenogeneic homolog of PAP was used as the immunogen. To explore the potential role of xenoantigen immunization in cancer patients, we performed a phase I clinical trial using dendritic cells pulsed with recombinant mouse PAP as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly vaccinations of xenoantigen-loaded dendritic cells with minimal treatment-associated side effects. All patients developed T cell immunity to mouse PAP following immunization. Eleven of the 21 patients also developed T cell proliferative responses to the homologous self-Ag. These responses were associated with Ag-specific IFN-gamma and/or TNF-alpha secretion, but not IL-4, consistent with induction of Th1 immunity. Finally, 6 of 21 patients had clinical stabilization of their previously progressing prostate cancer. All six of these patients developed T cell immunity to human PAP following vaccination. These results demonstrate that xenoantigen immunization can break tolerance to a self-Ag in humans, resulting in a clinically significant antitumor effect.

Idiotype vaccination following ABMT can stimulate specific anti-idiotype immune responses in patients with B-cell lymphoma.

Vaccination with the idiotype (Id) protein derived from B-cell malignancies can produce Id-specific immune responses that correlate with improved remission duration and survival rates in patients with follicular non-Hodgkin's lymphoma (NHL). A state of minimal or no residual disease correlates strongly with the laboratory detection of a cellular or humoral immune response. High-dose cytotoxic therapy (HDCT) with autologous stem cell support (autologous bone marrow transplantation [ABMT]) can provide profound cytoreduction of B-cell NHL, but the potential immune suppression associated with myeloablative therapy may compromise a patient's ability to mount a specific immune response. To determine whether patients with NHL could mount detectable immuneresponses following ABMT, Id vaccines were administered at 2 to 12 months following myeloablative therapy to a series of patients with relapsed or resistant B-cell NHL. Two different vaccination strategies produced robust immune responses against KLH in all patients, supporting the capacity of the reconstituted immune system following HDCT to react against a strong antigen. Combining the results from both vaccination strategies, 10 of 12 patients mounted Id-specific humoral or cellular responses. Vaccinations were consistently well tolerated. Of the 12 patients, 7 have experienced prolonged remissions with a follow-up from HDCT ranging from 3 to more than 11 years. Our experience serves to document the ability of the recovering immune system to react against both self and xenotypic antigens and supports the feasibility and safety of antigen-specific vaccination following myeloablative therapy in patients with B-cell NHL.

Dendritic cell development from common myeloid progenitors.

Dendritic cells (DCs) are professional antigen-presenting cells which both initiate adaptive immune responses and control tolerance to self-antigens. It has been suggested that these different effects on responder cells depend on subsets of DCs arising from either myeloid or lymphoid hematopoietic origins. In this model, CD8 alpha+ Mac-1- DCs are supposed to be of lymphoid while CD8 alpha- Mac-1+ DCs are supposed to be of myeloid origin. Here we summarize our findings that both CD8 alpha+ and CD8 alpha- DCs can arise from clonogenic common myeloid progenitors (CMPs) in both thymus and spleen. Therefore CD8 alpha expression DCs does not indicate a lymphoid origin and differences among CD8 alpha+ and CD8 alpha- DCs might rather reflect maturation status than ontogeny. On the basis of transplantation studies, it seems likely that most of the DCs in secondary lymphoid organs and a substantial fraction of thymic DCs are myeloid-derived.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy.

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA(+)CD27(-)CCR7(-)). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer(+) T cells, confirming the role of CD8 T cells in this treatment strategy.

Negative interaction of dopamine D2 receptor antagonists and GBR 12909 and GBR 12935 dopamine uptake inhibitors in the nucleus accumbens.

The objective of this study was to examine the interaction of dopamine D2 receptor antagonists and dopamine uptake inhibitors on the regulation of extracellular dopamine release in the nucleus accumbens of Wistar rats employing in vivo microdialysis and in vitro dopamine uptake studies. Application of the D2 receptor antagonists raclopride (100 microm) or sulpiride (100 microm) alone through the microdialysis probe in the nucleus accumbens for 60 min increased the extracellular levels of dopamine in the nucleus accumbens to 150% and 200% of basal, respectively. Perfusion of the nucleus accumbens for 60 min with the dopamine uptake inhibitors, 1-[2-[bis(4-Fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride (GBR 12909; 100 microm) or 1-[2-(Diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine dihydrochloride (GBR 12935; 100 microm) alone, increased the extracellular levels of dopamine in the nucleus accumbens to 400% and 350% of basal, respectively. Co-perfusion of 100 microM GBR 12909 or GBR 12935 with either 100 microM sulpiride or raclopride produced a significant reduction in the GBR 12909 or GBR 12935 induced increase in the extracellular levels of dopamine to basal levels. In vitro, GBR 12909 (1-9 nM) dose-dependently inhibited active uptake of [3H]dopamine in homogenates of the nucleus accumbens. Addition of 100 microm sulpiride had little effect on GBR 12909 inhibition of [3H] dopamine uptake, suggesting that dopamine D2 receptor antagonists are not blocking the actions of the GBR-type dopamine uptake inhibitors at the dopamine transporter. Overall, the data suggest that complex interactions occur in vivo between D2 antagonists and GBR-type dopamine uptake inhibitors, which negate their effects on elevating the extracellular levels of dopamine in the nucleus accumbens.

Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice.

Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.

Dendritic cells injected via different routes induce immunity in cancer patients.

Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-gamma production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.

Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity.

The combination of angiostatin and endostatin has been shown to have synergistic antiangiogenic and antitumor effects when the genes for these proteins are delivered to tumor cells by retroviral gene transfer. Here we report the construction of a murine angiostatin-endostatin fusion gene (Statin-AE) which shows enhanced antiangiogenic activity on human umbilical vein endothelial cell (HUVEC) tube formation in vitro compared with angiostatin or endostatin alone. Similarly, the fusion gene demonstrates antiangiogenic effects in vivo and antitumor activity in a B16F10 melanoma model when co-delivered by retroviral packaging cell inoculation in mice. The fusion gene demonstrates significantly greater inhibition of tumor growth compared with angiostatin, endostatin or the combination of genes.