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This paper summarizes a discussion that took place at the 52nd Annual DIA Meeting in Philadelphia, PA, on June 30, 2016, titled “Hot-Button Protocol and Operational Issues between Sponsors and Sites in Clinical Pharmacology Studies.” The symposium was a moderated panel of phase 1 clinical research experts representing the sponsor, and investigational sites. Conference attendees of similar experience joined in the discussion after commentary by each panelist. The learning objectives of the symposium were (1) to recognize issues that can provoke sponsor/site conflict or diminish conduct efficiency when they arise in the course of preparing to conduct or execution of phase 1 clinical studies, (2) to discuss how to handle such issues with counterparts when they arise and describe ways to negotiate and formulate a successful resolution. Sponsors and sites both have challenges in executing clinical trials on time and within budget. Both need to set and maintain realistic expectations and communicate with honesty, transparency, and timeliness. Achieving this goal will advance the more important take-away message, that developing new drugs requires sound execution of clinical trials.

New therapeutic approaches for progressive forms of multiple sclerosis (MS) will be the challenge for the future. While we now have 10 approved medicines for relapsing-remitting MS (RRMS), the major unmet need for RRMS is for predictive biomarkers to enable us to decide for a given individual which of the 10 drugs we should use in a given patient. For the progressive forms of MS, the major need centers on providing therapies to arrest disability. While therapy with monoclonal antibodies targeting B cells and even plasma cells presents promise, other therapies with small molecules, especially those that either cross the blood–brain barrier or those that at least meet the blood–brain barrier, may be attractive new therapeutic entities. Another approach that is worth exploring is the arming of monocytic cells with protective cytokines, while disarming them from release of pathogenic mediators. After all, monocytic cells possess brilliant mechanisms for traversing from the blood to the brain, and these mechanisms might be harnessed for benefit. While much worthy attention has been given to implantation of stem cells in the brain, the exploitation of mechanisms whereby protective immune cells diapedese into brain, may ultimately provide a solution for a disease that is triggered via the transmigration of pathogenic immune cells. This would represent a true reversal of misfortune.

Studies of informed consent for neonatal research suggest that there are gaps in parental understanding of key aspects of research. Few studies have examined parental understanding and investigator disclosure simultaneously. This pilot study evaluated the content and processes of 21 audio-recorded consent discussions with parents of eligible newborns, as well as parents' self-reported comprehension of research elements following these discussions. Gaps in both investigator disclosure and parental understanding were found. Nevertheless, parents reported comprehension of a greater number of research elements than were actually disclosed by investigators. Our preliminary results indicate the feasibility and potential utility of jointly examining parental understanding and investigator behavior to characterize consent for neonatal research. Directions for future research are suggested.

Vaginocervical stimulation (VS) significantly elevated the concentration of oxytocin (OT) in spinal cord superfusates of 8 intact urethane-anesthetized rats measured 10–15 min after VS (median [interquartile range]: 1.7 [1.00–3.37] pg/ml) compared to that measured 10–15 min before VS (1.1 [1.01–1.40] pg/ml). When VS was administered once (n = 8), it produced a 55% increase over baseline values; when administered a second time 45 min later (n = 6), it produced only a 22% increase over pre-VS values. The effects of estrogen on the VS-induced release of OT were then investigated using ovariectomized rats that were treated either with estradiol benzoate (EB; 10 µg/100 g bw) (n = 6) or with an oil vehicle (n = 6) subcutaneously for 3 days. The EB treatment significantly elevated the basal levels of OT released into spinal cord superfusates above vehicle control levels. Within 5–10 min after the onset of VS, OT concentrations in the superfusates were significantly higher in EB-treated than in vehicle-treated rats. The vehicle-treated rats did not show a significant elevation in OT concentration following VS. To rule out the possibility that the posterior pituitary gland was the source of this OT, the effect of hypophysectomy (HYPOX) was assessed on the VS-induced release of OT into spinal cord superfusates and plasma. The concentration of OT in spinal cord superfusates of both the HYPOX (n = 5) and intact rats (n = 6) increased significantly from 5.8 [4.4–6.5] pg/ml pre-VS to 7.9 [6.7–10.3] pg/ml immediately after VS, and from 4.4 [3.8–5] pg/ml pre-VS to 5.1 [4.6–5.7] pg/ml immediately after VS, respectively. There was no significant difference in baseline levels of OT in cerebrospinal fluid between the two groups. By contrast, plasma OT levels, while significantly elevated in response to VS from 3.42 [2.9–5.34] pg/ml baseline to 7.25 [5.33–15.77] pg/ml in the intact group, failed to respond significantly to VS in the HYPOX group (n = 5). The present findings provide evidence of a direct estrogen-dependent release of OT within the spinal cord in response to VS, presumably via descending oxytocinergic neurons.

Clinical Pharmacology Units (CPUs) are a special class of clinical research units for investigating the pharmacokinetic, pharmacodynamic, and toxicologic properties of drugs in man. The Association of Clinical Pharmacology Units (ACPU) is a national professional society with a membership drawn from academic and in-house pharmaceutical industry CPUs. It was established to provide a forum facilitating cooperation and the exchange of information and ideas between CPUs. One of its major objectives has been to promote the establishment of voluntary minimum standard of practice and guidelines for clinical pharmacology units.In October 1992, ACPU's Standards Committee undertook to draft a set of principles which would guide CPUs in the development of local standards and procedures. The product of this work was adopted by the membership in October 1994 after several review and revision cycles. ACPU invites comments from the clinical research community concerning its Voluntary Standards for the Operation of Clinic...

Corticotropin-releasing factor (CRF) exerts a major role in the stress response. Both CRF and urocortin, a newly discovered neuropeptide homologous to CRF, suppressed experimental autoimmune encephalomyelitis (EAE). Suppression of paralysis with CRF involved stimulation of the hypothalamic-pituitary-adrenal axis and inhibitory effects on an encephalitogenic T cell line. While CRF increased glucocorticoid production, which is known to block EAE, it also suppressed EAE in adrenalectomized rats, where glucocorticoid stimulation via CRF plays no role. Moreover, the encephalitogenicity of a T cell line exposed to CRF in vitro was reduced. Stress may influence autoimmune disease through the hypothalamic-pituitary-adrenal axis and directly via the immune system.

Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, prevented the clinical development of experimental autoimmune encephalomyelitis (EAE) with a reduction in inflammation and demyelination. Administration of AG reduced the expression of nitrosotyrosine in inflammatory lesions in the central nervous system. Cytokine expression, determined by semiquantitative PCR, revealed increased expression of IFN-gamma, IL-10, and TGF-beta, which was associated with protection from EAE, and reduced TNF-alpha, associated with the development of EAE. Furthermore, AG blocked the secretion of nitric oxide, TNF-alpha, and PGE2 in astrocyte cultures. AG did not influence the proliferation response of T cells to a pathogenic epitope of myelin basic protein. Down-regulation of nitric oxide by AG has widespread consequences for cytokine production in central nervous system inflammation and prevents EAE.

T cells are considered to be of prime importance in immune regulation of both B and T cell functions. The targets of recognition in T-T cell interactions are not clear. Most recent experimental work has focused on the idiotypic regulatory interactions mediated by TCR peptides. There is experimental evidence that regulatory cells exist that do not recognize the TCR. This type of regulation is selectively induced by activated T cells. Therefore, we designed this study to examine the possible role of cytokine receptors as targets of immune regulation. We tested two peptides of IL-2R alpha-chain, 2 of IL-2R beta-chain, and one of TNFR (p60). All peptides were found to be immunogenic at inducing T cell proliferation and four induced Abs in Lewis rats. We generated T cell lines to these five peptides, and tested them both in vitro and in vivo. We found that the T cells exhibited a proliferative response when cultured with activated, irradiated stimulator cells that were augmented upon addition of the cytokine receptor peptide. The cytokine profile of the lines was characterized as well as the Vbeta gene composition. One of the lines significantly protected against active encephalomyelitis. These results point at cytokine receptors as possible targets of immune regulation and T-T cell interactions.

The Open Unified Technical Framework (OpenUTF) provides an External Modeling Framework (EMF) that offers support for all of its standard modeling constructs within an encapsulated object that can be created and used by non-OpenUTF applications. Acting like a proxy to simulations executing in the OpenUTF, the EMF coordinates robust and repeatable event processing and state management in logical time between external applications and the core parallel and distributed OpenUTF simulation. Like the High Level Architecture (HLA),applications can optionally use their own simulation engines to coordinate their internal event processing with OpenUTF simulations using the EMF. Applications can also integrate with standard OpenUTF-compliant models that execute directly within the EMF. Because the EMF and HLA provide similar functionality, it is straightforward to implement an HLA interface as a wrapper for the EMF to facilitate direct HLA interoperability between external systems and simulations executing within the OpenUTF. This paper first provides historical background on previous EMF implementations that were developed for the Synchronous Parallel Environment for Emulation and Discrete Event Simulation (SPEEDES), leading to its current implementation within the WarpIV Kernel. The paper then describes the current set of EMF services that are provided for the OpenUTF, its high-level design, and its recent implementation. This topic is important to the Simulation Interoperability Standards Organization (SISO) because the EMF is a core architectural component of the OpenUTF that is being investigated by the Parallel and Distributed Modeling & Simulation Standing Study Group (PDMS-SSG) for future standardization.

The effect of bilateral pelvic, hypogastric, or pudendal neurectomy on oxytocin-induced facilitation of lordosis behavior was examined in ovariectomized, estrogen, and progesterone primed female rats. Oxytocin-induced facilitation of lordosis behavior was significantly decreased following bilateral pelvic, or combined bilateral pelvic and hypogastric neurectomy. Hypogastric only or pudendal only neurectomy had no effect on the facilitation of lordosis behavior after systemic administration of oxytocin. These results suggest that the integrity of the pelvic nerve is necessary and sufficient to mediate the stimulatory effects of systemically administered oxytocin on receptivity.

The present study was designed to elucidate the mechanism in the spinal cord by which vaginocervical stimulation (VS) attenuates responses to noxious stimulation. This was accomplished by testing the hypothesis that VS reduces noxious stimulation-induced release of substance P at the level of the spinal cord. Noxious foot shock significantly increased the release of substance P (measured using radioimmunoassay) into superfusates of the lumbosacral spinal cord region in urethane-anesthetized rats. VS applied concurrently with foot shock significantly attenuated the release of substance P compared to the foot shock-only condition. In addition, substance P levels were significantly lower after the VS-only condition than after the no stimulation or foot shock-only conditions. These findings indicate that VS may produce analgesia, at least in part, by suppressing the release of substance P within the spinal cord.

Neuropeptide Y (NPY) is an extremely potent orexigenic agent. These studies demonstrate that the effect of NPY on food and water intake are seen after infusion into either the third (3V) or fourth (4V) ventricle and that this is a specific effect, as it was not seen with the deaminated form. There was a nonsignificant tendency for lateral midbrain knife cuts to increase food intake. Both 3V and 4V NPY infusions showed an attenuated increases in food intake at 1 but not 2 h following NPY infusion in the lateral knife cut rats compared to the sham controls. Medical knife cuts resulted in significantly greater food intake in the basal state and a lesser increase in food intake in response to NPY infused into the 3V. These midbrain data suggest a role for both medical 3nd lateral fibers in mediating the effects of NPY on food intake. Lateral fibers appear to be more important, but their transection only delays the time of onset of the stimulating effect of NPY to the second hour. Lateral knife cuts virtually abolish the effect of 4V NPY on stimulating water intake. 3V NPY in the presence of NPY has a less clear effect at 1 h, but mildly attenuated the NPY effect on water intake at 2 h in lateral knife cut rats. Medial knife cuts slightly attenuate the effect of 3V NPY on water intake. However, medial knife cuts markedly increased basal water ingestion. These studies demonstrate the importance of neuronal communications between third and fourth ventricle associated structures in the modulation of ingestive behaviour. Further, it appears that NPY, when infused either into the 3V or 4V, stimulates more than one pathway to modulate ingestive behavior.

Improved molecular methods allow identification of the specific autoaggressive T cells involved in autoimmune inflammations. In this study of interphotoreceptor retinoid-binding protein-induced uveitis, TCR V beta usage was studied using RNA-polymerase chain reaction amplification of transcripts derived directly from ocular tissues and from T cell lines obtained from the spleen. Specific V beta 5' primers from the major murine TCR V beta families were coupled with a common 3' primer from the V beta C region. Amplification of rearranged TCR V beta-D beta-J beta-C beta sequences was confirmed by Southern blot analysis. In ocular tissue from sensitized mice, TCR V beta expression was limited mainly to one to three V beta families, with predominant expression of V beta 2, V beta 12, and V beta 15. In most animals there was similar, albeit limited, TCR gene usage in both the recognition of autoantigen in uveitogenic T cell lines and at the site of inflammation in the eye. Identification of a limited TCR V beta repertoire in Ag-reactive T cell lines correlated with TCR usage at the target site of autoimmune expression. The gene products of the restricted TCR V beta rearrangements found in lesions and in the cell lines may serve as the target for selective immunotherapy.

The diversity of Ag-specific receptors on T cells homing to an inflammatory infiltrate in the central nervous system has been analyzed. Experimental autoimmune encephalomyelitis, a T cell-mediated inflammatory disease of the central nervous system, was induced in Lewis rats with a CD4+, CD8- T cell line specific for peptide 68-86 of myelin basic protein. Within the line a wide array of TCR V beta genes was transcribed including the V beta 8, V beta 10, V beta 15, V beta 16, and V beta 19 families. Accumulation of T cells at the site of inflammation was determined by using RNA-polymerase chain reaction amplification of rearranged TCR V beta transcripts derived from brain. By 8 to 10 h after i.p. infusion of the pathogenic T cell line, TCR V beta transcripts, including mainly V beta families that were predominantly rearranged by the line, could be identified in brains. Restricted TCR V gene transcripts with predominance of the V beta 8 family were identified in brain 48 h after injection, before onset of disease. Paralysis was apparent by 4 to 5 days after injection. At this time diverse V beta gene transcripts were detected in brain, reaching a maximum by day 9, when paralyzed rats have recovered. By day 14 a second stage of limited heterogeneity in the T cell infiltrate could be identified with predominant expression of V beta 8, V beta 9, V beta 10, and V beta 19. Interestingly, three out of these four V beta families were predominantly expressed within the encephalitogenic line. Thus, T cell migration to brain in experimental autoimmune encephalomyelitis is characterized by a rapid penetration of T cells followed by a selective trapping of T cells before the clinical manifestations of disease. When clinical disease was present the T cell infiltrate was diverse, whereas in the post-acute phase of disease the T cells in the central nervous system had limited heterogeneity with selective accumulation of T cells transcribing the same V regions that were detected in the line that incited disease.

Forty-three CD3+4+8- TCR alpha beta+ Borrelia burgdorferi-reactive T cell clones isolated from the peripheral blood of a single patient with clinically active chronic Lyme arthritis were characterized. The spirochetal Ag recognized by 16 of these T cell clones was determined by reactivity with a panel of recombinant spirochetal Ag, which included the OspA, OspB, flagellin, Hsp60 and Hsp70 proteins. All three T cell clones reactive with heat shock proteins recognized a non-cross-reactive epitope unique to the spirochetal Ag. Analysis of the TCR V regions revealed preferential usage of V beta 5.1; 5 of 15 T cell clones that recognized an unidentified spirochetal Ag utilized this V beta gene segment. Most of the T cell clones recognized a given spirochetal Ag exclusively within the context of one HLA class II allele. However, two T cell clones, which recognized an unidentified Ag in the spirochetal lysate within the context of different HLA class II alleles, were both TCR V beta 5.1+, although each displayed a distinct alpha-chain. Moreover, in vitro incubation of this patient's PBMC with B. burgdorferi Ag resulted in a specific increase in the percentage of T cells expressing TCR V beta 5.1. These results indicate that B. burgdorferi has a V beta-selective factor influencing the cellular immune response in a patient with clinically active Lyme disease.

The repertoire of TCR V beta genes transcribed and expressed within the central nervous system was determined in mice with experimental allergic encephalomyelitis. Disease was induced in (PL/J x SJL/)F1 mice by immunizing with myelin basic protein-acetylated peptide 1-11, and mice were sacrificed at intervals from day 3 postimmunization to 3 wk after recovery from disease. Transcription of V beta genes was determined by reverse transcriptase polymerase chain reaction on RNA extracted from spinal cord, and expression of the V beta gene products was detected by immunohistochemistry with mAb specific for various V beta proteins. Multiple V beta genes were found to be transcribed and expressed in the central nervous system starting 7 days after immunization, and continuing up to 3 wk after clinical recovery. Preferential utilization of a single TCR V beta gene was not detected in the central nervous system at any time in the course of disease.

Superantigens have the ability to stimulate a subset of T cells based upon their expressed TCR beta-chain. It has been demonstrated that the administration of staphylococcal enterotoxin B (SEB) in mice leads to unresponsiveness in V beta 8+ T cells in vivo which are the same T cells that could be stimulated in vitro by this enterotoxin. We present here data on the effect of SEB administration in DBA/2 and (PL/J x SJL)F1 mice on their T cell response to two different T cell determinants, the responses against which are dominated by the use of V beta 8+ T cells. Treatment of mice with SEB not only diminished their primary T cell proliferative response to these determinants, but also was able to effectively reduce the memory T cell response. SEB treatment, however, showed only a modest effect in preventing Ac 1-11-induced experimental autoimmune encephalomyelitis in H-2u mice.

The pattern of vaginocervical stimulation-evoked expression of the proto-oncogene c- fos in lumbar 5-sacral 1 segments of the spinal cord of ovariectomized adult rats was mapped using immunocytochemistry. A calibrated force of mechanostimulation was applied to the vaginal cervix of experimental animals and to the perineum of control animals while they were gently restrained. The number of cells expressing c- fos was significant greater in the experimental than the control animals in laminae I, IV, V-VI and X. The implications of the present findings for elucidating the spinal pathways mediating the various behavioral, neuroendocrine and autonomic effects of vaginocervical stimulation (VS) are discussed.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory neurologic disease initiated by myelin basic protein-reactive CD4+ T cells, which are restricted by a particular MHC class II molecule. Recent studies have utilized inhibitor peptides that bind to restricting MHC class II molecules in order to inhibit EAE, presumably by means of competing with encephalitogenic epitopes. However, these studies leave open the possibility of alternative explanations, such as Ag-specific nonresponsiveness and immunodominance. In order to demonstrate that competition for MHC binding alone can inhibit EAE, the inhibitor peptide should ideally be structurally unrelated and nonimmunogenic yet physically associate with the MHC class II molecule. In this study, we show that the OVA-323-339 peptide, which is unrelated to the disease-inducing peptide, binds to A alpha uA beta u. However, although OVA-323-339 is extremely immunogenic in A alpha dA beta d-expressing BALB/c mice, it is nonimmunogenic in (PL/J x SJL)F1 and PL/J mice expressing A alpha uA beta u. When administered as a coimmunogen with Ac1-11, OVA-323-339 inhibited induction of EAE in (PL/J x SJL)F1 mice. Myelin basic protein-89-101, which does not bind A alpha uA beta u, had no effect on the disease process. This study provides evidence that MHC class II binding alone can modulate the induction of EAE. The use of a nonimmunogenic non-self peptide to modulate an autoimmune disease minimizes the potential complications of immunodominance or alternative regulatory mechanisms associated with immunogenic peptide therapies and further confirms the MHC-blocking model of immunosuppression.

Bilateral transection of the pelvic and/or hypogastric nerves, which convey afferent activity from the reproductive tract, was performed to ascertain the role of these nerves in the analgesic and motoric effects of vaginocervical mechanostimulation (VS) in rats. Two indices of analgesia were used: tail flick latency to radiant heat (TFL) and vocalization threshold to electrical shock of the tail (Voc-T). Nerve cuts were performed at least one week prior to behavioral testing. Bilateral transection of both the pelvic and hypogastric nerves eliminated the analgesic effects of VS on the TFL and Voc-T tests. Bilateral transection of only the pelvic nerves reduced the number of rats showing maximal VS-induced elevation in TFL, without altering the effect of VS on Voc-T. By contrast, bilateral transection of only the hypogastric nerves attenuated the Voc-T-elevating effect of VS, without reducing the effect of VS on elevating TFL. The effects of VS on producing immobility, hindlimb extension and blockage of hindlimb withdrawal to foot pinch were eliminated by combined bilateral pelvic and hypogastric neurectomy. However, bilateral transection of either nerve alone did not significantly alter the efficacy of VS in producing these effects. These findings indicate that the pelvic and hypogastric nerves contribute to the immobility- and extensor-inducing, and flexor-inhibiting effects of VS, and differentially mediate the analgesia-producing effects of VS.

EMC-M virus causes a monophasic paralytic syndrome characterized by encephalitic lesions in the brain and patchy demyelinating lesions in the spinal cord and nerve roots of BALB/c mice. Since the replication of EMC virus in vitro is inhibited by tumor necrosis factor (TNF)-alpha we have studied the effect of in vivo administration of this cytokine on the acute disease. Our studies show that periodic administration of TNF-alpha to animals infected with EMC-M reduces viral titers in the brain, and decreases the degree of clinical paralysis and the severity of the inflammatory lesions in the brain.

The pathogenesis of joint destruction in rheumatoid arthritis remains ill defined, although it is thought to be the result of tissue damage mediated by T cells. This prompted us to isolate and characterize in vivo activated T cells from rheumatoid arthritis synovial fluid in an attempt to determine their specificity. Heterogeneous synovial fluid cells, containing both adherent and non-adherent cell types, were recovered from joint aspirates and cultured in the presence of IL-2. After 2 weeks, the non-adherent cells were phenotyped as CD3-positive and TCR alpha beta-positive T cells. Polyclonal T cell lines were derived from four rheumatoid arthritis patients; of these, two proliferated, in a dose-dependent manner to only autologous synovial fluid in the presence of autologous or DR4Dw4 histocompatible antigen presenting cells. T cell proliferation to the synovial fluid could be inhibited by monomorphic anti-HLA-DR monoclonal antibody, but not by anti-DQ or anti-class I antibodies. T cell clones were established by limiting dilution of a synovial T cell line in the presence of autologous synovial fluid and DR4Dw4 histocompatible accessory cells. Examination of the antigen specificity of these T cell clones demonstrated that they were reactive with a component of synovial fluid. The results of these experiments suggest the presence of an MHC class II-restricted antigen in the rheumatoid arthritis synovial compartment that induces proliferation of in vivo activated T cells.

A failure of T lymphocytes to produce interleukin 2 (IL-2) on restimulation may, in part, account for the specific unresponsiveness that accompanies incomplete activation. The evidence to support this has been derived predominantly from the investigation of the molecular basis of anergy in murine type 1 T cells. In this study, the effects of different tolerogenic signals delivered by specific peptide or Staphylococcus aureus enterotoxin on the ability of antigen-specific human T cells to produce lymphokines, both in the induction phase and in established antigen-specific non-responsiveness, have been examined. Although T cell proliferation was decreased by supraoptimal concentrations of specific peptide in the presence or absence of antigen presenting cells, IL-2, IL-4, and interferon gamma (IFN-gamma) synthesis were comparable to that of activated T cells. The different tolerogenic signals, all capable of inhibiting phase of unresponsiveness. Restimulation of anergic T cells with an antigenic challenge failed to induce lymphokine production, with the exception of allergen-reactive T cells that secreted IFN-gamma. This latter observation is relevant to the desensitization of specific responsiveness in allergic disease.

A family of rat-mouse chimeric anti-murine CD4 antibodies was used to study the mechanisms of anti-CD4-mediated depletion and immunotherapy. The chimeric antibodies retain identical affinity and specificity as the therapeutically effective prototype antibody, rat GK1.5, but are of different mouse isotypes. GK1.5 gamma 1, GK1.5 gamma 2a, and GK1.5 gamma 2b are significantly more effective at CD4+ cell depletion than rat GK1.5 when low doses of antibody are administered. In contrast, no depletion is seen with GK1.5 gamma 3 at any dose. Depletion of CD4+ cells in vivo is not correlated with either the ability of the antibody to mediate C-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity in vitro, implying that additional antibody-mediated cytotoxic mechanisms occur in vivo. The chimeric antibodies were used to investigate the mechanism of GK1.5-mediated immunotherapy in a prototypic model of T cell-mediated autoimmunity, experimental allergic encephalomyelitis. Mice treated with a single dose of 100 micrograms of either GK1.5, GK1.5 gamma 1, or GK1.5 gamma 2a showed significant recovery within 72 h. In contrast, mice treated with 100 micrograms of GK1.5 gamma 3 showed only marginal improvement within the first 72 h and regressed within 5 days of treatment initiation. These data suggest that anti-CD4-mediated immunotherapy of murine experimental allergic encephalomyelitis is correlated with depletion of CD4+ cells.

Organ donation is affected by legal, cultural, religious, and racial factors, as well as by health considerations. Although organs in and of themselves are gender neutral and can be exchanged between the sexes, women account for up to two thirds of all organ donations. There are no clear reasons why women are more willing to undergo the risks of surgery than are men, nor is this gender disparity mirrored in the demand for donated organs. More men than women are recipients, and women are less likely to complete the necessary steps to receive donated organs. Internationally, ethical concern has been focused on possible human rights violations in the harvesting of organs from prisoners and, in poor countries, on the trafficking of organs from girls and women who are expected to financially help their families by selling their organs.

Molecular mimics of self-antigens can behave as altered peptide ligands and serve to ameliorate autoimmune disease. Analysis of experimental autoimmune encephalomyelitis with proteomic autoantibody microarrays reveals that there might exist a wide variety of microbes with features that mimic self-epitopes. Autoimmunity could therefore be modulated via microbial immunity, which may account for relapse and remission of ongoing disease.

Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, is an autoimmune disorder seen in mice and rats following immunization with myelin basic protein (MBP) or MBP-derived peptides. IFN-gamma, a cytokine produced by a variety of cells, is involved in many inflammatory and immune regulatory events. Contradictory results concerning exacerbations and the disease course were seen comparing injections of IFN-gamma in humans suffering from multiple sclerosis to studies using anti-IFN-gamma Abs in mice with EAE. To study the role of IFN-gamma and IFN-gamma-producing cells in EAE, we crossed IFN-gamma knockout mice (H-2b) (unable to produce IFN-gamma due to the disruption of the IFN-gamma gene) with an EAE-susceptible mouse strain, B10.PL (H-2u). EAE was seen in IFN-gamma knockout mice, heterozygotic (IFN-gamma +/-) mice, as well as wild-type littermates following immunization with MBP. Histologic analyses of the central nervous system of IFN-gamma knockout mice with EAE revealed massive infiltrates composed of lymphocytes, macrophages, and granulocytes. We conclude that the presence of IFN-gamma is not crucial to the induction or the clinical course of EAE.

The RADTRAN and RISKIND transportation risk analysis computer codes are the primary tools used to estimate dose consequences and risks associated with the transport of radioactive material. Over the years, some of the mathematical models used within the two computer codes have been updated and the methodologies to calculate input parameters have been improved. In addition, both codes have been evaluated for ease of use and appropriateness of application and verified against other computer codes that perform similar calculations. However, neither code has been validated against experimental data. This report discusses the results of five sets of experimental measurements used to partially validate the specific mathematical models used to predict the dose to an individual due to a passing shipment of radioactive material within the RADTRAN and RISKIND computer codes. Based on the comparisons it was found that RISKIND most closely predicted the measured dose in the majority of the investigated scenarios and that 12 out of 14 cases demonstrate the expected inverse relationship between the measured dose and the distance of closest approach. Only half of the data demonstrated the expected inverse relationship between dose and speed of travel.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

The development of antigen specific therapy for multiple sclerosis (MS) involves specifically suppressing undesired immune responses targeting the myelin sheath and underlying axon. We have recently reported some success with altered peptide ligands for a major target of the autoimmune response in MS. Antigen specific therapy has the potential to suppress undesirable autoimmunity, while leaving the rest of the immune system intact. Induction of an antigen specific Th1-to-Th2 shift could achieve this aim, once side effects, such as allergic responses, are minimized with optimal dosing.

Homopolymers or peptides containing a high percentage of cationic amino acids have been shown to have a unique ability to cross the plasma membrane of cells, and consequently have been used to facilitate the uptake of a variety of biopolymers and small molecules. To investigate whether the polycationic character of these molecules, or some other structural feature, was the molecular basis for the effect, the ability of a variety of homopolymers to enter cells was assayed by confocal microscopy and flow cytometry. Polymers of L- or D-arginine containing six or more amino acids entered cells far more effectively than polymers of equal length composed of lysine, ornithine and histidine. Peptides of fewer than six amino acids were ineffective. The length of the arginine side-chain could be varied without significant loss of activity. These data combined with the inability of polymers of citrulline to enter cells demonstrated that the guanidine headgroup of arginine was the critical structural component responsible for the biological activity. Cellular uptake could be inhibited by preincubation of the cells with sodium azide, but not by low temperature (3 degrees C), indicating that the process was energy dependent, but did not involve endocytosis.

Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive from of vaccination, with DNA encoding a minigene for residues 139-151 of myelin proteolipid protein (PLP139-151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-gamma, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-gamma were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139-151 reduced the capacity of a T cell line reactive to PLP139-151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.

We evaluated myoblast implantation in 10 boys with Duchenne muscular dystrophy (DMD) and absent dystrophin (age 5-10 years) who were implanted with 100 million myoblasts in the anterior tibial muscle of one leg and placebo in the other. Cyclosporine (5 mg/kg/day) was administered for 7 months. Pre- and postimplantation (after 1 and 6 months) muscle biopsies were analyzed. Force generation (tetanic tension and maximum voluntary contraction) was measured monthly in a double-blind design. There was increased force generation in both legs of all boys, probably due to cyclosporine. Using the polymerase chain reaction, evidence of myoblast survival and dystrophin mRNA expression was obtained in 3 patients after 1 month and in 1 patient after 6 months. These studies suggest a salutary effect of cyclosporine upon muscular force generation in Duchenne muscular dystrophy; however, myoblast implantation was not effective in replacing clinically significant amounts of dystrophin in DMD muscle.

Multiple sclerosis (MS) is a demyelinating disease during which an autoimmune reaction is directed against oligodendrocytes. Alterations of normal myelin structure or oligodendrocyte metabolism may be primary events that influence the susceptibility to MS. Once triggered, the immune system attacks and destroys myelin and the myelin forming cell. Evidence is presented that the oligodendrocyte responds to the attack by immune cells and their secreted products through modulation of its metabolism and gene expression. Cytokines, immunoglobulins, and complement complexes may elicit a survival response in the oligodendrocytes, involving the induction of heat shock proteins and other protective molecules. The possibility of manipulating these complex glial cell functions and controlling their pathologic interactions with immune cells will illuminate how myelin damage can be contained and how the injured tissue can be repaired.

The purpose of this study was to characterize the phenotype and clonality of the T cell population in patients who experience acute rejection (AR) following bone marrow transplantation (BMT) from a partially mismatched related donor (PMRD). Phenotypic analysis was performed using flow cytometry, assignment of donor/host lineage by cytogenetics or HLA-specific flow cytometry, and analysis of the T cell receptor (TCR) by reverse-transcriptase polymerase chain reaction (RT-PCR). We have previously reported the initial appearance in the blood of AR patients of host CD8+brightCD3low T cells that progressively express increasing amounts of CD3+ cells. We now report that this cell population can differentiate into either a cytotoxic T cell phenotype (CD3+CD8+HLA-DR+CD57-) usually associated with AR of grafts from matched unrelated donors or a suppressor T cell phenotype (CD3+CD8+CD57+HLA-DR-) usually associated with AR of grafts from matched sibling donors. Analysis of the TCR V beta subsets from two patients revealed sorted host CD3+CD8+ cells (purity 90-95%) from the first patient to express V beta 18 almost exclusively. In a second patient with late rejection (55 days post-BMT), the CD3+CD8+ cells were predominantly restricted to V beta 1, 5.1, 7, 9, and 18. Although CD3+CD8+ T cells are known to be associated with AR, cytotoxic and suppressor lineages in AR from the same type of BMT and clonal distribution of T cells in AR have not been reported. Preliminary results suggest that V beta expression in AR of PMRD grafts is restricted and host T cell phenotype may vary. Further studies will investigate whether specific mismatches correlate with specific V beta usage and/or host T cell phenotype.

CSF lymphocytes from patients with Coccidioides immitis meningitis exhibited a significant antigen-specific response to in vitro stimulation with C. immitis antigens. In some patients, lesser responses to control antigens (Candida and PPD) were also detected. Antigen-specific responses by CSF lymphocytes were seen early in the course of this disease as well as several years after patients had entered remission. When compared to CSF cells, the response of autologous peripheral blood mononuclear cells was similar but of a much smaller magnitude and at times undetectable. Fluorescence activated cell sorting revealed an increased percentage of CD3+ (T-cells), CD4+ (helper/inducer) and CD3+/HLA-DR+ (activated T-cell) cells in the CSF of C. immitis meningitis patients compared to their blood. Most of the antigen-specific proliferative response resided in the CD4+ lymphocyte subset. CSF T-cell proliferation assays may have a role in the diagnosis of C. immitis meningitis.

Mononuclear cells infiltrate degenerating muscles of Duchenne muscular dystrophy (DMD) patients. Using a quantitative PCR, we first characterized the T cells infiltrating muscle biopsies from six DMD patients. High levels of TCR V beta 2 transcripts were observed in DMD muscle tissue. TCR V beta 2 transcripts from seven DMD patients and five controls were sequenced, and the VDJ junctional region analyzed in 166 clones. One specific amino acid motif, RVSG, was found in the third complementary determining region (CDR3) of TCR V beta 2 chains in samples from five DMD patients, but not in controls. A specific immune reaction at the site of tissue degeneration may play an important role in the pathogenesis of DMD.

To evaluate the role of candidate genes in the susceptibility to multiple sclerosis (MS) and describe the role of T-cell receptor (TCR) gene rearrangements in the MS brain lesion in identifying a major target of the immune response in this disease.MEDLINE, bibliography review of published data, and unpublished studies.Published studies using novel molecular approaches to analyze the role of the major histocompatibility complex (MHC) and TCR gene complexes, as well as other candidate genes, in susceptibility to MS. We analyze epigenetic events involving TCR genes in individuals with MS and describe recent clinical trials in which immunotherapy has been attempted.Consistent with a polygenic model for disease predisposition, MHC and TCR gene associations with MS are relatively weak. Despite intensive research, no other putative "MS genes" have been firmly established. The analysis of TCR rearrangements in the brain lesion has helped to identify a major target of the immune response in MS.Understanding the genetic basis for autoimmune demyelination will offer new possibilities for the treatment of this illness.

Expression of the lymphokine genes in human astroglial cell lineage was studied. Primers for 9 different human lymphokines, from IL-1 alpha to IL-8, were used to analyze RNA transcripts in 5 cultured human astrocytoma, one neuroblastoma cell line and 4 fresh brain specimens by polymerase chain reaction (PCR). mRNA transcripts of neither IL-1 nor IL-3, the biological activities of which were observed in rat primary cultured astrocytes, could be detected within these cell lines. Two out of 5 unstimulated astrocytomas, U138 and U373, expressed IL-6 genes. IL-8 gene was detected within U87, U138, U251, U373 glioma cells. After stimulation with IL-1 beta, all astrocytoma and one neuroblastoma cell line expressed IL-6 and IL-8 genes. In addition to the cultured cells, we examined IL-6 and IL-8 gene expression within human malignant astrocytoma specimens. The result shows that three out of four glioma specimens expressed IL-6 and IL-8 genes. From these results, it is suspected that astroglial cell-derived IL-6 or IL-8 may participate in local immune reactions accompanying infection, degeneration and malignancies in the central nervous system.

Applications of PCR have revolutionized the field of immunogenetics particularly in studies of human leukocyte antigen class II polymorphism and more recently in the analysis of T cell receptor usage. The diversity of the variable region of the T cell receptor, however, has made it difficult to amplify the complete repertoire of T cell receptor transcripts. We have chosen to address this problem through the design of oligonucleotide primers specific for each of the known V alpha- and V beta-region T cell receptor families in order to characterize the T cell receptor repertoire. Using nonradioactive probes labeled with horse radish peroxidase, the system presented here allows for the rapid elucidation of the T cell receptor repertoire expressed in cells or tissue samples, such as those derived from autoimmune lesions. The identification of the T cell receptor repertoire involved in a pathogenic process can have therapeutic implications given the success of reversing experimental autoimmune disorders by directing specific forms of immunotherapy against V region gene products.