Donor-Specific Antibodies Are Produced Locally in Ectopic Lymphoid Structures in Cardiac Allografts.

Cardiac allograft vasculopathy (CAV) is a transplant pathology, limiting graft survival after heart transplantation. CAV arteries are surrounded by ectopic lymphoid structures (ELS) containing B cells and plasma cells. The aim of this study was to characterize the antigenic targets of antibodies produced in ELS. Coronary arteries and surrounding epicardial tissue from 56 transplant recipients were collected during autopsy. Immunofluorescence was used to identify antibody-producing plasma cells. Immunoglobulin levels in tissue lysates were measured by enzyme-linked immunosorbent assay and analyzed for donor-specific HLA antibodies by Luminex assay. Cytokine and receptor expression levels were quantified using quantitative polymerase chain reaction. Plasma cells in ELS were polyclonal and produced IgG and/or IgM antibodies. In epicardial tissue, IgG (p < 0.05) and IgM levels were higher in transplant patients with larger ELS than smaller ELS. In 4 of 21 (19%) patients with ELS, donor-specific HLA type II antibodies were detected locally. Cytokine and receptor expression (CXCR3, interferon γ and TGF-ß) was higher in large ELS in the epicardial tissue than in other vessel wall layers, suggesting active recruitment and proliferation of T and B lymphocytes. ELS exhibited active plasma cells producing locally manufactured antibodies that, in some cases, were directed against the donor HLA, potentially mediating rejection with major consequences for the graft.

De novo alloreactive memory CD8+ T cells develop following allogeneic challenge when CNI immunosuppression is delayed.

Allospecific memory T cells are a recognized threat to the maintenance of solid-organ transplants. Limited information exists regarding the development of alloreactive memory T cells when post-transplant immunosuppression is present. The clinical practice of delaying calcineurin inhibitor (CNI) initiation post-transplant may permit the development of a de novo allospecific memory population. We investigated the development of de novo allospecific memory CD8+ T cells following the introduction of CNI immunosuppression in a murine model using allogeneic cell priming. Recipient mice alloprimed with splenocytes from fully mismatched donors received cyclosporine (CyA), initiated at 0, 2, 6, or 10days post-prime. Splenocytes from recipients were analyzed by flow cytometry or enzyme-linked immunosorbent assay for evidence of memory cell formation. Memory and effector CD8+ T cell development was prevented when CyA was initiated at 0day or 2days post-prime (p<0.001), but not 6days post-prime. Following a boost challenge, these memory CD8+ T cells were capable of producing a similarly sized population of secondary effectors as recipients not treated with CyA (p>0.05). Delaying CyA up to 6days or later post-prime permits the development of functional de novo allospecific memory CD8+ T cells. The development of this potentially detrimental T cell population in patients could be prevented by starting CNI immunosuppression early post-transplant.

Activation of innate immunity to reduce lung metastases in breast cancer.

Breast cancer continues to be one of the leading causes of cancer death in women. Mortality is primarily due to the development of metastases. Although therapies exist, they lack efficacy in preventing metastatic growth. As a result, novel agents are being investigated. In particular, treatments that target the immune system are being examined as potential anti-neoplastic agents. Cordyceps sinensis (Cs) is a fungus that has been used for over 2,000 years in China as a treatment for a variety of conditions including neoplasms. The available evidence suggests that efficacy of Cs as an anti-neoplastic therapeutic agent is related to a role as an activator of innate immune responses. The objectives of this study were: to investigate the ability of Cs to activate macrophages to produce factors that will induce protective responses against tumour growth; to study the ability of Cs to reduce primary tumour growth in vivo; and to examine the ability of Cs to reduce lung metastasis growth in vivo. We found that oral Cs does not reduce primary tumour growth but can reduce lung metastasis occurrence in a surgical excision model of metastatic mammary carcinoma. The evidence we have shown to date suggests that the reduction in metastases growth may be due to the effects of macrophage-derived factors on tumour cell cycle.

Selection of natural health products for clinical trials: a preclinical template.

In preparation for a clinical trial on the efficacy of Echinacea products with a pediatric population, a rational method for selection of test products was developed, based on phytochemical and bioassay evaluation. Ten currently available commercial products of Echinacea angustifolia (EA) or Echinacea purpurea (EP) were selected, and 3 bottles of each of 2 different lots were purchased for each product. Investigators were blinded to product identity before phytochemical analysis. Lot-to-lot variation was small, but product variation due to species and formulation was large. Products derived from ethanol extracts had low polysaccharide content and high levels of alkamides (EA), echinacoside (EA), cynarin (EA), cichoric acid (EP), and caftaric acid (EP). These products possessed high antiviral activities that differed between EA and EP products, but limited immune activation properties. In contrast, products derived without ethanol extraction had higher polysaccharide levels, but low levels of other components. These aqueous compounds showed immunostimulant activity as measured in a mouse macrophage model and a somewhat different antiviral profile. The choice of Echinacea product for clinical trial must therefore consider the impact of immune enhancement, the specific viral infection targeted, and the potential to reduce symptoms via antiinflammatory activity. Product selection may also depend on whether the intent of the trial is prophylaxis or treatment.

C. sinensis ablates allograft vasculopathy when used as an adjuvant therapy with cyclosporin A.

Immunosuppressive treatments are available to suppress acute cardiac rejection; however, no viable treatment exists for long-term cardiac graft failure. Moreover, extended use of calcineurin inhibitor immunosuppressants, the mainstay of the current therapeutic for cardiac transplantation, leads to significant associated pathologies such as nephrotoxicity and increased risk of cardiac disease. For the last ten years alternatives to calcineurin inhibitors, or adjuvant therapies designed to complement their activities, have been explored. In tandem with this development, there has been considerable interest in Traditional Chinese Medicines (TCM) as sources for novel therapeutics. Our study examines the ability of the TCM Cordyceps sinensis to reduce acute and chronic rejection associated with cardiac transplantation. The objectives of this study were to first determine if oral delivery of the extract could reduce acute rejection in a rat heterotopic heart model of transplantation. The second objective was to determine, in vitro, if a sterile, aqueous extract of C. sinensis could decrease CD8+ T cell activity. The third objective was to determine if oral delivery of the extract could ablate allograft vasculopathy in a mouse abdominal aortic transplant model. We found that oral delivery of the extract demonstrated a reduction in acute rejection when used in conjunction with a sub-therapeutic dose of Cyclosporine. Further, we found, using a mixed lymphocyte reaction, that the extract was able to significantly reduce CD8+ T cell activity. Finally, we demonstrate that oral delivery of the extract, used with a therapeutic dose of Cyclosporine to suppress acute rejection, ablates allograft vasculopathy.

Immune activation by a sterile aqueous extract of Cordyceps sinensis: mechanism of action.

Cordyceps sinensis is a fungus that has been used for over 2,000 years in China as a treatment for a variety of conditions including infectious diseases. The available evidence suggests a hypothesis that any efficacy of C. sinensis as an anti-infective therapeutic would be related to a role as an activator of innate immune responses. The objectives of this study were first to investigate the ability of C. sinensis to activate pro-inflammatory responses in macrophages in vitro and induce protective responses against intracellular pathogens in vivo, and second to characterize a method of action. We found that C. sinensis activates murine macrophages to produce a variety of pro-inflammatory cytokines. IFN-gamma synergizes with C. sinensis to amplify this response. Bacterial endotoxin contamination was ruled out as a potential artefact. The evidence presented in this study supports a hypothesis that C. sinensis activates macrophages by engaging Toll-like receptors and inducing mitogen-activated protein kinase (MAPK) pathways characteristic of inflammatory stimuli.

Efficacy of a standardized echinacea preparation (Echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Recently, echinacea has regained popularity as one of the treatments chosen most commonly by consumers with the expectation that it will reduce the severity and duration of the common cold. However, the results from a limited number of clinical trials for this application have thus far been inconclusive. This incongruity may be the result of investigators utilizing poorly standardized echinacea products, likely devoid of sufficient quantities of active constituents necessary to exert a definitive clinical effect. Therefore, a formulation containing alkamides, cichoric acid, and polysaccharides at concentrations of 0.25, 2.5, and 25 mg/mL, respectively, was prepared from freshly harvested Echinacea purpurea plants (commercially available as Echinilin, Natural Factors Nutritional Products, Inc., Vancouver, BC, Canada). The objective of this study was to test the efficacy of this highly standardized formulation in reducing the severity and duration of symptoms of a naturally acquired common cold. METHODS: In a randomized, double-blind, placebo-controlled trial, 282 subjects aged 18-65 years with a history of two or more colds in the previous year, but otherwise in good health, were recruited. The subjects were randomized to receive either echinacea or placebo. They were instructed to start the echinacea or placebo at the onset of the first symptom related to a cold, consuming 10 doses the first day and four doses per day on subsequent days for 7 days. Severity of symptoms (10-point scale: 0, minimum; 9, maximum) and dosing were recorded daily. A nurse examined the subjects on the mornings of days 3 and 8 of their cold. RESULTS: A total of 128 subjects contracted a common cold (59 echinacea, 69 placebo). The total daily symptom scores were found to be 23.1% lower in the echinacea group than in placebo in those who followed all elements of the study protocol (P<0.01). Throughout the treatment period, the response rate to treatments was greater in the echinacea group. A few adverse event profiles were observed in both groups. CONCLUSIONS: Early intervention with a standardized formulation of echinacea resulted in reduced symptom severity in subjects with naturally acquired upper respiratory tract infection. Further studies with larger patient populations appear to be warranted.