Toxicity profile of the immunomodulatory thalidomide analogue, lenalidomide : Phase I clinical trial of three dosing schedules in patients with solid malignancies

Thalidomide is an anti-angiogenic agent currently used to treat patients with malignant cachexia or multiple myeloma. Lenalidomide (CC-5013) is an immunomodulatory thalidomide analogue licensed in the United States of America (USA) for the treatment of a subtype of myelodysplastic syndrome. This two-centre, open-label phase I study evaluated dose-limiting toxicities in 55 patients with malignant solid tumours refractory to standard chemotherapies. Lenalidomide capsules were consumed once daily for 12 weeks according to one of the following three schedules: (I) 25 mg daily for the first 7 d, the daily dose increased by 25 mg each week up to a maximum daily dose of 150 mg; (II) 25 mg daily for 21 d followed by a 7-d rest period, the 4-week cycle repeated for 3 cycles; (III) 10 mg daily continuously. Twenty-six patients completed the study period. Two patients experienced a grade 3 hypersensitivity rash. Four patients in cohort I and 4 patients in cohort II suffered grade 3 or 4 neutropaenia. In 2 patients with predisposing medical factors, grade 3 cardiac dysrhythmia was recorded. Grade 1 neurotoxicity was detected in 6 patients. One complete and two partial radiological responses were measured by computed tomography scanning; 8 patients had stable disease after 12 weeks of treatment. Fifteen patients remained on treatment as named patients; 1 with metastatic melanoma remains in clinical remission 3.5 years from trial entry. This study indicates the tolerability and potential clinical efficacy of lenalidomide in patients with advanced solid tumours who have previously received multi-modality treatment. Depending on the extent of myelosuppressive pre-treatment, dose schedules (II) or (III) are advocated for large-scale trials of long-term administration. © 2006 Elsevier Ltd. All rights reserved.

Oral immunization with a novel lipid-based adjuvant protects against genital Chlamydia infection

Oral immunization is attractive as a delivery route because it is needle-free and useful for rapid mass vaccination programs to target pandemics or bioterrorism. This potential has not been realized for human vaccination, due to the requirement of large antigen doses and toxic (to humans) adjuvants to overcome the induction of oral tolerance and potential degradation of antigens in the stomach. To date, only oral vaccines based on live attenuated organisms have been approved for human use. In this study we describe the use of a lipid-based delivery system/adjuvant, Lipid C, for oral immunization to protect mice against genital tract chlamydial infection. Lipid C is formulated from food-grade purified and fractionated triglycerides. Bacterial shedding following vaginal challenge with Chlamydia muridarum was reduced by 50% in female mice orally immunized with the chlamydial major outer membrane protein (MOMP) formulated in Lipid C, protection equivalent to that seen in animals immunized with MOMP admixed with both cholera toxin (CT) and CpG oligodeoxynucleotides (CpG-ODN). Protection was further enhanced when MOMP, CT and CpG were all combined in the Lipid C matrix. Protection correlated with production of gamma interferon (IFN) by splenic T cells, a serum MOMP-specific IgG response and low but detectable levels of MOMP-specific IgA in vaginal lavage.

Transcutaneous immunization with a novel lipid-based adjuvant protects against Chlamydia genital and respiratory infections

Mucosal adjuvants are important to overcome the state of immune tolerance normally associated with mucosal delivery and to enhance adaptive immunity to often-weakly immunogenic subunit vaccine antigens. Unfortunately, adverse side effects of many experimental adjuvants limit the number of adjuvants approved for vaccination. Lipid C is a novel, non-toxic, lipid oral vaccine-delivery formulation, developed originally for oral delivery of the live Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine. In the present study, murine models of chlamydial respiratory and genital tract infections were used to determine whether transcutaneous immunization (TCI) with Lipid C-incorporated protein antigens could elicit protective immunity at the genital and respiratory mucosae. BALB/c mice were immunized transcutaneously with Lipid C containing the chlamydial major outer membrane protein (MOMP), with and without addition of cholera toxin and CpG-ODN 1826 (CT/CpG). Both vaccine combinations induced mixed cell-mediated and mucosal antibody immune responses. Immunization with Lipid C-incorporated MOMP (Lipid C/MOMP), either alone or with CT/CpG resulted in partial protection following live challenge with Chlamydia muridarum as evidenced by a significant reduction in recoverable Chlamydia from both the genital secretions and lung tissue. Protection induced by immunization with Lipid C/MOMP alone was not further enhanced by the addition of CT/CpG. These results highlight the potential of Lipid C as a novel mucosal adjuvant capable of targeting multiple mucosal surfaces following TCI. Protection at both the respiratory and genital mucosae was achieved without the requirement for potentially toxic adjuvants, suggesting that Lipid C may provide a safe effective mucosal adjuvant for human vaccination.

Immunity against a Chlamydia infection and disease may be determined by a balance of IL-17 signaling

Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG- oligodeoxynucleotide–CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.

Immunization with a MOMP-based vaccine protects mice against a Pulmonary Chlamydia challenge and identifies a disconnection between infection and pathology

Chlamydia pneumoniae is responsible for up to 20% of community acquired pneumonia and can exacerbate chronic inflammatory diseases. As the majority of infections are either mild or asymptomatic, a vaccine is recognized to have the greatest potential to reduce infection and disease prevalence. Using the C. muridarum mouse model of infection, we immunized animals via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, with recombinant chlamydial major outer membrane protein (MOMP) combined with adjuvants CTA1-DD or a combination of cholera toxin/CpG-oligodeoxynucleotide (CT/CpG). Vaccinated animals were challenged IN with C. muridarum and protection against infection and pathology was assessed. SL and TC immunization with MOMP and CT/CpG was the most protective, significantly reducing chlamydial burden in the lungs and preventing weight loss, which was similar to the protection induced by a previous live infection. Unlike a previous infection however, these vaccinations also provided almost complete protection against fibrotic scarring in the lungs. Protection against infection was associated with antigen-specific production of IFNγ, TNFα and IL-17 by splenocytes, however, protection against both infection and pathology required the induction of a similar pro-inflammatory response in the respiratory tract draining lymph nodes. Interestingly, we also identified two contrasting vaccinations capable of preventing infection or pathology individually. Animals IN immunized with MOMP and either adjuvant were protected from infection, but not the pathology. Conversely, animals TC immunized with MOMP and CTA1-DD were protected from pathology, even though the chlamydial burden in this group was equivalent to the unimmunized controls. This suggests that the development of pathology following an IN infection of vaccinated animals was independent of bacterial load and may have been driven instead by the adaptive immune response generated following immunization. This identifies a disconnection between the control of infection and the development of pathology, which may influence the design of future vaccines.

Langerhans cells in the cornea of diabetic patients : relevance to diabetic neuropathy

Purpose The presence of a lymphocytic infiltration in autonomic ganglia and an increased prevalence of autoantibodies and iritis in diabetic patients with autonomic neuropathy suggests a role for autoimmune mechanisms in the development of diabetic and perhaps somatic neuropathy. Corneal Langerhans cells are antigenpresenting cells which can be identified in corneal immunologic conditions using in-vivo confocal microscopy. The aim of this study was to assess the presence and density of Langerhans cells (LCs) in Bowman’s layer of the cornea in diabetic patients with varying degrees of neuropathy compared to healthy control subjects. Method 128 diabetic patients aged 58±1 years with differing severity of neuropathy (NDS – 4.7±0.28) and 26 control subjects aged 53±3 years were examined with in-vivo corneal confocal microscopy to quantify the density of “Langerhans cells” (LCs). Results LCs were observed more often in diabetic patients (73.8%) compared to control subjects (46.1%), P = 0.001. The LC density (number/mm2) was also significantly increased in diabetic patients (17.73±1.45) compared to control subjects (6.94±1.58, P = 0.001). There was a significant correlation between the density of LCs with age (r = 0.162, P = 0.047) and severity of neuropathy assessed by NDS (r =−0.202, P = 0.02). Conclusions In vivo corneal confocal microscopy enables quantification of Langerhans cells in Bowman’s layer of the cornea. There is a relationship between density of LCs and the degree of nerve damage. Corneal confocal microscopy could be a valuable tool to establish the role of immune mediated corneal nerve damage and provide insights into the pathogenesis of diabetic neuropathy.

Myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones include double-positive CD8+CD4+ T cells : evidence from myeloma-bearing mouse model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8alphaalpha and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-gamma and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.

Impaction of spray droplets on leaves: Influence of formulation and leaf character on shatter, bounce and adhesion

This paper combines experimental data with simple mathematical models to investigate the influence of spray formulation type and leaf character (wettability) on shatter, bounce and adhesion of droplets impacting with cotton, rice and wheat leaves. Impaction criteria that allow for different angles of the leaf surface and the droplet impact trajectory are presented; their predictions are based on whether combinations of droplet size and velocity lie above or below bounce and shatter boundaries. In the experimental component, real leaves are used, with all their inherent natural variability. Further, commercial agricultural spray nozzles are employed, resulting in a range of droplet characteristics. Given this natural variability, there is broad agreement between the data and predictions. As predicted, the shatter of droplets was found to increase as droplet size and velocity increased, and the surface became harder to wet. Bouncing of droplets occurred most frequently on hard to wet surfaces with high surface tension mixtures. On the other hand, a number of small droplets with low impact velocity were observed to bounce when predicted to lie well within the adhering regime. We believe this discrepancy between the predictions and experimental data could be due to air layer effects that were not taken into account in the current bounce equations. Other discrepancies between experiment and theory are thought to be due to the current assumption of a dry impact surface, whereas, in practice, the leaf surfaces became increasingly covered with fluid throughout the spray test runs.

Serum levels of soluble receptor for advanced glycation end products and of S100 proteins are associated with inflammatory, autoantibody, and classical risk markers of joint and vascular damage in rheumatoid arthritis

Introduction: The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptor molecules. High concentrations of three of its putative proinflammatory ligands, S100A8/A9 complex (calprotectin), S100A8, and S100A12, are found in rheumatoid arthritis (RA) serum and synovial fluid. In contrast, soluble RAGE (sRAGE) may prevent proinflammatory effects by acting as a decoy. This study evaluated the serum levels of S100A9, S100A8, S100A12 and sRAGE in RA patients, to determine their relationship to inflammation and joint and vascular damage. Methods: Serum sRAGE, S100A9, S100A8 and S100A12 levels from 138 patients with established RA and 44 healthy controls were measured by ELISA and compared by unpaired t test. In RA patients, associations with disease activity and severity variables were analyzed by simple and multiple linear regressions. Results: Serum S100A9, S100A8 and S100A12 levels were correlated in RA patients. S100A9 levels were associated with body mass index (BMI), and with serum levels of S100A8 and S100A12. S100A8 levels were associated with serum levels of S100A9, presence of anti-citrullinated peptide antibodies (ACPA), and rheumatoid factor (RF). S100A12 levels were associated with presence of ACPA, history of diabetes, and serum S100A9 levels. sRAGE levels were negatively associated with serum levels of C-reactive protein (CRP) and high-density lipoprotein (HDL), history of vasculitis, and the presence of the RAGE 82Ser polymorphism. Conclusions: sRAGE and S100 proteins were associated not just with RA inflammation and autoantibody production, but also with classical vascular risk factors for end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had the opposite effects to S100 proteins in that S100 proteins were associated with autoantibodies and vascular risk, whereas sRAGE was associated with protection against joint and vascular damage. These data suggest that RAGE activity influences co-development of joint and vascular disease in rheumatoid arthritis patients.

Specific IgE recognition of pollen allergens from subtropic grasses in patients from the subtropics

Background Pollens of subtropical grasses, Bahia (Paspalum notatum), Johnson (Sorghum halepense), and Bermuda (Cynodon dactylon), are common causes of respiratory allergies in subtropical regions worldwide. Objective To evaluate IgE cross-reactivity of grass pollen (GP) found in subtropical and temperate areas. Methods Case and control serum samples from 83 individuals from the subtropical region of Queensland were tested for IgE reactivity with GP extracts by enzyme-linked immunosorbent assay. A randomly sampled subset of 21 serum samples from patients with subtropical GP allergy were examined by ImmunoCAP and cross-inhibition assays. Results Fifty-four patients with allergic rhinitis and GP allergy had higher IgE reactivity with P notatum and C dactylon than with a mixture of 5 temperate GPs. For 90% of 21 GP allergic serum samples, P notatum, S halepense, or C dactylon specific IgE concentrations were higher than temperate GP specific IgE, and GP specific IgE had higher correlations of subtropical GP (r = 0.771-0.950) than temperate GP (r = 0.317-0.677). In most patients (71%-100%), IgE with P notatum, S halepense, or C dactylon GPs was inhibited better by subtropical GP than temperate GP. When the temperate GP mixture achieved 50% inhibition of IgE with subtropical GP, there was a 39- to 67-fold difference in concentrations giving 50% inhibition and significant differences in maximum inhibition for S halepense and P notatum GP relative to temperate GP. Conclusion Patients living in a subtropical region had species specific IgE recognition of subtropical GP. Most GP allergic patients in Queensland would benefit from allergen specific immunotherapy with a standardized content of subtropical GP allergens.

The enigma of IgE+ B-cell memory in human subjects

Our understanding of the origin and fate of the IgE-switched B cell has been markedly improved by studies in mouse models. The immediate precursor of the IgE-switched B cell is either a relatively naive nonswitched B cell or a mature IgG-switched B cell. These 2 routes are referred to as the direct and indirect pathways, respectively. IgE responses derived from each pathway differ significantly, largely reflecting the difference in time spent in a germinal center and thus time for clonal expansion, somatic hypermutation, affinity maturation, and acquisition of a memory phenotype. The clinical and therapeutic implications for IgE responses in human subjects are still a matter of debate, largely because the immunization procedures used in the animal models are significantly different from classical atopic sensitization to allergens from pollen and mites. On the basis of the limited information available, it seems likely that these atopic IgE responses are characterized by a relatively low IgG/IgE ratio, low B-cell memory, and modest affinity maturation, which fits well with the direct switching pathway. It is still unresolved how the IgE response evolves to cover a wide epitope repertoire involving many epitopes per allergen, as well as many different allergens from a single allergen source. © 2013 American Academy of Allergy, Asthma & Immunology.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Genetic aspects of susceptibility, severity, and clinical expression in ankylosing spondylitis

While twin studies have previously demonstrated high heritability of susceptibility to ankylosing spondylitis (AS), it is only recently that the involvement of genetic factors in determining the severity of the disease has been demonstrated. The genes involved in determining the rate of ankylosis in AS are likely to be different from those involved in the underlying immunologic events, and represent important potential targets for treatment of AS. This article will describe the progress that has been made in the genetic epidemiology of AS, and in identifying the genes involved.

Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.

The carbon dioxide hydration activity of the sulfonamide-resistant carbonic anhydrase from the liver of male rat: pH independence of the steady-state kinetics

The steady-state kinetic constants for the catalysis of CO2 hydration by the sulfonamide-resistant and testosterone-induced carbonic anhydrase from the liver of the male rat has been determined by stopped-flow spectrophotometry. The turnover number was 2.6 ± 0.6 × 103 s− at 25 °C, and was invariant with pH ranging from 6.2 to 8.2 within experimental error. The Km at 25 °C was 5 ± 1 mImage , and was also pH independent. These data are in quantitative agreement with earlier findings of pH-independent CO2 hydration activity for the mammalian skeletal muscle carbonic anhydrase isozyme III. The turnover numbers for higher-activity isozymes I and II are strongly pH dependent in this pH range. Thus, the kinetic status of the male rat liver enzyme is that of carbonic anhydrase III. This finding is consistent with preliminary structural and immunologic data from other laboratories.