Staphylococcus δ-toxin induces allergic skin disease by activating mast cells.

Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

A synthetic peptide vaccine directed against the 2ß2-2ß3 loop of domain 2 of protective antigen protects rabbits from inhalation anthrax.

The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.

Cancer Carepartners: Improving patients' symptom management by engaging informal caregivers.

BACKGROUND: Previous studies have found that cancer patients undergoing chemotherapy can effectively manage their own symptoms when given tailored advice. This approach, however, may challenge patients with poor performance status and/or emotional distress. Our goal is to test an automated intervention that engages a friend or family member to support a patient through chemotherapy. METHODS/DESIGN: We describe the design and rationale of a randomized, controlled trial to assess the efficacy of 10 weeks of web-based caregiver alerts and tailored advice for helping a patient manage symptoms related to chemotherapy. The study aims to test the primary hypothesis that patients whose caregivers receive alerts and tailored advice will report less frequent and less severe symptoms at 10 and 14 weeks when compared to patients in the control arm; similarly, they will report better physical function, fewer outpatient visits and hospitalizations related to symptoms, and greater adherence to chemotherapy. 300 patients with solid tumors undergoing chemotherapy at two Veteran Administration oncology clinics reporting any symptom at a severity of ≥4 and a willing informal caregiver will be assigned to either 10 weeks of automated telephonic symptom assessment (ATSA) alone, or 10 weeks of ATSA plus web-based notification of symptom severity and problem solving advice to their chosen caregiver. Patients and caregivers will be surveyed at intake, 10 weeks and 14 weeks. Both groups will receive standard oncology, hospice, and palliative care. DISCUSSION: Patients undergoing chemotherapy experience many symptoms that they may be able to manage with the support of an activated caregiver. This intervention uses readily available technology to improve patient caregiver communication about symptoms and caregiver knowledge of symptom management. If successful, it could substantially improve the quality of life of veterans and their families during the stresses of chemotherapy without substantially increasing the cost of care. TRIAL REGISTRATION: NCT00983892.

Genetic vaccines for anthrax based on recombinant adeno-associated virus vectors.

Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments encoding activated PA (PA63), or LF, were synthesized and cloned into optimized rAAV1 vectors containing a human cytomegalovirus (hCMV) promoter and synthetic optimized leader. Serum from rabbits immunized intramuscularly with rAAV1/PA (monovalent), rAAV1/LF (monovalent), rAAV1/PA + rAAV1/LF (bivalent), or rAAV1/enhanced green fluorescent protein (control) exhibited substantial PA- and LF-specific antibody responses at 4 weeks by both western blot (> 1:10,000 dilution) and enzyme-linked immunosorbent assay (ELISA) (mean end-point titer: 32,000-260,000), and contained anthrax LeTx-neutralizing activity in vitro, with peak titers approximating those of a rabbit hyperimmune antisera raised against soluble PA and LF. Compared to the monovalent groups (rAAV1/PA or rAAV1/LF), the bivalent group (rAAV1/PA + rAAV1/LF) exhibited marginally higher ELISA and neutralization activity with dual specificity for both PA and LF. The finding of robust neutralizing antibody responses after a single injection of these rAAV1-based vectors supports their further development as candidate anthrax vaccines.

A heterologous helper T-cell epitope enhances the immunogenicity of a multiple-antigenic-peptide vaccine targeting the cryptic loop-neutralizing determinant of Bacillus anthracis protective antigen.

We previously showed that a multiple antigenic peptide (MAP) displaying amino acids (aa) 305 to 319 from the 2beta2-2beta3 loop of protective antigen (PA) can elicit high-titered antibody that neutralizes lethal toxin (LeTx) in vitro and that this loop-neutralizing determinant (LND) specificity is absent in PA-immune rabbits. Some immune rabbits were, however, nonresponders to the MAP. We hypothesized that the immunogen elicited suboptimal major histocompatibility complex (MHC) class II-restricted T-cell help and that introduction of a functional helper T-cell epitope would increase MHC-restricted responsiveness and the magnitude and affinity of the antibody responses. In the current study, we characterized the T- and B-cell responses to LND peptides in mice, then designed second-generation MAP immunogens for eliciting LND-specific immunity, and tested them in rabbits. The 305-319 sequence was devoid of helper T-cell epitopes in three strains of mice; however, a T-B peptide comprising aa 305 to 319, colinearly synthesized with the P30 helper epitope of tetanus toxin, elicited robust LeTx-neutralizing immunity in mice. T-B MAPs displaying B-cell epitopes 304 to 319 (MAP304) or 305 to 319 (MAP305) elicited high-titer, durable antibody responses in rabbits which exhibited potent neutralization of LeTx in vitro. All MAP304-immune rabbits demonstrated neutralization titers exceeding that of hyperimmune sera of rabbits immunized with PA in Freund's adjuvant, with peak neutralization titers 23-, 6-, and 3-fold higher than that of the PA antiserum. Overall, immunization with MAPs containing the P30 epitope elicited higher antibody and toxin neutralization titers and peptide-specific affinity than immunization with an LND MAP lacking a helper epitope. P30-containing MAP304 represents a promising LND-specific vaccine for anthrax.

Synthetic peptide vaccine targeting a cryptic neutralizing epitope in domain 2 of Bacillus anthracis protective antigen.

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2beta2-2beta3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region. Overall, four out of six rabbits vaccinated with the MAP peptide in Freund's adjuvant developed high-titer, high-avidity antibody responses which cross-reacted with the immobilized peptide sequence comprising aa 305 to 319 and with PA, as determined by an enzyme-linked immunosorbent assay, and which displayed potent and durable neutralization of lethal toxin (LeTx) in vitro, with peak titers which were 452%, 100%, 67%, and 41% of the peak neutralization titers observed in positive-control rabbits immunized with PA. Importantly, analysis of sera from multiple cohorts of rabbits with high-titer immunity to PA demonstrated a virtual absence of this potent antibody specificity, and work by others suggests that this specificity may be present at only low levels in primate PA antiserum. These results highlight the potential importance of this immunologically cryptic neutralizing epitope from PA as a target for alternative and adjunctive vaccines for anthrax.

The predictive role of plasma TGF-beta1 during radiation therapy for radiation-induced lung toxicity deserves further study in patients with non-small cell lung cancer.

BACKGROUND: This study aimed to further investigate the role of circulating TGF-beta1 during radiation therapy (RT) in predicting radiation-induced lung toxicity (RILT). METHODS AND MATERIALS: Patients with stages I-III non-small cell lung cancer treated with RT based therapy were included in this study. Platelet poor plasma was obtained pre-RT, at 2 and 4 weeks during-RT, and at the end of RT. TGF-beta1 was measured using an enzyme-linked immunosorbent assay. The primary endpoint for RILT was >or=grade 2 radiation pneumonitis or fibrosis. RESULTS: Twenty-six patients with a minimum follow-up of 12 months were included. Six patients (23.1%) experienced >or=grade 2 RILT. There was no significant difference in absolute TGF-beta1 levels pre-RT, at 2 and 4 weeks during-RT, or at the end of RT between patients with and without RILT. The TGF-beta1 ratios (over the pre-RT levels) for patients with and without RILT at 2, 4 weeks during-, and the end of RT were 2.8+/-2.2 and 1.0+/-0.6 (P=0.123), 2.3+/-1.3 and 0.8+/-0.5 (P=0.001), 1.5+/-0.9 and 0.8+/-0.5 (P=0.098), respectively. Using 2.0 as a cut-off, the TGF-beta1 ratio at 4 weeks during-RT predicted RILT with a sensitivity and specificity of 66.7% and 95.0%, respectively. CONCLUSION: Elevation of plasma TGF-beta1 level 4 weeks during-RT is significantly predictive of RILT. The role of plasma TGF-beta1 in predicting RILT deserves further study.

Effect of Midtreatment PET/CT-Adapted Radiation Therapy With Concurrent Chemotherapy in Patients With Locally Advanced Non-Small-Cell Lung Cancer: A Phase 2 Clinical Trial.

IMPORTANCE: Our previous studies demonstrated that tumors significantly decrease in size and metabolic activity after delivery of 45 Gy of fractionated radiatiotherapy (RT), and that metabolic shrinkage is greater than anatomic shrinkage. This study aimed to determine whether 18F-fludeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) acquired during the course of treatment provides an opportunity to deliver higher-dose radiation to the more aggressive areas of the tumor to improve local tumor control without increasing RT-induced lung toxicity (RILT), and possibly improve survival. OBJECTIVE: To determine whether adaptive RT can target high-dose radiation to the FDG-avid tumor on midtreatment FDG-PET to improve local tumor control of locally advanced non-small-cell lung cancer (NSCLC). DESIGN, SETTING, AND PARTICIPANTS: A phase 2 clinical trial conducted at 2 academic medical centers with 42 patients who had inoperable or unresectable stage II to stage III NSCLC enrolled from November 2008, to May 2012. Patients with poor performance, more than 10% weight loss, poor lung function, and/or oxygen dependence were included, providing that the patients could tolerate the procedures of PET scanning and RT. INTERVENTION: Conformal RT was individualized to a fixed risk of RILT (grade >2) and adaptively escalated to the residual tumor defined on midtreatment FDG-PET up to a total dose of 86 Gy in 30 daily fractions. Medically fit patients received concurrent weekly carboplatin plus paclitaxel followed by 3 cycles of consolidation. MAIN OUTCOMES AND MEASURES: The primary end point was local tumor control. The trial was designed to achieve a 20% improvement in 2-year control from 34% of our prior clinical trial experience with 63 to 69 Gy in a similar patient population. RESULTS: The trial reached its accrual goal of 42 patients: median age, 63 years (range, 45-83 years); male, 28 (67%); smoker or former smoker, 39 (93%); stage III, 38 (90%). Median tumor dose delivered was 83 Gy (range, 63-86 Gy) in 30 daily fractions. Median follow-up for surviving patients was 47 months. The 2-year rates of infield and overall local regional tumor controls (ie, including isolated nodal failure) were 82% (95% CI, 62%-92%) and 62% (95% CI, 43%-77%), respectively. Median overall survival was 25 months (95% CI, 12-32 months). The 2-year and 5-year overall survival rates were 52% (95% CI, 36%-66%) and 30% (95% CI, 16%-45%), respectively. CONCLUSIONS AND RELEVANCE: Adapting RT-escalated radiation dose to the FDG-avid tumor detected by midtreatment PET provided a favorable local-regional tumor control. The RTOG 1106 trial is an ongoing clinical trial to validate this finding in a randomized fashion. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01190527.

Identification and validation of a linear protective neutralizing epitope in the ß-pore domain of alpha toxin.

The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric ß-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2ß2-2ß3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the ß-pore of S. aureus alpha toxin that share structural and functional homology to ß-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha toxin could facilitate the development of an epitope-focused vaccine against S. aureus.

Anthrax vaccine recipients lack antibody against the loop neutralizing determinant: A protective neutralizing epitope from Bacillus anthracis protective antigen.

BACKGROUND: Epitope-focused immunogens can elicit antibody against the loop neutralizing determinant (LND), a neutralizing epitope found within the 2ß2-2ß3 loop of protective antigen (PA), which can protect rabbits from high-dose inhalation challenge with Bacillus anthracis Ames strain. Interestingly, data suggests that this epitope is relatively immunosilent in rabbits and non-human primates immunized with full length PA. METHODS: To determine whether the LND is immunosilent among humans vaccinated with PA, we screened antisera from AVA- or placebo-vaccinees from a clinical trial for antibody reactive with the LND. RESULTS: AVA-vaccinee sera had significant PA-specific antibody compared to placebo-vaccinee sera; however, sera from the two cohorts were indistinguishable with regard to the frequency of individuals with antibody specific for the LND. CONCLUSIONS: AVA-vaccinees have a low frequency of antibody reactive with the LND. As with rabbits and non-human primates, the elicitation of LND-specific antibody in humans appears to require immunization with an epitope-focused vaccine.

Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax.

BACKGROUND: Anthrax represents a formidable bioterrorism threat for which new, optimized vaccines are required. We previously demonstrated that epitope-focused multiple antigenic peptides or a recombinant protein in Freund's adjuvant can elicit Ab against the loop neutralizing determinant (LND), a cryptic linear neutralizing epitope in the 2ß2-2ß3 loop of protective antigen from Bacillus anthracis, which mediated protection of rabbits from inhalation challenge with B. anthracis Ames strain. However, demonstration of efficacy using human-use adjuvants is required before proceeding with further development of an LND vaccine for testing in non-human primates and humans. METHODS: To optimize the LND immunogen, we first evaluated the protective efficacy and immune correlates associated with immunization of rabbits with mixtures containing two molecular variants of multiple antigenic peptides in Freunds adjuvant, termed BT-LND(2) and TB-LND(2). TB-LND(2) was then further evaluated for protective efficacy in rabbits employing human-use adjuvants. RESULTS: Immunization of rabbits with TB-LND(2) in human-use adjuvants elicited protection from Ames strain spore challenge which was statistically indistinguishable from that elicited through immunization with protective antigen. All TB-LND(2) rabbits with any detectable serum neutralization prior to challenge were protected from aerosolized spore exposure. Remarkably, rabbits immunized with TB-LND(2) in Alhydrogel/CpG had significant anamnestic increases in post-challenge LND-specific Ab and neutralization titers despite little evidence of spore germination in these rabbits. CONCLUSIONS: An LND-specific epitope-focused vaccine may complement PA-based vaccines and may represent a complementary stand-alone vaccine for anthrax.

In vivo mapping of a protective linear neutralizing epitope at the N-terminus of alpha hemolysin from Staphylococcus aureus.

Staphylococcus aureus is responsible for a large and diverse burden of human disease associated with significant morbidity and mortality. The dynamic challenge of this pathogen is exemplified by the emergence of highly virulent community-associated methicillin-resistant S. aureus strain USA300, which threatens both healthy and vulnerable individuals and constitutes a public health imperative in the United States. Though S. aureus employs many virulence factors that enable infectivity and evasion of host defenses, evidence suggests that the increased production of alpha hemolysin may be a critical contributor to the increased virulence of USA300. To enable and inform immunological targeting of alpha hemolysin, we sought to precisely map a neutralizing epitope that we hypothesized existed in the N-terminal domain. Using an in vivo mapping strategy employing peptide immunogens and an optimized in vitro toxin neutralization assay, we identified a linear neutralizing determinant in the N-terminal 19 amino acids of alpha hemolysin. Affinity purified rabbit antibody against this neutralizing epitope was shown to be highly effective at mitigating dermonecrosis in inbred and outbred mice challenged with USA300. To our knowledge, this is the first description of a linear neutralizing epitope in alpha hemolysin, and the delineation of this determinant should inform and facilitate the rational design and development of an efficacious, epitope-focused or multivalent vaccine against S. aureus.

Timing and intensity of changes in FDG uptake with symptomatic esophagitis during radiotherapy or chemo-radiotherapy.

PURPOSE: To study whether esophageal FDG activity changes by time of mid-course of fractionated radiotherapy (RT), and whether these changes are associated with radiation esophagitis in patients with non-small cell lung cancer (NSCLC). METHODS: Fifty patients with stage I-III NSCLC were enrolled prospectively and, all received ≥60 Gy RT. FDG-PET/CT scans were acquired prior to, and during-RT after delivery of 45 Gy. Normalized standardized uptake values (NSUV), defined by the esophageal maximum SUV relative to intravascular background level in the aortic arch, were sampled in the esophagus at the level of the primary tumor, sternal notch, aortic arch, carina, and gastro-esophageal junction. Symptomatic radiation esophagitis was defined as an event. RESULTS: Compared to baseline, esophageal NSUV increased significantly during-RT at the level of the primary tumor (1.09 ± 0.05 vs.1.28 ± 0.06, p = 0.001), but did not change at other levels in the esophagus. 16 patients had radiation esophagitis events and these patients had significantly higher during-RT to baseline NSUV ratios than those without esophagitis (1.46 ± 0.12, 95% CI 1.20-1.71; vs. 1.11 ± 0.05, 95% CI 1.01-1.21, p = 0.002). Maximum esophageal dose (p = 0.029), concurrent chemotherapy (p = 0.022) and esophageal FDG PET NSUV ratio (during-RT to baseline, p = 0.007), were independent factors associated with esophagitis and area under curves (AUC) were 0.76, 0.70 and 0.78, respectively. Combining esophageal maximum dose and FDG PET NSUV Ratio at the tumor level increased AUC to 0.85 (p = 0.016). CONCLUSION: FDG uptake increased in esophagus during-RT and this increase may predict radiation esphagitis during later course of treatment.

Recombinant vaccine displaying the loop-neutralizing determinant from protective antigen completely protects rabbits from experimental inhalation anthrax.

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2ß2-2ß3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD(50)) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines.

A CD20 tandem-epitope immunogen elicits antibody in mice that binds murine cell surface CD20 and depletes splenic B cells in vivo.

CD20 is an important target for monoclonal antibody therapy of B-cell malignancies and for some autoimmune disorders. Though there is interest in evaluating the induction of active immunity to CD20 in the mouse model, the CD20 extracellular domain (ECD) has significant secondary and tertiary structure which make it difficult to target using peptide immunogens. We constructed, expressed, and purified a recombinant immunogen, CD20ECD-6, which displays six tandemly repeated copies of the C-terminus of the murine CD20 ECD covalently linked to maltose-binding protein. Analysis of the purified protein suggested a complex conformation as the protein migrated in significantly retarded fashion by SDS-PAGE analysis. Immunization of mice and rabbits with the CD20ECD-6 led to the induction of antibodies reactive with the C-terminal ECD peptide sequence by ELISA and more importantly, with native cell surface CD20 on the murine B-cell lymphomas, 38C13 and A20. Immunoprecipitation using the rabbit antisera and non-denaturing detergent confirmed the identity of the bound cell surface protein as murine CD20 and suggested that the cell-binding antibodies were specific for the native, folded conformation. Finally, immunization of mice with the CD20ECD-6 using Freund's or QS-21 adjuvants was shown to exert significant biological effects in vivo with the pronounced depletion of splenic B cells. The tandem-epitope immunogen represents a promising tool for eliciting and studying active autoimmunity to CD20, as a basis for potential development of new immunotherapeutics for cancer and autoimmune diseases.

Comparative survival in patients with postresection recurrent versus newly diagnosed non-small-cell lung cancer treated with radiotherapy.

PURPOSE: To compare the survival of postresection recurrent vs. newly diagnosed non-small-cell lung cancer (NSCLC) patients treated with radiotherapy or chemoradiotherapy. METHODS AND MATERIALS: The study population consisted of 661 consecutive patients with NSCLC registered in the radiation oncology databases at two medical centers in the United States between 1992 and 2004. Of the 661 patients, 54 had postresection recurrent NSCLC and 607 had newly diagnosed NSCLC. Kaplan-Meier and Cox regression models were used for the survival analyses. RESULTS: The distribution of relevant clinical factors between these two groups was similar. The median survival time and 5-year overall survival rates were 19.8 months (95% confidence interval [CI], 13.9-25.7) and 14.8% (95% confidence interval, 5.4-24.2%) vs. 12.2 months (95% CI, 10.8-13.6) and 11.0% (95% CI, 8.5-13.5%) for recurrent vs. newly diagnosed patients, respectively (p = .037). For Stage I-III patients, no significant difference was observed in the 5-year overall survival (p = .297) or progression-free survival (p = .935) between recurrent and newly diagnosed patients. For the 46 patients with Stage I-III recurrent disease, multivariate analysis showed that chemotherapy was a significant prognostic factor for 5-year progression-free survival (hazard ratio, 0.45; 95% CI, 0.224-0.914; p = .027). CONCLUSION: Our institutional data have shown that patients with postresection recurrent NSCLC achieved survival comparable to that of newly diagnosed NSCLC patients when they were both treated with radiotherapy or chemoradiotherapy. These findings suggest that patients with postresection recurrent NSCLC should be treated as aggressively as those with newly diagnosed disease.

Low-dose intraperitoneal Freund's adjuvant: toxicity and immunogenicity in mice using an immunogen targeting amyloid-beta peptide.

Complete Freund's adjuvant (CFA) is effective for potentiating immune responses in mice when administered subcutaneously, and is often more potent when given intraperitoneally (i.p.). However, the the potential toxicity of i.p. administration in mice has led investigators and Institutional Animal Care and Use committees to increasingly view the use of CFA i.p. with reservation. We evaluated whether an 80% reduction in the dose of CFA administered i.p. to mice, compared to the i.p. doses used in a previous analysis, could abrogate the untoward effects associated with its use, while still maintaining adjuvanticity. Using a novel immunogen targeting the N-terminus of the 42-amino acid amyloid-beta peptide, we compared low dose CFA administered i.p., with three other commonly used adjuvants given i.p.: alum, incomplete Freunds adjuvant (IFA) and monophoshoryl lipid A + trehalose dicorynomycolate (MPL + TDM). The results of the study showed that, though the reduction in intraperitoneal dose of CFA mitigated transient weight loss and leukocytosis observed previously with higher doses of i.p. CFA, all mice administered CFA or IFA i.p. developed abdominal adhesions and granulomatous peritonitis. Mice from all adjuvant groups, however, appeared to tolerate the respective adjuvants well and excellent comparative immunogenicity was observed in mice immunized with the Freunds and MPL + TDM adjuvants. Consequently, we conclude that though a high-titered, humoral response may be generated using low dose CFA administered i.p., the accompanying toxicity remains significant, and thus alternative adjuvants and/or routes should be considered.