A Case Report of Cardiogenic Syncope Due to Loperamide Abuse: Acute Presentation and Novel Use of Buprenorphine.

INTRODUCTION: Loperamide is a non-prescription anti-diarrheal agent targeting µ-opioid receptors in the intestinal tract. At high doses it crosses the blood-brain barrier, where µ-opioid agonism can cause euphoric effects. Misuse has been increasing for both the euphoric effects and as an alternative treatment for opioid dependence and withdrawal. CASE REPORT: Here we report the case of a 30-year-old woman presenting with syncope, who was found to have severe myocardial conduction delays in the setting of chronic loperamide abuse. CONCLUSION: Treatment with sodium bicarbonate and hypertonic sodium resulted in improvement of her conduction abnormalities. Prior to discharge she was initiated on buprenorphine for her opioid use disorder.

Combination of cyclophosphamide, rituximab, and intratumoral CpG oligodeoxynucleotide successfully eradicates established B cell lymphoma.

Rituximab plus chemotherapy is standard therapy for patients with non-Hodgkin B cell lymphoma, but often complete response or cure is not achieved. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG) can improve antibody-dependent cellular cytotoxicity and adaptive antitumor immune responses. Using a syngeneic murine B cell lymphoma expressing human CD20 (38C13-huCD20), we previously demonstrated that rituximab plus intratumoral CpG, but not systemic CpG, could eradicate up to half of 7-day established 38C13-huCD20 tumors. However, larger 10-day established tumors could not be cured with this regimen. We thus hypothesized that cytoreduction with cyclophosphamide (Cy) before immunotherapy might permit eradication of these more advanced tumor burdens. Pretreatment with Cy resulted in tumor eradication from 83% of animals treated with rituximab/CpG, whereas Cy/CpG or Cy/rituximab treatments only cured 30% or 17%, respectively (P<0.005). Tumor eradication depended on natural killer cells, but not T cells, macrophages, or complement. Only mice treated with Cy/rituximab/CpG partially resisted rechallenge with tumor cells. Foxp3 Treg and CD11bGr1 myeloid suppressor cells persisted within lymphoid organs after therapy, possibly influencing the ability to establish adaptive tumor immunity. In conclusion, cytoreduction with Cy permitted the cure of large, established lymphomas not otherwise responsive to rituximab plus intratumoral CpG immunotherapy.

Analysis of Fcγ receptor IIIa and IIa polymorphisms: lack of correlation with outcome in trastuzumab-treated breast cancer patients.

PURPOSE: The mechanisms by which trastuzumab imparts clinical benefit remain incompletely understood. Antibody-dependent cellular cytotoxicity via interactions with Fcγ receptors (FcγR) on leukocytes may contribute to its antitumor effects. Single-nucleotide polymorphisms (SNP) in FCGR3A and FCGR2A genes lead to amino acid substitutions at positions 158 and 131, respectively, and affect binding of antibodies to FcγR such that 158V/V and 131H/H bind with highest affinity. This study aimed to determine whether high-affinity SNPs are associated with disease-free survival (DFS) among patients with HER2-positive nonmetastatic breast cancer. EXPERIMENTAL DESIGN: Genomic DNA was isolated from 1,286 patients enrolled in a trial of adjuvant trastuzumab-based chemotherapy. Genotyping was conducted using Sanger sequencing and Sequenom mass spectrometry. RESULTS: Patient samples (N = 1,189) were successfully genotyped for FCGR3A and 1,218 for FCGR2A. Compared with the overall results of the BCIRG006 study, in the subset of patients genotyped in this analysis, a less robust improvement in DFS was observed for the trastuzumab arms than control arm (HR, 0.842; P = 0.1925). When stratified for prognostic features, the HR in favor of trastuzumab was consistent with that of the overall study (HR, 0.74; P = 0.036). No correlation between DFS and FCGR3A/2A genotypes was seen for trastuzumab-treated patients (158V/V vs. V/F vs. F/F, P = 0.98; 131H/H vs. H/R vs. R/R, P = 0.76; 158V/V and/or 131H/H vs. others, P = 0.67). CONCLUSION: This analysis evaluating the association between FCGR3A/2A genotypes and trastuzumab efficacy in HER2-positive breast cancer did not show a correlation between FCGR3A-V/F and FCGR2A-H/R SNPs and DFS in patients treated with trastuzumab.

Programmed death ligand 1 is expressed by non-hodgkin lymphomas and inhibits the activity of tumor-associated T cells.

PURPOSE: Programmed death ligand 1 (PD-L1) is expressed on antigen-presenting cells and inhibits activation of T cells through its receptor PD-1. PD-L1 is aberrantly expressed on some epithelial malignancies and Hodgkin lymphomas and may prevent effective host antitumor immunity. The role of PD-L1 in non-Hodgkin lymphomas (NHL) is not well characterized. EXPERIMENTAL DESIGN: PD-L1 expression was analyzed in cell lines and lymphoma specimens by using flow cytometry and immunohistochemistry. Functional activity of PD-L1 was studied by incubating irradiated lymphoma cells with allogeneic T cells with or without anti-PD-L1 blocking antibody; T-cell proliferation and IFN-γ secretion served as measures of T-cell activation. Similar experiments were conducted using cultures of primary lymphoma specimens containing host T cells. RESULTS: PD-L1 was expressed uniformly by anaplastic large cell lymphoma (ALCL) cell lines, but rarely in B-cell NHL, confined to a subset of diffuse large B-cell lymphomas (DLBCL) with activated B-cell features (3 of 28 cell lines and 24% of primary DLBCL). Anti-PD-L1 blocking antibody boosted proliferation and IFN-γ secretion by allogeneic T cells responding to ALCL and DLBCL cells. In autologous cultures of primary ALCL and DLBCL, PD-L1 blockade enhanced secretion of inflammatory cytokines IFN-γ, granulocyte macrophage colony-stimulating factor, interleukin (IL)-1, IL-6, IL-8, IL-13, TNF-α, and macrophage inflammatory protein-1α. In establishing cell lines from an aggressive PD-L1(+) mature B-cell lymphoma, we also noted that PD-L1 expression could be lost under certain in vitro culture conditions. CONCLUSIONS: PD-L1 may thwart effective antitumor immune responses and represents an attractive target for lymphoma immunotherapy.

ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies).

Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V(L)-V(H) orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with (124)I for PET imaging, and biodistribution of (131)I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both (124)I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of (131)I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas.

Recombinant anti-CD20 antibody fragments for small-animal PET imaging of B-cell lymphomas.

UNLABELLED: The CD20 cell surface antigen is expressed at high levels by over 90% of B-cell non-Hodgkin lymphomas (NHL) and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific PET imaging of NHL, we sought to develop PET agents targeting CD20. METHODS: Two recombinant anti-CD20 rituximab fragments, a minibody (scFv-C(H)3 dimer; 80 kDa) and a modified scFv-Fc fragment (105 kDa), designed to clear rapidly, were generated. Both fragments were radiolabeled with (124)I, and the minibody was additionally labeled with (64)Cu (radiometal) after conjugation to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). The radioiodinated fragments and the radiometal-labeled minibody were evaluated in mice as small-animal PET imaging agents for the in vivo imaging of human CD20-expressing lymphomas. RESULTS: Rapid and specific localization to CD20-positive tumors was observed with the radioiodinated fragments. However, the tumor uptake levels and blood activities differed, resulting in different levels of contrast in the images. The better candidate was the minibody, with superior uptake (2-fold higher than that obtained with scFv-Fc) in CD20-positive tumors and low uptake in CD20-negative tumors. Ratios of CD20-positive tumors to CD20-negative tumors at 21 h were 7.0 +/- 3.1 (mean +/- SD) and 3.9 +/- 0.7 for the minibody and scFv-Fc, respectively. The ratio achieved with the (64)Cu-DOTA-minibody at 19 h was about 5-fold lower because of higher residual background activity in CD20-negative tumors. CONCLUSION: A radioiodinated minibody and a radioiodinated scFv-Fc fragment produced excellent, high-contrast images in vivo. These new immunoPET agents may prove useful for imaging CD20-positive lymphomas in preclinical models and in humans with NHL.

Intratumoral but not systemic delivery of CpG oligodeoxynucleotide augments the efficacy of anti-CD20 monoclonal antibody therapy against B cell lymphoma.

The anti-CD20 monoclonal antibody rituximab (Rituxan) has become a mainstay in the treatment of B cell non-Hodgkin lymphomas. The mechanisms of action for rituximab include antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Combination of anti-CD20 antibodies with immunostimulatory agents may improve their efficacy via enhancement of one or more of these mechanisms. Toll-like receptor 9 agonist CpG oligodeoxynucleotides administered systemically have been studied in clinical trials with and without rituximab. However, recent data suggest that intratumoral (IT) delivery of CpG has advantages in the treatment of tumors. Using a syngeneic murine B cell lymphoma line expressing human CD20, we found that IT, but not systemically administered CpG significantly improved the efficacy of rituximab against 7-day established tumors. Rituximab plus IT CpG could eradicate tumors from 42% of mice, whereas systemically administered CpG, with or without rituximab, did not achieve tumor eradication. Both natural killer cells and complement participated in the cure of tumors by rituximab plus IT CpG, apparently by increasing tumor cell sensitivity to complement and ADCC lysis, and by augmenting the cytotoxicity of ADCC effectors. No role for T cells in mediating tumor eradication was demonstrated in this model. These results suggest that previous clinical trials in B cell lymphoma combining systemic administration of CpG with rituximab may have employed suboptimal routes of CpG delivery. Future trials combining IT CpG with anti-CD20 antibodies or the antibody-mediated targeting of CpG directly to the sites of B cell lymphoma may thus be warranted.

Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells.

Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources.

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines.

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8(+) T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.

Sulfhydryl-based tumor antigen-carrier protein conjugates stimulate superior antitumor immunity against B cell lymphomas.

Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8(+) T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers.

Dendritic cells loaded with apoptotic antibody-coated tumor cells provide protective immunity against B-cell lymphoma in vivo.

The in vitro priming of tumor-specific T cells by dendritic cells (DCs) phagocytosing killed tumor cells can be augmented in the presence of antitumor monoclonal antibody (mAb). We investigated whether DCs phagocytosing killed lymphoma cells coated with tumor-specific antibody could elicit antitumor immunity in vivo. Irradiated murine 38C13 lymphoma cells were cocultured with bone marrow-derived DCs in the presence or absence of tumor-specific mAb. Mice vaccinated with DCs cocultured with mAb-coated tumor cells were protected from tumor challenge (60% long-term survival), whereas DCs loaded with tumor cells alone were much less effective. The opsonized whole tumor cell-DC vaccine elicited significantly better tumor protection than a traditional lymphoma idiotype (Id) protein vaccine, and in combination with chemotherapy could eradicate preexisting tumor. Moreover, the DC vaccine protected animals from both wild-type and Id-negative variant tumor cells, indicating that Id is not a major target of the induced tumor immunity. Protection was critically dependent upon CD8(+) T cells, with lesser contribution by CD4(+) T cells. Importantly, opsonized whole tumor cell-DC vaccination did not result in tissue-specific autoimmunity. Since opsonized whole tumor cell-DC and Id vaccines appear to target distinct tumor antigens, optimal antilymphoma immunity might be achieved by combining these approaches.

Recent gene conversions between duplicated glutamate decarboxylase genes (gadA and gadB) in pathogenic Escherichia coli.

Escherichia coli have evolved adaptive systems to resist strongly acidic habitats in part through the production of 2 biochemically identical isoforms of glutamate decarboxylase (GAD), encoded by the gadA and gadB genes. These genes occur in E. coli and other members of the genospecies (e.g., Shigella spp.) and originated as part of a genomic fitness island acquired early in Escherichia evolution. The present duplicated gad loci are widely spaced on the E. coli chromosome, and the 2 genes are 97% similar in sequence. Comparison of the nucleotide sequences of the gadA and gadB in 16 strains of pathogenic E. coli revealed 3.8% and 5.0% polymorphism in the 2 genes, respectively. Alignment of the homologous genes identified a total of 120 variable sites, including 21 fixed nucleotide differences between the loci within the first 82 codons of the genes. Twenty-three phylogenetically informative sites were polymorphic for the same nucleotides in both genes suggesting recent gene conversions or intergenic recombination. Phylogenetic analysis based on the synonymous substitutions per synonymous site indicated 2 cases in which specific gadA and gadB alleles were more closely related to one another than to other alleles at the corresponding locus. The results indicate that at least 3 gene conversion events have occurred after the gad gene duplication in the evolution of E. coli. Despite multiple gene conversion events, the upstream regulatory regions and the 5' end of each gene remains distinct, suggesting that maintaining functionally different gad genes is important in this acid-resistance mechanism in pathogenic E. coli.

Bordetella pertussis acquires resistance to complement-mediated killing in vivo.

In order to initially colonize a host, bacteria must avoid various components of the innate immune system, one of which is complement. The genus Bordetella includes three closely related species that differ in their ability to resist complement-mediated killing. Bordetella parapertussis and Bordetella bronchiseptica resist killing in naïve serum, a characteristic that may aid in efficient respiratory tract colonization and has been attributed to expression of O antigen. Bordetella pertussis lacks O antigen and is sensitive to naïve serum in vitro, yet it also efficiently colonizes the respiratory tract. Based on these observations, we hypothesized that B. pertussis may have an alternate mechanism to resist complement in vivo. While a number of reports on serum sensitivity of the bordetellae have been published, we show here that serum concentration and growth conditions can greatly alter the observed level of sensitivity to complement and that all but one strain of B. pertussis observed were sensitive to some level of naïve serum in vitro, particularly when there was excess complement. However, B. pertussis rapidly acquires increased resistance in vivo to naïve serum that is specific to the alternative pathway. Resistance is not efficiently acquired by B. parapertussis and B. bronchiseptica mutants lacking O antigen. This B. pertussis-specific mechanism of complement resistance does not appear to be dependent on either brkA or other genes expressed specifically in the Bvg(+) phase. This in vivo acquisition of alternative pathway resistance suggests that there is a novel O antigen-independent method by which B. pertussis evades complement-mediated killing.