Investigation of a Chlamydia pneumoniae outbreak in a Federal correctional facility in Texas.

BACKGROUND: Chlamydia pneumoniae illness is poorly characterized, particularly as a sole causative pathogen. We investigated a C. pneumoniae outbreak at a federal correctional facility. METHODS: We identified inmates with acute respiratory illness (ARI) from 1 November 2009 to 24 February 2010 through clinic self-referral and active case finding. We tested oropharyngeal and/or nasopharyngeal swabs for C. pneumoniae by real-time polymerase chain reaction (qPCR) and serum samples by microimmunofluorescence. Cases were inmates with ARI and radiologically confirmed pneumonia, positive qPCR, or serological evidence of recent infection. Swabs from 7 acutely ill inmates were tested for 18 respiratory pathogens using qPCR TaqMan Array Cards (TACs). Follow-up swabs from case patients were collected for up to 8 weeks. RESULTS: Among 33 self-referred and 226 randomly selected inmates, 52 (20.1%) met the case definition; pneumonia was confirmed in 4 by radiology only, in 9 by qPCR only, in 17 by serology only, and in 22 by both qPCR and serology. The prison attack rate was 10.4% (95% confidence interval, 7.0%-13.8%). White inmates and residents of housing unit Y were at highest risk. TAC testing detected C. pneumoniae in 4 (57%) inmates; no other causative pathogens were identified. Among 40 inmates followed prospectively, C. pneumoniae was detected for up to 8 weeks. Thirteen (52%) of 25 inmates treated with azithromycin continued to be qPCR positive >2 weeks after treatment. CONCLUSIONS: Chlamydia pneumoniae was the causative pathogen of this outbreak. Higher risk among certain groups suggests that social interaction contributed to transmission. Persistence of C. pneumoniae in the oropharynx creates challenges for outbreak control measures.

Glycan-Based Near-infrared Fluorescent (NIRF) Imaging of Gastrointestinal Tumors: a Preclinical Proof-of-Concept In Vivo Study.

PURPOSE: Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-concept in vivo study aims to validate the use of aberrant Lewis glycans as target for antibody-based, real-time imaging of gastrointestinal cancers. PROCEDURES: Immunohistochemical (IHC) staining with monoclonal antibody FG88.2, targeting Lewisa/c/x, was performed on gastrointestinal tumors and their healthy counterparts. Then, FG88.2 and its chimeric human/mouse variant CH88.2 were conjugated with near-infrared fluorescent (NIRF) IRDye 800CW for real-time imaging. Specific binding was evaluated in vitro on human gastrointestinal cancer cell lines with cell-based plate assays, flow cytometry, and immune-fluorescence microscopy. Subsequently, mice bearing human colon and pancreatic subcutaneous tumors were imaged in vivo after intravenous administration of 1 nmol (150 µg) CH88.2-800CW with the clinical Artemis NIRF imaging system using the Pearl Trilogy small animal imager as reference. One week post-injection of the tracer, tumors and organs were resected and tracer uptake was analyzed ex vivo. RESULTS: IHC analysis showed strong FG88.2 staining on colonic, gastric, and pancreatic tumors, while staining on their normal tissue counterparts was limited. Next, human cancer cell lines HT-29 (colon) and BxPC-3 and PANC-1 (both pancreatic) were identified as respectively high, moderate, and low Lewisa/c/x-expressing. Using the clinical NIRF camera system for tumor-bearing mice, a mean tumor-to-background ratio (TBR) of 2.2 ± 0.3 (Pearl: 3.1 ± 0.8) was observed in the HT-29 tumors and a TBR of 1.8 ± 0.3 (Pearl: 1.9 ± 0.5) was achieved in the moderate expression BxPC-3 model. In both models, tumors could be adequately localized and delineated by NIRF for up to 1 week. Ex vivo analysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. CONCLUSIONS: Using a novel chimeric Lewisa/c/x-targeting tracer in combination with a clinical NIRF imager, we demonstrate the potential of targeting Lewis glycans for fluorescence-guided surgery of gastrointestinal tumors.

Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan-Targeting Antibodies without the Need for Immune Effector Cells or Complement.

Murine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved ("i") chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286-306 and 339-378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa-targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential. SIGNIFICANCE: Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

Monoclonal Antibody Targeting Sialyl-di-Lewisa-Containing Internalizing and Noninternalizing Glycoproteins with Cancer Immunotherapy Development Potential.

Tumor glycans constitute attractive targets for therapeutic antibodies. The sialylated glycocalyx plays a prominent role in cancer progression and immune evasion. Here, we describe the characterization of the mAb, FG129, which targets tumor-associated sialylated glycan, and demonstrate its potential for multimodal cancer therapy. FG129, obtained through BALB/c mouse immunizations with liposomes containing membrane glycan extracts from the colorectal cancer cell line LS180, is an mIgG1κ that targets sialyl-di-Lewisa-containing glycoproteins. FG129, as well as its chimeric human IgG1 variant, CH129, binds with nanomolar functional affinity to a range of colorectal, pancreatic, and gastric cancer cell lines. FG129 targets 74% (135/182) of pancreatic, 50% (46/92) of gastric, 36% (100/281) of colorectal, 27% (89/327) of ovarian, and 21% (42/201) of non-small cell lung cancers, by IHC. In our pancreatic cancer cohort, high FG129 glyco-epitope expression was significantly associated with poor prognosis (P = 0.004). Crucially, the glyco-epitope displays limited normal tissue distribution, with FG129 binding weakly to a small percentage of cells within gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissues. Owing to glyco-epitope internalization, we validated payload delivery by CH129 through monomethyl auristatin E (MMAE) or maytansinoid (DM1 and DM4) conjugation. All three CH129 drug conjugates killed high-binding colorectal and pancreatic cancer cell lines with (sub)nanomolar potency, coinciding with significant in vivo xenograft tumor control by CH129-vcMMAE. CH129, with its restricted normal tissue distribution, avid tumor binding, and efficient payload delivery, is a promising candidate for the treatment of sialyl-di-Lewisa-expressing solid tumors, as an antibody-drug conjugate or as an alternative cancer immunotherapy modality.

A new anticancer glycolipid monoclonal antibody, SC104, which directly induces tumor cell apoptosis.

A novel monoclonal antibody was raised by immunization of mice with colorectal tumor cell lines. The fusion was screened by immunohistochemistry for binding to primary colorectal tumors. Subsequent analysis on primary disaggregated colorectal tumors show that the antibody recognizes a cell surface antigen expressed by the majority of colorectal tumors. Antigen characterization has shown that the antibody recognizes a sialyltetraosylceramide but does not bind to GM1, GD1a, GT1b, or sialyl Lewis(X) antigens. Binding to a frozen panel of tumor and normal tissue sections revealed that the antigen was also strongly expressed on esophageal, gastric, and endometrial tumors. Its normal tissue distribution was largely restricted to moderate staining of large intestine. Surprisingly, SC104 antibody directly induces tumor cell death without the need for immune effector cells or complement. This may be related in part to its homophilic binding properties that allow cross-linking of antibody and receptors on the cell surface. Caspase activation can be detected following SC104 treatment of colorectal cells, and cotreatment with caspase inhibitors has been shown to inhibit cell death. This suggests that SC104 induces death by a classic apoptotic pathway. Furthermore, SC104 antibody shows additive killing with complement and 5-fluorouracil/leucovorin in vivo, suggesting a new therapeutic approach for this class of antibodies.

High expression of Lewis y/b antigens is associated with decreased survival in lymph node negative breast carcinomas.

INTRODUCTION: There is sufficient evidence that blood group related Lewis antigens are tumour-associated molecules. The Lewisy and Lewisb antigens are complex carbohydrates that are over-expressed by breast, lung, colon and ovarian cancers. The SC101 mAb is a unique Lewisy/b binding antibody that binds to native and extended Lewisy and Lewisb haptens, displaying no cross reactivity with H type 1, H type 2, Lewisx or normal blood group antigens. METHODS: Immunohistochemical detection of Lewisy/b was performed on 660 formalin-fixed, paraffin embedded breast tumour specimens using a streptavidin-biotin peroxidase technique. Tissue from these patients had previously been included in tissue microarrays. This cohort comprises a well characterized series of patients with primary operable breast cancer diagnosed between 1987 and 1992, obtained from the Nottingham Tenovus Primary Breast Carcinoma Series. This includes patients 70 years of age or less, with a mean follow up of 7 years. RESULTS: Of the breast carcinomas, 370 of 660 (56%) were negative for Lewisy/b expression, 110 (17%) cases showed a low level of expression (<25% of positive cells) and only 54 cases (8%) showed extensive expression of Lewisy/b (>75% of positive cells). We found significant positive associations between histological grade (p < 0.001), Nottingham Prognostic Index (p = 0.016), tumour type (p = 0.007) and the level of Lewis y/b expression. There was a significant correlation between the proportion of Lewisy/b positive tumour cells and survival in lymph-node negative patients (p = 0.006). CONCLUSION: The unique epitope recognised by SC101 mAb on Lewisy/b hapten is over-expressed on breast tumour tissue compared with normal breast. In this large series of invasive breast cancers, higher expression of Lewisy/b was more often found in high grade and poor prognosis tumours compared to good prognosis cancers. Moreover, in lymph node negative breast carcinomas, over-expression of Lewisy/b hapten was associated with significantly decreased patient survival.

Monoclonal Antibodies Targeting LecLex-Related Glycans with Potent Antitumor Activity.

PURPOSE: To produce antitumor monoclonal antibodies (mAbs) targeting glycans as they are aberrantly expressed in tumors and are coaccessory molecules for key survival pathways. EXPERIMENTAL DESIGN: Two mAbs (FG88.2 and FG88.7) recognizing novel tumor-associated Lewis (Le) glycans were produced by immunizations with plasma membrane lipid extracts of the COLO205 cell line. RESULTS: Glycan array analysis showed that both mAbs bound Le(c)Le(x), di-Le(a), and Le(a)Le(x), as well as Le(a)-containing glycans. These glycans are expressed on both lipids and proteins. Both mAbs showed strong tumor reactivity, binding to 71% (147 of 208) of colorectal, 81% (155 of 192) of pancreatic, 54% (52 of 96) of gastric, 23% (62 of 274) of non-small cell lung, and 31% (66 of 217) of ovarian tumor tissue in combination with a restricted normal tissue distribution. In colorectal cancer, high FG88 glyco-epitope expression was significantly associated with poor survival. The mAbs demonstrated excellent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), in addition to direct tumor cell killing via a caspase-independent mechanism. Scanning electron microscopy revealed antibody-induced pore formation. In addition, the mAbs internalized, colocalized with lysosomes, and delivered saporin that killed cells with subnanomolar potency. In vivo, the mAbs demonstrated potent antitumor efficacy in a metastatic colorectal tumor model, leading to significant long-term survival. CONCLUSIONS: The mAbs direct and immune-assisted tumor cell killing, pan-tumor reactivity, and potent in vivo antitumor efficacy indicate their potential as therapeutic agents for the treatment of multiple solid tumors. In addition, internalization of saporin conjugates and associated tumor cell killing suggests their potential as antibody drug carriers.

Therapeutic targeting of Lewis(y) and Lewis(b) with a novel monoclonal antibody 692/29.

BACKGROUND: Several monoclonal antibodies (mAbs) recognising Lewis(y), such as BR96, have reached the clinic but have failed to show good anti-tumour responses with an acceptable level of toxicity. No Lewis(b) mAbs have been trialled in patients. In this study we compare the specificity of three mAbs; BR96 (Lewis(y)), 2-25 LE (Lewis(b)) and 692/29 that recognises a unique facet of both Lewis(y) and Lewis(b). We then assessed the in vivo therapeutic effect of 692/29 using xenograft models. METHODOLOGY/PRINCIPAL FINDINGS: Using a glycan array, each mAb was shown to display a different binding pattern with only 692/29 binding to both Lewis(y) and Lewis(b). 692/29 was able to kill tumour cells over-expressing Lewis(y/b) directly, as well as by antibody and complement mediated cytotoxicity (ADCC/CDC), but failed to kill cells expressing low levels of these haptens. In contrast, BR96, directly killed cells expressing either high or low levels of Lewis(y) perhaps explaining its toxicity in patients. 2-25 LE failed to cause any direct killing but did mediate ADCC/CDC. Both 692/29 and BR96 bound to >80% of a panel of over 400 colorectal tumours whereas 2-25 LE showed lower reactivity (52%). 692/29 demonstrated more restricted normal tissue reactivity than both BR96 and 2-25 LE. 692/29 anti-Lewis(y/b) mAb also showed good in vivo killing in xenograft models. CONCLUSIONS/SIGNIFICANCE: MAbs targeting both Lewis(y) and Lewis(b) may have a therapeutic advantage over mAbs targeting just one hapten. 692/29 has a more restricted normal tissue distribution and a higher antigen threshold for killing which should reduce its toxicity compared to a Lewis(y) specific mAb. 692/29 has an ability to directly kill tumours whereas the anti-Lewis(b) mAb does not. This suggests that Lewis(y) but not Lewis(b) are functional glycans. 692/29 showed good anti-tumour responses in vivo and is a strong therapeutic candidate.

Providing misleading and reinstatement information a year after it happened: effects on long-term memory.

The question addressed here is whether misleading suggestions made to children a year after target events had occurred will alter long-term recall. One group (3-13 years old when injured and treated in a hospital Emergency Room) were given both misleading and accurate reinstating information a year later, and recall of target events assessed both 1 week and another year later (i.e., 2 years post-injury). A control group had recall assessed both 1 and 2 years post-injury. Misleading had little effect on children's recall 1 week later, although a few misled details were reported. However, a year later virtually none of the misleading information was incorporated into long-term recall. Rather, children were more, not less, accurate when recalling details about which they had been misled. Results were attributed to target events having been highly memorable and well rehearsed via previous recalls, and detection of discrepancies between memory and misleading information focusing attention on targeted details.