Correction: Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non-small-cell lung carcinoma and rodent tumor models.

[This corrects the article DOI: 10.1371/journal.pone.0234268.].

Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non-small-cell lung carcinoma and rodent tumor models.

Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.

Antibody Fragment F(ab')2 Targeting Caveolae-Associated Protein PV1 for Selective Kidney Targeting and Retention.

Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.

Targeting p16-induced senescence prevents cigarette smoke-induced emphysema by promoting IGF1/Akt1 signaling in mice.

Senescence is a mechanism associated with aging that alters tissue regeneration by depleting the stem cell pool. Chronic obstructive pulmonary disease (COPD) displays hallmarks of senescence, including a diminished stem cell population. DNA damage from cigarette smoke (CS) induces senescence via the p16 pathway. This study evaluated the contribution of p16 to CS-associated lung pathologies. p16 expression was prominent in human COPD lungs compared with normal subjects. CS induces impaired pulmonary function, emphysema, and increased alveolar epithelial cell (AECII) senescence in wild-type mice, whereas CS-exposed p16-/- mice exhibit normal pulmonary function, reduced emphysema, diminished AECII senescence, and increased pro-growth IGF1 signaling, suggesting that improved lung function in p16-/- mice was due to increased alveolar progenitor cell proliferation. In conclusion, our study suggests that targeting senescence may facilitate alveolar regeneration in COPD emphysema by promoting IGF1 proliferative signaling.

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

Tumor uptake of pegylated diabodies: Balancing systemic clearance and vascular transport.

The accumulation, dissemination and clearance of monoclonal antibody-based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)-modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG-modified diabodies improved the PK of the parental diabody from a half-life of 40 min to over 40 h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody-PEG5K showing peak accumulation early on, but with the larger diabody-PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody-PEG20K-B with a hydrodynamic radius (Rh) of 6 nm had superior tumor uptake than the larger diabody-PEG40K-B with Rh of 12 nm, indicating that beyond 6 nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ~6 nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides.

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models.

Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging.

In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.

MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.

From bench to cageside: Risk assessment for rodent pathogen contamination of cells and biologics.

Many newly developed animal models involve the transfer of cells, serum, or other tissue-derived products into live rodents. These biologics can serve as repositories for adventitious rodent pathogens that, when used in animal studies, can alter research outcomes and result in endemic outbreaks. This review includes a description of some of the biologics that have inadvertently introduced infectious agents into in vivo studies and/or resulted in endemic outbreaks. I also discuss the points of potential exposure of specific biologics to adventitious rodent pathogens as well as the importance of acquiring a complete developmental and testing history of each biologic introduced into a barrier facility. There are descriptions of specific cases of mycoplasma and lactate dehydrogenase-elevating virus (LDHV), two of the most common organisms that contaminate cells and cell byproducts. The information in this article should help investigators and animal resource program personnel to perform an appropriate risk assessment of biologics before their use in in vivo studies that involve rodents.

Development of molecular and cellular biomarkers of pain.

Observation of physiologic and behavioral responses is the main method used to assess pain in people and animals. These approaches are often difficult to objectively measure in laboratory rodents and provide no insight into associated molecular and cellular changes in the organism. To identify CNS markers for pain, we analyzed the gene expression profiles of midbrain sections of mice that had experienced either adjuvant injections in the footpad or partial sciatic nerve ligation (PSL), which are recognized models of inflammatory and neuropathic pain, respectively. The potential for pain-associated factors to be present in the blood and to affect other tissues was analyzed by monitoring the growth of various cell lines that were exposed to serum from these mice and to plasma from rats experiencing surgical pain and their respective controls. Adjuvant injection increased the transcription of 12 genes and decreased that of 38 genes by at least 2-fold, whereas PSL increased the transcription of 2 genes and decreased that of 23, with no overlap. Serum from mice with PSL stimulated the growth of the rat mammary tumor cell line RMT50. Similarly, plasma collected from rats after a painful surgical procedure promoted the growth of RMT50 and MDA-MB-235 cells. These results demonstrate that the gene expression profiles of brain tissue from mice exposed to painful stimuli vary depending on the nature of the stimulus, and that the growth of some mammary tumor cell lines can be affected by blood collected from rodents exposed to these stimuli.

Characterization of the effects of Bcl-2 and Bcl-xl deletion mutant expression in cell lines used for antibody production.

Strategies to maximize monoclonal antibody (MAb) yields by in vitro production methods entail that hybridoma cells be maintained at high density. Approaches to increase culture density and antibody yields from hybridomas by inhibiting apoptosis through over-expression of exogenous Bcl-2 family genes have produced variable results. In order to determine if expression of mutant forms of Bcl-2 and Bcl-xl could increase viable culture densities and batch MAb yields when compared to parental cell lines, recombinant delta loop deletion mutant of these apoptotic inhibitory genes were expressed in a myeloma and two hybridoma cell lines. Expression of either Bcl-2-delta or Bcl-xl-delta in P3x63Ag8.653 myeloma cells did not significantly increase viable cell densities in cultures over time. However, the rapid post-peak decline in viable cell density was significantly reduced in Bcl-xl-delta-expressing hybridoma cell lines 552 and 7.16.4 and in Bcl-2-expressing hybridoma 7.16.4. Significant increases in MAb yield were only observed in cultures of Bcl-xl-delta-expressing hybridoma 7.16.4. Annexin staining in hybridoma 7.16.4 confirmed that apoptosis was the primary means of cell death in this cell line, and expression of Bcl-2-delta and Bcl-xl-delta inhibited programmed cell death. These results suggest that cell viability in cultures can be improved by transfection and selection of hybridomas that express delta loop deletion mutant forms of Bcl-2 family genes; however, improvements in MAb yields are dependent upon the genetic background of each manipulated cell line.

Advances in monoclonal antibody technology: genetic engineering of mice, cells, and immunoglobulins.

The ability to produce antibodies that are directed against specific antigens has played a crucial role in advancing scientific discoveries. Recombinant technologies have extended the application of antibodies beyond the research laboratory and into the clinic for the treatment of cancer and other diseases. Creative approaches using these technologies have been used to reduce the antibody to its minimal functional size, and/or make them bifunctional (immunotoxins), bispecific, or less immunoreactive (humanized). Additionally, mice that are engineered to generate antibodies of human genomic origin have been used to produce therapeutic antibodies and are being further developed. As the research and clinical demands for antibodies continue to increase, the development of improved resources (cell lines and animals) to improve production efficiency, generate larger repertoires, and deliver greater yields of antibodies is being explored, and advances in this area are discussed further in this review.

Tamoxifen resistance and Her2/neu expression in an aged, irradiated rat breast carcinoma model.

Clear links have been established between occupational or therapeutic radiation exposure and breast cancer. Tamoxifen chemoprevention following radiation exposure may be able to reduce the risk of developing breast cancer later in life. In order to model carcinogenesis in this setting, an in vivo model of tamoxifen chemoprevention and tamoxifen failure in a radiation-induced rat mammary carcinoma model was characterized. Two hundred and twenty-seven 60-day-old female rats received whole body or sham exposure to ionizing radiation. Thirty days later long-term, continuous, tamoxifen chemoprevention was initiated in half the population and all animals were monitored over three and a half years for the development of mammary tumors. Mammary tumors were surgically removed and carcinomas were histologically identified and characterized. Results showed that tamoxifen chemoprevention decreased the incidence and prolonged the latency of radiation-induced mammary carcinomas. However, many individuals receiving tamoxifen chemoprevention developed their first carcinoma very late in life. These carcinomas shared morphological features distinct from the majority of carcinomas that developed in the absence of tamoxifen chemoprevention. Analyses of cell lines established from these carcinomas and immunohistochemistry of tumor sections revealed that the highest levels of Her2/neu expression were associated with in vivo tamoxifen exposure. Treatment of rat mammary carcinoma cells with an anti-rat Her2/neu monoclonal antibody (MAb 7.16.4) inhibited cell growth and this effect was more pronounced in the presence of tamoxifen. These studies suggest that carcinoma growth driven by the Her2/neu pathway may be associated with tamoxifen chemoprevention failure in the rat mammary carcinoma model. Additionally, strategies combining targeted Her2/neu antibodies, vaccines or drugs with estrogen pathway modification may be more effective in reducing breast cancer chemoprevention failures.

Chronic stress accelerates ultraviolet-induced cutaneous carcinogenesis.

BACKGROUND: Physical and emotional stressors have been found to mediate a wide variety of biological changes including the facilitation of tumor progression; however most of these paradigms utilized artificial sources of neoplasms and stress. METHODS: Skh mice were exposed to carcinogenic doses of ultraviolet light (UV). The stressed group was subjected to the close proximity of fox urine as a source of stress from the presence of the odor of their natural predator, while the control group remained stress free. RESULTS: A significant acceleration in the development of cutaneous neoplasms was observed in mice that had been exposed to the stressor. The first tumor appeared in the group after 8 weeks, whereas nonstressed mice began to develop these by week 21. CONCLUSION: These results suggest that stress plays a role in potentiating cutaneous carcinogenesis.

The susceptibility of wild caught sand flies to infection by a subspecies of Leishmania mexicana isolated from proechimys iheringi denigratus (Rodentia, echimyidae)

Várias espécies de flebotomíneos silvestres foram coletados no mesmo local onde o roedor Proechimys iheringi denigratus foi encontrado infectado com uma subespécie de Leishmania mexicana. A ausência de infecçäo natural desses flebotomíneos nos permitiu testar, com relativa segurança, a susceptibilidade de algumas dessas espécies à infecçäo por esse parasito. O sucesso obtido nas infecçöes experimentais sugere que uma ou mais das espécies de flebotomíneos encontradas em alta densidade nesse local podem ser um vetor, em potencial, dessa subespécie de L. mexicana

Leishmania mexicana in proechimys ihering denigratus moojen (Rodentia, echimyidae) in a region endenic for American cutaneous Leishmaniasis

Três isolados de Leishmania foram obtidos de cinco entre 27 exemplares do roedor Proechimys iheringi denigratus capturados na regiäo de Três Braços, na mata atlântica do Estado da Bahia, Brasil. O isolamento desse parasito foi feito através de inoculaçäo de triturado de pele, braço e fígado em patas de hamsters. Em pelo menos um dos casos, (MTB-574), o parasito foi isolado da pele. Metástase foi observada nos hamsters inoculaçäos, os parasitos cresceram abundantemente em meios artificiais de cultura e um padräo suprapapilário típico foi obtido em Lutzomyia longipalpis, indicando que o parasito pertence ao complexo L. mexicana. Todos os isolados reagiram positivamente com anticorpos monoclonais de L. m. mexicana e L. m. amazonensis. A análise isoenzimática diferenciou o parasito de isolados padröes de L. m. mexicana, L. m. amazonensis, L. m. aristedesi, L. m. pifanoi, L. m. garnhami e L. m. ssp(Goias-W. Barbosa). O parasito parece ser uma subespécie de L. mexicana muito próxima a L. m. amazonensis, da qual difere pela menor mobilidade eletroforética de GPI, PEP e ALAT. Este é o primeiro registro do isolamento de um parasito do gênero Leishmania em um roedor capturado no Estado da Bahia

Chave para identificação de mamíferos da região amazônica brasileira com exceção dos quirópteros e primatas

Summary A key to the mammals, other than bats and monkeys, believed to be found in the Brazilian Amazon Region is presented. This key uses a minimum number of technical terms, and is intended to be used by persons untrained in mammalogy as well as by mammalogists. It includes 107 species: 17 marsupials, 16 edentates, 1 lagomorph, 41 rodents, 2 cetaceans, 20 carnivores, 2 sirenians, 1 perissodactyl and 7 artiodactyls. It should be of special use in identifying mammals collected in faunal surveys and in epidemiological studies.

Resumo Apresenta-se uma chave para identificar os mamíferos encontrados na região amazônica brasileira, excluídos os morcegos e macacos. Esta chave utiliza quantidade mínima de termos técnicos e poderá ser usada tanto por pessoas sem conhecimentos básicos, como também, por conhecedores de mastozoologia. São relacionadas 107 espécies de mamíferos compreendendo: 17 marsupiais, 16 edentatos, 1 lagomorfo, 41 roedores, 2 cetáceos, 20 carnívoros, 2 sirênios, 1 perissodáctilo e 7 artiodáctilos. Esta chave será especialmente útil para identificar mamíferos coletados em levantamentos da fauna e em estudos epidemiológicos.