Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8⁺ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34⁺ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that arises from break point cluster region/Abelson murine leukemia viral oncogene homolog 1 (BCR/ABL)–transformed hematopoietic stem (HSCs) or early progenitor cells known as leukemia stem cells (LSCs; Kavalerchik et al., 2008). LSCs have been first characterized as the tumor-initiating cells in acute myeloid leukemia (Lapidot et al., 1994) and have also been defined in other hematopoietic neoplasms since then (Cox et al., 2004; Matsui et al., 2004).BCR/ABL-specific tyrosine kinase inhibitors (TKIs) such as Imatinib mesylate (Glivec) have revolutionized the therapy of CML (Druker et al., 2001a,b; Baccarani et al., 2006). Nevertheless, LSCs seem resistant to TKIs and traditional chemotherapy (Weiden et al., 1979; Deininger et al., 2000; Savona and Talpaz, 2008) and CML inevitably progresses to incurable acute leukemia (Faderl et al., 1999). Quiescent, self-renewing LSCs remain in the BM and are responsible for refractoriness and relapse of CML after treatment (Hughes et al., 2003). Therefore, novel cytotoxic agents that selectively target LSCs are under investigation (Jin et al., 2006; Guzman et al., 2007; Neviani et al., 2007; Ito et al., 2008; Bellodi et al., 2009; Majeti et al., 2009; Wang et al., 2010; Schürch et al., 2012).Another promising approach in the treatment of CML is immunotherapy. In fact, currently, the only curative treatment for CML remains allogeneic stem cell transplantation (alloSCT). The graft-versus-leukemia effect of alloSCT is most likely executed by donor CD8⁺ effector CTLs specific for minor histocompatibility antigens (Weiden et al., 1979; Kolb et al., 1990; Gale et al., 1994; Druker et al., 2002). Patients who receive T cell–depleted alloSCT grafts have a higher risk of disease relapse, and donor lymphocyte infusions are able to induce complete remission after relapse (Thomas et al., 1979; Horowitz et al., 1990; Kolb et al., 1995; Sehn et al., 1999). Furthermore, endogenous CTLs directed against leukemia antigens have been detected in the peripheral blood of chronic phase CML patients (Molldrem et al., 2000; Butt et al., 2005). Several proteins may potentially act as potent leukemia-specific antigens for T cells, including BCR/ABL, Wilms’ tumor 1 protein (WT1), and proteinase 3 (Pr3; Van Driessche et al., 2005). Peptides from the junctional region of BCR/ABL are not present in healthy individuals and therefore are leukemia-specific. Yotnda et al. (1998) identified a BCR/ABL junctional nonapeptide that binds to human leukocyte antigen (HLA)-A2.1 and elicits specific CTL responses in vitro and in vivo. Additional studies confirmed and extended the finding of immunogenic BCR/ABL junction peptides (Bocchia et al., 1996; Clark et al., 2001).CTLs have been shown to kill CML target cells in vitro via Fas-receptor triggering (Selleri and Maciejewski, 2000). In a BCR/ABL-induced murine CML model, we have shown that CD8⁺ T cells crucially contribute to disease control in vivo. However, programmed death ligand 1 (PD-L1) expression by the malignant cells induced T cell dysfunction leading to disease progression (Mumprecht et al., 2009b).Despite these advances in the understanding of the immunosurveillance of CML and the development of immunotherapy strategies, the interaction of effector CTLs with the disease-originating LSCs has not been analyzed so far. In the present study, we analyzed the immunogenicity of LSCs in vitro and in vivo using the glycoprotein of lymphocytic choriomeningitis virus (LCMV) as model leukemia antigen. LSCs expressed MHC and co-stimulatory molecules. LSCs isolated from CD8⁺ T cell–depleted CML mice were more immunogenic than LSCs from control CML mice, indicating that CD8⁺ T cells interact with LSCs in vivo and select for low immunogenic variants. To analyze whether LSCs can be recognized and lysed by activated leukemia-specific effector CTLs, we treated CML mice with large numbers of specific T cells. Although these effector CTLs efficiently lysed LSCs in vitro, adoptive immunotherapy in vivo failed to eradicate LSCs but paradoxically increased LSC numbers. LSC proliferation was induced by CTL-secreted IFN-γ. Specific effector CTLs secreted high amounts of IFN-γ when transferred to CML mice in advanced disease stage with high antigen load, but not after transfer to CML mice in early stage of disease. Importantly, our results were confirmed in a humanized CML model using HLA-A2.1 transgenic mice and a BCR/ABL b3a2 junctional peptide. In addition, IFN-γ induced the proliferation of primary CD34⁺ stem/progenitor cells from newly diagnosed CML patients.     

Multiple Extrauterine Pregnancy with Early and Near Full-Term Mummified Fetuses in a New Zealand White Rabbit (Oryctolagus cuniculus)

Extrauterine pregnancy (EP) is infrequent in mammalian species and occurs when fertilized ova implant and develop outside the uterus. A common outcome is abdominal pregnancy resulting in mummified fetuses (lithopedia). Here we describe an unusual case of abdominal pregnancy with early and near full-term lithopedia. Macroscopic findings supported the diagnosis of lithopedia with distinct age differences and facilitated further characterization of primary ectopia and risk factors leading to this occurrence.Abbreviation: EP, extrauterine pregnancyExtrauterine pregnancy (EP) occurs infrequently in most mammalian species.¹² The term derives from the Latin prefix meaning ‘outside’ or ‘beyond’ and refers to the implantation of a fertilized ovum outside the uterine cavity. Extrauterine pregnancy was first recognized more than 900 y ago² among other discoveries with a hereditary nature.¹³ Early reports compared EP in women, cats, dogs, and rabbits⁷ and described the presence of mummified fetuses in laboratory rabbits.^16,35EP is a serious obstetric complication that occurs asymptomatically in most cases.¹⁷ There are 4 classifications of EP: tubal, ovarian, abdominal–peritoneal, and cervical. The fallopian tube is the most common location and leads to tubal pregnancy. When gestation occurs in the abdominal–peritoneal cavity, abdominal pregnancy results and is subdivided as primary, when fertilization occurs outside the uterus after an oocyte is accidentally released from the fimbria, and secondary, when an oocyte is released due to direct tubal trauma.⁴⁵ A rare form of EP associated with high maternal morbidity and fetal mortality is called heterotopic (or combined) pregnancy, which occurs when 2 fertilized eggs coexist, one outside the uterus and the other inside.^18,33,44Undetected EP is frequently associated with fatal outcomes to the dam and offspring, including the formation of mummified fetuses, which may eventually become calcified and are called lithopedia (from the Greek: lithos, stone; paidion, child).^11,51,53 The condition is infrequent, and the factors that influence the unexpected outcomes of this pathology are not well understood.^26,34,35 Epizootiologic investigations are few,¹² although a recent report outlined the prevalence of EP in large NZW rabbit breeding colonies.⁵¹Examples of EP have been documented in dogs,¹⁷ cats,^14,39,42,49 rabbits,^20,29,45,51 hamsters,^9,46 rats,²⁶ mice,^8,12 guinea pigs,^3,30 lambs,⁴⁰ nonhuman primates,^10,34,38,50 and other species including humans.^11,12 However, despite the number of documented species, the majority of reports failed to note detailed clinical symptoms that interfered with reproduction even in instances that led to the formation of lithopedions.⁴²Experimentally, mouse embryos have successfully been transferred to a variety of sites including the peritoneal cavity, kidney, spleen, muscles, testis, and the anterior chamber of the eye.^1,6,21,31 The aim of the current report is to describe a rare case of abdominal pregnancy in which lithopedia developed clinically silently and coexisted with multiple pregnancies in a healthy doe rabbit.     

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease.Chronic myeloid leukemia (CML) is a chronic myeloproliferative neoplasm, resulting from a reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11). This lesion was the first recurrent chromosomal abnormality described in cancer (Nowell and Hungerford, 1960; Rowley, 1973) and generates the BCR-ABL oncoprotein, a constitutively activated protein tyrosine kinase (TK; Deininger et al., 2000). Mouse models and human data have demonstrated BCR-ABL expression to be causative in CML (Daley et al., 1990; Heisterkamp et al., 1990; Zhao et al., 2001; Ramaraj et al., 2004; Koschmieder et al., 2005), and this observation has led to the paradigmic development of potent small molecule inhibitors that selectively target ABL enzymatic function and interrupt its oncogenic TK activity. Imatinib mesylate, the prototypic ABL tyrosine kinase inhibitor (TKI), and subsequent second and third generation TKIs, have revolutionized CML treatment (Druker et al., 1996; 2006; Carroll et al., 1997; Heinrich et al., 2000; O’Brien et al., 2003), significantly improving cytogenetic and molecular response rates, keeping the majority of patients in chronic phase, and prolonging overall survival (Druker et al., 2001, 2006; Sawyers et al., 2002; Hughes et al., 2003). However, despite this vast improvement, significant clinical challenges still remain in CML therapy. CML stem cells appear relatively resistant to the effects of TKIs (Copland et al., 2006; Jørgensen et al., 2007; Konig et al., 2008) such that, in the majority of patients, CML is controlled rather than cured. In addition, resistance occurs and this, together with stem cell persistence, facilitates disease transformation. Three distinct phases of the disease have been described. The initial phase, in which ∼85–90% of patients are diagnosed, is the indolent chronic phase (CP), which is readily amenable to treatment. However, without adequate therapy, this almost inevitably progresses to an aggressive acute leukemia of myeloid or lymphoid phenotype (70 and 30%, respectively), termed blast crisis (BC), which may be preceded by an ill-defined intermediate or accelerated phase (AP; during which the levels of myeloblasts in the BM or peripheral blood (PB) are increased but remain <20%). 10–15% of patients present beyond CP and a small percentage of CP cases continue to transform even on TKI therapy. The frequency of transformation is recorded at 3–5% within the first few years of TKI therapy but drops to ∼1% per year thereafter in randomized trials (Druker et al., 2006), although these values have been found to be higher in population-based studies (de Lavallade et al., 2008; Gallipoli et al., 2011). Treatment options for AP and BC are very limited, with response rates to TKIs lower and much less durable. Other options involve highly toxic therapies, such as combination chemotherapy and BM transplantation, and are not available or appropriate for many patients with progression. Therefore, even in the TKI era, the median survival of patients with BC is still dismal at around 6 mo (Hehlmann and Saussele, 2008; Silver et al., 2009), defining it as an unmet clinical need.Although the chronic phase of CML appears almost entirely dependent on BCR-ABL and CML is regarded as an invaluable model of leukemic evolution, the molecular mechanisms underlying disease progression are still poorly annotated. It is generally accepted that additional mutations cooperate with BCR-ABL during progression to BC (Calabretta and Perrotti, 2004), as is demonstrated by the observation that >75% of BC patients harbor additional cytogenetic abnormalities (Mitelman and Levan, 1978; Radich, 2007). There is also good evidence that the BCR-ABL protein itself contributes to the acquisition of further mutations, through its effects on reactive oxygen species induction, DNA damage, DNA repair, apoptosis, and cellular growth (Perrotti et al., 2010; Nieborowska-Skorska et al., 2012; Bolton-Gillespie et al., 2013), and the levels of BCR-ABL protein can indeed increase in the transition from CP to BC (Gaiger et al., 1995). However, to date, only a small number of mutations in specific pathways have been associated with disease progression in CML. For example, mutations or deletions in TP53, ASXL1, and RUNX1 are commonly described in myeloid BC at frequencies ranging between 3 and 25% (Ahuja et al., 1989; Grossmann et al., 2011), 15 and 20% (Boultwood et al., 2010; Grossmann et al., 2011), and 13 and 33% (Grossmann et al., 2011; Zhao et al., 2012) of cases, respectively. Similarly, mutations or deletions in the CDKN2A/B and IKAROS genes have been reported in up to 50 and 80% of patients in lymphoid BCs, respectively (Sill et al., 1995; Mullighan et al., 2008). Modern sequencing technologies and lowered costs have refined the mutational landscape for many tumors (Pleasance et al., 2010a,b; Curtis et al., 2012; Cancer Genome Atlas Research Network, 2013), but as yet have only been used in a directed fashion in CML (Piccaluga et al., 2009; Boultwood et al., 2010; Grossmann et al., 2011). Therefore, the spectrum of mutations that cooperate with BCR-ABL and the majority of pathways and processes that are corrupted by these mutations during the progression of CML to advanced phases, particularly for myeloid transformation, have yet to be fully described.Mouse models have greatly informed cancer biology in general and CML in particular. Several models have been previously generated, in which transgenic BCR-ABL expression is driven by several different promoters after either germline or retroviral integration (Hariharan et al., 1989; Castellanos et al., 1997; Honda et al., 1998; Huettner et al., 2000, 2003; Koschmieder et al., 2005). However, many of these models have failed to recapitulate the human disease by either generating predominantly acute lymphoid leukemias that lacked a preceding chronic phase, or a very rapidly fatal myeloproliferative neoplasm (MPN)–like disease not resembling the human counterpart (Daley et al., 1990; Honda et al., 1998; Huettner et al., 2000; Huettner et al., 2003). Models of BC have also been reported, where BCR-ABL expression has been combined with a known second hit, such as p53 or Dok1/Dok2 loss, or NUP98-HOXA9 or Hes1 overexpression (Skorski et al., 1996; Honda et al., 2000; Dash et al., 2002; Yasuda et al., 2004; Neering et al., 2007). Although confirmatory of the cooperation of specific mutations with BCR-ABL, these models have not informed the broader biology of BC due to their directed nature. Previous attempts to model random secondary mutations using retroviral insertional mutagenesis have also proven of limited value, with two reported studies only documenting three common insertions (Notch 1, Zfp423, and BCR-ABL; Mizuno et al., 2008; Miyazaki et al., 2009). Furthermore, the majority of these models have generated lymphoid leukemias, mainly T-ALL, thereby reducing their relevance for human disease.We therefore set out to generate a novel mouse model of CML progression that would allow us to identify mechanisms of BC progression in an unbiased and tractable manner. Here, we have combined a mouse transposon-based insertional mutagenesis system with a published transgenic mouse model of chronic phase CML (Koschmieder et al., 2005). For the first time, we report a BC model that closely mimics the natural progression of human CML and faithfully recapitulates the cellular and molecular aspects of its biology. We have identified known and novel candidate genes and pathways that, in combination with BCR-ABL, drive disease progression and could act as potential therapeutic targets in BC. Our novel model therefore defines mechanisms of CML progression, identifies therapeutic targets and provides a translational resource to improve clinical outcomes in this aggressive disease.     

PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

Somatic mutations in the PIK3CA gene have been discovered in many human cancers, and their presence correlates to therapy response. Three “hotspot” mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity, and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous, and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies, the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group), 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas, we report one PIK3CA mutation for the first time. In the selected group, 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive, reliable, cost-effective, and easy-to-perform, and therefore can be used as a screening test in a high-throughput pharmacodiagnostic setting.Somatic mutations and gene amplification for the gene encoding for phosphatidylinositol 3-kinase (PI3K) p110α catalytic subunit, PIK3CA, located in chromosome 3, have been discovered in many different human cancers.^{1,2,3,4,5,6,7,8,9} PIK3CA mutations in exons 9 and 20 account for more than 90% of mutations reported for this gene, according to COSMIC Database (Catalogue Of Somatic Mutations In Cancer Database, v41 release; Wellcome Trust Genome Campus, Hinxton, Cambridge; Accessed May 2009; http://www.sanger.ac.uk/perl/genetics/CGP/cosmic?action=gene&ln=PIK3CA). There are three recurrent or “hotspot” mutations within these exons: c.1624G>A(E542K), c.1633G>A(E545K) in exon 9 (helical domain), and c.3140A>G(H1047R) in exon 20 (kinase domain). These hotspot mutations have been shown to have oncogenic effects.^1,10,11PIK3CA mutations appear to have a clinical significance.¹² Recent studies have shown that PIK3CA mutations can independently hamper the therapeutic response to anti-EGFR biological therapies (panitumumab or cetuximab) in metastatic colorectal cancer¹³ and demonstrate resistance to dietary restriction therapies.¹⁴ Moreover, several efforts are underway nowadays to target the PI3K pathway with therapeutic inhibitors.Additionally, keeping in mind that not all patients with HER2-overexpressing metastatic breast cancer respond to Trastuzumab (Herceptin), activated PI3K signaling has been proposed to predict Trastuzumab resistance.^15,16 Loss of the PTEN (Phosphatase and Tensin Homolog) protein has been suggested as a key factor for the development of resistance to this drug.^15,16 However, PTEN loss alone,^16,17 and in combination with phosphorylated AKT expression,¹⁷ proved inadequate to predict response to therapy. Conversely, combining PTEN loss and gain-of-function mutations of the PIK3CA gene, resistance to Trastuzumab was successfully predicted.¹⁶The introduction of High-Resolution Melting Analysis (HRMA) technique in 2003¹⁸ came with several advantages. HRMA, a closed-tube probe-free technique, with genotyping and mutation scanning capabilities, is rapid, simple, cost-effective, and nondestructive. Especially, HRMA of small amplicons (<100bp) overcomes the difficulty of classic HRMA analysis to detect mutant homozygotes, and demonstrates a higher diagnostic sensitivity in detecting homozygotes and heterozygotes,^19,20,21,22 with an increase in rapidity.²⁰ Moreover, small amplicons display a higher analytical sensitivity compared with larger amplicons.^23,24 This is a major advantage when scanning for somatic mutations, where the tumor tissue is contaminated with adjacent normal tissue.Archival tissue specimens, such as formalin-fixed paraffin-embedded (FFPE) tissues, represent a vast source of tissue genomic DNA (gDNA), easily obtained from clinical archives. However, they present difficulties in amplification due to DNA degradation, therefore demanding a reduction of PCR amplicons’ length. In addition, use of FFPE tissue decreases diagnostic specificity when used as a template for HRMA.²⁵Until recently, several techniques have been used for scanning and/or genotyping somatic mutations of the PIK3CA gene. These include DNA sequencing,^1,2,3,5,6,8 denaturing High Performance Liquid Chromatography (dHPLC), Single Strand Conformation Polymorphism (SSCP),⁵ multiplex PCR amplification and primer extension,²⁶ and Amplification Refractory Mutation System (ARMS) PCR.²⁷ ARMS PCR has the highest analytical sensitivity reported; however, it does not allow mutation scanning capabilities and the use of Scorpion probes carries a high cost. A HRMA method for hotspot regions of the PIK3CA gene has been reported, using amplicons larger than 100bp and not applied in archival tissue specimens.²⁸Herein, we report a novel methodology for the detection of PIK3CA hotspot mutations using HRMA of small amplicons. By performing novel in silico primer design and extensive optimization studies we maximized analytical sensitivity and obtained very accurate results for FFPE samples, as verified by DNA sequencing. After validation using a group of 129 breast tissue samples, the method was applied on a selected group of 75 patients who underwent Trastuzumab treatment.     

The Src family kinases Hck,Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment

Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell–mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in β₂ integrin–deficient (CD18^−/−) mice, Hck^−/−Fgr^−/−Lyn^−/− neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells.Src family kinases are best known for their role in malignant transformation and tumor progression, as well as signaling through cell surface integrins (Parsons and Parsons, 2004; Playford and Schaller, 2004). Due to their role in cancer development and progression, Src family kinases have become major targets of cancer therapy (Kim et al., 2009; Zhang and Yu, 2012). Src family kinases are also present in immune cells with dominant expression of Lck and Fyn in T cells and NK cells; Lyn, Fyn, and Blk in B cells and mast cells; and Hck, Fgr, and Lyn in myeloid cells such as neutrophils and macrophages (Lowell, 2004).The best known function of Src family kinases in the immune system is their role in integrin signal transduction. Indeed, Hck, Fgr, and Lyn mediate outside-in signaling by β₁ and β₂ integrins in neutrophils and macrophages (Lowell et al., 1996; Meng and Lowell, 1998; Mócsai et al., 1999; Suen et al., 1999; Pereira et al., 2001; Giagulli et al., 2006; Hirahashi et al., 2006), Lck participates in LFA-1–mediated T cell responses (Morgan et al., 2001; Fagerholm et al., 2002; Feigelson et al., 2001; Suzuki et al., 2007), and Src family kinases are required for LFA-1–mediated signal transduction and target cell killing by NK cells (Riteau et al., 2003; Perez et al., 2004).Src family kinases also mediate TCR signal transduction by phosphorylating the TCR-associated immunoreceptor tyrosine-based activation motifs (ITAMs), leading to recruitment and activation of ZAP-70 (van Oers et al., 1996; Zamoyska et al., 2003; Palacios and Weiss, 2004). However, their role in receptor-proximal signaling by the BCR and Fc receptors is rather controversial. Although the combined deficiency of Lyn, Fyn, and Blk results in defective BCR-induced NF-κB activation, receptor-proximal BCR signaling (ITAM phosphorylation) is not affected (Saijo et al., 2003). Genetic deficiency of Lyn, the predominant Src family kinase in B cells, even leads to enhanced BCR signaling and B cell–mediated autoimmunity (Hibbs et al., 1995; Nishizumi et al., 1995; Chan et al., 1997). Similarly, both positive (Hibbs et al., 1995; Nishizumi and Yamamoto, 1997; Parravicini et al., 2002; Gomez et al., 2005; Falanga et al., 2012) and negative (Kawakami et al., 2000; Hernandez-Hansen et al., 2004; Odom et al., 2004; Gomez et al., 2005; Falanga et al., 2012) functions for Fyn and Lyn during Fc receptor signaling in mast cells have been reported. In addition, Hck^−/−Fgr^−/− neutrophils respond normally to IgG immune complex–induced activation (Lowell et al., 1996) and Fc receptor–mediated phagocytosis of IgG-coated red blood cells is delayed but not blocked in Hck^−/−Fgr^−/−Lyn^−/− macrophages (Fitzer-Attas et al., 2000; Lowell, 2004). The differential requirement for Src family kinases in TCR, BCR, and Fc receptor signaling is thought to derive from the fact that Syk, but not ZAP-70, is itself able to phosphorylate ITAM tyrosines (Rolli et al., 2002), making Src family kinases indispensable for signaling by the ZAP-70–coupled TCR but not by the Syk-coupled BCR and Fc receptors.Autoantibody production and immune complex formation is one of the major mechanisms of autoimmunity-induced tissue damage. In vivo models of those processes include the K/B×N serum transfer arthritis (Korganow et al., 1999) and autoantibody-induced blistering skin diseases (Liu et al., 1993; Sitaru et al., 2002, 2005), which mimic important aspects of human rheumatoid arthritis, bullous pemphigoid, and epidermolysis bullosa acquisita. Activation of neutrophils or macrophages (Liu et al., 2000; Wipke and Allen, 2001; Sitaru et al., 2002, 2005; Solomon et al., 2005), recognition of immune complexes by Fcγ receptors (Ji et al., 2002; Sitaru et al., 2002, 2005), and β₂ integrin–mediated leukocyte recruitment (Watts et al., 2005; Liu et al., 2006; Chiriac et al., 2007; Monach et al., 2010; Németh et al., 2010) are indispensable for the development of those in vivo animal models.The role of Src family kinases in β₂ integrin signaling and the requirement for β₂ integrins during autoantibody-induced in vivo inflammation prompted us to test the role of Src family kinases in autoantibody-induced inflammatory disease models. We found that Hck^−/−Fgr^−/−Lyn^−/− mice were completely protected from autoantibody-induced arthritis and inflammatory blistering skin disease. Surprisingly, this was not due to a cell-autonomous defect in β₂ integrin–mediated leukocyte migration but to defective generation of an inflammatory microenvironment, likely due to the role of Src family kinases in immune complex–induced neutrophil and macrophage activation.     

Soiled Bedding Sentinels for the Detection of Fur Mites in Mice

Identification and eradication of murine fur mite infestations are ongoing challenges faced by many research institutions. Infestations with Myobia musculi and Myocoptes musculinus can lead to animal health problems and may impose unwanted research variables by affecting the immune and physiologic functions of mice. The purpose of this study was to evaluate the utility and efficacy of soiled bedding sentinels in the detection of fur mite infestations in colony mice. Female young-adult CRL:CD1(ICR) mice (n = 140) were exposed over a 12-wk period to various volume percentages of soiled bedding (11%, 20%, 50%, and 100%) from fur-mite–infested animals. Mice were tested every 2 wk with the cellophane tape test to identify the presence of fur mite adults and eggs. At the end of 12 wk, all mice exposed to 11%, 20%, and 50% soiled bedding tested negative for fur mites. One of the 35 mice (3%) receiving 100% soiled bedding tested positive for fur mites at the end of the 12-wk follow-up period. These findings suggest that the use of soiled bedding sentinels for the detection of fur mite infestations in colony mice is unreliable.Ectoparasite infestations present an ongoing threat to barrier facilities. Murine acariasis in laboratory mice frequently is caused by Myobia musculi, Myocoptes musculinus, and Radfordia affinis.^{1,13,17,40,41} These infestations can be challenging to identify and control and often lead to animal health problems and research complications. For this reason, many institutions strive to exclude these parasites from their barrier facilities.^1,17,18,41 Infestations can further compromise ongoing research by disrupting collaboration with institutions affected by sporadic or endemic mite infestations in their facilities.¹⁸Myocoptes musculinus is the most common fur mite identified among laboratory mice, although mixed infections with Myobia musculi are common.¹⁷ The life cycles of Myocoptes and Myobia are 14 and 23 d, respectively.^2,17 Myobia mites most frequently are found to inhabit the head and neck of mice, whereas Myocoptes are reported to have a predilection for the back, ventral abdomen, and inguinal regions.^2,17 Mite infestations in live animals are often diagnosed by using cellophane tape tests.^5,14,25 A clear piece of cellophane tape is pressed against the fur of the mouse, affixed to a slide, and examined microscopically for the presence of eggs or adult mites. Pelage collection and examination and skin scraping are 2 other common diagnostic methods. These tests have been shown to have increased sensitivity when compared with the tape test, but they have the disadvantage of requiring an anesthetized or recently euthanized animal.^2,5,17Fur mites feed on the superficial skin tissues and secretions of the animals they infest.^1,2,17 Mite infestations in mice have been associated with numerous health problems. Common clinical manifestations of acariasis include alopecia, pruritis, and scruffiness.^{1,2,10,15,17-20,22,26,31,42,44} Severe health problems including ulcerative dermatitis, hypersensitivity dermatitis, and pyoderma can develop also.^1,2,10,17,41 Infested mice may also be prone to secondary infections, reduced life span, and decreased body weight.^2,17,42 Several studies have analyzed the potential research complications associated with murine acariasis.^{10,15,18-20,22,26,31,42,44} Mite infestations have been shown to cause elevations in IgE, IgG, and IgA levels; mast cell degranulation; increased levels of inflammatory cytokines; and lymphocytopenia.^{18-20,22,26,31,44} The changes in the immunologic function of affected mice can persist even after mite eradication.¹⁸Multiple chemical treatment modalities have been proposed for the eradication of fur mites in infested animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} Conflicting information exists regarding the success of many of these treatment regimes. In addition, several of the proposed treatments have been associated with toxicity, adverse health effects in mice, and alterations in the physiologic or immune function of the animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} The complications associated with identifying an effective treatment for murine acariasis while minimizing toxicity and the introduction of unknown research variables highlight the importance of rapid and effective detection of mite infestations in barrier facilities.Many institutions rely on soiled bedding sentinels for their primary source of information on colony health status.^9,21,33,35 Several studies have demonstrated the efficacy of soiled bedding sentinels to detect common murine pathogens such as mouse hepatitis virus, mouse norovirus, Helicobacter spp., and pinworms.^{4,7,24,28,37,38} However, not all pathogens are easily transmitted through soiled bedding exposure. Agents that are not routinely identified through soiled bedding sentinels include those that are shed in low numbers, are susceptible to environmental factors, or are not easily transmitted through the fecal–oral route.^6,21,33 Examples of pathogens that are not easily transmitted or detected through soiled bedding exposure include mouse Sendai virus, Pasteurella pneumotropica, lymphocytic choriomeningitis virus, and cilia-associated respiratory bacillus.^7,9,11,16,35 In addition, the sensitivity of soiled bedding sentinel programs varies with the number of animals affected within the colony.^27,38In 2008, our institution faced a fur-mite outbreak that affected more than 25 rooms in a single barrier facility. Animals positive for Myobia musculi, Myocoptes musculinus, or both were identified through either health check requests for itching and scratching animals and by testing of animals scheduled for export to other institutions. Despite the extent of this outbreak, the soiled bedding sentinels in all mite-positive rooms consistently tested negative on cellophane tape tests for fur mites.To our knowledge, only one study has specifically examined the efficacy of soiled bedding sentinels in the detection of fur mites in mice.³⁴ A separate study, examining the transmission of mouse hepatitis virus to soiled bedding sentinels,³⁸ demonstrated that 75% of cages (3 of 4) exposed to soiled bedding from colony animals tested positive for fur mites after 19 wk of exposure. That previous study used 8 cages of 12 mice each; 4 cages received soiled bedding from colony animals, whereas the other 4 cages received clean nonsoiled bedding. In that study,³⁸ 56.3% of colony mice were known to be mite-positive. Other literature suggests that spread of mites to naïve animals requires direct contact and that soiled bedding does not serve as an effective mechanism for transmission.^1,17,23,39 However, we were unable to identify any research or experiments that substantiated these conclusions.The purpose of the present study was to evaluate whether CRL:CD1(ICR) mice housed in static microisolation caging on soiled bedding from mice with Myobia and Myocoptes infestations can be used as sentinels for the detection of fur mites and to determine how the efficacy of these soiled bedding sentinels for fur-mite detection varies with the prevalence of fur-mite infestation among colony animals.     

Myoblasts and Embryonic Stem Cells Differentially Engraft in a Mouse Model of Genetic Dilated Cardiomyopathy

The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.Cell therapies are progressively emerging as promising tools for the treatment of heart failure. In an attempt to achieve cardiac cell-based replacement therapy in the setting of postischemic cardiomyopathies (ICM), a variety of adult cell types have been tested up to preclinical stages in small and large animal models, including skeletal myoblasts (Mb), muscle-derived stem cells, adipose-derived stem cells, bone marrow mononuclear cells, hematopoietic stem cells, circulating endothelial progenitors, mesenchymal stem cells, smooth muscle cells, cardiac stem cells, and most of these approaches have demonstrated some degree of efficacy.^{1,2,3,4,5,6,7} Except for some specific populations of cardiac stem cells, most categories of adult stem cells show partial or complete inability to produce bona fide cardiomyocytes and to participate to true myocardial tissue formation, with respect to homogeneity of electrical conduction.⁸ Their functional benefits would be linked, essentially, to the mechanical strengthening of the scar tissue, and/or to the promotion of myocardial cell survival through paracrine synthesis of trophic factors and/or improved local angiogenesis.^{1,4,7,8,9,10,11} Indeed, phase II randomized clinical trials developed using adult stem cells have provided encouraging but still limited results.^12,13 However, the applicability and therapeutic relevance of cell therapies remain under-explored for nonischemic heart failure (dilated cardiomyopathy (DCM), myocarditis), probably due to the progressive nature of the disease and extension of fibrotic remodeling, which make the targeting of a specific area more difficult than when considering a delineated scar formed upon myocardial infarction. A few preclinical studies have been carried out using Mb,^14,15 smooth muscle cells or ventricular heart cells¹⁶ in cardiomyopathic hamsters, or mesenchymal stem cells,¹⁷ mixed mesenchymal stem cells and Mb,¹⁸ or bone marrow cells in rat models of DCM.¹⁹ Among those studies, Mb seem to have the best potential of integration in the dilated myocardium, and represent a “gold standard” for cell-based therapy, although these cells are not able to differentiate into cardiomyocyte lineage.In contrast, embryonic stem cells (ESC) are pluripotent and can be readily committed towards the cardiogenic lineage in vitro. There is also increasing evidence that cardiac-committed ESC can engraft into the scar tissue within the infarcted myocardium and differentiate into cardiomyocytes, thereby operating a regeneration of the myocardium, eliminating fibrotic scar tissue, and promoting sustained improvement of left ventricular function.^{7,8,11,20,21,22,23,24,25,26} This confers the potential ability to rebuild true cardiac tissue, to replenish areas that have been depopulated following ischemic accidents or the progression of fibrosis.⁸ In contrast, this positive benefit is limited by the formation of teratomas²⁷ or if too many cells are in uncommitted state at the time of injection.²⁴In the present study, we have compared the integration and functional efficacy of the CGR8 line of murine ESC with the D7LNB1 line of murine Mb, in the myocardium of Lmna^H222P/H222P mice. This genetic model of laminopathy reproduces a human missense mutation in the Lamin A/C gene causing Emery-Dreifuss muscular dystrophy. This model exhibits a rapidly progressive and lethal DCM,²⁸ showing pathophysiological evolution and conduction defects comparable to the human situation. Of note, these animals are immunocompetent.The CGR8 cell line of ESC was chosen because it can be grown feeder-free, and it is efficiently committed toward cardiogenic differentiation in vitro upon treatment with bone morphogenic protein 2 (BMP-2),^23,24,29,30 a treatment that indirectly lowers the risk of teratoma formation in vivo by decreasing the proportion of pluripotent cells.^24,27 The committed CGR8 cells, whether selected or not, have been previously shown to efficiently improve cardiac function following injection into the scar tissue in animal models of postischemic heart failure.^7,8,23,24,25 The time window for the addition of BMP-2 is of crucial importance,³⁰ therefore we pretreated CGR8 ESC for a short period of time, and we designed the experiments using limited amounts of cells to reach a compromise between myocardial differentiation and risk of teratoma formation (3 × 10⁵ per heart, at four different sites).The Mb have been assayed, in the present study, as a gold standard for validating the injection procedure, the efficacy of the immunosuppression regimen, the natural evolution of the implanted cells, the immunohistological procedures. Comparisons between Mb and ESC in murine models of postischemic heart failure have pinpointed important intrinsic differences in the efficacies and persistence of these two cell types, which now deserve a comparison in a DCM model.¹¹ The D7 Mb cell line was originally derived from the dy/dy mouse model of laminin-2 deficient congenital muscular dystrophy.³¹ It was engineered to express β-Galactosidase (β-Gal) constitutively and named D7LNB1. It showed no modification in its ability to form myotubes in vitro and in vivo. In addition, unlike C2 or G8 murine Mb, D7LNB1 Mb cell line did not produce carcinomas in vivo.³² CGR8 ESC and D7 Mb originate from close parental strains of 129 mice derived from the same animals in the 1970''s (129P/Ola background for ESC, 129/ReJ background for D7), and they share identical markers.³³ To avoid immune rejection triggered by murine ESC³⁴ or Mb³⁵ further expressing β-Gal in allogenic contexts, recipient mice were immunosuppressed using Tacrolimus.³⁶ We compared the engraftment of committed ESC and D7LNB1 Mb in terms of functional benefits and fate of engrafted cells in vivo. Our results suggest that cardiac-committed ESC failed to engraft into the dilated myocardium of the Lmna^H222P/H222P mice model, whereas Mb have a higher transplantation efficiency and greater functional improvement of cardiac function in this genetic model of dilated heart failure.     

Efficacy of Enrofloxacin in a Mouse Model of Sepsis

We examined the efficacy of enrofloxacin administered by 2 different routes in a mouse model of sepsis. Male CD1 mice were infected with a bioluminescent strain of enteropathogenic Escherichia coli and treated with enrofloxacin either by injection or in drinking water. Peak serum levels were evaluated by using HPLC. Mice were monitored for signs of clinical disease, and infections were monitored by using bioluminescence imaging. Serum levels of enrofloxacin and the active metabolite ciprofloxacin were greater in the group treated by injection than in controls or the groups treated by administration in drinking water. Survival of the group treated with enrofloxacin injection was greater than that of controls and groups treated with enrofloxacin in the drinking water. Bioluminescence in the group treated with enrofloxacin injection was less than that in the groups treated with oral administration at 12 h and in the groups treated orally and the control group at 16 h. According to these findings, we recommend the use of injectable enrofloxacin at 5 mg/kg SC for mice with systemic infections.Abbreviation: MIC, minimal inhibitory concentrationSepsis is defined as the systemic inflammatory response to the presence of bacterial infection.⁶ It affects approximately 750,000 people annually in the United States, with a mortality rate of 30% to 50% and a financial burden of US$16.7 billion dollars.¹ Septic shock is persistent hypotention with hypoperfusion abnormalities or organ dysfunction secondary to sepsis and kills 10 times more people than myocardial infarction in the United States.^7,38 Bacterial isolates from patients with gram-negative infections most commonly include Escherichia coli, Klebsiella species, or Enterobacter species.⁶ Due to the widespread and severe nature of this disease, this is an active area of scientific research.^14,17Enrofloxacin is a frequently used antibiotic approved for use in dogs, cats, cattle, and pigs in the United States. It is a fluoroquinolone antibiotic that leads to bacterial cell death through the inhibition of topoisomerase II, which controls supercoiling of bacterial DNA.³³ The bactericidal effect is dependent on the concentration of antibiotic present in tissues,³⁵ both of the parent compound and of the active metabolite ciprofloxacin.¹⁶ Enrofloxacin has been studied in a wide range of laboratory animal species including bovines,^16,26,27 dogs,^5,36 frogs,^20,25 horses,⁴¹ macaques,⁴ marmosets,⁴⁴ mice,^{22,32,34,36,40,49} rabbits,¹⁹ rats,⁸ sheep,¹⁵ and swine.^15,37,52 Published doses in veterinary formularies range from 2.5 to 20 mg/kg for enteral and parenteral bolus dosing and for rodents at 0.05 to 0.2 mg/mL in drinking water.^11,45,46 In laboratory animal medicine, fluoroquinolone antibiotics are sometimes administered as a therapeutic agent to populations of animals. One example of this practice is for eradication of an opportunistic pathogen such as Pneumocysitis carinii in genetically modified mice.³² In addition, fluroquinolones are used on a large scale prophylactically in research protocols involving bone marrow transplantation in mice.³⁹ When large groups of animals require treatment, the route of administration can affect the personnel time required to conduct the research.Bioluminesence imaging is a noninvasive imaging modality that has been used to study cell trafficking, tumor development, gene expression, gene therapy, inflammation and infection, protein–protein interactions, and protein stability and function in laboratory rodents.¹³ Bioluminescent imaging systems use specialized charge-coupled cameras to capture low amounts of light. Organisms can be genetically engineered to express firefly luciferase or other types of luciferase enzymes, which emit light when the organisms are provided an exogenous substrate, such as luciferin, in the presence of ATP and oxygen.²⁹ In addition, specialized bacteria have been engineered to carry genes for both the luciferase and the substrate, a long-chain fatty aldehyde, and thus do not require an exogenous source of substrate.⁴⁷ These types of modified bacteria have been used to study the progression and treatment of bacterial infections of the gastrointestinal tract,^10,21 wounds,^23,47 and surgical implants.^30,51,53 Advantages of bioluminescence imaging include the ability to assess the spatial and temporal distribution of bacteria after intervention in the same animal, thus reducing animal numbers.The purpose of the current study was to refine a model of sepsis by evaluating the efficacy of different routes of administration of prophylactic antibiotics. This work was part of a larger study evaluating the pathogenesis of hospital-acquired infection in patients receiving blood transfusions during concurrent antibiotic therapy. With an effective model system, research results can be translated to treatment of human diseases. Serial noninvasive optical imaging of a bioluminescently engineered strain of enteropathogenic Escherichia coli was used as a biomarker of bacterial infection in the presence of mitigating drug regimens. We hypothesized that there would be no difference in the severity of infection, evaluated as photons per second emitted from a region of interest over the abdomen, between animals that received enrofloxacin via different routes of administration in this model of sepsis. Our goal was to establish an effective method of preventing or treating gram-negative sepsis through antibiotic administration that minimized animal handling and manipulation, promoted animal wellbeing, and maximized the information obtained from the animals used in our research.     

Eliminating Animal Facility Light-at-Night Contamination and Its Effect on Circadian Regulation of Rodent Physiology, Tumor Growth, and Metabolism: A Challenge in the Relocation of a Cancer Research Laboratory

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR^–) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O₂, glucose, total fatty acid and CO₂ production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine–threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.Abbreviation: 13-HODE, 13-hydroxyoctadecadienoic acid; γH2AX, histone 2AX; AKT, serine–threonine protein kinase; ERK1/2, extracellular signal-regulated kinase p44/46; GSK3β, glycogen synthase kinase 3β: LAN light at night; MEK, mitogen-activated protein kinase kinase, PCNA, proliferating cell nuclear antigen; SR^–, steroid-receptor–negativeRelocating laboratory animal research from one institution to another can be a daunting task for both scientists and animal care personnel with regard to control of lighting and elimination of light-at-night (LAN) contamination. Appropriate facility lighting and lighting protocols, as outlined in the Guide for the Care and Use of Laboratory Animals,³⁰ are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations.^16-18,22 The profound effect of light on circadian behavior and physiology is well established.^{2,3,5,9,11,12,16-18,22,29,32,44,46,49,52,55-58,64}Minor alterations in light intensity,¹¹ spectral quality,¹² and duration⁹ at any given time of day can alter or disrupt chronobiologic rhythms markedly in all mammals.^{6,17,26,44,55-59} Light information, which initially is detected by a small group of intrinsically photosensitive retinal ganglion cells containing the blue light-sensitive photopigment melanopsin,^6,26 is transmitted through the retinohypothalamic tract⁵⁹ to a central molecular clock located in the suprachiasmatic nucleus of the hypothalamus.^32,57 The suprachiasmatic nucleus, the activity of which is entrained by the light:dark cycle,^32,57 sends projections over a polysynaptic pathway to the pineal gland driving a series of molecular events leading to the production of the pineal neurohormone melatonin (N-acetyl-5 methoxytryptamine), primarily during the night.^29,46 The daily rhythmic melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions including the sleep–wake^23,46,66 and reproductive cycles,^51,55 immune function,^38,41,56 hormone levels,^{19,31,45,47,68} temperature regulation,²³ electrolyte balance,⁶⁹ neural protein synthesis,⁶⁵ and redox states.^24,53Dark-phase LAN exposure suppresses endogenous melatonin concentrations and may lead to various disease states,^42,58 including carcinogenesis,^7,8,16,18,33 and metabolic syndrome.^{17,34-37,39,70} Previous in vivo studies in our former laboratory (at the Bassett Research Institute, Cooperstown, NY) demonstrated that animal room LAN of as little as 0.2 lx (0.08 µW/cm²; rodent eye level) during an otherwise normal dark-phase suppressed normal physiologic nighttime melatonin levels, leading to markedly disrupted circadian regulation of physiology and metabolism in nontumor-bearing host animals^16,18 and a stimulation in metabolism and proliferation of both tissue-isolated MCF7 steroid-receptor–negative (SR^–) human breast cancer xenografts and syngeneic grafts of rodent hepatoma 7288CTC in rats.^7,17 This effect was mediated by melatonin receptor-mediated suppression of cAMP, leading to inhibition of tumor linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminated in downregulation of epidermal growth factor and insulin-like growth factor 1 pathways.^7,8,16-19,62Exposure to LAN is likely to exert pervasive and problematic effects on mammalian behavior and physiology in laboratory animal facilities around the world. During the past decade, improved facility design and better adherence to animal room lighting protocols certainly has helped to reduce the problem. In moving to our laboratory animal facility at Tulane University School of Medicine (New Orleans, LA), we discovered considerable preexisting LAN contamination that had to be eliminated before we could resume our human cancer research.The current study was performed to monitor the effects of the elimination of animal room LAN contamination over time on animal health and wellbeing, tumor growth, and metabolic profiles by assessing well-established circadian parameters in physiology and metabolism.^7,8,16-18 We measured light-induced suppression of melatonin, an accepted and sensitive marker of the effects of light on the circadian system in all mammals,^{2,3,5,9,11,12,15,16,18,20,21,29,44,46,49,52,55-58,64} before and after tumor implantation and growth. Tissue-isolated MCF7(SR^–) human breast cancer xenografts and 7288CTC rat hepatomas have been well-characterized over the years in our former light-tight facilities^7,8,16-18 and provided us with unique markers and measures of the extent to which LAN contaminated our new animal quarters. In tumor-bearing animals exposed to even minimal LAN, the latency-to-onset of tumor growth and proliferation rates of these tumors increase markedly in direct proportion to LAN intensity. As improvements were made to eliminate LAN contamination in the new location over the course of more than 20 generations of tumor passages, we measured the changes and reestablishment of normal rat and host–tumor circadian regulation. The information from this study may assist investigators and animal care personnel in addressing this important influence on the health and wellbeing of laboratory animals and consequent effects on the outcome of scientific investigations.     

KIR2DS4 is a product of gene conversion with KIR3DL2 that introduced specificity for HLA-A*11 while diminishing avidity for HLA-C

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between “full-length” and “deleted” forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1⁺ and C2⁺ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4⁺ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline–valine motif at positions 71–72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.NK cells respond early to infection by killing infected cells and secreting cytokines (Lanier, 1998). Such activation involves integration of signals from a variety of activating and inhibitory receptors, including several that recognize MHC class I molecules (Moretta et al., 1996). Members of the killer cell Ig-like receptor (KIR) family recognize epitopes of HLA-A, -B, and -C. The inhibitory KIRs comprise KIR2DL and KIR3DL, and the activating receptors comprise KIR2DS and KIR3DS.KIRs with HLA-A, -B, and -C specificity comprise two phylogenetic lineages (Khakoo et al., 2000). In lineage II, KIR3DL1 recognizes the subset of HLA-A and -B allotypes having the Bw4 epitope (Gumperz et al., 1995), and KIR3DL2 recognizes HLA-A3 and -A11 (Döhring et al., 1996; Pende et al., 1996). In lineage III, KIR2DL1 recognizes the subset of HLA-C allotypes having the C2 epitope (HLA-C2) defined by lysine 80, whereas KIR2DL2/3 recognizes the alternative subset having the C1 epitope (HLA-C1) defined by asparagine 80 (HLA-C1; Mandelboim et al., 1996). Unlike the inhibitory KIRs, functions and ligands for the lineage II and III activating KIRs are poorly understood. Few KIR genes are fixed, and activating KIR genes are less common than inhibitory KIR genes (Abi-Rached and Parham, 2005). KIR2DS1 has similar C2 specificity as 2DL1 but much reduced avidity (Biassoni et al., 1997; Stewart et al., 2005; Chewning et al., 2007). Ligands for KIR2DS2, 2DS3, 2DS5, and 3DS1 remain elusive (Kim et al., 1997; Valés-Gómez et al., 1998; Winter et al., 1998; Carr et al., 2007; Della Chiesa et al., 2008; VandenBussche et al., 2009).KIR2DS4, the most prevalent lineage III–activating KIR, is also the oldest and most divergent, being the only human lineage III KIR with an orthologue in another species: chimpanzee Pt-KIR2DS4 (Khakoo et al., 2000). Before rationalization of the KIR nomenclature (Marsh et al., 2003), KIR2DS4 was alternatively termed p50.3 (Bottino et al., 1996), clone 39 (Wagtmann et al., 1995), NKAT8 (Colonna and Samaridis, 1995; Campbell et al., 1998), and KAR-K1 (Kim et al., 1997). Several early studies failed to detect interactions between 2DS4 and HLA class I (Bottino et al., 1996; Kim et al., 1997; Valés-Gómez et al., 1998; Winter et al., 1998), but two detected weak but potentially significant interactions with HLA-C*03 (Campbell et al., 1998) and HLA-C*04 (Katz et al., 2001). Overall, the weak and ambiguous interactions of activating KIRs with HLA class I led to the physiological relevance of the activating human KIRs being questioned and, in the case of KIR2DS4, to a search for non–MHC class I ligands (Katz et al., 2004).Epidemiological studies implicate activating KIR genes, often in combination with HLA class I, in a variety of clinical associations (for review see Kulkarni et al., 2008). Although KIR haplotypes differ widely in KIR gene content, they divide into two groups: A, which has mainly inhibitory KIR genes, and B, which has additional activating KIR genes (Uhrberg et al., 1997). All populations examined have both haplotype groups but their relative frequencies vary, and they are likely maintained by balancing selection (Norman et al., 2007). Furthermore, many clinical associations with KIR can be attributed to A and B haplotype differences (Parham, 2005). Overall, the epidemiological studies point to the activating KIRs as having significant influence on the physiology of the human immune response. Of particular importance in this regard is 2DS4, the only activating KIR of A haplotypes. For these compelling reasons, we reinvestigated the HLA class I specificity of KIR2DS4 and its functional implications.     

Nonsurgical Repair of a Pseudoaneurysm in a Cynomolgus Macaque (Macaca fascicularis)

A cynomolgus macaque presented with an ecchymotic and edematous left leg approximately 1 wk after a blood sample had been collected from the left femoral vein. Ecchymosis was noted in the femoral triangle, prepuce, and scrotum. The animal was not febrile or exhibiting signs of pain or distress. Duplex Doppler ultrasound imaging was used to evaluate the area. An arteriovenous fistula between the femoral artery and vein, accompanied by a pseudoaneurysm arising from the femoral artery, was identified. Various invasive and noninvasive treatment options for the pseudoaneurysm, including surgical repair, thrombin injection, stent placement, and ultrasound-guided compression repair (UGCR), were considered. UGCR was chosen as the first option for treatment. After a total of 20 min of UGCR at the neck of the pseudoaneurysm, complete thrombosis was achieved. Subsequent imaging of the lesion revealed resolution of the pseudoaneurysm. Because of the risks involved with invasive management techniques for this vascular lesion, UGCR is a valuable noninvasive treatment option for the repair of pseudoaneurysms.Abbreviation: CDI, color Doppler imaging; UGCR, ultrasound-guided compression repairVascular malformations have been reported to occur in a wide variety of species.^{2,3,5-7,10,11,13,15-22,24,25,27-35} These abnormalities include arteriovenous fistulas, vascular shunts, hemangiomas, vascular atresias, aneurysms, pseudoaneurysms, telangectasia, and lymphangiomas, with the overwhelming majority of reports describing arteriovenous fistulas.^{2,3,5,6,11,15-19,25,27,30,33,35} Aneurysms have occurred in several primates including chimpanzees, gorillas, squirrel monkeys, howler monkeys, owl monkeys, African green monkeys, spider monkeys, patas monkeys, and capuchin monkeys.²⁴ Pseudoaneurysms are reported infrequently. Pseudoaneurysms (or false) aneurysms are the result of leakage of blood from an artery into a defined space. A pseudoaneurysm associated with the femoral artery in a black and white colobus monkey was detected 6 d after manual restraint for routine blood collection from the femoral artery.³² The pseudoaneurysm was excised, and the resulting vascular defect was closed with an autologous graft. Another report¹⁰ describes a pseudoaneurysm associated with the femoral artery in a rhesus monkey. The diagnosis was obtained interoperatively, and excision of the defect was successful. Because surgical treatment of pseudoaneurysm has inherent risks and requires considerable surgical skill for a successful outcome, a noninvasive approach would be a valuable and cost-effective treatment option. This report is the first to describe the clinical presentation, diagnosis, and nonsurgical treatment of a pseudoaneurysm in a cynomolgus macaque.     

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.B lymphocytes can respond rapidly to nonself-antigens, yet even at mature stages of development can be rendered tolerant if they encounter self-antigen (Goodnow et al., 2005). How B cells distinguish self from nonself has been explained in part by Bretscher and Cohn’s associative recognition (“two-signal”) hypothesis (Bretscher and Cohn, 1970), which posits that B cells can only achieve activation after a second signal is delivered, the first being recognition of antigen by the BCR. Without this second signal, tolerance is induced. In response to T-dependent antigens, activated helper T cells provide this second signal. In a T-independent type 1 response, the second signal might come from the B cells’ Toll-like receptors (TLRs) recognizing conserved microbial motifs attached to the antigen (e.g., lipopolysaccharide; Coutinho et al., 1974). This model, however, fails to explain how T-independent type 2 (TI-2) responses occur, as TI-2 antigens require neither T cells (Mond et al., 1995) nor recognition by known innate immune receptors (Gavin et al., 2006), and can elicit antibody responses in cultures of single B cells (Nossal and Pike, 1984). Although we do not dispute contributory roles of innate immune receptors, cytokines, or accessory cells in amplifying their responses (Mond et al., 1995; Vos et al., 2000; Hinton et al., 2008), TI-2 antigens appear to have only two surprisingly simple properties, high molecular weight and ≥20 closely spaced BCR epitopes (Dintzis et al., 1976), and are thus unlikely to have innate receptors specialized for their recognition.Alternatively, B cells might be capable of “missing self”–recognition (Parish, 1996; Nemazee and Gavin, 2003) similar to that originally observed in NK cells (Kärre et al., 1986). In NK cell recognition, the decision to lyse a target cell depends on integration of opposing signals from activating and inhibitory receptors (Lanier, 2008). Activating receptors trigger recruitment of tyrosine kinases to immunotyrosine activating motifs of associated adapter molecules but are kept in check by inhibitory receptors recognizing classical MHC I molecules expressed on target cells (Lanier, 2008). Inhibitory receptors carry immunotyrosine inhibitory motifs (ITIMs), which serve as docking sites for phosphatases, such as SHP-1, that counteract activation (Ravetch and Lanier, 2000). Target cells that down-regulate MHC I are lysed owing to unopposed activation, hence missing self–recognition.Extrapolating from this model, we hypothesize that besides their BCR epitopes, self-antigens carry self-markers that can engage inhibitory receptors on B cells, preventing antiself TI-2–like responses and rendering activation dependent on second signals. The concept that self-markers might facilitate self-tolerance was first suggested many years ago by Burnet and Fenner (1949) but has garnered little experimental support with respect to lymphocyte tolerance. According to our model, antigens that simultaneously cross-link the BCRs and inhibitory receptors should prevent or blunt B cell responses. Conversely, antigens that bind only the BCR and not inhibitory receptors are predicted to elicit a TI-2 response, provided that they carry the appropriate number and spacing of epitopes. This missing self–model of self-/nonself-discrimination would explain why B cells constitutively express so many inhibitory receptors that recognize ubiquitous self-components, and why null mutations in those receptors or their signaling machinery can lead to autoantibody formation (Nishimura et al., 1998; Pan et al., 1999; Ravetch and Lanier, 2000; Nemazee and Gavin, 2003).In this study, we chose to test if self-/nonself-discrimination is regulated by self-markers through the roles of the sialic acid–binding Ig-like lectins (siglecs) CD22 and Siglec-G in B cells. The siglec family consists of 9 members in mice and 13 members in humans (for review see Crocker et al., 2007). In mice, mature B cells express CD22 (Siglec 2) and Siglec-G, which bind to host sialic acids carried on glycans of glycoproteins and glycolipids and have properties of inhibitory receptors. They carry ITIMs capable of recruiting the tyrosine phosphatase SHP-1 and attenuating BCR signaling (Campbell and Klinman, 1995; Doody et al., 1995; Cornall et al., 1998). Mice carrying null mutations in either CD22 or Siglecg exhibit B cell hyperactivity, variable responses to T-independent antigens, and a tendency toward autoantibody formation (O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; Cornall et al., 1998; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Mouse CD22 exhibits a strong preference for sialoside ligands with the disaccharide sequence NeuGcα2-6Gal (Collins et al., 2006a; Crocker et al., 2007), whereas Siglec-G, before this study, has had an unknown ligand specificity. Their disaccharide ligands represent terminal sugars commonly carried on N- and O-linked glycans of glycoproteins and are found on virtually all cells, including B cells (Crocker et al., 2007). It is well documented that CD22 binds to glycans on endogenous B cell glycoproteins in cis, and masks the ligand binding site from binding synthetic polymeric ligands (Hanasaki et al., 1995; Razi and Varki, 1998; Razi and Varki, 1999; Collins et al., 2002; Han et al., 2005). Yet, CD22 is able to recognize native ligands on glycoproteins of apposing cells in trans, causing it to redistribute to the site of cell contact (Lanoue et al., 2002; Collins et al., 2004). Although mutations of the ligand binding domain of CD22 (Jin et al., 2002; Poe et al., 2004) and ablation of enzymes involved in the synthesis of its glycan ligands (Hennet et al., 1998; Poe et al., 2004; Collins et al., 2006b; Ghosh et al., 2006; Grewal et al., 2006; Naito et al., 2007; Cariappa et al., 2009) document the importance of siglec ligands in the regulation of CD22 function, a unifying role for CD22 ligand interactions in B cell biology has not yet emerged (Crocker et al., 2007; Walker and Smith, 2008). Although less is known about the specificity of Siglec-G and its interaction with ligands, it is assumed that similar concepts regarding cis and trans ligands will apply to the modulation of Siglec-G function (Hoffmann et al., 2007; Chen et al., 2009).Because siglecs see sialylated glycans that are usually absent from microbes, with the notable exceptions of some pathogenic microbes (Crocker et al., 2007; Carlin et al., 2009a), one possible role of siglecs is to discriminate self from nonself. Though CD22 and Siglec-G have been implicated to play roles in B cell tolerance, evidence has been indirect, inferred from the facts that they possess ITIMs able to recruit SHP-1 and dampen Ca²⁺ signaling (Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Hypomorphic or null alleles of CD22 and SHP-1 (Ptpn6) have been correlated with anti-DNA production and development of lupus erythematosus (Shultz et al., 1993; O’Keefe et al., 1999; Mary et al., 2000). CD22 mutations also lead to increased in vivo B cell proliferation and turnover (Otipoby et al., 1996; Nitschke et al., 1997; Poe et al., 2004; Haas et al., 2006; Onodera et al., 2008). However, studies designed to directly assess tolerance induction in antigen-specific CD22^−/− or SHP-1 mutant B cells found, paradoxically, a more robust tolerance relative to unmutated controls (Cyster and Goodnow, 1995; Cornall et al., 1998; Ferry et al., 2005). This suggests that the autoimmune phenotypes of siglec and SHP-1 null mutants could be caused by abnormal B cell selection and development rather than failure of tolerance. It is generally assumed that physical association of CD22 with the BCR will allow CD22 to exert a maximal inhibitory response (Pezzutto et al., 1987; Doody et al., 1995; Lanoue et al., 2002; Courtney et al., 2009), but evidence to support this has been garnered only from in vitro experiments (Ravetch and Lanier, 2000; Lanoue et al., 2002; Tedder et al., 2005; Courtney et al., 2009). In this paper, we show in wild-type mice with unaltered B cell selection and development that decorating a TI-2 antigen with siglec ligands not only prevents its immunogenicity but can also tolerize B cells to subsequent challenges with the unsialylated, immunogenic form. The results suggest that one function of B cell inhibitory receptors like siglecs is to assist B cells in distinguishing self from nonself.     

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless, fungal infections are often life-threatening, and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture, is sensitive to the level of one fungal cell per milliliter of blood or other tissue types, and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis, in combination with more sensitive, inclusive, and appropriately positioned panfungal primers, to address these needs. PCR-based amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species, including mixed and novel species, can be identified directly in vaginal samples. This tool can potentially detect, count, and identify fungi in hundreds of samples per day without further manipulation, costs, or delays, offering a major step forward in fungal molecular diagnostics.Rapid and economical detection, identification, and quantification of fungal species directly from clinical samples is a long-sought goal of clinicians that has still not been fulfilled.^1,2,3,4,5,6 Culture-based diagnosis of fungal infections is inadequate in that many species do not culture efficiently or require unacceptably long incubations.⁷ Antigen-based tests for galactomannan or β-glucan are improvements over culture, but are either too specific, too insensitive, plagued by false positives, or not yet validated by widespread testing.^{8,9,10,11,12,13} Identification of C. albicans and C. glabrata by Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is in clinical use. However, this tool requires an initial culture step to increase fungal titer to detectable levels and is limited in the number of species it can identify.^14,15,16,17PCR-based strategies are the most likely solutions to challenges posed by fungal diagnostics. However, clinical diagnosis of fungal infections by PCR is perhaps its most challenging application, due to low cell numbers, potentially <1 cell/ml sample, to the added problems in lysing fungal walls, and to the similarity in rDNA sequences to human. It is clear that PCR is sufficiently sensitive and specific by in vitro testing, but sample processing under these extreme demands remains problematic. Reviews from 2002 to 2008 indicate that both the promise and problems are great.^6,8,18,19 Most approaches detect positives in clinical samples at their limits of detection, meaning they lack the level of robustness needed to avoid false negatives when widely applied.^1,20PCR strategies using panfungal primers that complement conserved regions of rDNA but span the variable internal transcribed spacer regions (ITS1 and ITS2) have the strong advantage that any and all fungal species will be captured in a single reaction.^{21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47} Traditionally, these amplicons are then sequenced to identify species, using standard, automated capillary sequencing, pyrosequencing, or sequencing-grade microarrays.^{27, 29, 32,33,34,35, 37, 44, 47,48,49,50} Alternatively, precise determination of the base composition of the amplicons by electrospray mass spectroscopy may identify species.⁵¹ Less precise but adequate resolution may be achieved by restriction enzyme analysis of the amplicon.^24,30 Repetitive sequence-PCR (REP-PCR), a version of randomly amplified polymorphic DNA (RAPD) in which primers target repetitive sequence elements, have been used for fungal identification.^52,53 However, this requires pure cultures as the starting material, which is useful in some applications but is not an acceptable precondition for a clinical fungal diagnostic tool. An alternative is to identify species with probes, either standard hybridization after PCR, or during amplification using Taqman, Beacon, or Scorpion probes,^{22,25,28,38,41,45} or hybridization-based fluorescence resonance energy transfer (FRET) probes.⁵⁴An alternative is the use of species-specific PCR, which is typically more sensitive and does not require sequencing of product. Species that are certain to be seen with reasonable frequencies can be detected by species-specific PCR. Approximately 80% of these are species of Candida (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, and C. lusitaniae), or Aspergillus (A. fumigatus, A. flavus, A. terreus, A. niger). The remaining ∼20% include Fusarium, Sporothrix schenckii, zygomycetes (Absidia corymbifera, Rhizomucor pusillus, Rhizopus arrhizus, Mucor, and Cunninghamella). Some of the less common species are also the most problematic in terms of resistance or virulence. There are a number of publications reporting a variety of primers for this approach, with widely varying levels of rigor in their validation.^{55,56,57,58,59,60,61,62} In general, this approach has the disadvantage that multiple assays have to be run on each sample, adding cost and labor. Multiplexing is a possible alternative, but this is widely associated with reduced sensitivity. A further limitation is that many clinical samples will have novel species that may be missed by these primers.High resolution melt analysis is likely to provide an even simpler, faster, and cheaper identification tool sufficiently specific for fungal speciation. This approach more fully exploits the shape of the melting curve of an amplicon, which is a much richer source of information than melting temperature alone. Short, regional sequences denature to form single stranded regions, which release double-stranded DNA-binding fluorescent dyes, before reaching the temperature at which the entire amplicon denatures. This influences the shape of the melt curve, to generate nuances that reflect species-specific sequence differences. Resolution can be further enhanced or normalized by several methods.^63,64 This has enabled identification of bacterial and viral species.⁶⁵Our application of this tool to species of Candida shows that the separation between species is great enough to call species without any postamplification handling.     

Recurrent Idiopathic Gingival Enlargement in an Olive Baboon (Papio anubis)

We here describe a case of recurrent gingival enlargement in an olive baboon (Papio anubis). This baboon (a male breeder that had not undergone any experimental procedures) also had shown mild gingival enlargement the 2 y prior to the current lesion. Clinical and histopathologic findings confirmed a diagnosis of idiopathic gingival enlargement.The term ‘gingival enlargement’ describes abnormal, excessive growth of the periodontal tissues.¹¹ Although gingival enlargement is seen and studied in nonhuman primates only rarely, it has been extensively evaluated in humans.¹⁵ The etiology in humans is multifactorial and includes age, genetic predisposition, and induction due to drugs (for example, cyclosporine, dihydropyridines, calcium channel blockers, sodium valproate, erythromycin) or plaque.^{6,13,14,22,29}Numerous case reports of gingival overgrowth in humans have been described during the past decade, but case reports from nonhuman primates are rare. For example, papillary gingival enlargement occurred in baboons used to study the physiologic effects of estrogen and progesterone during pregnancy.²⁰ The oral anatomy and radiographic presentation of baboons exhibits a striking similarity to human dentition.¹ Similar to that in humans, gingivitis in baboon is characterized by the presence of plaque, calculus, and proliferation of pocket epithelium.^1,9,18 Baboons also exhibit an age-associated increase in prevalence and severity of periodontitis.⁹ There are also case reports of gingival enlargement from rhesus macaques (Macaca mulatta),³⁰ mustache guenon monkeys (Cercopithecus cephus),²³ stump-tailed macaques (Macaca arctoides),^25,26 baboons (Papio ssp.),⁹ gorillas (Gorilla gorilla),⁵ ferrets (Mustela putorious furo),^27,28 and dogs (Canis familiaris).²¹ Most of these reported cases involved animals that had undergone experimental treatments, which induced the lesions.We present a case report of recurrent idiopathic gingival enlargement in a male olive baboon (Papio anubis) that has not undergone experimental manipulation.