A murine DC-SIGN homologue contributes to early host defense against Mycobacterium tuberculosis

The C-type lectin dendritic cell−specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.Innate immune defense against pathogens involves interactions between conserved microbial molecular motifs and pattern recognition receptors (PRRs; Janeway and Medzhitov, 2002), such as the Toll-like receptors (TLRs; Ishii et al., 2008) and C-type lectins (Gordon, 2002; Geijtenbeek et al., 2004; Robinson et al., 2006), expressed on host phagocytic cells. The binding of microbial ligands to PRR results in various immune effector functions, including phagocytosis, oxidative burst (Saijo et al., 2007; Taylor et al., 2007), secretion of antimicrobial peptides (Liu et al., 2006), autophagy (Delgado et al., 2008), and the production of inflammatory cytokines, which help the body to resist pathogens either directly or by shaping the adaptive immune response (Medzhitov, 2007). Conversely, inappropriate innate responses, caused by mutations in PRR-encoding genes, for example, may lead to pathogen persistence and profound effects on host susceptibility to infection (Quintana-Murci et al., 2007).C-type lectins constitute an extensive family of receptors involved in the recognition of endogenous and microbial carbohydrates in a calcium-dependent manner (Weis et al., 1998). DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is a prototypic member of the C-type lectin family (Geijtenbeek et al., 2000). This molecule facilitates broad immune surveillance by DCs and some subsets of macrophages, through interactions with fucose- and mannose-containing glycans on many viruses, bacteria, and parasites (Robinson et al., 2006). In particular, DC-SIGN is an important receptor for M. tuberculosis, the pulmonary bacterial pathogen responsible for tuberculosis (TB; Geijtenbeek et al., 2003; Tailleux et al., 2003, 2005; Torrelles et al., 2008). DC-SIGN recognizes mannose residues in glycoproteins and lipoglycans, such as mannosylated lipoarabinomannan (ManLAM), in the mycobacterial cell envelope (Geijtenbeek et al., 2003; Maeda et al., 2003; Tailleux et al., 2003; Pitarque et al., 2005). A transgenic knockin model of “humanized” mice expressing DC-SIGN (Schaefer et al., 2008) has suggested that this lectin may be involved in protection against TB pathogenesis. Other in vitro studies have suggested that DC-SIGN may be exploited by M. tuberculosis as a means of evading immune surveillance and persisting in the host (Geijtenbeek et al., 2003). Genetic association studies in TB patients and healthy contacts have led to conflicting results on whether DC-SIGN promotes protection or increases susceptibility to TB (Barreiro et al., 2006; Vannberg et al., 2008); nevertheless, both studies clearly suggested a role for DC-SIGN in the course of the disease. This role remains unclear, particularly because of the lack of an appropriate animal model.The mouse DC-SIGN locus (Park et al., 2001; Powlesland et al., 2006) encompasses seven genes, Signr1-5 (also called Cd209b, Cd209c, Cd209d, Cd209e, and Cd209a) and Signr7-8 (also called Cd209g and Cd209f), and one pseudogene, Signr6, which is also called Cd209h (Fig. S1). SIGNR5 (also termed mDC-SIGN or CIRE) was initially described as the mouse orthologue of human DC-SIGN (Park et al., 2001). However, further analyses clearly showed that SIGNR5 differs functionally from human DC-SIGN (Caminschi et al., 2006; Gramberg et al., 2006; Powlesland et al., 2006). Amino acid sequence comparison and reconstruction of the phylogeny of these lectins revealed that mouse SIGNR1, SIGNR3, and SIGNR4 were the homologues most closely related to human DC-SIGN (Fig. S1). In human DC-SIGN, Val³⁵¹ is instrumental in sugar recognition, particularly in binding fucose residues (Guo et al., 2004). The only mouse homologues containing this residue are SIGNR3 and SIGNR4. However, SIGNR4 is unable to bind sugars in vitro, probably because the critical calcium-binding amino acids, Glu³⁴⁷ and Glu³⁵⁴, which are present in all other related molecules, have been replaced by Gln residues (Powlesland et al., 2006). SIGNR1 does not contain Val³⁵¹, and is thus different from human DC-SIGN, as confirmed by the sugar recognition profiles of the two lectins. Mouse SIGNR1, like human DC-SIGN, recognizes mannose residues (Geijtenbeek et al., 2002), but it recognizes these residues as terminal monosaccharides rather than branched glycans, a property common to other C-type lectins outside the DC-SIGN family, such as the mannose-binding lectin (Powlesland et al., 2006). SIGNR1 binds terminal mannose motifs in mycobacterial ManLAM (Koppel et al., 2004), but SIGNR1-deficient mice have no phenotype upon M. tuberculosis infection (Wieland et al., 2007). SIGNR3 is the only one of the seven mouse homologues that, like human DC-SIGN, shows dual specificity for both glycans with high mannose content and fucose-containing oligosaccharides, including the Lewis^X antigen (Powlesland et al., 2006). Furthermore, like human DC-SIGN, SIGNR3 forms tetramers, mediates endocytosis, and releases bound ligands in acidic conditions (Powlesland et al., 2006). Collectively, these data suggest that SIGNR3 is the best candidate for a functional orthologue of human DC-SIGN.We investigated the role of members of the DC-SIGN family in TB in vivo in more detail by generating mice lacking three homologues of human DC-SIGN, namely SIGNR1 and SIGNR3, the homologues that are biochemically most similar to human DC-SIGN, and the more divergent SIGNR5. KO animals were generated directly in a C57BL/6 genetic background so that backcrosses were not required for background purification. We assessed the susceptibility of these mice to M. tuberculosis and compared the results obtained with those for WT animals. We report that only SIGNR3-deficient mice have impaired pulmonary defenses against the bacillus, whereas SIGNR1- and SIGNR5-deficient animals show no particular phenotype upon infection. Consistent with this finding, we report that, like human DC-SIGN in patients with TB (Tailleux et al., 2005), SIGNR3 is expressed in lung (myeloid) cells during the course of M. tuberculosis infection, whereas it is not expressed in naive animals. Mycobacterial recognition by SIGNR3 induces a signaling cascade requiring the tyrosine kinase Syk and involving a YxxI motif in the cytoplasmic tail of SIGNR3. This motif resembles the immunoreceptor tyrosine–based activation motif (ITAM)-like YxxI/L found in other C-type lectins, such as dectin-1 (Brown, 2006). SIGNR3 signaling activates macrophages, inducing the production of inflammatory cytokines, including TNF, in an NF-κB– and Raf1–ERK-dependent pathway, with these cytokines being key components of protection against TB (Flynn et al., 1995; Ladel et al., 1997; Saunders et al., 2000).     

Detection of Spironucleus muris in Unpreserved Mouse Tissue and Fecal Samples by Using a PCR Assay

The purpose of this study was to develop a rapid DNA isolation method and a sensitive and specific PCR assay for detecting Spironucleus muris in mouse tissue and fecal samples. A PCR assay based on the carboxy terminus of the elongation factor 1α gene was developed; the PCR product was confirmed by nucleic acid sequencing and nested PCR. The new PCR assay then was used to test feces from animals that had been screened for S. muris by using direct intestinal examination and histology. The PCR assay was determined to be a more sensitive test than either direct intestinal examination or intestinal histology. To our knowledge, this assay represents the first use of a PCR-based diagnostic screening method to confirm the presence of S. muris in murine tissue and fecal samples.Abbreviation: bp, basepairSpironucleus muris is a flagellated protozoan found in the intestinal lumen of mice, rats, and hamsters.^23,25 The elongated, bilaterally symmetrical trophozoites measure 3 to 4 × 10 to 15 μm and have 6 anterior and 2 posterior flagella. The trophozoites are found in the small intestine, mainly the duodenum, whereas 4 × 7 μm cysts appear in the large intestine and feces.^6,15 The organism has a prepatent period of 5 d.²⁴Although numerous mouse strains have been infected with S. muris,^2,5,15 often spironucleosis does not result in clinical signs. Instead, the infection is identified histologically as accumulations of trophozoites in the intestinal lumen, between villi, and in the crypts of Luberkuhn.⁸ Trophozoites have also been detected in the glands of the pyloric region of the stomach.^19,20 A variety of intestinal lesions have inconsistently been reported, including damaged, degenerating and hyperplastic villus epithelial cells and occasional crypt abscesses.^9,25,26 One study using electron microscopy to evaluate mice with severe spironucleosis revealed that protozoa coated the mucosal surface of the small intestine, perhaps leading to malabsorption and severe malnutrition in the affected mice.⁸Spironucleus muris reportedly has interfered with research by causing deaths among irradiated and cadmium-exposed mice,^9,16 altering macrophage activity and metabolism,^4,12 and depressing lymphocyte responsiveness to tetanus toxoid in the mouse.^18,22 As a result of these findings, S. muris is now considered a facultative pathogen.In the past, several techniques have been used to detect S. muris, including direct smear of intestinal contents (direct examination), histologic examination of intestinal samples, fecal smears, fecal smear immunochemical techniques, and light and electron microscopy. With the exception of direct smears of intestinal contents for trophozoites, these techniques are impractical for screening large numbers of animals due to time constraints, expense, or the need for highly trained personnel. In addition, individual research mice could not reliably be shown to be free of infection because the techniques required euthanized animals or had poor sensitivity.²⁰ The purpose of the current study was to develop a sensitive and specific PCR technique for detection of S. muris in fecal and tissue samples so that large numbers of and individual live mice could be screened rapidly.     

Mycobacterium tuberculosis infection induces il12rb1 splicing to generate a novel IL-12Rβ1 isoform that enhances DC migration

RNA splicing is an increasingly recognized regulator of immunity. Here, we demonstrate that after Mycobacterium tuberculosis infection (mRNA) il12rb1 is spliced by dendritic cells (DCs) to form an alternative (mRNA) il12rb1Δtm that encodes the protein IL-12Rβ1ΔTM. Compared with IL-12Rβ1, IL-12Rβ1ΔTM contains an altered C-terminal sequence and lacks a transmembrane domain. Expression of IL-12Rβ1ΔTM occurs in CD11c⁺ cells in the lungs during M. tuberculosis infection. Selective reconstitution of il12rb1^−/− DCs with (mRNA) il12rb1 and/or (mRNA) il12rb1Δtm demonstrates that IL-12Rβ1ΔTM augments IL-12Rβ1-dependent DC migration and activation of M. tuberculosis-specific T cells. It cannot mediate these activities independently of IL12Rβ1. We hypothesize that M. tuberculosis-exposed DCs express IL-12Rβ1ΔTM to enhance IL-12Rβ1-dependent migration and promote M. tuberculosis–specific T cell activation. IL-12Rβ1ΔTM thus represents a novel positive-regulator of IL12Rβ1-dependent DC function and of the immune response to M. tuberculosis.The control of M. tuberculosis infection occurs through an acquired antigen-specific CD4⁺ T cell response and the IL12B gene is essential to this response (Cooper et al., 2007; Cooper, 2009). Although IL-12 plays an expected role in modulating Th1 responses to M. tuberculosis, we have also shown that IL-12(p40)₂ is required for DCs to migrate in response to chemokines after exposure to mycobacterial and other pathogenic stimuli (Khader et al., 2006; Robinson et al., 2008). That this migration may be important in initiating T cell responses is suggested by the observation that depletion of CD11c⁺ cells before infection delays T cell activation and influences the outcome of infection (Tian et al., 2005).IL-12 family members mediate their biological activities through specific, high-affinity dimeric receptors. All these receptors share IL-12Rβ1, a 100-kD glycosylated protein that spans the plasma membrane and serves as a low-affinity receptor for the IL-12p40 subunit of IL-12 family members (Chua et al., 1994, 1995); coexpression with IL-12Rβ2 or IL-23R results in high-affinity binding of IL-12 and IL-23, respectively, and confers biological responsiveness to these cytokines (Presky et al., 1996; van Rietschoten et al., 2000; Parham et al., 2002). Polymorphisms in IL12B or IL12RB1 are associated with psoriasis (Capon et al., 2007), atopic dermatitis, and other allergic phenotypes (Takahashi et al., 2005) and, importantly, nonfunctional IL12RB1 alleles predispose to mycobacterial susceptibility (Altare et al., 1998; de Jong et al., 1998; Filipe-Santos et al., 2006; Fortin et al., 2007). Thus, understanding how IL-12Rβ1 expression and IL-12Rβ1–dependent signaling is regulated has important implications for tuberculosis and may impact other diseases.Given that CD11c⁺ cells contribute to the control of M. tuberculosis infection (Tian et al., 2005) and that IL-12(p40)₂ is required for their migration in response to pathogenic stimuli (Khader et al., 2006; McCormick et al., 2008; Robinson et al., 2008), we sought to determine if IL-12Rβ1 is required for DC migration after exposure to this organism. In the course of these investigations we not only confirmed this hypothesis but also discovered that DCs express both IL-12Rβ1 and a novel IL-12Rβ1 splice variant (IL-12Rβ1ΔTM) in response to M. tuberculosis. This splice variant can be seen at the mRNA level in CD11c⁺ cells from the lungs of M. tuberculosis–infected mice and as a protein in the membrane of DCs. Importantly, we have determined that IL-12Rβ1ΔTM functions to enhance IL-12Rβ1–dependent DC migration and promote CD4⁺ T cell activation. This finding not only impacts our understanding of DC migration and IL-12Rβ1–dependent mycobacterial immunity it also reveals a previously unknown positive regulator of IL-12Rβ1–dependent events.     

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.     

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.     

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.     

Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds

Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks’ air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection.Corynebacterium bovis is an opportunistic pathogen of immunodeficient mice and is primarily recognized as the causative agent of hyperkeratotic dermatitis (scaly skin disease) in athymic nude (Foxn1, nu/nu) mice. With a worldwide distribution,^2,10,14 C. bovis causes a clinical illness of short duration followed by what is believed to be lifelong subclinical skin colonization.^2,4 Despite the limited duration of clinical signs, C. bovis is thought to have significant effects on xenograft tumor development, leading to delayed, slowed, or failed xenograft and allograft tumor growth.^7,8Eradication of C. bovis from infected nude mouse colonies has proven to be challenging. Variable success has been demonstrated even with an ideal remediation plan of depopulation, decontamination, and repopulation.^14,16 Additional challenges face institutions that attempt phased decontamination, including efficient horizontal spread of infection despite modern husbandry practices and the ineffectiveness of antibiotics to cure clinically and subclinically infected nude mice.^2,3 In addition, C. bovis is known to produce diffuse environmental contamination throughout facilities by airborne deposition of bacterially populated skin flakes.^1,3 Airborne transmission has even been documented within biosafety cabinets, which should be considered one of the primary methods of cage-to-cage transmission.³Early detection is crucial to maintain nude mice colonies free of C. bovis, given the potential for rapid spread through experimental manipulations, general animal care practices, and extensive equipment and environmental contamination.^3,16 After rapid detection, prompt restriction of animal manipulations and movement would allow time for the identification and removal of infected cage(s), followed by localized decontamination of housing and research equipment. However, colony-based C. bovis detection is neither rapid nor efficient, with ubiquitous soiled-bedding sentinel programs that are based on a paradigm of serologic response, in which soiled bedding typically is gathered every 1 to 2 wk at the time of a cage change and is followed by a traditional 3-mo monitoring interval. Although the monitoring interval might be shortened to enhance surveillance, only a fraction of the cages on an IVC rack can contribute bedding to the sentinel cage at any specific time point, due to volume limitations.^5,9 Therefore, multiple cage-change cycles must occur for all cages on a rack to equally contribute to the sentinel cage. Furthermore, little is known about the duration that immunocompetent mice will carry C. bovis on the haircoat to facilitate detection. To more accurately represent the mice under surveillance, nude mice have been used as soiled-bedding sentinels, with the successful detection of C. bovis within nude mouse colonies.³ However, according to the cited report,³ the inherent limitations of a soiled-bedding sentinel program were not overcome, given that only about half of the cages on a rack contributing soiled bedding during weekly cage changes, with a maximal surveillance interval of 1 mo. Moreover, concerns remain that nude sentinel mice may aid in propagating disease, because the potential for environmental contamination from a C. bovis-infected sentinel nude mouse would mirror that of naturally infected nude mice.⁶ Finally, an additional obstacle to the use of soiled-bedding sentinels is data that suggests that soiled-bedding accumulation points in biologic safety cabinets during cage changing may aid in the horizontal spread of infection.³PCR-based diagnostic surveillance of IVC rack air exhaust for mouse pathogens has yielded some success, through the use of 2 sample collection methods primarily.^5,9,11 Small pieces of filter fabric placed in front of exhaust-air rack filters successfully led to the detection of Helicobacter muridarum, Sendai virus, mouse hepatitis virus, and mouse parvovirus but failed to detect Helicobacter hepaticus and mouse rotavirus in experimentally infected mice.⁵ More recently, direct sampling of a rack system''s horizontal exhaust manifold (HEM, Figure 1) with a sterile swab for PCR has been used successfully to detect the fur mites Myobia musculi and Radfordia affinis from naturally infected mice.⁹ However, in another study using HEM sampling, the mouse pinworm Aspiculuris tetraptera was not detected from naturally infected mice.¹¹ Despite the mixed results, we were interested in using quantitative PCR (qPCR) techniques to evaluate the HEM for C. bovis, given the known distribution of C. bovis-contaminated skin flakes by air currents.³ In addition, this method would preclude entering individual cages for sample collection, subsequently decreasing the potential for cross-contamination between cages. Furthermore, surveillance intervals would not be limited by cage-change frequency, and all cages on an IVC rack could be monitored simultaneously. We also wanted to determine the effects of cage location, mouse cage density, and stage of infection on how quickly C. bovis could be detected by PCR analysis of HEM swabs. To further aid in the practical implementation of this surveillance technique, we also evaluated whether our standard rack-sanitation procedure eliminated C. bovis DNA from the HEM of racks that housed infected nude mice.Open in a separate windowFigure 1.Cut-view illustration of the air supply and exhaust plenums of an IVC rack system (Allentown) viewed from the rear. HEPA-filtered supply air is forced into each cage from the horizontal supply air plenum. Negative pressure draws air from each IVC cage into the horizontal exhaust plenum of the rack. Exhaust air from all cages on the row passes through the row''s HEM to enter the common vertical exhaust plenum flowing in the direction of the red arrow.     

Evaluation of Diagnostic Methods for Myocoptes musculinus According to Age and Treatment Status of Mice (Mus musculus)

Detecting and controlling murine fur mites continues to be challenging. Here we compared the efficacy of fur-pluck, cage PCR, and fur PCR testing of mice naturally infested with Myocoptes musculinus and make recommendations regarding the application of these diagnostic strategies in aged or treated mice. We compared all 3 diagnostic methods in groups of infested and noninfested control mice over time. For fur plucks, we used a scoring system to quantitatively compare mite infestations across ages. Mice that were 4 wk old had higher egg and mite scores than did older mice, with average scores at 4 wk corresponding to 40 to 100 individual fur mites and eggs per sample. Furthermore, 15% and 20% of samples from infested mice at 24 and 28 wk of age, respectively, lacked all fur mites and eggs. Cage PCR results varied as mice grew older. Fur PCR testing was the most sensitive and specific assay in untreated infested mice, particularly when mite densities were low. In addition, we compared fur-pluck and fur PCR tests for evaluating the efficacy of selamectin treatment. Two treatments with selamectin eliminated Myocoptes fur-mite infestations. At 8 wk after treatment, all fur-pluck samples were negative, but one-third of treated infested cages remained positive by fur PCR assay; at 16 wk after treatment, all cages were negative by fur PCR assay. Because offspring of infested mice were invariably heavily infested, breeding of suspected infested mice with subsequent testing of offspring was the definitive testing strategy when fur-pluck and PCR results conflicted.Despite advances in laboratory animal colony management, murine fur mites continue to be challenging to detect and control in laboratory mice, with as many as 40% of facilities self-reporting the presence of fur mites in their rodent colonies.⁴ Murine acariasis is commonly caused by Myocoptes musculinus, Myobia musculi, and Radfordia affinis. All 3 species frequently are excluded from laboratory mouse colonies because of their deleterious effects on animal health and potential to confound ongoing research. Specifically, fur mite infestations provoke a Th2 immune response, alter inflammatory cytokines, and elevate serum IgE.^12,13,18 Pathologic changes due to fur mite infestation include lymphadenopathy, hypergammaglobulinemia, secondary amyloidosis, lymphocytopenia, and splenic hypertrophy.¹ Clinical manifestations vary from none to severe dermatitis with ulceration and pyoderma, depending on mouse strain^5,6 and the species of mite, with M. musculi more commonly causing clinical disease.¹ Interestingly, the severity of lesions does not appear to be directly related to the number of mites,²⁵ but lesions may be more severe in older mice.⁸ Although clinical conditions may resolve with treatment, immunologic and pathologic changes due to fur mite infestation can persist even after treatment.^8,11 Therefore, the existence of fur mites within many laboratory mouse colonies is a cause for concern. Because the failure to detect and treat even a few fur mites represents a source for reinfestation,¹ the need for clear recommendations regarding accurate surveillance and effective treatment of fur mite infestations is evident.Fur mites persist in modern laboratory mouse colonies largely because of difficulties in detection. One source of error is the diagnostic technique. Microscopic examination of impressions, fur plucks, and skin scrapes prepared in mineral oil or on cellophane tape; sticky paper applied postmortem; pooled fecal floats; and direct examination of the pelt of either anesthetized or euthanized mice have all been used.^{1,3,14,16,20,24,26} However these traditional methods, even when conducted by commercial laboratories, have the potential for false-negative results due to factors including the selection of the wrong test animal, low mite yield, wrong sampling site, hair overlap, and technical error.^2,16,20,24 Recently, diagnosis by PCR assay has become commercially available, but information regarding its efficacy compared with that of traditional techniques is limited.²⁶ Fur pluck remains a common diagnostic screening tool at many institutions, likely owing to its low cost, relative technical ease, and minimal deleterious effect on tested subjects. Here we report our investigation regarding whether PCR analysis of fur swabs or environmental cage samples was more sensitive in detecting mice infested with M. musculinus than was the fur-pluck technique customarily used at our institution.Another potential source of error is the failure to select appropriate animals for testing. Soiled-bedding sentinels frequently are used for colony surveillance, but their effectiveness is highly controversial, with some reports describing successful detection of fur mites and others showing that using sentinels is quite ineffective in this context.^14,15,20,24 An alternative strategy is to sample colony mice, but information regarding which animals are most likely to yield positive results is limited. It is known that monoinfestations of both M. musculi and M. musculinus increase rapidly in neonatal mice.^6,14 Furthermore, M. musculi populations decrease in numbers concurrent with the development of host immunity and then subsequently equilibrate and then demonstrate cyclical fluctuations (cycle length of 20 to 25 d, presumably related to the 23-d life cycle of mites) in population size.^6,7 However, only sparse information on population fluctuations in M. musculinus is available: a single previous report suggested that population densities varied and that group-housed animals were more likely to yield positive results than were than single-housed mice.¹⁶ Here we investigated how M. musculinus populations varied with age and determined the most appropriate age of mice for sampling.Posttreatment testing may be confounded by false-positive results if lingering eggs of unknown viability that are cemented to hair shafts are detected. Indeed, it may take more than 8 mo for the entire fur coat to be replaced completely.²⁰ Similarly, false-positive PCR tests after treatment could result from the persistence of mite DNA in the haircoat. Here we documented how soon diagnostic testing by fur-pluck and by fur PCR analysis correctly identified mice that had been treated successfully with selamectin. Finally, we evaluated whether the mite status of mice with low or undetectable populations of mites could be identified by breeding them and evaluating their offspring.     

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.     

Assessment of Immune Activation in Mice before and after Eradication of Mite Infestation

Mite infestation of mice remains a persistent problem for many institutions, leading to numerous health problems and creating unknown and unwanted variables for research. In this study, mice with mite infestation demonstrated significantly higher levels of inflammatory cytokines, both at draining lymph nodes (axillary) and systemically, as compared with mice without mites. In addition, histologic evaluation revealed significant inflammation in mite-infested mice. Inflammatory changes were still present in the skin of mice at 6 to 8 wk after treatment, despite absence of detectable infestation at that time. Because these significant and lasting local and systemic changes have the potential to alter research findings, eradication of mites infestations should be an important goal for all institutions.Abbreviation: KC, keratinocyte-derived chemokine; MIP, macrophage inflammatory proteinLaboratory mice can harbor several species of acarids (fur mites), including Myobia musculi, Radfordia affinis, Myocoptes musculinus, and Psorergates simplex.^{11,14,29,40,45} Fur mites are an excluded pathogen in most research facilities, particularly within barrier suites, and in order to control or avoid mite infestations, many facilities, including those with ongoing infestations, will not accept infested animals from outside sources. Such policies can prevent or halt collaborative research between investigators in different institutions because mite infestation is a sporadic or endemic problem in many facilities that house mice under conventional conditions, despite attempts at eradication.^{12,22,25,43,62,69}Mite infestations cause several health problems in mice, including ulcerative dermatitis, amyloidosis, and other immune system alterations.^{2,12,22,27,29-31,37,44,45,61} For example, mite infestations are associated with increased serum concentrations of IgE and IgG in mice.^30,44,48 Alterations in immune responses could alter research data and thereby perhaps alter the associated conclusions.^36,65,66,70 Mice with mite infections often develop dermatitis, which can lead to bacterial infection and additional changes in immune status.^{15,30,31,45,61} Because any pathogenic infection can cause variability and alter basal measures of immune function, clinical chemistry, and behavior in mice, maintaining laboratory rodents in a disease-free state is crucial to their use for the collection of valid research data.⁵¹The eradication of external parasites is a difficult process. Many reports have been published that attempt mite eradication using various drug treatments,^{5-7,17,18,23,24,35,39,41-43,46,47,49,50,57,59,67} with each method having distinct advantages and disadvantages. Some, but not all, of these treatment regimens have been compared directly.¹⁰ The mite life cycle complicates treatment, because eggs and larvae can be less susceptible to drugs than are adult parasites.^2,19,20,55 In addition, mite eggs can contaminate the environment, providing a source for re-infection of treated animals.^20,63,64 Some drugs (for example, ivermectin) have been associated with toxicity and death in mice, especially among specific transgenic lines.^{8,12,28,53,55,69} Other drugs may require frequent or repeated treatment of the mice. Furthermore, the drugs themselves may have properties that alter physiology or immune function in animals.^2,13,60 The development of new veterinary drugs for treatment of parasites has increased the available therapies for rodent acariasis. Compounds such as fipronil and selamectin provide good efficacy against external parasites with limited side effects in mammals.^9,22,68Our facility housed a large colony of mice that occupied several rooms and were infested with Myocoptes musculinus and Myobia musculi. Although the majority of mite-infested mice had mild or no dermatitis, some infected mice had severe dermatitis. The goal of this study was to evaluate the local and systemic immune response in mice infested with mites. To our knowledge, this study is the first to comprehensively compare cytokine levels and histologic findings in mite-infested, treated, and mite-negative mice. We hypothesized that the immune response would be altered in mite-infested mice as demonstrated by significantly elevated cytokine levels in the draining lymph nodes or spleen as compared with mice that had never been infested with mites. In addition, we hypothesized that significant pathologic changes in the epidermis, dermis, and subcutaneous tissues would be present in response to mite infestation.     

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.     

Dual-reactive B cells are autoreactive and highly enriched in the plasmablast and memory B cell subsets of autoimmune mice

Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG⁺ cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG⁺ cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.While developing in the BM, B cells undergo stochastic rearrangement of Ig heavy (IgH) and Ig light (IgL) chain V(D)J gene segments resulting in the random expression of Ig H and L (κ and λ) chains in the emerging B cell population (Schlissel, 2003; Nemazee, 2006). During V(D)J recombination, allelic and isotypic exclusion at the Ig loci are also established, leading to the expression of a unique H and L chain pair and, therefore, of BCRs with unique specificity in each B cell (Langman and Cohn, 2002; Nemazee, 2006; Vettermann and Schlissel, 2010). These mechanisms ensure that developing B cells expressing BCRs reactive with self-antigens (i.e., autoreactive B cells) undergo tolerance induction, whereas those expressing BCRs specific for a foreign antigen or a peripheral self-antigen proceed in differentiation and selection into the periphery (Burnet, 1959). Autoreactive B cells are silenced by central tolerance in the BM via receptor editing and, less frequently, clonal deletion (Halverson et al., 2004; Ait-Azzouzene et al., 2005), whereas peripheral B cell tolerance proceeds via anergy and clonal deletion (Goodnow et al., 2005; Pelanda and Torres, 2006, 2012; Shlomchik, 2008). Despite these tolerance mechanisms, small numbers of autoreactive B cells are detected in peripheral tissues of healthy mice and humans (Grandien et al., 1994; Wardemann et al., 2003) and their numbers are increased in autoimmunity (Andrews et al., 1978; Izui et al., 1984; Warren et al., 1984; Samuels et al., 2005; Yurasov et al., 2005, 2006; Liang et al., 2009).A small population of dual-reactive B cells expressing two types of L chains (or more rarely H chains) has been observed both in mice and humans (Nossal and Makela, 1962; Pauza et al., 1993; Giachino et al., 1995; Gerdes and Wabl, 2004; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007; Kalinina et al., 2011). These allelically and isotypically (overall haplotype) included B cells are <5% of all peripheral B cells in normal mice (Barreto and Cumano, 2000; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007), but they are more frequent in Ig knockin mice in which newly generated B cells are autoreactive and actively undergo receptor editing (Li et al., 2002a,b; Liu et al., 2005; Huang et al., 2006; Casellas et al., 2007). B cells that coexpress autoreactive and nonautoreactive antibodies can escape at least some of the mechanisms of central and peripheral B cell tolerance and be selected into the mature peripheral B cell population (Kenny et al., 2000; Li et al., 2002a,b; Gerdes and Wabl, 2004; Liu et al., 2005; Huang et al., 2006), sometimes with a preference for the marginal zone (MZ) B cell subset (Li et al., 2002b).Furthermore, dual-reactive B cells observed within a normal polyclonal Ig repertoire exhibit characteristics of cells that develop through the receptor editing process, including delayed kinetics of differentiation and more frequent binding to self-antigens (Casellas et al., 2007). Hence, dual-reactive B cells might play a role in autoantibody generation and autoimmunity. However, the contribution of these B cells to autoimmunity has not yet been established. Our hypothesis is that haplotype-included autoreactive B cells are positively selected within the context of genetic backgrounds that manifest defects in immunological tolerance and contribute to the development of autoimmunity.Until recently, the analysis of dual-reactive B cells was impaired by the inability to detect dual-κ cells, which are the most frequent among haplotype-included B cells (Casellas et al., 2007; Velez et al., 2007). To overcome this issue, we took advantage of Igk^h mice that bear a gene-targeted human Ig Ck allele in the context of a wild-type Ig repertoire (Casellas et al., 2001) and crossed these to MRL-Fas^lpr/lpr (MRL/lpr) and MRL mice that develop an autoimmune pathology with characteristics similar to human lupus (Izui et al., 1984; Rordorf-Adam et al., 1985; Theofilopoulos and Dixon, 1985; Cohen and Eisenberg, 1991; Watanabe-Fukunaga et al., 1992). MRL/lpr mice, moreover, display defects in receptor editing (Li et al., 2002a; Lamoureux et al., 2007; Panigrahi et al., 2008) and reduced tolerance induction (Li et al., 2002a), which could potentially contribute to higher frequency of haplotype-included autoreactive B cells.We found that the frequency of dual-κ cells increased with age and progression of disease in autoimmune-prone mice and independent of the expression of Fas. Dual-κ B cells exhibited higher prevalence of autoreactivity than single-κ B cells and were frequently selected into the antigen-activated cell subsets in MRL/lpr and MRL mice where up to half of the plasmablasts and memory B cells were dual-κ B cells. Moreover, disruption of Fas expression appeared to mediate increased survival of dual-reactive memory B cells. Overall, these data indicate that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in the development of autoimmunity.     

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.     

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4⁺ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.Within 8 d after peroral infection with Toxoplasma gondii, susceptible C57BL/6 mice develop massive necrosis in the ileum, leading to death (Liesenfeld et al., 1996). T. gondii–induced ileitis is characterized by a CD4 T cell–dependent overproduction of proinflammatory mediators, including IFN-γ, TNF, and NO (Khan et al., 1997; Mennechet et al., 2002). Activation of CD4⁺ T cells by IL-12 and IL-18 is critical for the development of small intestinal pathology (Vossenkämper et al., 2004). Recently, we showed that LPS derived from gut flora via Toll-like receptor (TLR)–4 mediates T. gondii–induced immunopathology (Heimesaat et al., 2006). Thus, the immunopathogenesis of T. gondii–induced small intestinal pathology resembles key features of the inflammatory responses in inflammatory bowel disease (IBD) in humans and in models of experimental colitis in rodents (Liesenfeld, 2002). However, most animal models of IBD assessed inflammatory responses in the large intestine, and models of small intestinal pathology are scarce (Kosiewicz et al., 2001; Strober et al., 2002; Olson et al., 2004; Heimesaat et al., 2006).IL-12 shares the p40 subunit, IL-12Rβ1, and components of the signaling transduction pathways with IL-23 (Parham et al., 2002). There is strong evidence that IL-23, rather than IL-12, is important in the development of colitis (Yen et al., 2006). The association of IL-23R encoding variant Arg381Gln with IBD (Duerr et al., 2006) and the up-regulation of IL-23p19 in colon biopsies from patients with Crohn''s disease (Schmidt et al., 2005) underline the importance of IL-23 in intestinal inflammation. Effector mechanisms of IL-23 include the up-regulation of matrixmetalloproteinases (MMPs; Langowski et al., 2006), a large family of endopeptidases that mediate homeostasis of the extracellular matrix. MMPs were significantly up-regulated in experimental models of colitis (Tarlton et al., 2000; Medina et al., 2003) and in colonic tissues of IBD patients (von Lampe et al., 2000).Studies in mouse models of autoimmune diseases have associated the pathogenic role of IL-23 with the accumulation of CD4⁺ T cells secreting IL-17, termed Th17 cells (Aggarwal et al., 2003; Cua et al., 2003). Moreover, increased IL-17 expression was reported in the intestinal mucosa of patients with IBD (Fujino et al., 2003; Nielsen et al., 2003; Kleinschek et al., 2009).In addition to IL-17, Th17 cells also produce IL-22, a member of the IL-10 family (Dumoutier et al., 2000). IL-22, although secreted by certain immune cell populations, does not have any effects on immune cells in vitro or in vivo but regulates functions of some tissue cells (Wolk et al., 2009). Interestingly, IL-22 has been proposed to possess both protective as well as pathogenic roles. In fact, IL-22 mediated psoriasis-like skin alterations (Zheng et al., 2007; Ma et al., 2008; Wolk et al., 2009). In contrast, IL-22 played a protective role in experimental models of colitis (Satoh-Takayama et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008; Zheng et al., 2008), in a model of Klebsiella pneumoniae infection in the lung (Aujla et al., 2007), and against liver damage caused by concanavalin A administration (Radaeva et al., 2004; Zenewicz et al., 2007). IL-22 has been reported to be produced by CD4⁺ T cells (Wolk et al., 2002; Zheng et al., 2007), γδ cells (Zheng et al., 2007), CD11c⁺ cells (Zheng et al., 2008), and natural killer cells (Cella et al., 2008; Luci et al., 2008; Sanos et al., 2009; Satoh-Takayama et al., 2008; Zheng et al., 2008). The role of IL-22 in small intestinal inflammation remains to be determined.In the present study, we investigated the role of the IL-23–IL-17 axis in T. gondii–induced small intestinal immunopathology. We show that IL-23 is essential in the development of small intestinal immunopathology by inducing local MMP-2 up-regulation that could be inhibited by prophylactic or therapeutic chemical blockage. Interestingly, IL-23–dependent IL-22 production was markedly up-regulated and essential for the development of ileal inflammation, whereas IL-17 production was down-regulated after T. gondii infection. IL-22 was mostly produced by CD4⁺ T cells in the small intestinal lamina propria.     

Safety and Efficacy of Topical Lime Sulfur in Mice Infested with Myocoptes musculinus

Current treatment options for murine fur mites have limitations in safety and efficacy. This study evaluated whether topical lime sulfur (LS) is an adjunct or alternative to traditional treatment options for Myocoptes musculinus. To evaluate the safety of topical LS, mice were dipped in a 3% LS solution at 34 and 41 d of age. Mice were observed daily for side effects and mortality, with blood work and necropsy at 42 d of age to evaluate for pathologic changes. To determine the efficacy of topical LS, postweanling mice infested with M. musculinus were treated with LS once weekly for 2 wk and then housed with uninfested sentinel mice for 4 wk. Weekly tape tests and postmortem tape tests and skin scrapings were performed on all mice. Treated postweanling mice had significantly lower Hgb levels and higher BUN levels than did control animals. In mite-infested mice, the number of positive cages at euthanasia was the same between treated and control animals. Although topical LS did not cause gross or microscopic changes to organ systems, it may cause clinicopathologic changes, and topical LS is not effective as a sole treatment for M. musculinus infestation of postweanling mice.Abbreviation: LS, lime sulfurNumerous treatment protocols have been described for murine fur mites, with drugs in the avermectin family (ivermectin, selamectin) and the related compound moxidectin being used commonly.^{2-4,8,9,13,19,29,34,37,40,51} The avermectins and related drugs, although effective and safe for use in many mice, can be toxic to neonatal mice and adults of some strains.^{24,25,38,42,43} CF1 mice, which are naturally deficient in P-glycoprotein, are susceptible to ivermectin toxicity, and the Mdr1a genetic mutation has been reported to affect as much as 25% of a random CF1 population.²⁴ Ivermectin must be used cautiously in transgenic mice, because serum levels of the drug can vary among strains, and mutations causing increased blood–brain barrier permeability or P-glycoprotein deficiency can lead to toxicity in valuable transgenic mice.^13,42 Ivermectin has been reported to cause toxicity to suckling mice of various strains, likely due to the incomplete formation of the blood–brain barrier in young pups; moxidectin was shown to be toxic in strains of senescence-accelerated mice.^25,38,43 In addition, ivermectin may affect behavior and immune function.^7,15 Topical products that can be applied directly to the animal or distributed in the bedding, such as organophosphates and permethrins, may not be as successful mitocides as are avermectins; these topical products can have harmful side effects, and exposure may be dangerous to animal care personnel.^12,20,52 A safer treatment protocol is needed for mice in which avermectin use is associated with lethal consequences and for investigators who are concerned about the effects of avermectins on research outcomes.Lime sulfur (LS) is an antiparasitic that has been used safely in many species and therefore might offer a safer treatment option in ivermectin-sensitive mouse strains. LS has a long history of use in agriculture for the control of fungi and insects, and LS dips have been used in companion animals to treat mites and fungal infection.^{5,11,17,30,32,33,41,44,49} LS is a mixture of calcium and sulfur that can be applied topically as a rinse or dip. As an antiparasitic, the exact mechanism of action of LS is unknown. Its use has been described in dogs, cats, guinea pigs, horses, and tigers for treatment of dermatitis caused by demodicosis, ringworm, and sarcoptic mange.^{27,31,33,35,45,49} In one report, topical application of LS weekly for 6 wk was successful at eliminating the guinea pig sarcoptid mite, Trixacarus caviae.²⁷ Adverse effects in animals have not been reported with as-labeled use of LS, but it potentially can cause skin and eye irritation and inhalational injury, as well as gastrointestinal irritation if ingestion occurs.⁴⁷ Intentional ingestion in humans has resulted in severe toxicity; case reports from the United States and Japan describe metabolic acidosis and caustic injury to the gastrointestinal tract, including chemical burns, mucosal bleeding, and gastric perforation.^18,23 To our knowledge, the safety of LS has not previously been evaluated in mice.Myocoptes musculinus is one of the most common murine fur mite species reported in contemporary rodent research facilities.^1,10,36 M. musculinus is a surface-dwelling, ambulatory mite that tends to infest the caudal half of the body, but mites can be found on the head and neck of its host. Eggs are laid distally on the hair shaft, hatching on day 5 onto the hair coat and completing a full life cycle in 14 d. The parasite spreads rapidly with direct close contact and can be present on neonates as young as 4 to 5 d of age.^1,36 Clinical signs can include alopecia, pruritus, dermatitis, erythema, weight loss, and cutaneous allergy manifestations.^1,22,31,36The goal of the current study was to evaluate whether LS is safe when applied topically to mice and whether this treatment is effective in eliminating M. musculinus from postweanling mice. Postweanling mice were treated twice with LS and evaluated for acute toxicity by using blood work and necropsy. The efficacy study was designed according to the life cycle of M. musculinus. Because LS appears to be adulticidal only and because M. musculinus eggs hatch after 5 d, mice received 2 applications 1 wk apart. Mite remnants and egg casings can remain on a mouse even after effective treatment; with many diagnostic techniques, this situation complicates determining whether an animal is truly negative. Treated postweanling mice were housed with sentinel mice after treatment to evaluate for active infestations, which should be transmissible to the sentinels, and to mimic a ‘real world’ situation in which mice would be treated and returned to group housing. We hypothesized that LS would be a safe and effective treatment for M. musculinus in mice. To our knowledge, no previous prospective, controlled studies have evaluated LS use in mice.     

Successful Treatment of Cryptosporidiosis in 2 Common Marmosets (Callithrix jacchus) by Using Paromomycin

Two pair-housed, 1-y-old common marmosets (Callithrix jacchus) had intermittent loose feces and weight loss for approximately 2 mo. Cryptosporidum parvum was identified by ELISA in the feces of both animals. CBC and blood chemistry values, including liver enzymes, were within normal range. Both marmosets were treated with the antibiotic paromomycin (15 mg/kg PO) twice daily for 28 d. Resolution of clinical signs coincided with treatment. Three follow-up samples, taken 2 wk apart after treatment was finished, were negative for cryptosporidium ELISA in both animals. Paromomycin should be considered for treatment of cryptosporidiosis in marmosets.Cryptosporidium was first described in laboratory mice by Tyzzer in 1907.²⁰ However, the organism was not considered to be pathogenic until the 1970s, when it was identified as causing disease in mammals, primarily calves.^15,18 Cryptosporidiosis continues to be a serious disease in dairy cattle, causing high morbidity and mortality in neonatal calves.²⁶ Cyptosporidiosis has been identified as a zoonotic disease, particularly among veterinary students caring for sick calves.^7,11,17 Cryptosporidiosis caused by Cryptosporidium parvum is now considered a serious pathogen in immunocompromised subjects and one of the most serious opportunistic infections complicating disease in AIDS patients.²² Several recent book chapters and review articles discuss the epidemiology, treatment, biology, and pathogenesis of human and veterinary cryptosporidiosis.⁵,^13,22,23Cryptosporidiosis is considered to be a self-limiting disease in immunocompetent human patients,²¹ nursery-reared infant macaques (Macaca mulatta),¹⁴ and other animals.¹⁰ The disease has an acute onset in people (3 to 7 d), and diarrhea and abdominal cramping generally lasts 7 to 10 d. Cryptosporidiosis may be asymptomatic in marmosets (Callithrix jacchus)¹⁰ but has been reported to cause enterocolitis in immunocompetent marmosets, for which supportive therapy is the only published treatment.¹²Paromomycin is an aminoglycocide antibiotic that has been one of the most widely used agents to treat cryptosporidiosis in immunosuppressed human patients.^3,4,13,19 The drug has shown efficacy in experimentally infected animal models,^1,8,24 for prophylaxis in outbreaks of ruminants,⁶ and in cats with clinical cryptosporidiosis,² although paromomycin toxicity in cats has been reported.⁹Here we describe the use of paromomycin to treat cryptosporidiosis in 2 marmosets, with subsequent resolution of clinical signs.     

Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium

Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4⁺ T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.Inducing specific immunity and maintaining tolerance requires cells of the mononuclear phagocyte lineage. This lineage is comprised of three closely related cell types: DCs, monocytes, and macrophages (Shortman and Naik, 2007; Geissmann et al., 2010a,b; Liu and Nussenzweig, 2010; Yona and Jung, 2010; Chow et al., 2011). DCs are essential to both immunity and tolerance (Steinman et al., 2003); however, the role monocytes and macrophages play in these processes is not as well defined (Geissmann et al., 2008).In mice, DCs and monocytes arise from the same hematopoietic progenitor, known as the macrophage–DC progenitor (MDP; Fogg et al., 2006). Their development diverges when MDPs become either common DC progenitors (CDPs) that are Flt3L-dependent, or monocytes, which are dependent on CSF1 (M-CSF; Witmer-Pack et al., 1993; McKenna et al., 2000; Fogg et al., 2006; Waskow et al., 2008). CDPs develop into either plasmacytoid DCs or preDCs that leave the bone marrow to seed lymphoid and nonlymphoid tissues, where they further differentiate into conventional DCs (cDCs; Liu et al., 2009). In contrast, monocytes circulate in the blood and through tissues, where they can become activated and develop into several different cell types, including some but not all tissue macrophages (Schulz et al., 2012; Serbina et al., 2008; Yona et al., 2013).Despite their common origin from the MDP, steady-state lymphoid tissue cDCs can be distinguished from monocytes or macrophages by expression of cell surface markers. For example, cDCs in lymphoid tissues express high levels of CD11c and MHCII, but lack the expression of CD115 and F4/80 found in monocytes and macrophages, respectively. However, this distinction is far more difficult in peripheral tissues, like the intestine or lung, or during inflammation when monocytes begin to express many features of DC including high levels of MHCII and CD11c (Serbina et al., 2003; León et al., 2007; Hashimoto et al., 2011).The function of cDCs in immunity and tolerance has been explored extensively using a series of different mutant mice to ablate all or only some subsets of cDCs (Jung et al., 2002; Liu and Nussenzweig, 2010; Chow et al., 2011). In contrast, the methods that are currently available to study the function of monocytes and macrophages in vivo are far more restricted and less specific (Wiktor-Jedrzejczak et al., 1990; Dai et al., 2002; MacDonald et al., 2010; Chow et al., 2011). For example, Ccr2^−/− and Ccr2^DTR mice (Boring et al., 1997; Kuziel et al., 1997; Serbina and Pamer, 2006; Tsou et al., 2007) have been used to study monocytes (Boring et al., 1997; Peters et al., 2004; Hohl et al., 2009; Nakano et al., 2009). However, CCR2 is also expressed on some subsets of cDCs, activated CD4⁺ T cells, and NK cells (Kim et al., 2001; Hohl et al., 2009; Egan et al., 2009; Zhang et al., 2010). Thus, it is challenging to dissect the precise role of monocytes as opposed to other cell types in immune responses in Ccr2^−/− or Ccr2^DTR mice. Inducible DTR expression in CD11c^Cre x CX₃CR1^LsL-DTR mice is far more specific (Diehl et al., 2013), but restricted to a small subset of mononuclear phagocytes.Here, we describe a genetic approach to targeting monocytes and macrophages that spares cDCs and lymphocytes, and we compare the effects of monocyte and macrophage ablation to cDC depletion on the adaptive immune response to intestinal infection with Citrobacter rodentium.