Hydroxyl functionalized multi-walled carbon nanotubes modulate immune responses without increasing 2009 pandemic influenza A/H1N1 virus titers in infected mice.

Growing use of carbon nanotubes (CNTs) have garnered concerns regarding their association with adverse health effects. Few studies have probed how CNTs affect a host's susceptibility to pathogens, particularly respiratory viruses. We reported that exposure of lung cells and mice to pristine single-walled CNTs (SWCNTs) leads to significantly increased influenza virus H1N1 strain A/Mexico/4108/2009 (IAV) titers in concert with repressed antiviral immune responses. In the present study, we investigated if hydroxylated multi-walled CNTs (MWCNTs), would result in similar outcomes. C57BL/6 mice were exposed to 20 µg MWCNTs on day 0 and IAV on day 3 and samples were collected on day 7. We investigated pathological changes, viral titers, immune-related gene expression in lung tissue, and quantified differential cell counts and cytokine and chemokine levels in bronchoalveolar lavage fluid. MWCNTs alone caused mild inflammation with no apparent changes in immune markers whereas IAV alone presented typical infection-associated inflammation, pathology, and titers. The co-exposure (MWCNTs + IAV) did not alter titers or immune cell profiles compared to the IAV only but increased concentrations of IL-1ß, TNFα, GM-CSF, KC, MIPs, and RANTES and inhibited mRNA expression of Tlr3, Rig-i, Mda5, and Ifit2. Our findings suggest MWCNTs modulate immune responses to IAV with no effect on the viral titer and modest pulmonary injury, a result different from those reported for SWCNT exposures. This is the first study to show that MWCNTs modify cytokine and chemokine responses that control aspects of host defenses which may play a greater role in mitigating IAV infections.

Mycotic Rhinitis and Sinusitis in Florida Horses.

Rhinitis and sinusitis caused by fungal pathogens were studied in biopsy samples submitted from 52 horses distributed throughout subtropical and tropical regions of Florida. Methods included routine histopathology as well as polymerase chain reaction (PCR) with panfungal/panoomycete primers and DNA sequencing on extracted DNA (DNA barcoding). Granulomatous, pyogranulomatous, and fibrinopurulent lesions in nasal and sinus mucosa were associated with signs of upper airway obstruction and noise as well as nasal discharge. Morphologic and histochemical assessment of cases identified 31 cases of zygomycosis/pythiosis plus 1 mixed infection case, 16 cases of phaeohyphomycosis with 2 additional mixed infection cases, and 3 cases caused by other fungi. Morphologic evidence of Aspergillus sp. infection as a superficial copathogen was found in 2 of the mixed fungal infection cases. PCR and DNA sequencing facilitated identification of fungal pathogens in 11 of 52 cases (21%). No evidence of oomycete infection was found. Histomorphologic features of previously unrecognized forms of equine rhinitis/sinusitis were described, including those caused by Flavodon flavus, Curvularia lunata, Exserohilum rostrata, Alternaria alternata, Alternaria sp., Cladophialophora bantiana, Fusarium solani, and Toxicocladosporium irritans. PCR and DNA sequencing using panfungal and oomycete primers with DNA from formalin-fixed and paraffin-embedded specimens successfully identified the pathogen in phaeohyphomycosis (7/18 cases, 39%), zygomycosis/pythiosis (1/32 cases, 3%), and other nonpigmented fungal infections (3/3 cases, 100%). Zygomycosis and phaeohyphomycosis were the most common forms of fungal rhinitis found in Florida horses.

Single-walled carbon nanotubes modulate pulmonary immune responses and increase pandemic influenza a virus titers in mice.

BACKGROUND: Numerous toxicological studies have focused on injury caused by exposure to single types of nanoparticles, but few have investigated how such exposures impact a host's immune response to pathogen challenge. Few studies have shown that nanoparticles can alter a host's response to pathogens (chiefly bacteria) but there is even less knowledge of the impact of such particles on viral infections. In this study, we performed experiments to investigate if exposure of mice to single-walled carbon nanotubes (SWCNT) alters immune mechanisms and viral titers following subsequent influenza A virus (IAV) infection. METHODS: Male C57BL/6 mice were exposed to 20 µg of SWCNT or control vehicle by intratracheal instillation followed by intranasal exposure to 3.2 × 104 TCID50 IAV or PBS after 3 days. On day 7 mice were euthanized and near-infrared fluorescence (NIRF) imaging was used to track SWCNT in lung tissues. Viral titers, histopathology, and mRNA expression of antiviral and inflammatory genes were measured in lung tissue. Differential cell counts and cytokine levels were quantified in bronchoalveolar lavage fluid (BALF). RESULTS: Viral titers showed a 63-fold increase in IAV in SWCNT + IAV exposed lungs compared to the IAV only exposure. Quantitation of immune cells in BALF indicated an increase of neutrophils in the IAV group and a mixed profile of lymphocytes and neutrophils in SWCNT + IAV treated mice. NIRF indicated SWCNT remained in the lung throughout the experiment and localized in the junctions of terminal bronchioles, alveolar ducts, and surrounding alveoli. The dual exposure exacerbated pulmonary inflammation and tissue lesions compared to SWCNT or IAV single exposures. IAV exposure increased several cytokine and chemokine levels in BALF, but greater levels of IL-4, IL-12 (P70), IP-10, MIP-1, MIP-1α, MIP-1ß, and RANTES were evident in the SWCNT + IAV group. The expression of tlr3, ifnß1, rantes, ifit2, ifit3, and il8 was induced by IAV alone but several anti-viral targets showed a repressed trend (ifits) with pre-exposure to SWCNT. CONCLUSIONS: These findings reveal a pronounced effect of SWCNT on IAV infection in vivo as evidenced by exacerbated lung injury, increased viral titers and several cytokines/chemokines levels, and reduction of anti-viral gene expression. These results imply that SWCNT can increase susceptibility to respiratory viral infections as a novel mechanism of toxicity.

Comparison of oseltamivir and α-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus.

Influenza virus infections are a major cause of respiratory disease in humans. Neuraminidase inhibitors (NAIs) are the primary antiviral medication used to treat ongoing influenza infections. However, NAIs are not always effective for controlling virus shedding and lung inflammation. Other concerns are the emergence of NAI-resistant virus strains and the risk of side effects, which are occasionally severe. Consequently, additional anti-influenza therapies to replace or combine with NAIs are desirable. Here, we compared the efficacy of the NAI oseltamivir with the invariant natural killer T (iNKT) cell superagonist, α-galactosylceramide (α-GalCer), which induces innate immune responses that inhibit influenza virus replication in mouse models. We show that oseltamivir reduced lung lesions and lowered virus titers in the upper respiratory tract of pigs infected with A/California/04/2009 (CA04) pandemic H1N1pdm09. It also reduced virus transmission to influenza-naïve contact pigs. In contrast, α-GalCer had no impact on virus replication, lung disease, or virus transmission, even when used in combination with oseltamivir. This is significant as iNKT-cell therapy has been studied as an approach for treating humans with influenza.

Unaltered influenza disease outcomes in swine prophylactically treated with α-galactosylceramide.

Influenza A viruses (IAV) are a major cause of respiratory diseases in pigs. Invariant natural killer T (iNKT) cells are an innate-like T cell subset that contribute significantly to IAV resistance in mice. In the current work, we explored whether expanding and activating iNKT cells with the iNKT cell superagonist α-galactosylceramide (α-GalCer) would change the course of an IAV infection in pigs. In one study, α-GalCer was administered to pigs intramuscularly (i.m.) 9 days before infection, which systemically expanded iNKT cells. In another study, α-GalCer was administered intranasally (i.n.) 2 days before virus infection to activate mucosal iNKT cells. Despite a synergistic increase in iNKT cells when α-GalCer i.m. treated pigs were infected with IAV, neither approach reduced disease signs, lung pathology, or virus replication. Our results indicate that prophylactic use of iNKT cell agonists to prevent IAV infection is ineffective in pigs. This is significant because this type of approach has been considered for humans whose iNKT cell levels and IAV infections are more similar to those of pigs than mice.

Influenza A virus (H3N8) in dogs with respiratory disease, Florida.

In 2004, canine influenza virus subtype H3N8 emerged in greyhounds in the United States. Subsequent serologic evidence indicated virus circulation in dog breeds other than greyhounds, but the virus had not been isolated from affected animals. In 2005, we conducted virologic investigation of 7 nongreyhound dogs that died from respiratory disease in Florida and isolated influenza subtype H3N8 virus. Antigenic and genetic analysis of A/canine/Jacksonville/2005 (H3N8) and A/canine/Miami/2005 (H3N8) found similarity to earlier isolates from greyhounds, which indicates that canine influenza viruses are not restricted to greyhounds. The hemagglutinin contained 5 conserved amino acid differences that distinguish canine from equine lineages. The antigenic homogeneity of the canine viruses suggests that measurable antigenic drift has not yet occurred. Continued surveillance and antigenic analyses should monitor possible emergence of antigenic variants of canine influenza virus.

Gastric Intussusceptions in a Red Corn Snake (Pantherophis guttatus) Associated with Cryptosporidiosis.

A 3-year-old female red corn snake (Pantherophis guttatus) was presented for a three-week history of anorexia and decreased defecations. On physical examination, a soft midbody intracoelomic swelling was palpated. Transcutaneous coelomic ultrasound revealed a target-like mass on a transverse section of the stomach, suggesting the presence of a gastrointestinal intussusception. On exploratory coeliotomy, a double compounded esophagogastric and gastroduodenal intussusception was diagnosed and reduced surgically. A gastropexy was also performed to prevent recurrence. On histopathology, the gastric glandular mucosa showed moderate to marked proliferation. Diffusely lining the luminal surface of glandular epithelium and free within the lumen were a myriad of protozoa consistent with Cryptosporidium sp. A diagnosis of chronic proliferative gastritis due to Cryptosporidium sp. was made based on these findings. Intussusceptions are rare in reptiles and are infrequently reported in snakes. This is the first report of a double compounded intussusception in a nonmammalian species and the first report of an intussusception involving the stomach in a snake with gastritis due to Cryptosporidium sp.

α-Galactosylceramide protects swine against influenza infection when administered as a vaccine adjuvant.

Natural killer T (NKT) -cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide array of immune responses with many promising immunotherapeutic applications, including the enhancement of vaccines against infectious diseases and cancer. In the current study, we evaluated whether α-GalCer generates protective immunity against a swine influenza (SI) virus infection when applied as an intramuscular vaccine adjuvant. Immunization of newly weaned piglets with UV-killed pandemic H1N1 A/California/04/2009 (kCA04) SI virus and α-GalCer induced high titers of anti-hemagglutinin antibodies and generated virus-specific T cells that localized in intrapulmonary airways and in alveolar walls. Vaccination with α-GalCer resulted in a systemic increase in NKT-cell concentrations, including in the respiratory tract, which was associated with complete inhibition of viral replication in the upper and lower respiratory tract and much reduced viral shedding. These results indicate that NKT-cell agonists could be used to improve swine vaccine formulations in order to reduce the clinical signs of SI infection and limit the spread of influenza viruses amongst commercial pigs.

Ionic conjugates of lidocaine and sweeteners as better tasting local anesthetics for dentistry.

Lidocaine is the most widely utilized intraoral injected dental anesthetic, used for more than 500 million dental injections per year. Local anesthesia is essential for pain-free dentistry, yet intraoral injections are often considered painful and a source of anxiety for many patients. Any new anesthetics that will reduce the stress and anxiety of dental injection are expected to be beneficial. A novel chemical approach to taste modulation is proposed, in which the lidocaine cation is coupled with anionic sweeteners such as saccarinate and acesulfamate. The ionic conjugates synthesized using anion exchange techniques, were much less bitter, demonstrated a high local anesthetic potential in animal studies, and were as safe as the original hydrochloride. Based on the currently robust market for lidocaine it is expected that the resulting anesthetics will be in high demand in clinical practices worldwide.

Increased IFN-gamma protein in bronchoalveolar lavage fluid of anti-IP-10 antibody-treated F344 rats following Sendai viral infection.

A previous study showed that virus-resistant F344 rats had higher levels of interferon-gamma (IFN-gamma)-inducible protein 10 (IP-10) than did virus-susceptible BN rats early after Sendai viral infection. The initial goal of this study was to determine if an early high expression of IP-10 in F344 rats contributes to their resistance to virus-induced airway injury. Infected F344 rats were treated with anti-IP-10 rabbit serum or normal rabbit serum. Results indicated that blocking of IP-10 protein did not significantly change the resistance of F344 rats. However, we observed that neutralization of IP-10 increased IFN-gamma protein in bronchoalveolar lavage (BAL) fluid of F344 rats 7 days after inoculation compared with rats that received normal rabbit serum. The pulmonary IFN-gamma mRNA abundance remained comparable. This effect was not caused by fluctuation of the viral titer in the lung. This interesting phenomenon suggests that expression of IFN-gamma protein can be modulated by treatment with anti-IP-10 antibody at the posttranscriptional or translational level in this model.

A comparison of two methods of endoscopic dilation of acute subglottic stenosis using a ferret model.

OBJECTIVES/HYPOTHESIS: Balloon dilation is accepted as a first line treatment of acute subglottic stenosis, but its effects on the subglottic tissue remain largely unknown. We aimed to develop an animal model of acute subglottic stenosis using endoscopic techniques. Once developed, this model was used to compare the immediate effects of balloon dilation and endotracheal tube dilation on subglottic tissue. STUDY DESIGN: Prospective randomized animal study. METHODS: Acute subglottic injury was induced in 10 ferrets by endoscopic cauterization with silver nitrate. After 48-72 hours of observation, eight animals were randomized to undergo subglottic dilation with either a 5-mm balloon or endotracheal tubes of increasing diameter. These eight ferrets were euthanized within 10 minutes after dilation. The other two ferrets served as controls and were euthanized following observation only. The larynx from each ferret was harvested, and the subglottis was examined histologically by a pathologist blinded to the treatment arms. RESULTS: Acute subglottic stenosis was induced in all 10 ferrets using the endoscopic technique. Both balloon and endotracheal tube dilation resulted in comparable improvement in the subglottic airway diameter. A decreased thickness of submucosa/lamina propria was seen in the balloon dilation group. CONCLUSIONS: Acute subglottic stenosis can be reliably induced in ferrets using endoscopic techniques. Multiple dilation methods can be used to relieve acute obstruction. Balloon dilators seem to improve airway patency, in part, by decreasing the thickness of the submucosa and lamina propria. Further research is needed to determine how this impacts later stages of wound healing and final outcomes.

Comparative evaluation of differential laser-induced perturbation spectroscopy as a technique to discriminate emerging skin pathology.

Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre- and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy. First, the differential technique eliminates significant variations in absolute fluorescence response within subject populations. Second, UV perturbations alter the extracellular matrix (ECM), directly coupling the DLIPS response to the biological structure. Improved biosensing with DLIPS is demonstrated in vivo in a murine model of chemically induced skin lesion development. Component loading analysis of the data indicates that the DLIPS technique couples to structural proteins in the ECM. Analysis of variance shows that DLIPS has a significant response to emerging pathology as opposed to other population differences. An optimal likelihood ratio classifier for the DLIPS dataset shows that this technique holds promise for improved diagnosis of epithelial pathology. Results further indicate that DLIPS may improve diagnosis of tissue by augmenting fluorescence spectra (i.e. orthogonal sensing).

Experimental infection of neonatal foals with Rhodococcus equi triggers adult-like gamma interferon induction.

Rhodococcus equi is a facultative intracellular pathogen that causes pneumonia in young foals but does not induce disease in immunocompetent adult horses. Clearance of R. equi depends mainly on gamma interferon (IFN-gamma) production by T lymphocytes, whereas the predominance of interleukin 4 (IL-4) is detrimental. Young foals, like neonates of many other species, are generally deficient in the ability to produce IFN-gamma. The objective of this study was to compare the cytokine profiles, as well as cell-mediated and antibody responses, of young foals to those of adult horses following intrabronchial challenge with R. equi. The lymphoproliferative responses of bronchial lymph node (BLN) cells to concanavalin A were significantly higher in foals than in adult horses. In contrast, adult horses had significantly higher lymphoproliferative responses to R. equi antigens than did foals. Infected foals had significantly lower IL-4 mRNA expression but significantly higher IFN-gamma expression and IFN-gamma/IL-4 ratio in R. equi-stimulated BLN lymphocytes than did infected adults. Infection with R. equi in foals resulted in a significant increase in the percentage of T lymphocytes and CD4(+) T lymphocytes in bronchoalveolar lavage fluid in association with a significant decrease in the percentage of these cell populations in BLNs. Infection of foals also resulted in a marked increase in serum immunoglobulin Ga (IgGa) and IgGb levels, resulting in concentrations in serum that were significantly higher than those of adult horses. This study demonstrates that the immune response to R. equi in foals is not biased toward IL-4 and is characterized by the predominant induction of IFN-gamma.

Transmission of equine influenza virus to dogs.

Molecular and antigenic analyses of three influenza viruses isolated from outbreaks of severe respiratory disease in racing greyhounds revealed that they are closely related to H3N8 equine influenza virus. Phylogenetic analysis indicated that the canine influenza virus genomes form a monophyletic group, consistent with a single interspecies virus transfer. Molecular changes in the hemagglutinin suggested adaptive evolution in the new host. The etiologic role of this virus in respiratory disease was supported by the temporal association of rising antibody titers with disease and by experimental inoculation studies. The geographic expansion of the infection and its persistence for several years indicate efficient transmission of canine influenza virus among greyhounds. Evidence of infection in pet dogs suggests that this infection may also become enzootic in this population.

Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury.

Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-gamma-inducible protein 10 (IP-10), Mx1, and guanylate-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.

IL-12 reduces the severity of Sendai virus-induced bronchiolar inflammation and remodeling.

The goal of this research was to determine whether differential pulmonary IL-12 gene expression controls susceptibility to Sendai virus-induced chronic airway inflammation and fibrosis in inbred rat strains. Sendai virus-resistant F344 rats and susceptible BN rats were studied from 1 to 14 days following virus inoculation. F344 rats had 3.4-fold higher IL-12 mRNA levels detected by real-time PCR in lung than BN rats as early as two days following inoculation. This increase in mRNA was associated at two days with increased total IL-12 protein and with a 2-fold increase in numbers of bronchiolar, OX-6-positive dendritic cells and an increased number of IL-12 p40-positive, bronchiolar macrophages and dendritic cells (p<0.05). Virus-susceptible BN rats treated with 3 mug of recombinant, mouse IL-12 intraperitoneally at the time of virus inoculation had a 22.1% decrease in severity of chronic bronchiolar inflammation and a 23.8% decrease in fibrosis compared to virus-inoculated BN rats treated with saline. IL-12 treatment induced increased IFN-gamma mRNA and protein expression after virus inoculation (p<0.05). The results demonstrate that there is differential pulmonary IL-12 gene expression between virus-susceptible and resistant rat strains and that IL-12 treatment can provide significant protection from virus-induced chronic airway inflammation and remodeling during early life.

Simultaneous congenital and acquired extrahepatic portosystemic shunts in two dogs.

Two dogs with simultaneous congenital and acquired portosystemic shunts are reported. The first dog was an eight-month-old, male Golden Retriever with a history of peritoneal effusion, polyuria/polydipsia, and stunted growth. The dog had a microcytic, hypochromic anemia, a mildly elevated AST, and a moderate to severely elevated preprandial and postprandial serum bile acids. Transcolonic portal scintigraphy confirmed the presence of a portosystemic shunt. An intraoperative mesenteric portogram was performed. Two conjoined congenital extrahepatic portosystemic shunts and multiple acquired extrahepatic portosystemic shunts were identified. The second dog was a five-month-old, mixed breed with two week history of peritoneal effusion. Abdominal ultrasound and transcolonic scintigraphy were used to diagnose a portosystemic shunt. A single extrahepatic portosystemic shunt, portal hypertension, and multiple acquired collateral shunts were identified at surgery. The histologic alterations observed in these dogs were consistent with a portosystemic shunt. In these dogs, the presence of congenital and acquired portosystemic shunts and histopathologic findings are considered to represent a combination of congenital portosystemic shunts and noncirrhotic portal hypertension or portal vein hypoplasia.

Chronic postbronchiolitis airway instability induced with anti-IFN-gamma antibody in F344 rats.

Analogous to childhood-onset asthma in humans, rats may develop a chronic asthmalike phenotype, depending on their genetic background and the age at which they experience a viral airway injury. Brown Norway rats develop a postbronchiolitis asthmalike phenotype that may be prevented with supplements of interferon-gamma (IFN-gamma); we hypothesized that the normally resistant F344 rat strain would develop the asthmalike phenotype if the IFN-gamma response were suppressed during viral illness. Weanling F344 rats were pretreated with anti-IFN-gamma or control antibody, and inoculated with Sendai virus or vehicle. Anti-IFN-gamma treatment reduced lung IFN-gamma and increased IL-4 mRNA during the infection. Physiologic studies performed 8 wk later revealed premature airway closure (p = 0.03) and elevated specific pulmonary resistance (p < 0.01) in the postbronchiolitis anti-IFN-gamma group compared with noninfected controls and untreated postbronchiolitis rats. However, unlike the postbronchiolitis asthmalike phenotype in Brown Norway rats, bronchiolar inflammation and fibrosis were absent in the F344 rats. Lung elastic recoil and alveolar surface density also were unchanged compared with noninfected control rats. We conclude that there is an interactive effect of a weak IFN-gamma response and viral bronchiolitis at an early age that may result in persistent postbronchiolitis airway dysfunction. The presence of premature airway closure that is independent of airway wall inflammation or changes in lung elastic recoil suggests peripheral airway instability as a mechanism for the airway obstruction.