As mentioned previously this analysis applies to slices greater than

As mentioned previously, this analysis applies to slices greater than 10 times the knife radius. This is because the curved knife edge changes the effective knife angle during the cutting process. In order to estimate the forces for ultra-thin slices (<30nm), the rake angle was estimated from the following relationship:where r is the radius of the diamond knife edge. From this relationship, with 3nm thickness slicing, the rake angle becomes 0°. The TIC10 of the thickness increases the effective rake angle and, consequently, reduces the shear angle. Thus, selecting very thin slices increases the thrust and friction forces, which lead to an increase in damage to the block face. Slice thicknesses of 2.5nm have been reported [27], whereas sectioning below 2.5nm has not yet been successful. This might indicate the need for a minimum shear angle or maximum knife angle to enable the material to be sliced. In such cases, the thrust and friction forces may become greater than the cutting force and, consequently, the knife edge would rub along the block face instead of cutting it. Further, the slicing involves plastic deformation ahead of the diamond knife tip and elastic recovery of the block face after the knife has passed, resulting in a slight upward movement of material at the block face near the diamond knife tip. Importantly, the shear plane appears to start at a point above the diamond knife tip due to an increase of the thrust force as a consequence of reduction of the rake angle. Fig. 8 shows a schematic diagram of the force relations whilst taking account of the influence of the radius of the diamond knife. We assume that the difference between the thicknesses of the shear force starting point and the recovering layer is an atomic layer, as indicated by the following relationships:where a is the thickness of the recovering layer, ω is the angle between the shear force starting point and the cutting direction on the edge of knife, and β2 is the angle from the perpendicular to the cutting direction on the knife tip and the recovering layer touching the knife tip. The shear angle can then be determined from the following relationship:

Optimising the cutting conditions
The most important strategy for optimising the cutting conditions is to reduce the forces applied to the block face since these lead to the deformation. The stress at the block face arises from a combination of the cutting force, Fc, and the normal force, Ft, as indicated in the following expression:
This stress is strongly influenced by the rake angle. Fig. 9 shows the relationship between the compression rate, the shear and rake angles, and the applied stress. Reduction of the rake angle results in an increase in the shear angle and a reduction of the stress. When, the rake angle is 50°, the shear and normal stresses become equal, while for a rake angle of 76° the normal stress becomes zero. Consequently, if the knife and clearance angles sum to 14°, the damage on the block face is minimised. Further, when the normal stress exceeds the ultimate tensile strength the chip will form by ripping rather than by slicing. When the rake angle exceeds 50°, the normal stress becomes less than the tensile yield stress of the AA 2024 alloy. Hence, damage to the block face might be reduced. However, it is also dependent on the dislocation density, which is readily influenced by deformation on the block face.
Currently, the minimum rake angle of a commercially available diamond knife is 35° however, it is possible to minimise the normal stress. In order to minimise the stresses, the knife can be oscillated to minimise the apparent knife angle [46]. The apparent knife angle (mean knife angle during the oscillation of the knife), α2, is given by the following equation:where vs is the traverse speed of the knife and ve is the velocity normal to the cutting direction. The apparent angle can be applied in the Merchant cutting model and it determines the ultramicrotomy operating conditions for minimum damage on the block face. Notably, the oscillating knife reduces the apparent angle and, thereby, the associated stresses. Additionally, the transverse movement of the knife also causes the local temperature of the material in contact with the knife edge to increase because of the friction between the knife edge and the material, causing the material to soften. The effect of oscillation on reducing chatter and generally improving the surface finish is evident in Fig. 10.

Of course it is apparent that the indications identified

Of course it is apparent, that the indications identified in the present test experiment regarding secondary and inelastically scattered Chloroquine momentum mapping as a possible source of information, are not sufficient for a detailed and theoretically complex analysis of scattering processes. However we hope to have demonstrated an interesting capability of our cathode lens based spectromicroscope equipped with “in-lens” electron sample illumination system for near simultaneous, energy selective real imaging and “energy-loss”/secondary electron momentum mapping, and therefore for investigating the different scattering phenomena. They are intended to be the subject of further, more detailed experimental and theoretical studies exploiting the electron energy-loss (plasmon) imaging and momentum mapping in the high energy resolution regime. It\’s importance for the band structure analysis and -imaging in the case of electron photoemission [43,44] has been demonstrated recently by the application of full-field high resolution momentum microscopy: (1) time-of-flight imaging of the d-like surface resonances on Mo(110) [45] and (2) spin resolving high resolution energy selective imaging of the momentum distribution of photoelectrons [46].

I would like to express my gratitude to Professor Ernst Bauer for his encouragement and stimulation to this experiment, to Janusz Krajniak for his invaluable R&D work on the electronics and software, to Jerzy Dora for his unique concepts in electronics, to Bartosz Czaban for the same in imaging software and to Dariusz Mirecki for discussions and help. Financial support from the National Centre for Research and Development in Warsaw under Grant No. INNOTECH-K1/H11/26/159076/NCBR/12 is gratefully acknowledged.

A crucial step towards the optimization of the

A crucial step towards the optimization of the optoelectronic device structures is the investigation of the defect formation mechanisms. Hence, the detailed atomic study of the local structure at the interface between the semiconductor epilayers and the Si substrates is of particular interest. For this, transmission electron microscopy (TEM) is ideally suited as it provides the spatial resolution to study the structure of materials on the atomic level. Over the past 30 years, extended defects in semiconductor materials have been intensively studied by conventional and high-voltage high-resolution TEM [1,9–12]. However, it was experimentally challenging to resolve the exact atomic-scale structure due to the lack in spatial resolution and/or image delocalization in conventional phase contrast micrographs. Thus, “holographic” reconstruction techniques were indispensable to increase the spatial resolution [13–15]. Alternatively, image simulations based on structural models were performed to validate the image interpretation [11,12].
More recently, the implementation of spherical-aberration correctors in scanning transmission electron microscopes (STEM) [16–18] has lead to dramatic improvements in lateral resolution. These instruments are now capable of routinely producing images in the deep sub-Ångström range [19]. Thus, the dumbbell structure in crystalline Si along and samples can be clearly resolved, which is particularly useful for the characterization of defects [20]. Additionally, by using a high-angle annular dark-field (HAADF) detector it is possible to produce images which show contrast that approximately scales with the square of the atomic number, hence the name Z-contrast imaging. This allows to discriminate, for example, between the lighter Cd atomic columns from the heavier Te columns in the CdTe dpn semiconductor. Recent studies of low-dimensional semiconductor structures and devices by aberration-corrected STEM clearly demonstrate the power of this technique for investigating defects and interfaces [3,21–29]. It has proven to be decisive both in the detection of novel types of defects but also in the advancement of our understanding of seemingly basic crystal-structure defects.

Experimental details
Unless otherwise stated, all experimental images were taken using a double spherical aberration-corrected JEOL JEM-ARM200F microscope equipped with a cold field-emission electron source operating at 200kV. In STEM mode, a convergence semiangle of 25mrad was used in combination with an annular dark field (ADF) detector with inner and outer collection semiangles of 90 and 370mrad, respectively. A Gaussian low-pass filter for noise reduction was applied to all images.
Samples for the HAADF-STEM analysis were prepared by means of either a FEI Helios NanoLab 600i or FEI Helios NanoLab 450S focussed ion beam (FIB) operated at accelerating voltages of 30 and 5kV. Additionally, Si/Ge specimens [30] were prepared by mechanical polishing and dimple grinding, followed by ion-milling with Ar+ ions using a Fischione Model 1050 TEM-Mill operating at low voltages and grazing incidence to achieve electron transparency.

Configuration of planar defects
The most commonly observed planar (2D) defects in cubic semiconductors by using high-resolution TEM are twin boundaries (TBs) and stacking faults (SFs) [31]. They are caused by discontinuities in the …AaBbCcAaBbCc… stacking sequence of 111 -type close-packed layers in diamond or, its ordered variant, zincblende structure. Additionally, compound semiconductor layers grown on elemental semiconductor substrates, like Si or Ge, are also susceptible to form antiphase boundary (APB) defects. In the following sections, the characteristics of these defects are discussed.

Configuration of dislocations
Fig. 4 illustrates the presence of dislocations both inside a Ge crystal and at a Ge/Si interface, together with their corresponding ε strain field maps obtained by geometrical phase analysis (GPA) [53]. In particular, Fig. 4a and b show two parallel dislocations lying on perpendicular slip planes in close proximity within a Ge crystal and the strain field interaction between them: both dislocation cores exhibit a compression region (in blue) and a tensile region (in yellow) [30]. Similar butterfly-like shapes are observed at the Si/Ge interface (Fig. 4c and d). They are MDs (marked with white arrows) resulting from the 4.2% lattice mismatch between Ge and Si, and are identified as pairs of perfect glissile 60° and perfect sessile 90° MDs (a more detailed description of these type of dislocations is given in the following sections). The number of atomic planes between the MDs is not constant. It varies between 20 and 40 planes depending on the area of observation. Theoretically, for the total relaxation of the misfit strain one MD should be introduced every 24 planes [, n=number of planes, a=lattice constant, a=0.5658nm and a=0.5431nm]. Experimentally, the number of 111 planes between the MDs is slightly larger with an average of 26±0.5 planes between dislocations. Therefore, not enough MDs are incorporated to fully relax the strain plastically at the selected growth temperatures [54].

To test the feasibility of determining defocus and astigmatism within

To test the feasibility of determining defocus and astigmatism within the error limits discussed above from the diffractogram of the side band image I used the tool CTFIT from the EMAN1 package [9]. Based only on the first zero I determined a defocus of about 195nm and an additional defocus in the y-direction of about 45nm (rather than 200 and 40nm). I did not try to evaluate the accuracy of the direction of astigmatism. Obviously, due to the nature of the diffractogram, it\’s not possible to use an existing automatic procedure for determining the CTF-parameters.

I present a novel design for a single side band aperture which allows determining defocus and astigmatism from the recorded images. I present the image formation theory and a method of correcting the transfer function of such an imaging system and test both in simulations. One clear advantage of single side band imaging is that the modulus of the complex valued transfer function is 1 for all spatial frequencies (outside the gaps). This means that images can be recorded at an arbitrary defocus value without introducing zeros in the transfer function. Therefore single side band imaging with such an aperture could be very suitable for imaging small proteins (100kDa and below). Thus this imaging mode could be an attractive alternative to imaging with phase plates.


Strongly correlated MLN4924 materials exhibit various intriguing and drastic phenomena such as the metal-insulator transition, high- superconductivity, and colossal/giant magneto resistance [1]. When multiple phases are adjacent and competing in the vicinity of a first-order phase transition for instance, there emerges self-organized electronic inhomogeneity with various types and length scales [2]. In order to find deeper insight into the macroscopic phenomena in these materials, it is indispensable to understand local electronic structures with relevant spatial resolution.
Scanning photoemission microscopy (SPEM) is one of the primary spectroscopic techniques for studying electronic states with spatial inhomogeneity [3]. Recently, this type of instrument has been developed to incorporate the capabilities of angle-resolved photoemission spectroscopy (ARPES), mainly at third-generation synchrotron facilities, often referred to as µ-ARPES or nano-ARPES [4–6]. These techniques have successfully revealed various electronic inhomogeneities in strongly correlated electron materials, such as metallic and insulating phase separation with a length scale of 10 µm in Cr-doped V2O3[7], electronic and structural inhomogeneities with a length scale of 100 µm in high- cuprate YBa2Cu4O8[8] as well as bilayer manganites LaSrMn2O7[9]. However, it is often inevitable to lower energy resolution to get higher count rates since photon flux is considerably reduced to achieve high spatial resolution due to low efficiency of focusing optics.
On the other hand, a major focus of conventional ARPES has been to study the density-of-states, band-dispersions, and Fermi surfaces of solids [10]. Modern ARPES with high energy and momentum resolutions, usually referred as high-resolution ARPES, can precisely determine quasiparticle\’s dispersion relations and lifetimes [11,12]. However, detailed microscopic information in the smaller area has not been sufficiently pursued by means of high-resolution ARPES so far.
By maximizing the merits of high energy and spatial resolutions, we have developed a new laser-based µ-ARPES system at the Hiroshima Synchrotron Radiation Center (HiSOR). High brilliance and monochromaticity of laser light is suited for high-resolution ARPES [13–15], and furthermore, its spatial coherence/directionality can be applicable to SPEM. In this paper, we present the design and typical performance of our µ-ARPES system equipped with a vacuum ultraviolet (VUV) laser tunable from 5.90 eV to 6.49 eV. Based on considerations on the commercially achievable specifications of laser light source, optics and focusing systems, we realized the compatibility with high spatial resolution better than 5 µm as well as the state-of-the-art energy and momentum resolutions. We have also examined spatial dependence of fine spectral features, which enables us to find sample area suitable for obtaining intrinsic electronic states. Present µ-ARPES holds the promise for uncovering intrinsic and fine details of electronic features that may have been overlooked by conventional high-resolution ARPES.

The production and accumulation of the virally encoded

The production and accumulation of the virally encoded proteins signals a switch in the polymerase function, from viral mRNA transcription to genome replication, in which N plays a critical role. An essential step in the viral replication of the nascent positive-sense genome (antigenome) relies on its encapsidation, a process facilitated by cis-acting conserved sequences located on the 3′ ends of viral genome and antigenome (Whelan and Wertz, 1999; Li and Pattnaik, 1999). Additionally, N and P proteins are critical in promoting genome replication, as the N/P complex provides the structural and chaperone support for the nascent RNA to bind via sugar-phosphate interactions to the N protein (Albertini et al., 2006). The bound antigenome will then function as template for the synthesis of encapsidated negative-sense genomes, which will be assembled into progeny virions.
Virion assembly is a staggered process where the various components [nucleocapsid core (RNP), G and M proteins] are sequestered in different cellular compartments and converge in the final steps of the process. The nucleocapsid is assembled during RNA replication in the cytoplasm, as is observed for members of the genera Vesiculovirus, Lyssavirus, Ephemerovirus and Novirhabdovirus. Viral G protein is inserted into the endoplasmic reticulum where chaperones (BiP and calnexin)(Hammond and Helenius, 1994) facilitate its proper folding and assembly into trimers (Doms et al., 1988), prior to transport and fusion into the Golgi complex. As it traffics through the cell it undergoes further posttranslational modifications including glycosylations (Schmidt and Schlesinger, 1979), prior to its transport to cholesterol- and sphingolipid-rich lipid rafts in the baso-lateral plasma membrane. M protein is synthesized mostly as a soluble protein in the KN-93 hydrochloride (McCreedy et al., 1990) and is also membrane bound, albeit at lower amounts (Ogden et al., 1986). However both forms of the M protein are recruited for assembly of nucleocapsid/M complexes at the host plasma membrane from where virions will bud (Odenwald et al., 1986). This budding process is facilitated by the interaction of M with host-encoded proteins responsible for the formation of multivesicular bodies (MVB), and their release from the plasma membrane (Harty et al., 2001).

‘Classical’ vertebrate rhabdoviruses
For historical reasons any reference to classical vertebrate rhabdoviruses denotes members of the genera Vesiculovirus and Lyssavirus, represented by the prototype species vesicular stomatitis Indiana virus (VSIV) and rabies virus (RABV), respectively. Vesiculoviruses have a wide host range among mammals and are transmitted by hematophagous insects (sandflies and/or mosquitoes). Lyssaviruses utilize mostly bats as their principal reservoir hosts as well as various terrestrial carnivores as terminal hosts. Viruses of each genus form a monophyletic clade in a maximum likelihood (ML) tree inferred from complete L protein sequences (Dietzgen et al., 2011; Walker et al., 2015). Structurally both demonstrate the classic rhabdovirus enveloped bullet-shaped virions (Fig. 1) packaging a genome consisting of five genes (3′-N-P-M-G-L-5′), each separated by a short gene junction (intergenic region), and flanked by highly conserved 3′ leader (le) and 5′ trailer (tr) sequences (Fig. 3). In vesiculoviruses the P gene mRNA contains 2 additional alternate start codons that initiate translation at alternative open reading frames (ORFs) that encode two small basic proteins C and C’ (55-aa and 65-aa, respectively) of unknown function (Spiropoulou and Nichol, 1993; Peluso et al., 1996). Suppression of C/C’ expression has no apparent effects in virus replication or pathogenicity in vivo (Kretzschmar et al., 1996). Of note is that not all members of the genus express alternative ORFs in P [e.g. vesicular stomatitis Alagoas, Maraba, Malpais Spring, Morreton viruses] (Walker et al., 2015), and additional ORFs KN-93 hydrochloride (≥150nt) may be present in alternative reading frames in other genes than P (Walker et al., 2015).

The aim of the present

The aim of the present study was to assess the effect of different inflammatory stimuli on eBM-MSCs immunoregulatory ability and immunogenicity, studying the expression of immunogenic and immunomodulation-related molecules. Firstly, the influence of allogeneic inflammatory SF on eBM-MSCs was investigated, and subsequently, the effect of priming eBM-MSCs with a combination of the two pro-inflammatory molecules IFNγ and TNFα, was tested at two different doses. SF and CK inflammatory conditions were chosen according to previous studies (Ren et al., 2008; Leijs et al., 2012; van Buul et al., 2012; Vézina Audette et al., 2013; Zimmermann and McDevitt, 2014). This work contributes to understand the effects of inflammatory exposure on eBM-MSCs, as a previous step to enhance their use in vivo for equine joint diseases.

Materials and methods


The cultured SEA0400 showed capacity for attachment to plastic and the ability of differentiation into osteoblast, adipocyte and chondrocyte, as criteria established to define human MSCs (Dominici et al., 2006). Equine BM-MSCs displayed a gene and cell surface expression pattern similar to previous reports for this species and showed a normal growth pattern, with a proliferation rate and viability similar to other studies (Ranera et al., 2011). Inflammatory conditioned media were prepared based on recent publications on this field. Inflammatory SF was obtained from an allogeneic donor and added at a concentration considered likely to act as an enhancer of the immunoregulatory potential of MSCs (Leijs et al., 2012). SAA and Hp were used to determine the inflammatory status of the SF. Both APP were elevated with regard to described ranges (Basile et al., 2013; Jacobsen et al., 2006) in agreement with the expected changes for these proteins (Jacobsen and Andersen, 2007). For CK-conditioned media, the synergy displayed by the pro-inflammatory cytokines TNFα and IFNγ (Zimmermann and McDevitt, 2014) supported the decision of using them to stimulate the eBM-MSCs. The induction of immunoregulatory properties of BM-MSCs by synergistic cytokine priming is not previously reported in horses. Equine BM-MSCs has been stimulated with 100ng/ml of IFNγ alone in some studies (Paterson et al., 2014; Schnabel et al., 2014), but their gene and surface expression of immunoregulation-related molecules were not studied after the stimulation. Therefore, the tested doses (20ng/ml and 50ng/ml) were chosen according to previous studies in human and mouse MSCs showing induction of immunoregulatory factors expression or secretion (Ren et al., 2008; Waterman et al., 2010; Hegyi et al., 2012; van Buul et al., 2012) to determine if the same conditions also operate similar changes in the behaviour of MSC from equine species. The available volume of inflammatory SF did not allow a time course to be performed and thus a single time-point of 72h was chosen to maximize potential effects of exposure to SF. This was longer than previous studies on the effects of inflammatory SF (Leijs et al., 2012; Vézina Audette et al., 2013) but longer exposure to individual cytokines has been reported (Paterson et al., 2014) and immunosuppressive effects of IFNγ-activated MSCs have been shown to depend on the exposure time to IFNγ (Chan et al., 2006). Our results are similar to previous reports in other species using shorter times of cytokine stimulation, as it will be further discussed. However, the exposure of eBM-MSCs to SF did not produce remarkable effects despite of using an exposition time longer than the previously described (Leijs et al., 2012; Vézina Audette et al., 2013).
Several mechanisms have been proposed to participate in the immunoregulatory function of MSCs, including the participation of chemokine axis, adhesion molecules and soluble factors (Ma et al., 2014). The co-culture of MSCs in the presence of T cells triggers the expression of VCAM-1 (Ren et al., 2010), according to the results obtained in our CK20 conditions. This finding supports the implication of cell-to-cell contact in the MSC immunoregulatory mechanism. Adhesion molecules are also related to MSC migration, a mechanism which could be critical for the recruitment of MSCs into wound sites for tissue regeneration. This process is complex, especially in inflammatory environments, and whereas some studies describe the enhancement of MSC migratory property under inflammatory stimulation (Ries et al., 2007; Shi et al., 2007), others report a decrease in this property depending on the time of exposure (Waterman et al., 2010). The MFI of the hyaluronan receptor CD44, related to cell migration through the extracellular matrix (Zhu et al., 2006), was increased under CK50 conditions, indicating an enhancement in the expression level of this marker, despite the number of positive cells remained similar. However, the gene expression of the chemokine receptor CXCR4, also implied in the MSC migration (Honczarenko et al., 2006), was decreased in both CK Experiments, suggesting that MSC migration might be diminished in the tested inflammatory conditions.

br Conclusions Collectively extensive in vivo

Collectively, extensive in vivo data have demonstrated that orally administered low-dose IFN has rapid and systemic beneficial biological effects in animals and humans. In the present study, it was demonstrated that oral IFN statistically modulated the expression of 12 genes within a specific KEGG pathway (cytokine–cytokine receptor interactions). In turn, these data suggest that low-dose oral IFN exerts systemic and largely beneficial anti-viral effects by up- or down-regulation of specific immune response genes and their products likely needed for recovery from any viral disease, including FMDV. The accumulated evidence from this and other studies suggests that adoption of orally administered bovine IFNα or HuIFNα as a therapeutic modality to suppress viral replication in livestock at risk for FMDV exposure is an inexpensive, easily dispensed and widely available alternative and adjunct to vaccinations for FMDV. In addition to the obvious antiviral effects of oral IFN, systemic immune modulating effects are also part of the overall beneficial responses to IFN and it is the latter that may well provide prolonged systemic beneficial effects in virus-susceptible livestock populations. The results are intended 5 aminolevulinic acid to stimulate further testing using delivery in feed or water, potentially helping the livestock industry manage viral respiratory disease and protect against an FMDV outbreak.


Microbial colonization begins immediately after birth with facultative anaerobes, such as lactobacilli, enterococci and enterobacteria, being the first colonizers. Colonization by anaerobic microorganisms follows, including Bifidobacterium, Bacteroides and Clostridium, resulting in a gradual decrease of the ratio of facultative anaerobes to strict anaerobes over time (Arboleya et al., 2012). Bifidobacteria, along with lactobacilli, are an important part of normal intestinal microbiota of various mammalian species and are also the best characterized and widely commercialized probiotics. Both lactobacilli and bifidobacteria are non-spore-forming, gram-positive, lactic 5 aminolevulinic acid producing bacteria (LAPB). Lactobacilli have limited biosynthetic abilities and ferment refined sugars, generating lactic acid as the major end product (Wells, 2011), whereas bifidobacteria are important producers of short chain fatty acids (SCFA) (Tojo et al., 2014). Despite some common properties, lactobacilli and bifidobacteria belong to two taxonomically distinct groups: the genus Lactobacillus in the phylum Firmicutes and the genus Bifidobacterium in the phylum Actinobacteria, respectively. In adults, Firmicutes and Bacteroidetes phyla usually dominate the intestinal microbiota, whereas Actinobacteria, Proteobacteria and Verrucomicrobia are considerably less abundant. However, in naturally delivered, breast-fed infants, bifidobacteria (Actinobacteria) appear between days 2 and 5 after birth and reach a maximum of up to 99% of all bacteria within one week becoming the predominant bacterial component of the infant fecal microbiota (Kurokawa et al., 2007; Mitsuoka and Kaneuchi, 1977; Turroni et al., 2012; Yatsunenko et al., 2012). Some studies report that Bifidobacterium infantis and Bifidobacterium breve were the most common species found in healthy infants (He et al., 2001).
Although not the most dominant in adulthood, lactobacilli and bifidobacteria remain stable elements of the normal intestinal microbiota, maintaining their important functions throughout life, and their dysbiosis is associated with a plethora of pathological conditions (Gerritsen et al., 2011). Numerous studies with different strains of Lactobacillus and Bifidobacterium have been performed in vitro and in vivo, in humans and animal models to investigate their immunomodulatory properties and probiotic potential to treat various infectious, allergic and inflammatory conditions (Grimm et al., 2014; Picard et al., 2005; Tojo et al., 2014; Wells, 2011) (Fig. 1). While not always conclusive, most of them emphasized the beneficial effects of these probiotic bacteria, that appear to be pathogen/condition, bacteria and sometimes host species-specific. In most clinical trials, lactobacilli and bifidobacteria probiotics were demonstrated to be safe with the rare exception of probiotic-associated infections in immunosuppressed patients (Saarela et al., 2002). Historically, the most usual application of probiotics is to treat gastrointestinal disorders, including infectious diarrhea (de Vrese and Marteau, 2007).

Recent work from our laboratory showed that encoding porcine G

Recent work from our laboratory showed that encoding porcine G-CSF in a replication-defective adenovirus and delivering a single dose to pigs induced a neutrophilia for more than two weeks (Loving et al., 2013). As secondary bacterial infection is a common occurrence following PRRSV infection, particularly following challenge with HP-PRRSV, the aim of the current study was to evaluate the effectiveness of a single prophylactic dose of adenovirus-encoded G-CSF to mitigate secondary bacterial disease associated with HP-PRRSV infection.

Materials and methods


Administration of adenovirus vectored G-CSF induced a neutrophilia in all 40 pigs in the G-CSF treated groups (Ad5-G-CSF n=10, Ad5-G-CSF/HP-PRRSV n=30). In the Ad5-G-CSF/non-challenged group neutrophilia peaked at 7days post injection and remained elevated for the duration of the experiment, which is consistent with a previous report from our group (Loving et al., 2013). In contrast, HP-PRRSV infection depleted >60% of the Ad5-G-CSF induced neutrophilia within 2days of intranasal virus challenge in each of the 30 pigs in the Ad5-G-CSF/HP-PRRSV group, indicating that viral infection had a role in the rapid and dramatic GM6001 in circulating neutrophils. Interestingly, the neutrophil counts became similar between the Ad5-empty and Ad5-G-CSF virus challenge groups suggesting the neutrophils generated in response to Ad5-G-CSF stimulation were uniquely affected by HP-PRRSV infection. This study was initiated concurrent to the 2013 published report comparing the neutrophilia induced by adenovirus encoding wild-type versus mutated porcine G-CSF, and the current study used only the mutated porcine G-CSF presuming the response in swine would be synonymous to the response in humans when using the mutated form of G-CSF (Sarkar, 2002). However, the wild-type G-CSF induced a more profound neutrophilia (Loving, 2013) and thus, future studies should utilize the replication-defective adenovirus encoding the wild-type G-CSF for inducing neutrophilia.
Both HP-PRRSV challenged groups had a similar clinical response to infection based on rectal temperature response, weight loss, virus replication, macroscopic lesion profile, lymphopenia and mortality. The clinical response observed in this experiment was comparable to our previous studies with the JXwn06 HP-PRRSV isolate (Guo et al., 2013a,b) and the original reports for this virus (Li et al., 2007; Tian et al., 2007; Tong et al., 2007). In addition, the manifestation of secondary bacterial infection was similar between the groups and mirrored our previous experience (Guo et al., 2013a,b). The magnitude of secondary bacterial infection in the Ad5-G-CSF treated group indicated the prophylactic administration of an immunomodulator that induced a neutrophilia prior to the time of challenge had minimal impact on subsequent bacterial disease. This was unexpected because 1) the prophylactic use of G-CSF in cattle has reduced clinical disease associated with mastitis (Hassfurther et al., 2015; Kehrli et al., 1991a) and bacterial pneumonia (Youssef et al., 2004), 2) in humans G-CSF administration and restoration of circulating neutrophils reduces the incidence of febrile neutropenia disease in patients undergoing cancer treatments, and 3) a previous experiment testing different Ad5-G-CSF constructs in swine demonstrated the expanded neutrophil population consisted of functional neutrophils (Loving et al., 2013) that we presumed would mitigate the secondary bacterial diseases induced by HP-PRRS. However, given the negative impact of HP-PRRSV infection on circulating neutrophils, it’s difficult to clearly determine if enhancing neutrophil numbers through the use of G-CSF could be of benefit.
In addition to the marked and sustained neutrophilia observed in this study following Ad5-G-CSF administration, a monocytosis was induced as well. Interestingly, the effect of HP-PRRSV on monocytes paralleled the effect on neutrophils, with the increase in circulating numbers followed by a precipitous decrease within 2days of HP-PRRSV challenge. In addition, the magnitude of increase in both cell populations following Ad5-G-CSF administration was similar. The increase in circulating monocytes following Ad5-G-CSF administration to pigs has been reported by our group previously (Loving et al., 2013), and in other species following G-CSF administration (Kehrli et al., 1991b; Lord et al., 2001), and is referred to as a “bystander effect” (Lord et al., 2001). The mechanism for this phenomenon is not understood, but the increase in monocytes may be mediated by a non-specific signal released in response to the administration of G-CSF. The acute response to G-CSF should be related to movement of neutrophils and monocytes from bone marrow to peripheral circulation, which occurs rapidly in response to G-CSF administration (Lord et al., 2001).

In addition to that conditions of chronic oxidative stress are

In addition to that, conditions of chronic oxidative stress are likely to cause poor homeostatic control of the inflammatory responses. In particular, oxidative stress results in the release of enzymes, named “redoxkines”, that amplify signals of the TNF-α-induced inflammatory response and have also other cytokine-like activities, linking oxidative stress to innate immunity (Salzano et al., 2014).

Immune response to stress AMG-900 manufacturer and correlates of protection
The above data imply the existence of high levels of innate immune response to damage-associated molecular patterns (DAMPs) like uric acid, ATP, oxydative stress products, etc., released by stressed cells. Interestingly, oxidative stress may be the common final pathway of the inflammasome reaction, unifying a plethora of stress-related events (Martinon et al., 2009). Also, neo-antigens expressed on stressed cells like MIC, HSP 60, T10, T22, CD1c, described in mice, humans and also cattle, are recognized by the “Lymphoid Stress Surveillance System” (Hayday, 2009). This high level of baseline immune response often co-exists with further inflammatory stimuli as a result of poor farm hygiene and associated infectious pressure of common environmental pathogens. In pig farms, such an outcome is actually eased by common management practices like:
The above features (to name a few) do increase the infectious pressure in pig farms, as shown by the high levels of IFN-γ in many plasma samples of apparently healthy and thriving pigs in the post-weaning and back grounding phases (AmadoriM., unpublished results). In this scenario, the Type I IFN response of pigs at 5–6days after weaning (Razzuoli et al., 2011) represents an important indication of a homeostatic control action vis-à-vis the harmful effects of the early weaning stress (Amadori et al., 2012). Interestingly, infectious pressure should be meant as exposure to both microbial agents and airborne LPS, which is definitely an occupational hazard AMG-900 manufacturer for pig farm personnel (Zhiping et al., 1996). It goes without saying that such levels of background response to infectious and non-infectious stressors may jeopardize the correct evaluation of recognized immune correlates of protection. Most important, non-specific cross-reactions are likely to be seen in vitro because of the constitutive expression of stress antigens (MIC, ULPB 1-3) in many established cell lines of epithelial and endothelial origin used for propagation and in vitro testing of animal viruses (Tran et al., 2008). In our experience, established cell lines of pig (PK-15c28) and monkey (MARC-145) do express such stress antigens on their surface, as established by means of a NKG2D-human Fc chimera (Zanotti et al., 2015). This may have serious repercussions on immunoassays for viruses like PCV2 and PRRSV. Thus, PBMC of PCV2 vaccinated/infected pigs may show a higher response to a control cell cryolysate compared with the relevant virus grown in the same cells in an ELISPOT assay for IFN-γ−secreting cells (Fig. 5). Likewise, the presence of necrotic cell antigens in any assay for inflammatory cytokine responses to microbial agents is likely to raise non-specific responses to very high levels. Therefore, whenever purified viruses or viral proteins cannot be used in vitro, proper controls should include the potential DAMPs and stress antigens contained in the raw viral antigen.

Which kind of protective immune response?
There is a wide consensus in the scientific community that strong and long-lasting adaptive immune responses to microbial agents are generally beneficial to the naïve, uninfected host, with some minor exceptions like Respiratory Syncytial Virus (RSV) infections in cattle (Antonis et al., 2003). The picture is not as clear in animals experiencing an ongoing microbial infection. Thus, there are at least three microbial infections which question the beneficial role of vigorous adaptive immune responses to microbial agents in infected animals:

Stress induced disruption of the integrity of the gut

Stress-induced disruption of the integrity of the gut epithelium reduces the efficacy of the birds’ innate protective mechanisms and may increase the potential for pathogens, such as Salmonella spp., to bind to and colonize the intestinal epithelium (Burkholder et al., 2008). Quinteiro-Filho et al. (2012a) observed the combination of HS and Salmonella Enteritidis infection disrupts the intestinal barrier, which allows pathogenic bacteria to migrate through the intestinal mucosa to the spleen and generate an inflammatory infiltrate in the gut, decreasing performance parameters. However, in these previous reports, we did not analyze the role of the chicken\’s immune system status on heat stress associated with a pathogenic bacterial challenge.
The aim of this study was to analyze the effects of heat stress on different parameters of the immune system of broiler chickens experimentally infected with S. Enteritidis. The colonization of S. Enteritidis into the crop and caecum and the invasion of this pathogen to immunocompetent organs, such as the liver, spleen and bone marrow, were analyzed; in addition, the mRNA Sirtinol manufacturer of cytokines in the spleen and cecal tonsil, including avian β-defensin (AVBD) 4, AvBD-6, Toll-like receptor (TLR) 2, and TLR-4, and the plasmatic levels of IgA, IgG and IFN-γ were also investigated.

Materials and methods


Stress is an important topic in the poultry production. Studies on chronic stress and the immune system usually focus the occurrence of immunosuppression with subsequent failures in the development of immune system activity, particularly when the animals are challenged with different pathogens, such as Salmonella spp. and C. perfringens (Quinteiro-Filho et al., 2012a; Calefi et al., 2016b). Scientists worldwide agree that the interactions among the central nervous, endocrine and immune systems could explain the effects of stress in animals (Calefi et al., 2016a; Costa-Pinto and Palermo-Neto, 2010; Kaiser et al., 2009; Kelly et al., 2016). One of the most important systems that integrate within the body during stressful situations and/or homeostatic disturbances is the hypothalamus–pituitary–adrenal axis (HPA) (Besedovsky and DelRey, 1996; McEwen, 2000). The activation of the HPA axis releases corticosterone, the key hormone in stress situations. The present findings showed increased levels of corticosterone in heat-stressed birds, a finding that corroborates and reinforces the data reported in our previous work (Quinteiro-Filho et al., 2010, 2012a).
Different authors have shown that heat stress modulates the humoral immunity of chickens (Khajavi et al., 2003; Star et al., 2009; Honda et al., 2015). Mashaly et al. (2004) observed that chronic heat stress (35°C for 5 weeks) decreases the number of blood leucocytes and SRBC antibody production. Stressors via corticosterone inhibit antibody production by B cells (Gross, 1992; Glaser and Kiecolt-Glaser, 2005), which might explain the inverse relationship reported in this study for the corticosterone levels and immunoglobulin results. The current data show that heat stress decreases IgA plasmatic levels; however, when heat stress was applied in the course of S. Enteritidis infection (PHS31°C group), the reduction in IgA levels was more pronounced. IgA is responsible for local protection against bacterial pathogens and parasitic and viral infection (Fagarasan, 2006). We believe that the observed decrease in IgA levels could impair the first barrier mechanism used by the immune system in an attempt to respond to S. Enteritidis invasion.
In the context of heat stress and the S. Enteritidis challenge, we observed the evident presence of S. Enteritidis in the crop, caecum, liver and spleen of broiler chickens. Interestingly, Salmonella was also observed in the bone marrow of heat-stressed chickens (PHS31 group). In the same experimental protocol, heat stress increased S. Enteritidis migration to the spleen of broiler chickens, a fact that points towards a decrease in chickens host resistance to Salmonella-induced infection (Quinteiro-Filho et al., 2012a). Kiank et al. (2008) reported that commensal bacteria migrate to lymphoid organs and the liver in stressed mice. These authors also reported that chronically stressed mice spontaneously suffer from increased bacterial load in the liver and lungs, (Kiank et al., 2008). Moreover, induced stress in rats and mice increases their susceptibility to Salmonella infection (Kuriyama et al., 1996). If bacterial replication is not controlled by innate immunity, Salmonella is able to reach and replicate within several organs (e.g., the spleen) (Humphrey, 2006). In fact, 31°C heat stress applied from ED35 to ED42 decreases macrophage activity (Quinteiro-Filho et al., 2010), and herein, we showed that it decreased IgA and IFN-γ plasmatic levels.