Acyloxyacyl hydrolase modulates pelvic pain severity.

Chronic pelvic pain causes significant patient morbidity and is a challenge to clinicians. Using a murine neurogenic cystitis model that recapitulates key aspects of interstitial cystitis/bladder pain syndrome (IC), we recently showed that pseudorabies virus (PRV) induces severe pelvic allodynia in BALB/c mice relative to C57BL/6 mice. Here, we report that a quantitative trait locus (QTL) analysis of PRV-induced allodynia in F2CxB progeny identified a polymorphism on chromosome 13, rs6314295 , significantly associated with allodynia (logarithm of odds = 3.11). The nearby gene encoding acyloxyacyl hydrolase ( Aoah) was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited increased vesicomotor reflex in response to bladder distension, consistent with spontaneous bladder hypersensitivity, and increased pelvic allodynia in neurogenic cystitis and postbacterial chronic pain models. AOAH deficiency resulted in greater bladder pathology and tumor necrosis factor production in PRV neurogenic cystitis, markers of increased bladder mast cell activation. AOAH immunoreactivity was detectable along the bladder-brain axis, including in brain sites previously correlated with human chronic pelvic pain. Finally, AOAH-deficient mice had significantly higher levels of bladder vascular endothelial growth factor, an emerging marker of chronic pelvic pain in humans. These findings indicate that AOAH modulates pelvic pain severity, suggesting that allelic variation in Aoah influences pelvic pain in IC.

Pseudorabies virus US3 triggers RhoA phosphorylation to reorganize the actin cytoskeleton.

The conserved alphaherpesvirus serine/threonine kinase US3 causes dramatic changes in the actin cytoskeleton, consisting of actin stress fibre breakdown and protrusion formation, associated with increased virus spread. Here, we showed that US3 expression led to RhoA phosphorylation at serine 188 (S188), one of the hallmarks of suppressed RhoA signalling, and that expression of a non-phosphorylatable RhoA variant interfered with the ability of US3 to induce actin rearrangements. Furthermore, inhibition of cellular protein kinase A (PKA) eliminated the ability of US3 to induce S188 RhoA phosphorylation, pointing to a role for PKA in US3-induced RhoA phosphorylation. Hence, the US3 kinase leads to PKA-dependent S188 RhoA phosphorylation, which contributes to US3-mediated actin rearrangements. Our data suggest that US3 efficiently usurps the antagonistic RhoA and Cdc42/Rac1/p21-activated kinase signalling branches to rearrange the actin cytoskeleton.

Pseudorabies virus tegument protein Us2 recruits the mitogen-activated protein kinase extracellular-regulated kinase (ERK) to membranes through interaction with the ERK common docking domain.

The pseudorabies virus (PRV) Us2 protein binds to the extracellular-regulated kinase (ERK) and inhibits the activation of ERK nuclear targets by sequestering cytoplasmic ERK on cellular membranes. Utilizing a series of Us2 truncations, we determined that the minimal portion of Us2 required for interaction with ERK is contained within its amino-terminal 214 amino acids. The loss of the ability of Us2 to bind to ERK in coimmunoprecipitation experiments was accompanied by a failure of Us2 to form oligomers, raising the possibility that higher-order Us2 structures are required for ERK interaction. To map the Us2 interaction site on ERK, we introduced mutations into the region of ERK that interacts with the ERK kinase, MEK, or into the common docking (CD) domain that mediates interactions with many ERK substrates. ERK carrying mutations within the MEK binding region maintained the ability to bind Us2, whereas ERK carrying mutations within the CD domain did not. Furthermore, the ERK CD domain was required for the Us2-mediated recruitment of ERK to membranes. Taken together, these findings suggest that Us2 regulates ERK activity by spatially restricting ERK localization and also by interfering with select ERK-substrate interactions.

Pseudorabies virus induces a rapid up-regulation of nitric oxide synthases in the nervous system of swine.

Nitric oxide (NO) is a free radical gas with important roles in the host's immune response against viral infections. In this study, we examined the kinetics and distribution of nitric oxide synthase (NOS) expression during the early steps of infection of the porcine nervous system by the alphaherpesvirus pseudorabies virus (PRV). To this end, we examined changes in the expression of the three major NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), by immunohistochemistry in the trigeminal ganglia and brain of pigs inoculated intranasally with a virulent PRV strain. The results obtained show that infection of the porcine nervous system by PRV induced a rapid and progressive increment in NOS expression that coincided in timing, location, and magnitude with those of virus propagation in the nervous tissue. A major finding of this study was that PRV caused not only nNOS and iNOS induction in a variety of cell types, but also eNOS up-regulation in endothelial cells and neurons; therefore, all possible sources of NO are activated and probably contribute to the overproduction of NO during infection with the neurotropic alphaherpesvirus PRV in its natural host.

Inducible nitric oxide synthase expression in porcine immune cells.

Porcine immune cells were examined for the ability to produce inducible nitric oxide synthase following in vitro or in vivo stimulation. Enzyme activity and product formation were not detected following stimulation of porcine peripheral blood mononuclear cells (PBMC), splenocytes, or alveolar macrophages with a combination of ConA and lipopolysaccharide (LPS) or recombinant porcine interferon gamma and LPS. In vitro engulfment of Haemophilus parasuis by macrophages also failed to induce inducible nitric oxide synthase (iNOS) activity or nitrite formation. Swine Herpes Virus infection led to a small but significant increase in level of nitrite detected in lung lavage fluid, whereas the infection of pigs with Porcine Respiratory and Reproductive Syndrome Virus did not alter the lavage fluid nitrite levels. iNOS mRNA was detected in both stimulated and unstimulated porcine immune cells and in macrophages from both control and infected animals suggesting that it is constitutively expressed with little or no upregulation following cellular stimulation. The results presented in this paper indicate that the reactive nitrogen intermediate pathway is not an vital innate immune response in the pig.

Transneuronal labeling of neurons in the adult rat brainstem and spinal cord after injection of pseudorabies virus into the urethra.

Transneuronal tracing techniques were used to identify sites in the central nervous system involved in the neural control of urethral function. The distribution of virus-infected neurons was examined in the spinal cord and brainstem at various intervals (56-96 hours) following pseudorabies virus (PRV) injection into the urethra. In the lumbosacral (L6-S1) spinal cord at 56 hours, neurons containing PRV immunoreactivity (PRV-IR) were located in the region of the sacral parasympathetic nucleus (SPN), around the central canal, and in the dorsal commissure. Some animals also exhibited PRV-IR in cells in the L6 dorsolateral motor nucleus. At longer survival times (72-96 hours), PRV-IR cells were observed in the superficial and deeper laminae of the dorsal horn, and increased numbers of PRV-IR cells were consistently detected in the region of the SPN, around the central canal, and in the dorsal commissure. PRV-IR fiber-like staining also occurred along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the SPN. In rostral lumbar segments (L1-L2), PRV-IR cells were located in the region of the dorsal commissure and the intermediolateral cell nucleus (IML), around the central canal, and in the dorsal horn. After 72-84 hours, PRV-IR cells were also noted at more rostral levels of the neuraxis including the medulla, pons, midbrain, and diencephalon. At 72 hours, PRV-IR cells were consistently observed in Barrington's nucleus (pontine micturition center), nucleus raphe magnus (RMg), parapyramidal reticular formation, and the A5 and A7 regions. At 78-84 hours, additional regions exhibited PRV-IR cells, including the periaqueductal gray, locus coeruleus, the dorsal and ventral subcoeruleus alpha, and the red nucleus. A few cells were also located in the lateral hypothalamic area. This distribution of PRV-labeled cells in the spinal cord and brainstem is similar in many respects to the distribution of cells labeled in previous studies by PRV injection into the urinary bladder. This overlap of urethra and bladder neurons is consistent with the results of physiological experiments indicating a close coordination between the central nervous control of bladder and urethral activity.

Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus.

Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.

[Cytological and cytochemical studies of cell cultures infected with the avirulent viral mutant MK 35 of the pseudorabies virus]. / Tsitologichni i tsitokhimichni prouchvaniia na kletuchni kulturi, zarazeni s avirulentniia virusen mutant MK 35 na Pseudorabies virus.

Cytological and cytochemical studies were carried out of cell cultures of chick embryo fibroblasts infected with the vaccinal mutant strain MK 35 (4.8 X 10(7) PFU) of the Pseudorabies virus. It was found that the first cytologic changes in the infected cultures presented definite focal character. In the first hours of infection several rounded cells were included only, while later on an increasing number of cells were involved, and the foci grew in size. The cytoplasma of the infected cells contained inclusions which were small, spherical, basophilic, and Foelgen positive; six hours later the cytoplasmatic inclusions became larger, oval, acidophilic, surrounded by a brighter area, and were Fölgen-negative. Parallel to the cytologic changes in the infected cell cultures there set in metabolic disturbances and changes in the enzymatic activity. The infected cells showed enhanced lactate dehydrogenase, diphosphopyridin-nucleotide diaphorase, thiamine pyrophosphatase, and alkaline phosphatase activity and suppressed succinate dehydrogenase and acid phosphatase activity. These studies were said to reveal new biologic properties of the vaccinal mutant virus.

[Enzyme activity in cell cultures infected with herpesvirus suis]. / Enzymaktivität bei mit Herpesvirus suis infizierten Zellkulturen

The activity of succinate dehydrogenase (SDH) and lactage dehydrogenase (LDH) was studied in chick-embryo fibroblast cultures after inoculation of the virulent strain "A2" and the avirulent strain "MK" of herpesvirus suum. Strain "A2" reduced SDH activity, and so did strain MK, but here the decrease of enzyme activity was slower, and it did not become evident until the 24th hour. LDH activity fluctuated after "A2" infection but was generally increased, while there was no change in LDH activity, compared with uninfected control cells, after "MK" infection. When interaction of cell and virus took place in the presence of 5-iodo-2-desoxyuridine (IUDR), strain "A2" produced little change in the enzymes, but "MK" infection was accompanied by a definite fall in SDH and a slight increase in LDH. The presence of IUDR inhibited the proliferation of the virulent strain but had no apparent effect on proliferation of the attenuated strain "MK". Investigation of the enzyme activity of cells infected with Aujeszky's disease virus has revealed new biological properties of the virus, which might serve to distinguish between different strains of the virus.