Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis.

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105-310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.

N,N-Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance.

Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a significant cause of disease burden in pediatric and elderly populations. The rise in unencapsulated disease-causing strains and antimicrobial resistance in S. pneumoniae has increased the need for developing new antimicrobial strategies. Recent work by our laboratory has identified N,N-dimethyldithiocarbamate (DMDC) as a copper-dependent antimicrobial against bacterial, fungal, and parasitic pathogens. As a bactericidal antibiotic against S. pneumoniae, DMDC's ability to work as a copper-dependent antibiotic and its ability to work in vivo warranted further investigation. Here, our group studied the mechanisms of action of DMDC under various medium and excess-metal conditions and investigated DMDC's interactions with the innate immune system in vitro and in vivo. Of note, we found that DMDC plus copper significantly increased the internal copper concentration, hydrogen peroxide stress, nitric oxide stress, and the in vitro macrophage killing efficiency and decreased capsule. Furthermore, we found that in vivo DMDC treatment increased the quantity of innate immune cells in the lung during infection. Taken together, this study provides mechanistic insights regarding DMDC's activity as an antibiotic at the host-pathogen interface.

Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against Streptococcal species.

Despite the availability of several vaccines against multiple disease-causing strains of Streptococcus pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. N,N-dimethyldithiocarbamate (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including S. pneumoniae. Here, DMDCs efficacy against Streptococcal pathogens Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus anginosus was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives' effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against S. pneumoniae TIGR4 and tested in bactericidal and animal models. Two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an in vivo murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.

Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon.

Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

NMDA receptor-mediated activation of medullary pro-nociceptive neurons is required for secondary thermal hyperalgesia.

There is now direct evidence that a class of neurons in the rostral ventromedial medulla (RVM) exerts a net facilitatory influence on spinal nociception. The present experiments were designed to test whether activation of these neurons, referred to as "on-cells", is required as part of a positive feedback loop leading to secondary hyperalgesia in acute inflammation produced by topical application of mustard oil. Activity of a characterized RVM neuron and paw withdrawals to heat (plantar surface) were recorded in barbiturate-anesthetized rats. Following three baseline trials, mustard oil was applied to the skin above the knee. Cell activity and paw withdrawal latencies were monitored for an additional 45min. Application of mustard oil produced an increase in on-cell discharge that was associated with a substantial decrease in withdrawal latency of the ipsilateral paw. Blocking on-cell activation using local infusion of the NMDA-receptor antagonist AP5 into the RVM prevented hyperalgesia. Secondary thermal hyperalgesia following mustard oil was also associated with a significant decrease in the firing of "off-cells", a cell population thought to exert a net inhibitory influence on nociception. Depression of off-cell firing was unaffected by AP5 microinjection. The firing of "neutral cells", which have no documented role in nociceptive modulation, was unchanged following mustard oil and also unaffected by AP5 infusion in the RVM. Brainstem descending controls are receiving increasing attention in efforts to understand hyperalgesia and persistent pain states. The present experiments demonstrate that a novel, NMDA-mediated activation of on-cells is required for secondary thermal hyperalgesia in acute inflammation.

Role for medullary pain facilitating neurons in secondary thermal hyperalgesia.

The rostral ventromedial medulla (RVM) has recently received considerable attention in efforts to understand mechanisms of hyperalgesia and persistent pain states. Three classes of neurons can be identified in the RVM based on responses associated with nocifensive reflexes: on cells, off cells, and neutral cells. There is now direct evidence that on cells exert a net facilitating effect on spinal nociception and that off cells depress nociception. These experiments tested whether the secondary hyperalgesia produced by topical application of mustard oil involves an activation of on cells in RVM. Firing of a characterized RVM neuron and the latencies of withdrawal reflexes evoked by noxious heat were recorded in lightly anesthetized rats before and after application of mustard oil to the shaved skin of the leg above the knee. Mineral oil was applied as a control. Mustard oil produced a significant increase in ongoing and reflex-related discharge of on cells, as well as a decrease in the activity of off cells. neutral cell firing was uniformly unchanged after application of mustard oil. The alterations in on and off cell firing were associated with a significant decrease in the latency to withdraw the paw of the treated limb from the heat stimulus, and this hyperalgesia was blocked by microinjection of lidocaine within the RVM. Withdrawals evoked by heating the contralateral hindpaw, forepaw, and tail were unchanged after mustard oil application. These experiments support a pronociceptive role for on cells and suggest that these neurons contribute to secondary hyperalgesia in inflammation.

Nociceptive facilitating neurons in the rostral ventromedial medulla.

The role of the periaqueductal gray-rostral ventromedial medulla (RVM) system in descending inhibition of nociception has been studied for over 30 years. The neural basis for this antinociceptive action is reasonably well understood, with strong evidence that activation of a class of RVM neurons termed 'off-cells' exerts a net inhibitory effect on nociception. However, it has recently become clear that this system can facilitate, as well as inhibit pain. Although the mechanisms underlying the facilitation of nociception have not been conclusively identified, indirect evidence points to activation of a class of neurons termed 'on-cells' as mediating descending facilitation. Here we used focal infusion of the tridecapeptide neurotensin within the RVM in lightly anesthetized rats to activate on-cells selectively. Neurotensin has been shown in awake animals to produce a dose-related, bi-directional effect on nociception when applied within the RVM, with hyperalgesia at low doses, and analgesia at higher doses. Using a combination of single cell recording and behavioral testing, we now show that on-cells are activated selectively by low-dose neurotensin, and that the activation of on-cells by neurotensin results in enhanced nociceptive responding, as measured by the paw withdrawal reflex. Furthermore, higher neurotensin doses recruit off-cells in addition to on-cells, producing behavioral antinociception. Selective activation of on-cells is thus sufficient to produce hyperalgesia, confirming the role of these neurons in facilitating nociception. Activation of on-cells likely contributes to enhanced sensitivity to noxious stimulation or reduced sensitivity to analgesic drugs in a variety of conditions.

Prostaglandin E2 in the midbrain periaqueductal gray produces hyperalgesia and activates pain-modulating circuitry in the rostral ventromedial medulla.

Recent years have seen significant advances in our understanding of the peripheral and spinal mechanisms through which prostaglandins contribute to nociceptive sensitization. By contrast, the possibility of a supraspinal contribution of these compounds to facilitated pain states has received relatively little attention. One possible mechanism through which prostaglandins could act supraspinally to facilitate nociception would be by recruitment of descending facilitation from brainstem pain-modulating systems. The rostral ventromedial medulla (RVM) is now known to contribute to enhanced responding in a variety of inflammatory and nerve injury models. Its major supraspinal input, the midbrain periaqueductal gray (PAG), expresses prostanoid receptors and synthetic enzymes. The aim of the present study was to determine whether direct application of prostaglandin E(2) (PGE(2)) within the ventrolateral PAG is sufficient to produce hyperalgesia, and whether any hyperalgesia could be mediated by recruiting nociceptive modulating neurons in the RVM. We determined the effects of focal application of PGE(2) in the PAG on paw withdrawal latency and activity of identified nociceptive modulating neurons in the RVM of lightly anesthetized rats. Microinjection of PGE(2) (50 fg in 200 nl) into the PAG produced a significant decrease in paw withdrawal latency. The PGE(2) microinjection activated on-cells, RVM neurons thought to facilitate nociception, and suppressed the firing of off-cells, RVM neurons believed to have an inhibitory effect on nociception. These data demonstrate a prostaglandin-sensitive descending facilitation from the PAG, and suggest that this is mediated by on- and off-cells in the RVM.

Neural basis for the hyperalgesic action of cholecystokinin in the rostral ventromedial medulla.

The analgesic actions of opioids can be modified by endogenous "anti-opioid" peptides, among them cholecystokinin (CCK). CCK is now thought to have a broader, pronociceptive role, and contributes to hyperalgesia in inflammatory and neuropathic pain states. The aim of this study was to determine whether anti-opioid and pronociceptive actions of CCK have a common underlying mechanism. We showed previously that a low dose of CCK microinjected into the rostral ventromedial medulla (RVM) blocked the analgesic effect of systemically administered morphine by preventing activation of off-cells, which are the antinociceptive output of this well characterized pain-modulating region. At this anti-opioid dose, CCK had no effect on the spontaneous activity of these neurons or on the activity of on-cells (hypothesized to facilitate nociception) or "neutral cells" (which have no known role in pain modulation). In this study, we used microinjection of a higher dose of CCK into the RVM to test whether activation of on-cells could explain the pronociceptive action of this peptide. Paw withdrawal latencies to noxious heat and the activity of a characterized RVM neuron were recorded in rats lightly anesthetized with methohexital. CCK (30 ng/200 nl) activated on-cells selectively and produced behavioral hyperalgesia. Firing of off-cells and neutral cells was unaffected. These data show that direct, selective activation of RVM on-cells by CCK is sufficient to produce thermal hyperalgesia and indicate that the anti-opioid and pronociceptive effects of this peptide are mediated by actions on different RVM cell classes.