Chronic stress-induced mechanical hyperalgesia is controlled by capsaicin-sensitive neurones in the mouse.

BACKGROUND: Clinical studies demonstrated peripheral nociceptor deficit in stress-related chronic pain states, such as fibromyalgia. The interactions of stress and nociceptive systems have special relevance in chronic pain, but the underlying mechanisms including the role of specific nociceptor populations remain unknown. We investigated the role of capsaicin-sensitive neurones in chronic stress-related nociceptive changes. METHOD: Capsaicin-sensitive neurones were desensitized by the capsaicin analogue resiniferatoxin (RTX) in CD1 mice. The effects of desensitization on chronic restraint stress (CRS)-induced responses were analysed using behavioural tests, chronic neuronal activity assessment in the central nervous system with FosB immunohistochemistry and peripheral cytokine concentration measurements. RESULTS: Chronic restraint stress induced mechanical and cold hypersensitivity and increased light preference in the light-dark box test. Open-field and tail suspension test activities were not altered. Adrenal weight increased, whereas thymus and body weights decreased in response to CRS. FosB immunopositivity increased in the insular cortex, dorsomedial hypothalamic and dorsal raphe nuclei, but not in the spinal cord dorsal horn after the CRS. CRS did not affect the cytokine concentrations of hindpaw tissues. Surprisingly, RTX pretreatment augmented stress-induced mechanical hyperalgesia, abolished light preference and selectively decreased the CRS-induced neuronal activation in the insular cortex. RTX pretreatment alone increased the basal noxious heat threshold without influencing the CRS-evoked cold hyperalgesia and augmented neuronal activation in the somatosensory cortex and interleukin-1α and RANTES production. CONCLUSIONS: Chronic restraint stress induces hyperalgesia without major anxiety, depression-like behaviour or peripheral inflammatory changes. Increased stress-induced mechanical hypersensitivity in RTX-pretreated mice is presumably mediated by central mechanisms including cortical plastic changes. SIGNIFICANCE: These are the first data demonstrating the complex interactions between capsaicin-sensitive neurones and chronic stress and their impact on nociception. Capsaicin-sensitive neurones are protective against stress-induced mechanical hyperalgesia by influencing neuronal plasticity in the brain.

Contribution of platelet P2Y12 receptors to chronic Complete Freund's adjuvant-induced inflammatory pain.

Essentials The role of platelet P2Y12 receptors in the regulation of chronic inflammatory pain is unknown. Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain model was used in mice. Gene deficiency and antagonists of P2Y12 receptors attenuate hyperalgesia and local inflammation. Platelet P2Y12 receptors contribute to these effects in the chronic phase of inflammation. SUMMARY: Background P2Y12 receptor antagonists are widely used in clinical practice to inhibit platelet aggregation. P2Y12 receptors are also known to regulate different forms of pain as well as local and systemic inflammation. However, it is not known whether platelet P2Y12 receptors contribute to these effects. Objectives To explore the contribution of platelet P2Y12 receptors to chronic inflammatory pain in mice. Methods Complete Freund's adjuvant (CFA)-induced chronic inflammatory pain was induced in wild-type and P2ry12 gene-deficient (P2ry12-/- ) mice, and the potent, direct-acting and reversible P2Y12 receptor antagonists PSB-0739 and cangrelor were used. Results CFA-induced mechanical hyperalgesia was significantly decreased in P2ry12-/- mice for up to 14 days, and increased neutrophil myeloperoxidase activity and tumor necrosis factor (TNF)-α and CXCL1 (KC) levels in the hind paws were also attenuated in the acute inflammation phase. At day 14, increased interleukin (IL)-1ß, IL-6, TNF-α and KC levels were attenuated in P2ry12-/- mice. PSB-0739 and cangrelor reversed hyperalgesia in wild-type mice but had no effect in P2ry12-/- mice, and PSB-0739 was also effective when applied locally. The effects of both local and systemic PSB-0739 were prevented by A-803467, a selective NaV1.8 channel antagonist, suggesting the involvement of NaV1.8 channels in the antihyperalgesic effect. Platelet depletion by anti-mouse CD41 antibody decreased hyperalgesia and attenuated the proinflammatory cytokine response in wild-type but not in P2ry12-/- mice on day 14. Conclusions In conclusion, P2Y12 receptors regulate CFA-induced hyperalgesia and the local inflammatory response, and platelet P2Y12 receptors contribute to these effects in the chronic inflammation phase.

Brain injury induces specific changes in the caecal microbiota of mice via altered autonomic activity and mucoprotein production.

Intestinal microbiota are critical for health with changes associated with diverse human diseases. Research suggests that altered intestinal microbiota can profoundly affect brain function. However, whether altering brain function directly affects the microbiota is unknown. Since it is currently unclear how brain injury induces clinical complications such as infections or paralytic ileus, key contributors to prolonged hospitalization and death post-stroke, we tested in mice the hypothesis that brain damage induced changes in the intestinal microbiota. Experimental stroke altered the composition of caecal microbiota, with specific changes in Peptococcaceae and Prevotellaceae correlating with the extent of injury. These effects are mediated by noradrenaline release from the autonomic nervous system with altered caecal mucoprotein production and goblet cell numbers. Traumatic brain injury also caused changes in the gut microbiota, confirming brain injury effects gut microbiota. Changes in intestinal microbiota after brain injury may affect recovery and treatment of patients should appreciate such changes.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes.

The impact of intermediate-term alcohol abstinence on memory retrieval and suppression.

BACKGROUND: The nature of episodic memory deficit in intermediate-term abstinence from alcohol in alcohol dependence (AD) is not yet clarified. Deficits in inhibitory control are commonly reported in substance use disorders. However, much less is known about cognitive control suppressing interference from memory. The Think/No-think (TNT) paradigm is a well established method to investigate inhibition of associative memory retrieval. METHODS: Thirty-six unmedicated patients with AD and 36 healthy controls (HCs) performed the TNT task. Thirty image-word pairs were trained up to a predefined accuracy level. Cued recall was examined in three conditions: Think (T) for items instructed to-be-remembered, No-think (NT) assessing the ability to suppress retrieval and Baseline (B) for general relational memory. Premorbid IQ, clinical variables and impulsivity measures were quantified. RESULTS: AD patients had a significantly increased demand for training. Baseline memory abilities and effect of practice on retrieval were not markedly different between the groups. We found a significant main effect of group (HC vs. AD) × condition (B, T, and NT) and a significant difference in mean NT-B scores for the two groups. DISCUSSION: AD and HC groups did not differ essentially in their baseline memory abilities. Also, the instruction to focus on retrieval improved episodic memory performance in both groups. Crucially, control participants were able to suppress relational words in the NT condition supporting the critical effect of cognitive control processes over inhibition of retrieval. In contrast to this, the ability of AD patients to suppress retrieval was found to be impaired.

Caspase-1: is IL-1 just the tip of the ICEberg?

Caspase-1, formerly known as interleukin (IL)-1-converting enzyme is best established as the protease responsible for the processing of the key pro-inflammatory cytokine IL-1ß from an inactive precursor to an active, secreted molecule. Thus, caspase-1 is regarded as a key mediator of inflammatory processes, and has become synonymous with inflammation. In addition to the processing of IL-1ß, caspase-1 also executes a rapid programme of cell death, termed pyroptosis, in macrophages in response to intracellular bacteria. Pyroptosis is also regarded as a host response to remove the niche of the bacteria and to hasten their demise. These processes are generally accepted as the main roles of caspase-1. However, there is also a wealth of literature supporting a direct role for caspase-1 in non-infectious cell death processes. This is true in mammals, but also in non-mammalian vertebrates where caspase-1-dependent processing of IL-1ß is absent because of the lack of appropriate caspase-1 cleavage sites. This literature is most prevalent in the brain where caspase-1 may directly regulate neuronal cell death in response to diverse insults. We attempt here to summarise the evidence for caspase-1 as a cell death enzyme and propose that, in addition to the processing of IL-1ß, caspase-1 has an important and a conserved role as a cell death protease.

Rapid brain penetration of interleukin-1 receptor antagonist in rat cerebral ischaemia: pharmacokinetics, distribution, protection.

BACKGROUND AND PURPOSE: Limited data on the brain penetration of potential stroke treatments have been cited as a major weakness contributing to numerous failed clinical trials. Thus, we tested whether interleukin-1 receptor antagonist (IL-1RA), established as a potent inhibitor of brain injury in animals and currently in clinical development, reaches the brain via a clinically relevant administration route, in experimental stroke. EXPERIMENTAL APPROACH: Male, Sprague-Dawley rats [either naïve or exposed to middle cerebral artery occlusion (MCAo)] were given a single s.c. dose of IL-1RA (100 mg*kg(-1)). The pharmacokinetic profile of IL-1RA was assessed in plasma and CSF up to 24 h post-administration. Brain tissue distribution of administered IL-1RA was assessed using immunohistochemistry. In a separate experiment, the neuroprotective effect of the single s.c. dose of IL-1RA in MCAo was assessed versus a placebo control group. KEY RESULTS: A single s.c. dose of IL-1RA reduced damage caused by MCAo by 33%. This dose resulted in sustained, high concentrations in plasma and CSF, penetrated brain tissue exclusively in areas of blood-brain barrier breakdown and co-localized with morphologically viable neurones. CSF concentrations did not reflect massive parenchymal infiltration of IL-1RA in MCAo animals compared to naïve. CONCLUSIONS AND IMPLICATIONS: These data are the first to show that a potential treatment for stroke, IL-1RA, rapidly reaches salvageable brain tissue via an administration route that is clinically relevant. This allows confidence that IL-1RA, as a candidate for further clinical development, is able to confer its protective actions both peripherally and centrally.

Inflammation and brain injury: acute cerebral ischaemia, peripheral and central inflammation.

Inflammation is a classical host defence response to infection and injury that has many beneficial effects. However, inappropriate (in time, place and magnitude) inflammation is increasingly implicated in diverse disease states, now including cancer, diabetes, obesity, atherosclerosis, heart disease and, most relevant here, CNS disease. A growing literature shows strong correlations between inflammatory status and the risk of cerebral ischaemia (CI, most commonly stroke), as well as with outcome from an ischaemic event. Intervention studies to demonstrate a causal link between inflammation and CI (or its consequences) are limited but are beginning to emerge, while experimental studies of CI have provided direct evidence that key inflammatory mediators (cytokines, chemokines and inflammatory cells) contribute directly to ischaemic brain injury. However, it remains to be determined what the relative importance of systemic (largely peripheral) versus CNS inflammation is in CI. Animal models in which CI is driven by a CNS intervention may not accurately reflect the clinical condition; stroke being typically induced by atherosclerosis or cardiac dysfunction, and hence current experimental paradigms may underestimate the contribution of peripheral inflammation. Experimental studies have already identified a number of potential anti-inflammatory therapeutic interventions that may limit ischaemic brain damage, some of which have been tested in early clinical trials with potentially promising results. However, a greater understanding of the contribution of inflammation to CI is still required, and this review highlights some of the key mechanism that may offer future therapeutic targets.

The evolving role of alemtuzumab in management of patients with CLL.

New insights into prognostic markers and the pathophysiology of chronic lymphocytic leukemia (CLL) are beginning to change the concept of CLL treatment. Alemtuzumab has evolved as a potent and effective therapeutic option for patients with CLL. Specifically, alemtuzumab has demonstrated substantial efficacy in fludarabine-refractory patients and has shown impressive responses when administered subcutaneously in first-line therapy. A group of experts gathered to discuss new data related to the use of alemtuzumab in CLL and to assess its place in the rapidly changing approach to treating patients with this disease. The main goals of this program were to update the management guidelines that were previously developed for alemtuzumab-treated patients and to provide community oncologists with guidance on the most effective way to integrate alemtuzumab into a CLL treatment plan.

Central autonomic control of the bone marrow: multisynaptic tract tracing by recombinant pseudorabies virus.

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and beta-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4-7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

Phase I study of pegylated liposomal doxorubicin and the multidrug-resistance modulator, valspodar.

Valspodar, a P-glycoprotein modulator, affects pharmacokinetics of doxorubicin when administered in combination, resulting in doxorubicin dose reduction. In animal models, valspodar has minimal interaction with pegylated liposomal doxorubicin (PEG-LD). To determine any pharmacokinetic interaction in humans, we designed a study to determine maximum tolerated dose, dose-limiting toxicity (DLT), and pharmacokinetics of total doxorubicin, in PEG-LD and valspodar combination therapy in patients with advanced malignancies. Patients received PEG-LD 20-25 mg m(-2) intravenously over 1 h for cycle one. In subsequent 2-week cycles, valspodar was administered as 72 h continuous intravenous infusion with PEG-LD beginning at 8 mg m(-2) and escalated in an accelerated titration design to 25 mg m(-2). Pharmacokinetic data were collected with and without valspodar. A total of 14 patients completed at least two cycles of therapy. No DLTs were observed in six patients treated at the highest level of PEG-LD 25 mg m(-2). The most common toxicities were fatigue, nausea, vomiting, mucositis, palmar plantar erythrodysesthesia, diarrhoea, and ataxia. Partial responses were observed in patients with breast and ovarian carcinoma. The mean (range) total doxorubicin clearance decreased from 27 (10-73) ml h(-1) m(-2) in cycle 1 to 18 (3-37) ml h(-1) m(-2) with the addition of valspodar in cycle 2 (P=0.009). Treatment with PEG-LD 25 mg m(-2) in combination with valspodar results in a moderate prolongation of total doxorubicin clearance and half-life but did not increase the toxicity of this agent.

Phase I study of rubitecan and gemcitabine in patients with advanced malignancies.

BACKGROUND: Rubitecan (9-nitrocamptothecin, 9-NC, Orathecin) and gemcitabine have single-agent activity in pancreatic and ovarian carcinoma. We conducted a phase I trial to evaluate the maximum tolerated dose (MTD) and toxicities of this combination in advanced malignancies. PATIENTS AND METHODS: Twenty-one patients with refractory or recurrent malignancies were enrolled in this dose escalation trial. Dose escalation proceeded from a starting level of rubitecan at 0.75 mg/m(2)/day administered orally on days 1-5 and 8-12 in combination with gemcitabine 1000 mg/m(2) administered intravenously on days 1 and 8 of a 21-day cycle. RESULTS: The MTD was defined as rubitecan 1 mg/m(2) administered orally days 1-5 and 8-12, and gemcitabine 1000 mg/m(2) administered intravenously over 30 min days 1 and 8, given every 21 days. Dose-limiting toxicity was myelosuppression including neutropenia and thrombocytopenia. Other side effects included diarrhea, nausea, vomiting and fatigue. Five patients with stable disease were observed among 18 evaluable patients. CONCLUSIONS: The recommended phase II dose is rubitecan 1 mg/m(2) given orally on days 1-5 and 8-12 in combination with gemcitabine 1000 mg/m(2) as a 30-min intravenous infusion on days 1 and 8 of a 21-day cycle.

hADA3 is required for p53 activity.

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, p53 protein is stabilized and activated by post-translational modifications that enable it to induce either apoptosis or cell cycle arrest. Using a novel yeast p53 dissociator assay, we identify hADA3, a part of histone acetyltransferase complexes, as an important cofactor for p53 activity. p53 and hADA3 physically interact in human cells. This interaction is enhanced dramatically after DNA damage due to phosphorylation event(s) in the p53 N-terminus. Proper hADA3 function is essential for full transcriptional activity of p53 and p53-mediated apoptosis.

The management of depression: the implications for managed care--roundtable discussion: Part 3.

From the standpoint of managed care, the rising cost of depression can be addressed in multiple ways. In the final portion of the roundtable discussion, the faculty discuss not only disease management programs for depression, but other initiatives health plans (including at the pharmacy level) are undertaking to address the rising costs associated with depression. They also discuss the effect of mental health coverage "parity" laws, which can be expected to drive costs even higher.

Chronic lymphocytic leukemia remission following extra corporeal shock wave lithotripsy for urinary calculi.

Herein we report on a patient with chronic lymphocytic leukemia (CLL) who entered a long-term remission following shock wave lithotripsy (SWL) for a left proximal ureteral stone. In addition, we include data on the effect of SWL on in vitro and in vivo human lymphocyte subpopulations, and discuss the possible mechanisms of this observation.

DNA fingerprinting of Campylobacter fetus using cloned constructs of ribosomal RNA and surface array protein genes.

DNA fragments coding for the ribosomal RNA and the surface array proteins of Campylobacter fetus have been cloned from a genomic library constructed in Escherichia coli. They were used in the molecular characterization of C. fetus (subsp. fetus; subsp. venerealis) strains by restriction fragment length polymorphism (RFLP) method. Ribotyping results showed that all strains of the two subspecies can be classified under one ribogroup implying very close relatedness. The sapA gene DNA marker, however, discriminated all the strains regardless of the subspecies when chromosomal DNA was restricted with HindIII, HaeIII, XbaI or EcoRV. These results illustrate that the sapA probe is potentially useful in fingerprinting C. fetus strains and in determining the relationships of strains for epidemiological purposes.

Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs.

Campylobacter fetus subsp. venerealis isolated from a case of human vaginosis was inoculated into the uterus of a C. fetus-negative heifer. Isolates obtained weekly from the vaginal mucus exhibited variations in high-molecular-mass-protein profiles from that of the original inoculum, which had a dominant 110-kDa S-layer protein. Immunoblots of the weekly isolates with monoclonal antibody probes against the 110-kDa S-layer protein and other C. fetus S-layer proteins demonstrated antigenic shifts. Genomic digests of the isolates probed with a 75-mer oligonucleotide of the conserved sapA region also indicated that antigenic variation of the S-layer is accompanied by DNA rearrangement.

Prospective 5-year followup of recombinant interferon-gamma in rheumatoid arthritis.

OBJECTIVE: To define clinical and laboratory outcomes of longterm recombinant interferon-gamma (rIFN-gamma) treatment of patients with rheumatoid arthritis (RA). METHODS: Patients with RA (70) completing a 12-week multicenter double blind trial comparing rIFN-gamma with placebo were enrolled in a longterm prospective protocol evaluating rIFN-gamma in RA. RESULTS: The majority of patients sustained clinical improvement for one year. Eight (11%) patients with RA continued to receive rIFN-gamma after 5 years. One patient fulfilled remission criteria. rIFN-gamma was well tolerated with remarkably few suspected adverse drug reactions. Forty-seven (67%) patients discontinued rIFN-gamma because of lack of efficacy, 7 (10%) because of concurrent illnesses, 5 (7%) were not compliant to study protocol, 3 (4%) developed suspected adverse drug reactions for a total of 62 (89%) withdrawals over the 5 years of followup. Patients continuing rIFN-gamma treatment for 5 years had lower initial total leukocyte and neutrophil counts and higher hemoglobin and hematocrit levels than patients who discontinued rIFN-gamma during the 5-year followup. CONCLUSIONS: Longterm treatment of RA with rIFN-gamma was generally well tolerated. Although many patients maintained sustained clinical improvement for at least one year, the main reason for discontinuing the drug over 5 years was the lack of continued benefit.

Prospective two-year followup of recombinant interferon-gamma in rheumatoid arthritis.

Seventy patients with rheumatoid arthritis (RA) completing a 12-week multicenter double blind trial comparing recombinant human interferon-gamma (r-IFN-gamma) with placebo were enrolled in a longterm prospective protocol evaluating r-IFN-gamma in RA. Forty (57%) patients after 1 year and 26 (37%) patients after 2 years continued the drug with sustained clinical benefit. Over 2 years, r-IFN-gamma was discontinued in 44 patients (lack of efficacy--25, withdrawn consent--7, noncompliant--4, suspected adverse drug reactions--2, concurrent illness--6). Two years of treatment with r-IFN-gamma were well tolerated with sustained clinical benefit in some patients with few significant adverse drug reactions.