Targeting regulatory T cells for anticancer therapy.

Regulatory T-cells (Tregs) comprise a group of either thymically derived or peripherally induced suppressor CD4+ cells involved in the control of effector T-cells against both self- and foreign-antigens. They are found increased in tumor tissues and are thought to be involved in pathogenesis of cancer by providing tumors with a mechanism to evade immune detection and destruction. Despite the fact that mechanisms of Tregs regulation are still in progress, efforts are made aiming to develop approaches to deplete or inhibit tumor-associated Tregs function. This could lead to restore antitumor immunity and emerging strategies for therapeutic vaccination, and immunotherapeutic targeting of Tregs with specific drugs are underway.

Verbena officinalis essential oil and its component citral as apoptotic-inducing agent in chronic lymphocytic leukemia.

We evaluated the pro-apoptotic activity of Verbena officinalis essential oil and of its main component citral, on lymphocytes collected from normal blood donors and patients with chronic lymphocytic leukemia (CLL). The number of apoptotic cells was greater in CLL patients than in healthy subjects at all different times of incubation (4, 8 and 24 hours) for samples treated with Verbena officinalis essential oil (A) and citral (B) as well vs controls at different concentrations (0.1% and 0.01%). The greater pro-apoptotic ability was shown by both essential oil of Verbena officinalis and citral at lower concentrations (after 4 h A 0.1%: 17.8% vs 37.1%; A 0.01%: 15.8% vs 52%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%; after 8 h A 0.1%: 23% vs 38%; A 0.01%: 22.2% vs 55%; B 0.1%: 32% vs 42.2%; B 0.01%: 22% vs 54.3%; after 24 h A 0.1%: 5% vs 20.7%; A 0.01%: 25.8% vs 47.2%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%). Patients carrying deletion 17p13 (p53 mutation) showed a reduced ability to undergo apoptosis with respect to patients with other genomic aberrations or normal karyotype. The proapoptotic activity of Verbena officinalis essential oil and citral is thought to be due to a direct procaspase 3 activation. These data further support evidence that indicate natural compounds as a possible lead structure to develop new therapeutic agents.

Alteration of central excitation circuits in chronic headache and analgesic misuse.

Central excitatory circuits could be involved in the pathophysiology of pain; particularly, the genesis of chronic pain. The "second pain" is the sensation that follows the initial pain after an appropriate nociceptive stimulus. The second pain is amplified by repeating the stimulus after brief intervals (temporal summation). This phenomenon is the psychophysical correlate of the excitatory pain circuits. The temporal summation of the second pain was evaluated in four groups of subjects; one group affected by migraine without aura, one by episodic tension headache, one by chronic daily headache, and a group of healthy subjects. A percutaneous electrical shock was used as the nociceptive stimulus. The intensity of the second pain was significantly greater in the group of patients with chronic headache in comparison with the other groups. The patients with chronic headache were subdivided into three groups on the basis of their clinical history: a group with transformed migraine; a group with chronic headache ab initio, a form related to the first one; (both groups suffered from chronic daily headache with a frequent superimposition of episodes of migraine attacks) and the third group consisted of patients with chronic tension headache. The temporal summation of the second pain was altered in the first two groups. The patients with chronic migraine abused ergotamine given as a symptomatic drug. Those who were able to discontinue this drug were retested and reported a decrease of the second pain in comparison to the previous measurements. The results of the present study indicate that central excitatory circuits could be involved in the mechanism leading to the development of chronic daily headache.

Peppers and pain. The promise of capsaicin.

Capsaicin, the most pungent ingredient in red peppers, has been used for centuries to remedy pain. Recently, its role has come under reinvestigation due to evidence that the drug acts selectively on a subpopulation of primary sensory neurons with a nociceptive function. These neurons, besides generating pain sensations, participate through an antidromic activation in the process known as neurogenic inflammation. The first exposure to capsaicin intensely activates these neurons in both senses (orthodromic: pain sensation; antidromic: local reddening, oedema etc.). After the first exposure, the neurons become insensitive to all further stimulation (including capsaicin itself). This evidence led to the proposal of capsaicin as a prototype of an agent producing selective analgesia. This perspective is radically different from previous 'folk medicine' cures, where the drug was used as a counter-irritating agent (i.e. for muscular pain). The new concept requires that capsaicin be repeatedly applied on the painful area to obtain the desensitisation of the sensory neurons. Following this idea, capsaicin has been used successfully in controlling pain in postherpetic neuralgia, diabetic neuropathy and other conditions of neuropathic pain. Furthermore, evidence indicates that capsaicin could also control the pain of osteoarthritis. Finally, repeated applications of the drug to the nasal mucosa result in the prevention of cluster headache attacks. On the basis of this evidence, capsaicin appears to be a promising prototype for obtaining selective analgesia in localised pain syndromes.

Effect of hyperbaric oxygen on the immunoreactivity to substance P in the nasal mucosa of cluster headache patients.

Exposure to hyperbaric oxygen has been shown to be effective in cluster headache, but the mechanism of the action is still not clear. Primary nociceptive neurons, containing neuropeptides such as substance P and particularly those innervating the nasal mucosa, could be involved in the pathogenesis of cluster headache. The present study evaluated the effect of an exposure to hyperbaric oxygen on the content of substance P in the nasal mucosa of patients affected by cluster headache. The results were compared with those observed in another group of cluster headache patients who underwent a placebo procedure. The samples of nasal mucosa were analyzed by immunocytochemical methods. A qualitative analysis of the slides was carried out by an operator under "blinded conditions". A marked decrease in the content of immunoreactivity for substance P was found in the patients exposed to hyperbaric oxygen. The decrease was statistically significant when compared with the findings of the placebo procedure. The results of the present study indicate that an influence on the content of peripheral neuropeptides could be involved in the mechanism of action of the beneficial effect of hyperbaric oxygen in cluster headache.

Behavioral outcome of posterior parietal cortex injury in the monkey.

Recent studies from our laboratory have characterized the response properties of trigeminal nociceptive neurons located in the posterior parietal cortex of awake monkeys, particularly in the rostral portion of the inferior parietal lobule and parietal operculum within the lateral sulcus. The stimulus intensity-response functions of some nociceptive neurons were significantly correlated to the stimulus intensity-escape frequency functions. The present study provides evidence that trauma to the posterior parietal cortex alters pain sensibility to the contralateral face. Although thermal pain tolerance was dramatically altered, the discriminative aspect of thermosensitivity may have remained intact. Our results complement the recent findings of clinical studies concerned with pain and damage to the posterior parietal cortex and of experimental studies concerned with painful stimulation and changes in regional cerebral blood flow. The role of the posterior parietal cortex in nociception and pain is discussed in relation to the first somatosensory area and to unilateral spatial neglect (inattention).

"Capsaicin-sensitive" sensory neurons in cluster headache: pathophysiological aspects and therapeutic indication.

Capsaicin, when repeatedly applied to the nasal mucosa of cluster headache patients, has been shown to prevent the occurrence of pain attacks. In order to investigate the mechanism of the drug's action, we evaluated the effect of repeated nasal application of capsaicin on the contents of sensory fibres immunoreactive to substance P and CGRP in the rat nasal mucosa. Further, considering the possible involvement of the cerebral circulation, we verified the effect of a single application of capsaicin on the blood flow velocity of the internal carotid and middle cerebral arteries (of both sides) and the basilar artery, in a group of healthy humans. The measurements were taken using Doppler devices. In order to verify the reproducibility of therapeutic effect of capsaicin, we carried out a 2-year follow-up study on patients affected by cluster headache (17 by episodic form, 8 by chronic form) who responded positively to the first treatment with capsaicin. During this period they were treated again with capsaicin in case of re-occurrence of symptoms. Capsaicin depletes the fibers immunoreactive to substance P and CGRP in the rat nasal mucosa. In the healthy controls, a single application induced vasodilation in the internal carotid, whereas middle cerebral arteries and basilar artery were narrowed. The results of the follow-up study, demonstrates that in 65% of the patients, the beneficial effect of capsaicin was again present when the treatment was repeated. In the chronic patients the therapeutic effect was always transitory (lasting, at maximum one month).(ABSTRACT TRUNCATED AT 250 WORDS)

Function of the peripheral serotoninergic pathways in migraine: a proposal for an experimental model.

We propose a model for assessing the function of 5HT receptors in migraine by evaluating their expression on monocytes by means of double-labeling fluorocytometry (CD14-positive cells FITC-labeled). This model demonstrates that during headache induced in migraine without aura sufferers given isosorbide dinitrate followed by sumatriptan treatment 5HT expression on monocytes progressively increases. This increase may be due to the activation of 5HT turnover and to the increased availability of 5HT displaced by sumatriptan from cerebrovascular receptors during head pain.

Capsaicin-desensitization to the human nasal mucosa selectively reduces pain evoked by citric acid.

1. Kallidin (5-500 nmol), hypertonic saline (0.9-20% NaCl) or low pH medium (citric acid: pH 2.5-1) applied (50 microliters) to the human nasal mucosa produced a pain response (evaluated by a visual analogue scale) that was related to the concentration of the peptide, NaCl or hydrogen ions, respectively. 2. Application (50 microliters) of capsaicin (50 nmol) to the human nasal mucosa produced overt pain. After repeated administrations (once a day for 5-7 days) to one nostril this effect underwent almost complete desensitization, while in the contralateral nostril, treated with the vehicle, the response to capsaicin was unaffected. 3. The pain response produced in the human nasal mucosa by topical application (50 microliters) or kallidin (50-500 nmol), NaCl (10-20%) or citric acid (pH 1.5-1) solutions was then studied before and after local capsaicin desensitization. 4. The pain response to pH 1.5 or 1 citric acid was markedly reduced (by 60% and 75%, respectively) in the capsaicin-treated nostril. However, the pain response to 10% or 20% NaCl or the mild pain response to 50 or 500 nmol kallidin were unaffected by capsaicin pre-treatment. 5. The present results suggest that prolonged topical capsaicin treatment to the human nasal mucosa may lead to selective desensitization to certain algesic stimuli such as capsaicin itself and hydrogen ions.

Is the unresponsiveness to sumatriptan in cluster headache related to an alteration in the 5-HT receptors?

It is well established that cluster headache shows impaired functions at the neuroimmunomodulatory system level. Defects in the expression of receptors for 5-HT, IL-1 and IL-2 have been found in these patients. Sumatriptan, an agonist activity for 5-HT1D receptor, truncates cluster headache attack in 74% of patients. Flow cytometric analysis of monocytes expressing 5-HT receptor in cluster headache patients showed different trends clearly correlated with the clinical response to sumatriptan. Our findings strongly support the concept that cluster headache patients which are non-responders to sumatriptan could present a block in their 5-HT receptor expression possibly due to specific autoantibodies for this receptor site.

Analgesic effect of capsaicin in idiopathic trigeminal neuralgia.

Twelve informed and consenting patients were studied to determine the influence of capsaicin, the pungent component of the red pepper, on trigeminal neuralgia. All of these patients had idiopathic trigeminal neuralgia. These patients were followed up for 1 yr after the topical application over the painful area of 1.0 g of capsaicin three times a day for several days. Six patients had complete and four patients had partial relief of pain; the remaining two patients had no relief of pain. Of the 10 patients who were responsive to therapy, four had relapses of pain in 95-149 days. There were no relapses following the second therapy for the remainder of the year. We conclude that the topical application of capsaicin is frequently successful in relieving the pain from trigeminal neuralgia.

Differential effect on neuropeptide release of different concentrations of hydrogen ions on afferent and intrinsic neurons of the rat stomach.

In the muscle layer of the glandular portion of the rat stomach, in vivo capsaicin pretreatment markedly reduced calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) but did not affect substance P-like immunoreactivity (SP-LI). Accordingly, in vitro superfusion of slices of this tissue with capsaicin (10 mumol/L) released CGRP-LI but not SP-LI, whereas both neuropeptides were released by 80 mmol/L K+. Exposure to relatively low-pH (pH 6) physiological salt solution induced an increase in the CGRP-LI outflow that was reduced by 70% in a Ca(2+)-free medium and was completely abolished by a previous exposure to capsaicin. However, superfusion with pH-6 medium did not produce any detectable SP-LI release. After exposure to pH-6 medium, both capsaicin and high-K+ medium were still able to release a consistent quantity of CGRP-LI and SP-LI, respectively. Increased mucosal blood flow induced by acid back-diffusion is considered a protective mechanism against mucosal gastric lesion. The present findings suggest that hydrogen ions diffusing into the gastric wall may promote protective vasodilatation by activating the "efferent" function of capsaicin-sensitive nerves without affecting the secretory process of other intrinsic peptidergic neurons.

Idiopathic headache as a possible risk factor for phantom tooth pain.

Following tooth pulp extirpation, some subjects suffer from persistent pain which affects edentate sites in absence of any local pathology. As regards this peculiar pain, called phantom tooth pain (PTP), what is puzzling is the fact there is a low prevalence of PTP in a very large population showing identical conditions of tooth pulp extirpation. The present investigation indicates that PTP mainly affects migraine (M) and cluster headache (CH) sufferers, whereas it does not affect subjects who have a negative personal and family history for idiopathic headache (IH). These results circumscribe the presence of PTP to a specific section of the population. The present results, besides indicating that PTP may be the result of a peculiar neuronal predisposition relating to IH pathogenesis, suggests some practical therapeutic hints. In fact, successful anti- M and anti-CH prophylactic treatment greatly improve PTP syndrome.

Evidence for alpha 1-adrenoreceptor hyperresponsiveness in hypotensive cirrhotic patients with ascites.

Cirrhosis with ascites is often characterized by arterial hypotension associated with increased plasma norepinephrine levels. Clinical evidence suggests a decreased sensitivity to norepinephrine in this clinical setting, indicating a potential derangement of alpha 1 adrenoreceptors. The aim of the present study was to evaluate the reactivity of the alpha 1-adrenoreceptor, using phenylephrine, a selective alpha 1 receptor agonist. First, we evaluated the pupillar response to the intraconjunctival administration of the agonist. Hypotensive cirrhotic patients showed a trend to a more marked and prolonged response, compared with age-matched healthy controls. Second, we studied the response of alpha 1-adrenoreceptors in the peripheral vasculature by administering iv phenylephrine to a similar group of patients. A significantly greater and longer lasting pressor response was observed in hypotensive cirrhotic patients (p less than 0.0001 vs. healthy controls). Our results indicate that in this group of patients, there is a rather peculiar situation, characterized by alpha 1-adrenoreceptor hyperresponsiveness in the presence of high levels of circulating norepinephrine. This finding could be related to biochemical abnormalities within the peripheral sympathetic nervous system endings.

Kallidin applied to the human nasal mucosa produces algesic response not blocked by capsaicin desensitization.

Various kinins (dissolved in 50 microliters) were applied to the nasal mucosa of healthy human volunteers to test the algesic and proinflammatory effects of these peptides in an intact human tissue. [des-Arg9]-bradykinin (0.5 mumol) was found to be inactive, while bradykinin (0.05-0.5 mumol) and especially kallidin (0.005-0.5 mumol) induced: (a) a mild painful sensation described as burning and pricking (latency 30 s, duration 3-5 min), (b) perception of pulsatility and obstruction in the nasal cavity (onset 1 min, duration 6-8 min). Substance P (0.5 mumol) and neurokinin A (0.5 mumol) produced slight obstruction and weak pulsatile sensation but not pain. Capsaicin (0.05 nmol) produced pain and secretion of fluid, but not pulsatile sensation. The effects of kallidin were not affected by repeated (to induce desensitization) applications of capsaicin (0.5 mumol). Likewise, ipratropium bromide (80 mg in 100 microliters) did not affect responses to kallidin. In an intact human tissue, kallidin produces various effects, including an algesic response, that are apparently independent from activation of B1 receptors and from desensitization of capsaicin-sensitive primary afferents.

Beneficial effects of intranasal applications of capsaicin in patients with vasomotor rhinitis.

Capsaicin, a nonenamide derived from Capsicum plants, has proven useful in patients with vasomotor rhinitis. In the present study, we studied the effects of 15 micrograms capsaicin suspended in 100 microliters solution in patients with known vasomotor rhinitis. Drug was given 3 times/day for 3 days to each patient by means of a spray delivered to the nasal mucosa. Acute effects induced by the drug included painful sensation and secretion of nasal fluid but were no longer observed at the last capsaicin application. Patients recorded their symptoms over a 1-month period. The mean symptom score involving nasal obstruction and nasal secretion was markedly reduced by capsaicin treatment. We advance the hypothesis that the beneficial effect of drug treatment may be due to its specific action on the peripheral endings of primary sensory neurons leading to their functional blockade. Further randomized, double-blind, placebo-controlled trial is now needed.

In vivo pupillary constrictor effects of substance P in man.

The ocular effects of substance P (SP) were studied in 13 normal volunteers. Various concentrations of SP (0.135, 1.35 and 135 micrograms per 100 microliters) were instilled into the conjunctival sac and pupillary area changes were evaluated by means of an electronic pupillometer. The ability of SP to modify the mydriasis induced by pretreatment with 1% homatropine eyedrops was also studied. The instillation of SP produced miosis in a dose-dependent manner without provoking any ocular disturbances. Furthermore, the highest concentration tested was unable to reduce the homatropine-induced mydriasis. These findings indicate that SP exerts a pupillokinetic action in humans which probably occurs via a receptor mechanism. Since muscarinic blockade is not overcome by the peptide instillation, the results do not clarify whether SP causes miosis acting on iris muscles and/or cholinergic fibres.