Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms.

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Production and characterization of antibodies for the specific determination of the opioid peptide Met5-Enkephalin-Arg6-Phe7.

Endogenous opioids serve as modulators of neuroendocrine and immune system processes, the investigation of which calls for high-specificity radioimmunoassays (RIAs). This study focuses on the development and use of a specific radioimmunoassay for the opioid peptide Met5-Enkephalin-Arg6-Phe7 (MEAP), the C-terminus part of proenkephalin A. Antibodies were raised in four rabbits and investigated in terms of titre, avidity and specificity, followed by finding ideal conditions for these antibodies in RIA. MEAP concentrations were determined in crude extracts of rat hypothalamus, dorsal root ganglia, adrenals and ankle using this standardized assay after an oxidizing process. At reverse-phase high-pressure liquid chromatography (HPLC), the position of immunoreactive material from rat hypothalamus eluted as two peaks out of which one was compatible with that of synthetic MEAP. All rabbits exhibited individual differences in relative immune response and time of its onset. The avidity constant was 10 times higher on a molar basis for ab 4108 compared with ab 4182. There was no cross-reactivity for ab 4182 to related peptides, such as enkephalins and dynorphin B, and negligible background values for ab 4108. The relative levels ofimmunoreactive MEAP from the CNS versus peripheral tissues contrasted in accordance with current knowledge. It is suggested that reports with RIA results should include characterization of antibodies, extraction procedures, standard curves and compositions of buffers. Furthermore, the results should preferably be expressed in relation to total protein content.

Effects of acupuncture on skin temperature in children with neurological disorders and cold feet: an exploratory study.

In a single subject design, acupuncture effects on peripheral skin temperature were studied in six children with cold feet associated with cerebral palsy, progressive encephalopathy or Smith-Lemli-Opitz syndrome. Skin temperature was measured at fixed points on the forehead, suprasternal fossa, hand dorsum and palm, foot dorsum and sole from a distance of 3 cm with a Raytek Raynger ST2L. Acupuncture needles were inserted in arms or legs with or without electrical stimulation. Three children displayed a brief rise in temperature in the hands and one in the feet. A tendency towards a cumulative effect in improved skin temperature occurred in two of three children receiving additional sessions. One child did not respond to acupuncture. It is possible that acupuncture may increase skin temperature in some children with cold extremities of neurological origin. Studies in larger groups are needed.

Effects of electro-acupuncture on corticotropin-releasing factor in rats with experimentally-induced polycystic ovaries.

The aims of the present study were to investigate corticotropin-releasing factor (CRF) concentrations in the brain, the adrenal glands, and the ovaries in rats with estradiol valerate (EV) induced polycystic ovaries (PCO). The effect of 12 electro-acupuncture (EA) treatments on CRF concentrations was also investigated. The CRF concentrations in the median eminence (ME) were significantly increased in rats with PCO (both the PCO control group and the PCO group receiving EA) compared with the healthy control group (veichle control group), indicating increased activity in the hypothalamus-pituitary axis. The CRF concentrations in the ovaries were significantly reduced in the PCO group receiving EA compared with the PCO control group. Also, there was a decrease in comparison withthe healthy control group but the decrease was not as significant. This finding indicates that repeated EA treatments change the neuroendocrinological state in the ovaries, which may play an important role in reproductive failure.

Reliability and responsiveness of three different pain assessments.

The visual analogue scale (VAS) and ordered categorical scales, i.e. numeric rating scales (NRS), are commonly used in the assessment of pain. However, these scales are bounded by fixed endpoints and thus the range of measurement is limited. The disparity in repeated assessments of perceived pain intensity with the VAS, NRS, and electrical stimulation applied as a matching stimulus was studied in 69 patients (48 women and 21 men, 19-72 years) with chronic nociceptive or neurogenic pain. Responsiveness with transcutaneous electrical nerve stimulation (TENS) using the same measurement procedures was evaluated in the same patients. Comparison of results from the three pain assessments showed that the painmatcher is at least as reliable and responsive as VAS and NRS. None of the three measurements showed evidence for systematic disagreement and had only significant random individual disagreement. They also showed evidence for responsiveness.

Effects of electro-acupuncture on nerve growth factor and ovarian morphology in rats with experimentally induced polycystic ovaries.

Despite extensive research on the pathogenesis of polycystic ovary syndrome (PCOS), there is still disagreement on the underlying mechanisms. The rat model for experimentally induced polycystic ovaries (PCO)-produced by a single injection of estradiol valerate-has similarities with human PCOS, and both are associated with hyperactivity in the sympathetic nervous system. Nerve growth factor (NGF) is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types. Electro-acupuncture (EA) is known to reduce hyperactivity in the sympathetic nervous system. For these reasons, the model was used in the present study to investigate the effects of EA (12 treatments, approximately 25 min each, over 30 days) by analyzing NGF in the central nervous system and the endocrine organs, including the ovaries. The main findings in the present study were first, that significantly higher concentrations of NGF were found in the ovaries and the adrenal glands in the rats in the PCO model than in the control rats that were only injected with the vehicle (oil or NaCl). Second, that repeated EA treatments in PCO rats resulted in concentrations of NGF in the ovaries that were significantly lower than those in non-EA-treated PCO rats but were within a normal range that did not differ from those in the untreated oil and NaCl control groups. The results in the present study provide support for the theory that EA inhibits hyperactivity in the sympathetic nervous system.

Effects of electro-acupuncture on anovulation in women with polycystic ovary syndrome.

BACKGROUND: The present study was designed to evaluate if electro-acupuncture (EA) could affect oligo-/anovulation and related endocrine and neuroendocrine parameters in women with polycystic ovary syndrome (PCOS). METHODS: Twenty-four women (between the ages of 24 and 40 years) with PCOS and oligo-/amenorrhea were included in this non-randomized, longitudinal, prospective study. The study period was defined as the period extending from 3 months before the first EA treatment, to 3 months after the last EA treatment (10-14 treatments), in total 8-9 months. The menstrual and ovulation patterns were confirmed by recording of vaginal bleedings and by daily registrations of the basal body temperature (BBT). Blood samples were collected within a week before the first EA, within a week after the last EA and 3 months after EA. RESULTS: Nine women (38%) experienced a good effect. They displayed a mean of 0.66 ovulations/woman and month in the period during and after the EA period compared to a mean of 0.15 before the EA period (p=0.004). Before EA, women with a good effect had a significantly lower body-mass index (BMI) (p<0.001), waist-to-hip circumference ratio (WHR) (p=0.0058), serum testosterone concentration (p=0.0098), serum testosterone/sex hormone binding globulin (SHBG) ratio (p=0.011) and serum basal insulin concentration (p=0.0054), and a significantly higher concentration of serum SHBG (p=0.040) than did those women with no effect. CONCLUSION: Repeated EA treatments induce regular ovulations in more than one third of the women with PCOS. The group of women with good effect had a less androgenic hormonal profile before treatment and a less pronounced metabolic disturbance compared with the group with no effect. For this selected group EA offers an alternative to pharmacological ovulation induction.

Long-term follow-up of patients treated with acupuncture for xerostomia and the influence of additional treatment.

OBJECTIVE: To determine the long-term effects of acupuncture in patients with xerostomia of different etiologies and the influence of additional treatment. DESIGN: Retrospective study. SUBJECTS: Seventy patients, between the ages of 33 and 82, with xerostomia due to primary and secondary Sjögren's syndrome, irradiation and other causes were included. The median duration of xerostomia was 32 months. METHODS: Salivary flow rates (SFR) for whole unstimulated and stimulated saliva were used as indicators of effects of treatment. Data from 67/70 patients were analyzed 6 months following a baseline course of 24 acupuncture treatments using two-way ANOVA. Patients data up to 3 years were also compared by those who chose to receive additional acupuncture treatment vs those who did not. These data were analyzed descriptively. RESULTS: Statistically significant differences in unstimulated and stimulated salivary flow rates (P < 0.01) were found in all etiological groups after 24 acupuncture treatments and up to 6 months follow-up compared to baseline. Three years observation of these patients showed that patients receiving additional acupuncture treatment had a consistently higher median SFR in both unstimulated and stimulated saliva compared to patients who chose not to continue acupuncture. The upper limits of the interquartile range were also higher. CONCLUSIONS: This study shows that acupuncture treatment results in statistically significant improvements in SFR in patients with xerostomia up to 6 months. It suggests that additional acupuncture therapy can maintain this improvement in SFR for up to 3 years.

Sensory stimulation (massage) reduces blood pressure in unanaesthetized rats.

The objective of this study was to investigate how sensory stimulation by massage-like stroking influences blood pressure and heart rate in conscious rats. Also, the influence of different locations and durations of the stimulation were assessed. For this purpose, the ventral side of the abdomen or the dorsal side of the back was manually stroked at a speed of approximately 20 cm/s, with a frequency of 0.67 Hz and at an estimated pressure of 100 mm H2O. During the treatment, the rats were held across the scapula and the neck region. Blood pressure and heart rate were measured with the cuff technique before treatment and repeatedly during the post-stimulatory period. Massage-like stroking for 5 min of the abdominal area produced a maximum decrease of approximately 20 mm Hg in blood pressure and 60 beats/min in heart rate. This reduction remained significant at 3 and 4 h after stimulation, respectively. Stimulation of the abdominal area for 2 min produced a less pronounced decrease in blood pressure as compared to the 5-min stroking. Stroking of the back resulted in a short-lasting blood pressure increase that gradually returned to the baseline level within the post-stimulatory observation time. Control animals that were handled in the same way as the experimental animals except for the stroking showed an increase of approximately 20 mm Hg in blood pressure and 60 beats/min for about 1 h after the cessation of the handling. The responses of the blood pressure and heart rate to both abdominal and back massage were significantly inhibited as compared to the control animals. These results suggest that massage-like stroking of the skin produces an inhibitory effect on the cardiovascular excitatory responses in rats. Especially, the results of the present study demonstrate that massage-like stroking of the abdomen reduces both blood pressure and heart rate below the pre-stimulus baseline levels.

Altered levels of neuropeptides characterize the brain of lupus prone mice.

It has been reported that more than 50% of lupus patients show various forms of neurological deficits including impaired cognitive functions and psychiatric disorders. Using an animal model of lupus we investigated the production of neuropeptides in the brain of NZB/W F1 female hybrid mice and its parental strain NZB and NZW. Our results indicate that the alteration in learning and memory described in lupus mice are paralleled by a decrease in calcitonin gene-related peptide, substance P and neuropeptide Y (NPY) levels in the hippocampus and a significant decrease of NPY in the cortex. These findings are interesting in the light of previously reported results suggesting that these neuropeptides can play an important role in cognitive functions. We also observed a decrease of NPY and vasoactive intestinal polypeptide levels in the hypothalamus of lupus prone mice and these changes may be related to the disregulation of the hypothalamus-pituitary-adrenal axis observed in lupus prone mice.

Prognostic value of the pilocarpine test to identify patients who may obtain long-term relief from xerostomia by acupuncture treatment.

BACKGROUND: Xerostomia (dry mouth) is a clinical symptom due to a number of factors, including Sjögren syndrome and radiation treatment to the head and neck region. It has been reported that acupuncture increases the salivary flow rate (SFR) in healthy subjects and in patients with xerostomia. A prognostic tool that would allow the care provider to identify patients who may respond to acupuncture treatment will aid in early intervention and thus lead to normalized SFR or relief of symptoms. OBJECTIVES: To determine the prognostic value of a test using pilocarpine chloride to identify those patients with xerostomia who may achieve a long-term increase in SFR in response to acupuncture. DESIGN: Cohort clinical study of 10 months' duration. SETTING: School of dentistry in a large, urban, research institute. PATIENTS: Thirty-two consecutive patients with xerostomia due to radiation treatment (n = 21) or Sjögren syndrome (n=11). INTERVENTION: Salivary flow rates for unstimulated whole saliva and paraffin-chewing stimulated whole saliva were measured before and after the administration of individualized doses of pilocarpine. All patients were then given 24 acupuncture treatments and followed up at 1 and 6 months. The effects of acupuncture treatment on SFR were recorded and response compared with the results of the pilocarpine test. MAIN OUTCOME MEASURES: Sensitivity, specificity, and positive and negative predictive value of the pilocarpine test based on changes in SFR, defined as a 20% increase or greater, following acupuncture treatment, compared with response to the pilocarpine test. RESULTS: At the 1-month follow-up, 18 (72%) of 25 patients with a positive pilocarpine test result had defined significant changes in SFR; 4 (67%) of 6 patients with a negative pilocarpine test result had an unchanged SFR. At this point, the sensitivity of the pilocarpine test was 0.90 (95% confidence interval [CI], 0.68-0.99) and the specificity was 0.36 (95% CI, 0.11-0.69). The positive predictive value was 0.72 (95% CI, 0.51-0.88), and the negative predictive value was 0.67 (95% CI, 0.22-0.96). At the 6-month follow-up, 17 (74%) of 23 patients with a positive pilocarpine test result had defined significant changes in SFR; 3 (60%) of 5 patients with a negative pilocarpine test result had an unchanged SFR. At this point, the sensitivity of the pilocarpine test was 0.89 (95% CI, 0.67-0.99), and the specificity was 0.33 (95% CI, 0.07-0.70). The positive predictive value was 0.74 (95% CI, 0.52-0.90), and the negative predictive value was 0.60 (95% CI, 0.15-0.95). CONCLUSION: The pilocarpine test was found to have a high sensitivity and good positive predictive value in identifying patients who may respond to acupuncture for the treatment of xerostomia.

Sensory stimulation (acupuncture) increases the release of calcitonin gene-related peptide in the saliva of xerostomia sufferers.

Over the last decade, several patients afflicted with xerostomia have been treated with acupuncture. Their salivary flow rates increased significantly and the improvement lasted during a long observation period. We also found that the release of several neuropeptides in the saliva of healthy subjects can be increased by acupuncture stimulation. The concentration of vasoactive intestinal polypeptide increased significantly in the saliva of xerostomic patients after acupuncture treatment. The release of the neuropeptide calcitonin gene-related peptide (CGRP) was investigated in the saliva of xerostomic patients in order to elucidate further the mechanisms of the effect of sensory stimulation (acupuncture) on the salivary secretion. CGRP-like immunoreactivity was measured with radioimmunoassay (RIA) before and after a double series of acupuncture treatment, in stimulated saliva of 14 patients who suffered from xerostomia. The results showed that the concentration of CGRP increased significantly (P<0.001) in the saliva of these patients after the end of acupuncture treatment as compared to base-line levels. Taking into consideration the influence of CGRP on the salivary flow, as well as its trophic effect, we concluded that the increased release of CGRP could be one of the factors that affect positively the salivary flow rates of xerostomic patients who were treated with acupuncture.

The influence of sensory stimulation (acupuncture) on the release of neuropeptides in the saliva of healthy subjects.

In recent studies we have shown that xerostomia (dry mouth) can be treated successfully with sensory stimulation (acupuncture). The increase of saliva secretion lasted often for at least one year. Some neuropeptides have been found to influence the secretion of saliva. The aim of this study was to investigate the mechanisms behind the effect of acupuncture on salivary secretion by measuring the release of neuropeptides in saliva under the influence of sensory stimulation. VIP-like immunoreactivity (VIP-LI), NPY-LI, SP-LI, CGRP-LI and NKA-LI were analysed in the saliva of eight healthy subjects. Manual acupuncture and acupuncture with low-frequency electrical stimulation (2 Hz) were used. The saliva was collected during 20 minutes before the start of acupuncture stimulation, then during 20 minutes while the needles were in situ and then for another 20 minutes after the needles were removed. Four different saliva sampling techniques were used: whole resting saliva, whole saliva stimulated by paraffin-chewing, whole saliva stimulated by citric acid (1%), and parotid saliva, also stimulated with citric acid (1%). The results showed significant increases in the release of CGRP, NPY and VIP both during and after acupuncture stimulation, especially in connection with electro-acupuncture. SP showed only few increases, mainly in connection with electro-acupuncture, whereas NKA generally was unaffected by the acupuncture stimulation. The sensory stimulation-induced increase in the release of CGRP, NPY and VIP in the saliva could be an indication of their role in the improvement of salivary flow rates in xerostomic patients who had been treated with acupuncture.

Substance P-associated increase of intra-articular temperature and pain threshold in the arthritic TMJ.

Neuropeptides are considered mediators and modulators of inflammatory joint disease. Substance P (SP) has been proposed as a mediator of pain, and its vasoactive properties are well documented. In this study, the presence of SP-like immunoreactivity in the synovial fluid was correlated to intra-articular temperature (IAT) and pain from the arthritic temporomandibular joint (TMJ) 3 to 5 weeks after one intra-articular injection of glucocorticosteroids. Eighteen TMJs were investigated for IAT and the presence of SP-like immunoreactivity in the synovial fluid in 12 patients with systemic inflammatory joint disease. After arthrocentesis, the aspirates were analyzed for SP-like immunoreactivity by means of competitive radio immunoassay. A visual analogue scale and an algometer determining the pressure pain threshold and tolerance level assessed arthritic pain and hyperalgesia in the TMJ. Our results indicate that SP-like immunoreactivity is associated with IAT and that increased concentrations of joint fluid SP-like immunoreactivity correspond to increased pain threshold and tolerance and a concomitantly decreased visual analogue scale. These findings suggest that SP is implicated in the vascular and nociceptive response of the arthritic joint and that SP, possibly assisted by the antinociceptive effect of local corticosteroids, has a modulatory role in arthritic pain and hyperalgesia.

The effect of acupuncture in the treatment of patients with primary Sjögren's syndrome. A controlled study.

Twenty-one patients, 20 women and 1 man, participated in a controlled study. All patients were diagnosed with primary Sjögren's syndrome (primary SS) according to the Copenhagen and San Diego criteria. The patients were randomly assigned to either a group receiving acupuncture treatment or a control group with no active treatment. The patients in the control group received acupuncture after 10 weeks when the acupuncture treatment was completed in the first group. A majority of the patients subjectively reported some improvement after treatment, and a significant increase in paraffin-stimulated saliva secretion was found after treatment. No statistically significant differences between the acupuncture group and the control group were seen in unstimulated salivary secretion or most of the subjective variables. The study showed that acupuncture is of limited value for patients with primary SS.

Effects of physical activity and acupuncture on calcitonin gene-related peptide immunoreactivity in different parts of the rat brain and in cerebrospinal fluid, serum and urine.

Calcitonin gene-related peptide (CGRP) is a very potent vasodilator in the nervous system, and may be involved in hot flushes experienced by most women around menopause. Flushing post-menopausal women had higher urinary excretion of CGRP before than after successful treatment of their flushes with acupuncture. The prevalence of vasomotor symptoms is lower in physically active women. In a rat model we therefore intended to assess whether acupuncture and exercise affected CGRP concentrations in different parts of the brain and peripherally. The aim of the study was to elucidate the short- and long-term effects of exercise and acupuncture on CGRP concentrations in the nervous system of normal adult rats. In a rat model, we examined the effects of single interventions and long-term treatment with physical exercise and manual or electro-acupuncture on CGRP concentrations in urine, cerebrospinal fluid and serum and different parts of the brain. In all compartments studied, but significantly only in the cerebrospinal fluid, CGRP increased after a single session of physical exercise or electro-acupuncture. Manual acupuncture did not change CGRP concentrations in any compartment. Rats had the highest concentrations of CGRP in the pituitary and hypothalamus but the concentrations did not differ significantly between control rats and those subjected to long-term treatment with manual or electro-acupuncture or running rats. Rats treated with electro-acupuncture had twice the CGRP concentration in the frontal cortex compared to control rats, albeit the difference did not reach statistical significance. Evidently manual and electro-acupuncture have different effects, whereas electro-acupuncture and physical exercise have more similar effects on CGRP production and/or release. To elucidate the role of CGRP in vasomotor symptoms, further studies with older flushing rats should be performed.

Sensory stimulation (acupuncture) increases the release of vasoactive intestinal polypeptide in the saliva of xerostomia sufferers.

We have shown in earlier studies that xerostomia can be treated successfully with acupuncture. We also found that acupuncture stimulation can increase the concentration of neuropeptides in the saliva of healthy subjects. In this study, the concentration of the neuropeptide vasoactive intestinal polypeptide (VIP) was measured in the saliva of xerostomic patients in connection with acupuncture treatment (AP). Patients suffering from xerostomia caused by irradiation treatment, Sjögren's syndrome and other systemic disorders had been treated with acupuncture. Some of these patients showed an increase of their salivary flow rates after the AP was completed. Seventeen patients out of 65 were chosen due to their ability to produce enough saliva for the radio immunoassay (RIA) analyses to be conducted prior to the start of AP. VIP-like immunoreactivity (VIP-LI) was measured in the chewing stimulated saliva of these patients before and after the whole AP (24 sessions of 30 min each). The results showed that there was a significant increase of the concentration of VIP after the AP as compared to the measurements made before the start of the treatment (p<0.05). We concluded that the increase of neuropeptide VIP might be one of the mechanisms behind the positive effect of acupuncture on the salivary flow rates of the xerostomic patients.

Olfactory cues from an oxytocin-injected male rat can reduce energy loss in its cagemates.

Rats can recognize the odor of a stressed conspecific and react with stress themselves. Stress mobilizes energy, causing increased core temperatures and energy loss by radiation from the naked tail. Oxytocin administered in high doses (1 mg/kg, s.c.) reduces a rat's tail skin temperature and thereby the radiated energy loss. While administration of this high dose of oxytocin induces sedation low doses induce anxiolysis. This study demonstrates that the cagemates of an oxytocin-treated (1 mg/kg s.c.) rat, which themselves have not received any oxytocin-treatment, show energy conservation, apparent as reduced tail skin temperature. This effect was blocked by olfactory impairment. The temperature reduction in the cagemates probably reflects an oxytocin-mediated olfactorily activated stress inhibitory mechanism.