Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.

Although irritable bowel syndrome (IBS) can be considered a biopsychological disorder in which an association between life stress and physiological changes leading to bowel irregularity is present, there is a lack of data concerning possible modifications of the adrenal function during the disease. The aim of the present study was to measure biological and psychological variables related to the activity of the hypothalamo-pituitary-adrenal axis in IBS patients compared to healthy subjects. Cortisol was measured in the saliva (obtained by a stress-free, non invasive collection procedure) of 55 IBS outpatients and 28 matched controls. Moreover, each subject completed the following self-administered questionnaires: the Rome Burnout Inventory (RBI) in its physical (RBI-PE) and emotional-mental exhaustion (RBI-EME) components, Beck Depression Inventory, State and Trait Anxiety Inventory (STAI), Perceived Social Support Scale (PSSS) and a Scale for the Assessment of Perceived Actual Work-Non Work Stress. Compared with controls, IBS subjects showed significantly higher levels of cortisol in the morning and lower in the evening, while they maintained the physiological circadian fluctuation (i.e. cortisol morning level higher than in the evening). Moreover, IBS patients presented a significant difference from controls in RBI-PE scores, which confirms the presence of fatigue, a symptom frequently reported by the patients. Compared with controls, no differences were found in IBS patients with respect to other psychological parameters. These findings suggest a dysregulation of the adrenal activity in IBS patients. The results may be relevant considering that changes in cortisol levels have been shown to be sensitive indicators of psychosocial stress and coping patterns in both laboratory and life situations.

We determined circadian salivary cortisol levels in 18 outpatients affected by probable Alzheimer's disease (AD) and looked for a possible correlation with both cognitive impairment and brain CT scan findings. The diagnosis of probable AD was made according to the NINCDS-ADRDA criteria. The severity of cognitive impairment was quantified using the Mini Mental State Examination (MMSE) and the Global Deterioration Scale (GDS). Cortisol levels were measured on saliva samples collected at 08:00 AM and 08:00 PM. For each sample, a duplicate cortisol measurement was performed on 50 microl of saliva by means of a modified commercial radioimmunoassay kit. At the same time, 11 of the 18 AD patients enrolled also underwent a brain CT scan to estimate cerebral atrophy by using linear indexes. The mean value of cortisol levels was significantly higher in AD patients than in controls at both the morning and the evening measurements, and the circadian fluctuation of cortisol was less marked in AD patients than in controls, although this difference did not reach statistical significance. Morning cortisol levels were significantly correlated to both the MMSE and the GDS scores. A significant correlation was also found between morning cortisol levels and all the cerebral atrophy indexes. By contrast, no correlation was observed between evening cortisol levels or cortisol circadian fluctuations and either cognitive impairment or cerebral atrophy. In conclusion, despite the potential biases deriving from the small sample and the limitations of the CT scan study, our results suggest that, in AD patients, hypercortisolemia is correlated with severity of the disease.

Hypothalamus–pituitaryadrenal (HPA) axis activity was monitored in 20 women with chronic migraine (CM), previously affected by medication overuse headache (MOH), in comparison to healthy women (20 subjects) by measuring salivary cortisol, testosterone, dehydroepiandrosterone–sulphate (DHEA–S) levels, and their ratios, one week after the end of the MOH rehabilitation procedure. The participants were instructed how to collect saliva samples at home, a procedure that was performed twice a day (08:00 a.m. and 8:00 p.m.). Morning and evening levels of cortisol were significantly increased in CM patients with respect to controls. With regard to the cortisol/DHEA–S ratio, an inverse marker of psycho–physical wellbeing, CM women showed significantly higher values than controls. Moreover, testosterone/cortisol ratios (anabolic/catabolic index of physical performance) were significantly lower in CM patients than in controls. In the present study, CM appears not to be associated with an impairment of cortisol and DHEAS circadian fluctuation; however, CM patients present alterations in HPA axis function that might contribute to metabolic and psychological alterations that have also been associated with CM.

Amyotrophic lateral sclerosis (ALS) is a chronic progressive neuromuscular disorder of unknown etiology, characterized by weakness, muscle wasting, fasciculations and increased reflexes, with conserved intellect and higher function. The disease is due to degeneration of the motor neurons in the cerebral cortex, brainstem nuclei and anterior horns of the spinal cord. Although ALS poses an extreme burden on individual condition, data are missing concerning the regulation of adrenal function in the disease. In the present study we investigated cortisol levels in saliva of ALS patients as compared to healthy subjects. The results showed the loss of circadian rhythm of cortisol levels in ALS; in particular, levels of cortisol in the evening sample were significantly increased in ALS patients with respect to controls. Moreover, ALS patients did not show any physiological increase of cortisol levels following an unexpected mild stress (color-word Stroop test). These findings indicate the dysregulation of adrenal activity in the disease.