Psychoneuroendocrine modulation of regulatory T lymphocyte system: in vivo and in vitro effects of the pineal immunomodulating hormone melatonin.

BACKGROUND: At present, it is known that cancer-related immunosuppression would mainly depend on an immunosuppressive action mediated by a subtype of CD4+ lymphocytes, the so-called regulatory T lymphocytes (T-reg), which are identified as CD4+CD25+ cells. Moreover, it has been shown that anticancer immunity is under psychoneuroendocrine regulation, mainly mediated by the pineal hormone melatonin (MLT). This study was performed to investigate the in vivo and in vitro effects of MLT on T-reg generation. MATERIALS AND METHODS: We evaluated the in vivo effects of MLT (20 mg/daily orally in the evening) in 20 patients with untreatable metastatic solid tumor and the in vitro effects of MLT incubation (at 10 and 100 pg/ml) of pure lymphocyte cultures on T-reg cell count. RESULTS: MLT induced a statistically significant decline in mean T-reg cell numbers in patients who achieved disease control, whereas no effect was seen in those who had progressed. In contrast, no in vitro effect of MLT incubation was apparent. CONCLUSION: This preliminary study would suggest that MLT may exert in vivo an inhibitory action on T-reg cell generation in cancer patients which is associated with a control of the neoplastic progression, whereas no direct effect was seen in vitro on lymphocyte differentiation. This finding would suggest that MLT may counteract T-reg cell generation in vivo by inhibiting macrophage activity which is involved in stimulating T-reg cell production.

Synchronization of cortisol circadian rhythm by the pineal hormone melatonin in untreatable metastatic solid tumor patients and its possible prognostic significance on tumor progression.

BACKGROUND: Cancer progression has been associated with neuroendocrine alterations involved in the control of the circadian rhythms, particularly those of cortisol. Moreover, the evidence of an altered cortisol rhythm may predict a poor prognosis in cancer patients. Finally, cancer progression has been proven to be associated with alterations in the pineal gland, which plays a fundamental role in the control of circadian biological rhythms. On this basis, a study was planned to evaluate the effects of a chronic treatment with the pineal hormone melatonin (MLT) in advanced cancer patients with altered cortisol circadian rhythm. PATIENTS AND METHODS: The study included 14 untreatable metastatic cancer patients showing alterations of cortisol rhythm. They were treated by MLT at 20 mg/day orally, in the evening, for 3 consecutive months. RESULTS: a normalization of cortisol rhythm was achieved in 4/14 (29%) patients. Moreover, stable disease (SD) was obtained in 6/14 (43%) patients under MLT therapy, whereas the other 8 patients had progressive disease (PD). Finally, the percentage of cortisol rhythm normalization achieved in patients with SD was significantly higher than that observed in patients with PD. CONCLUSION: These results show that MLT may normalize cortisol rhythm in advanced cancer patients and this effect appears to be associated with SD, thus confirming the negative prognostic significance of cortisol rhythm alterations in cancer.

Enhancement of the efficacy of cancer chemotherapy by the pineal hormone melatonin and its relation with the psychospiritual status of cancer patients.

BACKGROUND: The anti-oxidant and immunomodulating natural agents may enhance the efficacy of cancer chemotherapy. One of the most important agents is the pineal hormone melatonin (MLT) which may exert both anti-oxidant and antiproliferative immunostimulating anticancer effects. This study was performed to evaluate the efficacy of a biochemotherapeutic regimen in metastatic cancer patients, and its therapeutic activity in relation to the psychospiritual status of patients. METHODS: The study included 50 metastatic non-small cell lung cancer (NSCLC) patients and a control group of 100 patients. Chemotherapy consisted of cisplatin plus gemcitabine. MLT was given orally at 20 mg/day in the evening. Patients were subdivided into 5 psychic profiles, as follows: spiritual faith, rationale faith, anxiety, apathy, and accusation behavior. RESULTS: Tumor response rate was significantly higher in patients treated by chemotherapy plus MLT than in those treated by chemotherapy alone (21/50 vs. 24/100, p < 0.001). However, the percentage of objective tumor regressions obtained in patients with spiritual faith was significantly higher than that found in the overall other patients concomitantly treated by chemotherapy plus MLT (6/8 vs. 15/42, p < 0.01). CONCLUSIONS: In conclusion, the efficacy of chemotherapy may be enhanced by the pineal hormone MLT, by representing a new promising biochemotherapeutic combination; also despite its objective ability to enhance chemotherapy efficacy, the activity of MLT is depending at least in part on the psychospiritual status of cancer patients, and it is maximal in the presence of a real spiritual faith.

HER2 expression in breast cancer: correlation with endocrine function and psychological status in operable and metastatic breast cancer.

BACKGROUND: Node involvement, negative estrogen receptor (ER) and HER2 expression are the main negative prognostic factors for breast cancer. Prolactin (PRL) is involved in the control of breast cancer growth and differentiation. Surgery-induced hyperprolactinemia seems to be a positive prognostic factor for operable breast cancer, whereas high PRL levels may predict a poor prognosis in women with metastatic breast cancer. In this study, we evaluated the relation between HER2 expression and PRL blood concentrations in women with metastatic breast cancer women and those whit operable breast cancer patients prior to before and 7 days after surgery. PATIENTS AND METHODS: The study included 50 women with breast cancer, 22 of whom had metastatic disease. HER 2 expression and serum levels of PRL were evaluated by fluorescence in situ hybridization (FISH) method and immunoradiometric assay (IRMA) method, respectively. RESULTS: HER2 expression occurred in 11/28 operable cases and in 8/22 metastatic cases. The percentage of surgery-induced hyperprolactinemia was significantly higher in HER2-negative patients than in those with its expression. Moreover, HER2-positive metastatic cases showed significantly higher mean serum PRL levels than in the negative group. CONCLUSION: These preliminary results show that metastatic cancer-related hyperprolactinemia and lack of surgery-induced hyperprolactinemia are statistically more frequent in HER2-positive patients, thus suggesting a link between PRL endogenous secretion and HER2 expression in breast cancer.

A randomized study of chemotherapy versus biochemotherapy with chemotherapy plus Aloe arborescens in patients with metastatic cancer.

BACKGROUND: The recent advances in the analysis of tumor immunobiology suggest the possibility of biologically manipulating the efficacy and toxicity of cancer chemotherapy by endogenous or exogenous immunomodulating substances. Aloe is one of the of the most important plants exhibiting anticancer activity and its antineoplastic property is due to at least three different mechanisms, based on antiproliferative, immunostimulatory and antioxidant effects. The antiproliferative action is determined by anthracenic and antraquinonic molecules, while the immunostimulating activity is mainly due to acemannan. PATIENTS AND METHODS: A study was planned to include 240 patients with metastatic solid tumor who were randomized to receive chemotherapy with or without Aloe. According to tumor histotype and clinical status, lung cancer patients were treated with cisplatin and etoposide or weekly vinorelbine, colorectal cancer patients received oxaliplatin plus 5-fluorouracil (5-FU), gastric cancer patients were treated with weekly 5-FU and pancreatic cancer patients received weekly gemcitabine. Aloe was given orally at 10 ml thrice/daily. RESULTS: The percentage of both objective tumor regressions and disease control was significantly higher in patients concomitantly treated with Aloe than with chemotherapy alone, as well as the percent of 3-year survival patients. CONCLUSION: This study seems to suggest that Aloe may be successfully associated with chemotherapy to increase its efficacy in terms of both tumor regression rate and survival time.

Interleukin-2 immunotherapy action on innate immunity cells in peripheral blood and tumoral tissue of pancreatic adenocarcinoma patients.

BACKGROUND AND AIMS: Innate immunity cells play a crucial role in host anticancer defense: cancer patients with high levels of natural killer (NK) cells and eosinophils have a better prognosis. Recombinant interleukin-2 (rIL-2) immunotherapy stimulates innate immunity cells. This study aims to evaluate the toxicity of pre- and postoperative rIL-2 treatment and the effects on innate immunity both in peripheral blood and in cancer tissue of patients with resectable pancreatic adenocarcinoma. MATERIALS AND METHODS: Seventeen patients received high dose rIL-2 preoperative subcutaneous administration and two low dose postoperative cycles. We evaluated NK cell and eosinophil count in blood and in pancreatic surgical specimens. RESULTS: Toxicity was moderate. In the early postoperative period, blood NK cells and eosinophils significantly increased compared to basal values (p < 0.02). Histopathological analysis did not find significant intratumoral infiltration of NK cells nor of eosinophils. CONCLUSIONS: Preoperative high dose rIL-2 administration is able to counteract surgery-induced deficiency of NK cells and eosinophils in peripheral blood in the early postoperative period, although it cannot overcome local mechanisms of immune tumor escape in cancer tissue. The amplification of innate immunity, induced by immunotherapy, may improve the control of metastatic cells spreading in the perioperative period.

Neuroimmunomodulation in medical oncology: application of psychoneuroimmunology with subcutaneous low-dose IL-2 and the pineal hormone melatonin in patients with untreatable metastatic solid tumors.

BACKGROUND: Anticancer immunity is under psychoneuroendocrine regulation, mainly via the pineal gland and brain opioid system, which may stimulate and inhibit antitumor immunity respectively. Cancer-related immuno-suppression does not depend only on functional damage of immune cells, but also on alterations of systems responsible for the neuroimmunomodulation, the most frequent of wich is a decline in blood levels of the pineal hormone melatonin (MLT). PATIENTS AND METHODS: A study was performed to evaluate the influence of an exogenous administration of MLT alone or MLT plus subcutaneous (SC) low-dose interleukin-2 on tumor progression and survival time in patients with untreatable metastatic solid tumors. The study included 846 patients with metastatic solid tumor (non-small cell lung cancer or gastrointestinal tract tumors) randomized to receive the best supportive care only, supportive care plus MLT (20 mg/day, orally in the evening), or MLT plus SC low-dose IL-2 (3 MIU/day for 5 days/week, for 4 consecutive weeks). RESULTS: The MLT alone was able to induce a significant increase of disease stabilization and survival time with respect to supportive care alone. The association of lL-2 with MLT provided a further improvement in the percentage of tumor regressions and of 3-year survival with respect to MLT alone. CONCLUSION: The administration of IL-2 and the pineal hormone MLT may induce control of neolplastic growth and a prolonged survival time in patients with metastatic solid tumors, for whom no other conventional anticancer therapy is available.

The endocannabinoid anandamide neither impairs in vitro T-cell function nor induces regulatory T-cell generation.

BACKGROUND: The cannabinoids have been proposed in the treatment of cancer. Generally, the cannabinoids are believed to be useful only in the palliative therapy of cancer-related symptoms, namely pain, anorexia and cachexia. However, preliminary experiments would also suggest an inhibitory effect of cannabinoids on cancer growth, whereas their influence on anticancer immunity is still controversial. The present study aimed to evaluate the influence of the endogenous cannabinoid anandamide (AEA) on T-cell phenotype and function. MATERIALS AND METHODS: The in vitro effects of AEA were evaluated at different concentrations on lymphocyte proliferation, cytotoxicity and differentiation, and in particular on T-regulator generation. RESULTS: AEA did not modify lymphocyte proliferation, neither under basal conditions, nor after IL-2 stimulation. Moreover, AEA did not induce the generation of regulatory T-lymphocytes nor the production of the immunosuppressive cytokine, IL-IO. CONCLUSION: The direct antitumor activity of AEA together with the absence of negative effects on T-cell functions might provide new insights into the potential use of cannabinoid agents in cancer immunotherapy.

[Immunotherapy in radical surgery of colorectal carcinoma]. / Immuno-modulazione nella chirurgia radicale del carcinoma del colon-retto.

Cancer-associated immunodeficiency is seriously worsened by surgical trauma. Short-term preoperative interleukin-2 (IL-2) immunotherapy abolishes postoperative immunodeficiency and can induce immunological control of the growth of minimal residual disease. Growth factors play an important role in oncological practice in treating neutropenia (G-CSF) or associated anaemia during chemotherapy (erythropoietin). Unfortunately, lymphocytopenia is not considered a biological marker with regard to survival. On the other hand, the role of the immune response to surgical trauma has been emphasised by many surgeons, and to counteract it immune nutrition (omega 3 fatty acid, mRNA, arginine) or thymic hormone have been tried. We believe that the obvious method of counteracting postoperative lymphocytopenia is the administration of the specific growth factor for T lymphocytes, i.e. IL-2. The aim of this study was to report on our experience with IL-2 preoperative immunoactivation in colorectal cancer and the long-term outcome of patients treated in comparison with a control group operated on without immunotherapy. In order to obtain activated lymphocytosis at the time of operation administration of IL-2 (6 million I.U. twice daily subcutaneously) for 3 preoperative days is sufficient, starting 4 days before surgery. The inclusion/exclusion criteria were histologically documented colorectal cancer, elective surgery, laparotomic surgery, no second tumour, age 20-80 years, no cardiovascular, hepatic or renal failure. From June 1992 to December 2005, 67 patients were treated (Dukes B/C: 46/21) with IL-2 immunotherapy. The clinical and biological results were compared with those of a control group of 173 patients (Dukes B/C 114/59) operated on in the same period and recruited with the same criteria. Dukes stage-C patients in both groups underwent adjuvant chemotherapy plus radiotherapy for rectal cancer. Data were statistically analysed using Fisher's exact test, Student's T-test and analysis of variance, as appropriate. The overall survival curves were plotted with the Kaplan-Mayer method. After a median follow-up of 69 months (range: 12-169) the progression rate was 15/67 (22%) vs 68/173 (39%) in controls (p = 0.02). Important results were obtained in Dukes-B patients: progression rate 7/46 (15%) vs 37/114 (32,4%) in controls (p = 0.03). We can conclude that immunotherapy is well tolerated. IL-2 is capable of counteracting surgery-induced immunodeficiency. The amplification of the immune response in the post-operative period is capable of controlling minimal residual disease after radical surgery, of reducing the progression rate, and of improving the prognosis and overall survival.

Impact of postoperative infections on survival in colon cancer patients.

PURPOSE: We investigated the prognostic significance of postoperative infections for the outcome of 192 patients with colon cancer. METHODS: The 5-year survival rates were analyzed by the Kaplan-Meier technique. Univariate and multivariate analyses were done to evaluate prognostic variables using Cox's proportional hazard model. RESULTS: Forty-three patients developed deep incisional or organ/space surgical site infections. The groups with and without infection were comparable. Multivariate analysis showed that only Dukes' stage (p=0.048) and postoperative infection (p=0.011) were independently associated with outcome. In patients with infective complications, the survival rate was significantly lower than in subjects without infection (log rank p=0.0004). CONCLUSIONS: These results stress the importance of evaluating variables other than the classical tumor stage in predicting long-term cancer outcome.

Pre-operative immunoprophylaxis with interleukin-2 may improve prognosis in radical surgery for colorectal cancer stage B-C.

Cancer-associated immunodeficiency is seriously worsened by surgical trauma. Short-term pre-operative interleukin-2 (IL-2) administration abolished post-operative immunodeficiency. The effects of a pre-operative IL-2 immunotherapy on the prognosis of colorectal cancer patients (Dukes' stages B and C), undergoing radical surgery, are reported. The study included, after post-operative stratification, 86 consecutive patients with colorectal cancer Dukes' stage B (57) and C (29), undergoing radical laparotomic surgery, randomised to be treated pre-operatively, with or without a short-term course of subcutaneous (s.c.) IL-2 immunotherapy. Human recombinant IL-2 was given s.c. at 6x10(6) I.U. twice daily pre-operatively for 3 consecutive days. Surgery was performed 36 hours after the last IL-2 injection. Dukes' C patients of both groups received standard adjuvant chemotherapy consisting of 5-FU plus folates and radiotherapy for rectal cancer patients. After a median follow-up of 54 months (range 18-86), the progression rate was significantly lower in patients pre-treated with IL-2 than in controls: 9/42 (21.4%) IL-2 group vs. 19/44 (43.1%) controls, (p <0.03). The positive effect of immunotherapy was detected both in the Dukes' B group, with 5/29 (17%) progression in the IL-2 group vs. 9/28 (32%) in controls, and Dukes' C patients with 4/13 (30%) vs. 10/16 (62%). This study shows that a 3-day pre-operative course of IL-2 immunotherapy may improve prognosis in patients with colorectal cancer at Dukes' stages B and C, as previously demonstrated in patients with more advanced disease. Therefore, the early activation of the antineoplastic immune system in the first post-operative days following a presurgical activation with IL-2 may counteract the growth of minimal residual disease and prevent late disease progression.

Endocrine effects of erythropoietin in cancer patients.

The recent advances in the knowledge of the psychoneuroimmunological pathogenesis of human neoplasms have demonstrated the existence of feed-back mechanisms operating between interleukins and endocrine secretions, which play an important role in the regulation of the immune responses, including the anticancer immunity. In contrast, few studies only have been performed to investigate the possible relation between endocrine activities and hematopoietic growth factors. The present study was performed to analyze the acute endocrine effects of erythropoietin-alpha (EPO) on the main endocrine secretions. The study was carried out in 10 advanced solid tumor patients. EPO was injected subcutaneously at a dose of 10,000 U, and venous blood samples were collected before and 2, 4 and 6 h after EPO administration. No significant changes in mean serum levels of FSH, LH and TSH were seen in response to EPO. Cortisol and DHEAS concentrations increased after EPO injection, whereas those of PRL decreased, but none of these differences was statistically significant. Finally, mean serum levels of both growth hormone (GH) and somatomedin-C (IGF-1) significantly decreased after EPO administration. This preliminary study shows that EPO may inhibit GH secretion from the pituitary gland and IGF-1 production. Since GH would stimulate EPO release, the results of this study may suggest the existence of feedback mechanism operating between GH secretion and EPO production, with inhibitory effect of EPO on GH secretion, and stimulatory action of GH on EPO production. Therefore, this study would describe the first example of hemato-endocrine feedback mechanisms. Moreover, this study, by showing an inhibitory effect of EPO on IGF-1 secretion, would suggest a possible use of EPO in the medical oncology not only for the treatment of cancer related anemia, but also to counteract tumor growth by blocking IGF-1 production, which has been proven to be a growth factor for several tumor histotypes. Obviously, IGF-1 is not the only tumor growth factor, but it could play a fundamental role in the regulation of production and activity of several other tumor growth factors. In any case, this study describes the only acute endocrine effects of EPO. Therefore, further studies, by evaluating the endocrine effects of a chronic treatment with EPO, will be required to establish which may be its effect on IGF-1 endogenous production, and its consequence on survival time.

Total pineal endocrine substitution therapy (TPEST) as a new neuroendocrine palliative treatment of untreatable metastatic solid tumor patients: a phase II study.

OBJECTIVES: It is known since many years that the pineal gland plays an anticancer role, and melatonin (MLT), the most investigated pineal hormone, has been proven to exert antitumor activity. However, MLT would not be the only hormone responsible for the antitumor action of the pineal gland. In fact, recent advances in the pineal investigations have shown that pineal indoles other than MLT may also exert anticancer activity, namely the three main indoles, consisting of 5-methoxytriptamine (5-MTT), 5-methoxytryptophol (5-MTP) and 5-methoxy-indole acetic acid (5-MIA). Cancer progression has appeared to be associated with a concomitant decline in the pineal endocrine function. Therefore, the replacement of a complete pineal function in the advanced cancer patients would require the exogenous administration of the overall four pineal indoles. Several clinical studies have shown that MLT alone at pharmacological doses may induce a control of the neoplastic progression in about 30% of untreatable metastatic solid tumor patients. The present study was performed in an attempt to evaluate the therapeutic of a total pineal endocrine substitution therapy with its four indole hormones in cancer patients, for whom no other conventional therapy was available. METHODS: The study included 14 metastatic solid tumor patients, who had failed to respond to the conventional anticancer therapies. The pineal indoles were given orally according to a schedule elaborated in an attempt to reproduce their physiological circadian secretion during the daily photoperiod. MLT was given at 20 mg/day during the night, whereas the other indoles were given at 1 mg/day, by administering 5-MIA in the morning, 5-MTP at noon and 5-MTT in the afternoon. RESULTS: A disease-control was achieved in 9/14 (64%) patients, consisting of partial response (PR) in one patient and stable disease (SD) in the other 8 patients. The median time of disease-control (PR + SD) was 6 months (range: 4-10). CONCLUSIONS: This preliminary study shows that a total pineal endocrine replacement therapy by an exogenous administration of the overall four pineal indoles may induce a disease-control in about 60% of untreatable metastatic solid tumor patients. Then, these results would be clearly superior with respect to those described with MLT alone, by confirming in humans that MLT is not the only hormone responsible for the anticancer property of the pineal gland. Since Cartesius was the first author who suggested the fundamental role of the pineal in the connection between consciousness and biological life, this therapy could be defined as a Cartesian therapy.

Effects of IL-2 preoperative immunotherapy on surgery-induced changes in angiogenic regulation and its prevention of VEGF increase and IL-12 decline.

BACKGROUND/AIMS: IL-2 preoperative immunotherapy has been proven to abrogate surgery-induced immunosuppression in cancer patients. In contrast, at present there are no data about the possible influence of IL-2 on angiogenesis-related molecular changes determined by the surgical operation. At present, it is known that VEGF (vascular endothelial growth factor) is the main endogenous angiogenic factor, whereas the antitumor cytokine IL-12 has appeared to play an anti-angiogenetic role. On this basis, a study was planned to evaluate the influence of IL-2 presurgical immunotherapy on the perioperative changes in VEGF and IL-12 secretions. METHODOLOGY: The study was performed on 30 colorectal cancer patients undergoing radical surgery, who were randomly chosen to be treated with or without preoperative immunotherapy of IL-2 (12 million IU/day subcutaneously for 3 consecutive days prior to surgery). Serum levels of VEGF and IL-12 were measured by ELISA for blood samples collected before surgery, and at days 3, 7 and 10 of the postoperative period. RESULTS: VEGF mean concentrations progressively and significantly increased during the postoperative period in patients treated with surgery alone. Mean values of VEGF were enhanced also in patients pretreated with IL-2, but VEGF increase observed in the IL-2 group was delayed, more transient and significantly lower with respect to that found in controls. IL-12 mean concentrations significantly decreased during the postoperative period only in the control patients, whereas in the IL-2-treated patients IL-12 postoperative mean values were not significantly lower than those found before surgery. CONCLUSIONS: This preliminary study would suggest that IL-2 preoperative immunotherapy may abrogate surgery decline in IL-12 levels and reduce, although not completely prevent, VEGF increase during the postoperative period in surgically treated cancer patients. These results would suggest that IL-2 presurgical immunotherapy may counteract surgery-induced stimulation of the angiogenesis, by either opposing the decline in blood levels of the anti-angiogenetic cytokine IL-12, or reducing the increase in those of the angiogenic factor VEGF.

Evidence for a neuroimmunomodulatory and a hematopoietic role of the Luschka's coccygeal body(3).

OBJECTIVES: In humans the glomus coccygeus was described in 1860 by Luschka. It is present at the coccyx tip and corresponds to a complex anastomosis between the median sacral artery and vein, and it is innervated by sympathetic fibers. In rats and mice it has been located in the tail ventral face. Its function is not known. According to our previous work, which demonstrated that hematopoiesis is under a noradrenergic control and based on the presence of epithelioid cells and sympathetic innervation, we assumed that the coccygeal gland might influence hematopoiesis via neuroendocrine or neural mechanisms. Therefore, the present study was undertaken to analyze the effect of glomus coccygeus on hematopoiesis. MATERIAL AND METHODS: Peripheral blood leukocyte and platelet concentrations as well as body temperature (BT) and body weight (BW), and norepinephrine (NE), adrenaline (A) and dopamine (DA) content in bone marrow of Luschkaectomized (LCGx), Sham LCGx operated (ShLCGx) and normal mice (Co) were investigated. RESULTS: We found that in LCGx vs. ShLCGx and Co, platelets and neutrophils increased while lymphocytes decreased. The effect of LCGx was significant from day 0 until day 65. Total leukocytes, monocytes, granulocytes, eosinophils and BT did not show any variation. Moreover, 22 days after the operation the amount of NE, A and DA seemed to be decreased in LCGx vs. ShLCGx while the difference was less evident between ShLCGx vs. Co. CONCLUSIONS: This study suggests for the first time a possible hematopoietic function and an immunomodulatory activity of the "Luschka's body" or Coccygeal body by a modulation of the sympathetic nervous system.

Evidence of pineal endocrine hypofunction in autistic children.

OBJECTIVE: The pineal hormone melatonin (MLT) has been proven to play a fundamental physiological regulatory role on both biological and psychic functions and alterations of the light/dark circadian rhythm of MLT have been described in several chronic immunoinflammatory diseases and in psychic disorders. Aim of the present biological explanatory study was the evaluation of MLT circadian rhythm in autistic children, in order to preliminary assess the pineal endocrine function in the autistic syndrome. METHODS: The study included 14 children suffering from classical infantile autism, who were investigated for the whole 24-hour circadian rhythm by collecting venous blood samples at 4-hour intervals. Serum levels of MLT were measured by the RIA method. The control group consisted of 20 age-matched healthy children. RESULTS: No autistic patient showed a normal MLT circadian rhythm. Moreover, autistic children showed significantly lower mean concentrations of MLT, mainly during the dark phase of the day, with respect to the values observed in the controls. CONCLUSION: The results of this preliminary study suggest the existence of a pineal endocrine hypofunction in autistic children, whose pathophysiological significance needs to be thoroughly investigated in successive clinical studies.