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Non-Celiac Gluten Sensitivity in patients with severe abdominal pain and bloating: The accuracy of ALCAT 5

Background and aims: Non-Celiac Gluten Sensitivity (NCGS) is a recently proposed clinical condition causing both intestinal and extra-intestinal symptoms, without gastrointestinal lesions, which improve on avoiding gluten intake, in the absence of celiac disease and wheat allergy. The prevalence of this condition is still a matter of debate, in part due to the very recent introduction of an accepted diagnostic test, a double-blind, placebo controlled gluten challenge. However, this is a lengthy and cumbersome procedure, theoretically burdened by a significant reduction of patient compliance. ALCAT 5 is an automated in vitro test evaluating the toxic effect of gluten on neutrophils by the exposure of these cells to a gluten-containing extract of gluten-containing cereals. The test is very simple to perform, the results are rapidly obtained, and might represent, if sufficiently accurate, a promising alternative to diagnose gluten intolerance. The aim of this study was the comparison of ALCAT 5 results with those of a double- blind, placebo-controlled, gluten challenge, in a group of patients with clinically-suspected NCGS. Methods: Twenty-five patients (M/F 3/22, mean age 32 ± 4 yrs) with severe functional abdominal pain and bloating, who had previously undergone the ALCAT 5 test, were enrolled. All the subjects reported their symptoms on a gluten-containing diet and considered gluten the causal agent. Following the Salerno Experts’ Criteria, they underwent a double-blind, placebo controlled trial with gluten vs placebo. A mean value during gluten ingestion >30% of the value during placebo was considered as indicative of gluten sensitivity.

Results: After blinded administration of gluten, 13 out of 25 (52%) patients showed an increase in the severity of abdominal pain, and 11 out of 25 (44%) showed an increase in the severity of abdominal bloating. Considering these two symptoms together, in 16 patients out of 25 (64%), blinded gluten administration induced an increase of abdominal pain and/or bloating. The ALCAT 5 test proved to be positive in 20 and negative in 5 patients. In sixteen patients out of 25 the result of ALCAT 5 agreed with the double-blind trial (64%). In particular, both tests were positive in 14 patients and negative in 2. Conclusions: In this subgroup of patients, ALCAT 5 could be used to support the clinical suspicion of the presence of NCGS and to address these patients to a blinded gluten challenge.

Introduction

Non-Celiac Gluten Sensitivity (NCGS) is a recently proposed clinical condition causing both intestinal and extra-intestinal symptoms which improve on avoiding gluten intake, in the absence of gastrointestinal lesions, celiac disease and wheat allergy. The prevalence of this condition is still a matter of debate. The first descriptions of NCGS were characterized by very high fre- quency reporting [2], but recently more realistic figures were described [3,4]. This discrepancy may be explained by the recent introduction of an accepted diagnostic test, a double-blind, placebo controlled gluten challenge [5], whereas the patient merely self- reporting the causative relationship between gluten ingestion and symptom occurrence was previously considered sufficient to diagnose the condition [6]. Apart from some criticisms on the interpretation of the results of the double-blind, placebo controlled food challenge, the proposed gluten challenge seems very prom- ising to describe the real prevalence of this condition. However, it is a lengthy and cumbersome procedure, theoretically burdened by a significant reduction in patient compliance during the three weeks of substrate intake.

ALCAT 5 is an in vitro test evaluating the toxic effect of gluten on neutrophils by the exposure of these cells to a gluten-containing extract of gluten-containing cereals, i.e., wheat, rye, barley, and oats. Results of the test are obtained through an automated mea- surement of neutrophil size and volume modifications following their incubation with gluten-containing cereal extracts [7]. The test is considered positive when the automated analysis reveals, in com- parison with the basal value, a change in volume and shape of neu- trophils after exposure to a test food substance. The test data are expressed as a volume distribution curve and the mean ± SD modi- fication of the area under the curve, in comparison with basal volume, is considered positive. An area change between the mean volume and 1 SD is considered positive. The test is very simple to perform, the results are rapidly obtained, and might represent, if sufficiently ac- curate, a promising alternative to diagnose gluten intolerance.

The aim of this study was the comparison of ALCAT 5 results to those of double-blind, placebo-controlled, gluten challenge, in a group of patients with suspected NCGS, in order to evaluate the performance of this test.

2. Patients and methods

Twenty-five patients (22 females, mean age 32 ± 4 yrs) with a long history of abdominal symptoms were enrolled. All the patients reported their symptoms on a gluten-containing diet, considered gluten to be the causal agent, and judged bloating and abdominal pain as the most severe. These characteristics of symptoms were confirmed by the completion of a questionnaire, based on a visual analogue scale, before entry the study. The mean duration of these two main symptoms were 10 ± 4 yrs. In all the patients, the pres- ence of organic conditions was excluded by endoscopic or radio- logic procedures, routine blood tests including thyroid function tests, coeliac disease associated serology, and abdominal ultra- sound. None of the patients suffered from wheat allergy. In this group of patients the following diagnoses were previously made: functional dyspepsia (n 1⁄4 14), irritable bowel syndrome (n 1⁄4 18), functional bloating (n 1⁄4 12), functional diarrhoea (n 1⁄4 8), functional constipation (n 1⁄4 6). In individual patients, more than one diag- nosis was frequently made. Rome IV criteria for functional bloating proved to be positive for all the patients, but for all the other conditions a positivity was present in only six patients (5 females, one male; 1 irritable bowel syndrome with constipation, 2 func- tional constipation, 1 irritable bowel syndrome with diarrhoea, 2 functional diarrhoea) [8,9].

All the patients underwent the ALCAT 5 test in the 3 months before the study on gluten-containing diet. However, the results of this test were blinded to the investigators during the next phases of the study. During the period from the ALCAT 5 test to the study entry, patients followed a gluten-containing diet.

None of the patients was following therapy known to interfere with intestinal function during the month prior to the study, such as antibiotics, prokinetics or laxatives. Constipated patients were advised to use a gentle water enema when needed and, in patients with diarrhoea, loperamide was allowed, if strictly necessary.

The protocol was approved by the local Ethical Committee and all the subjects gave their written informed consent.

2.1. NCGS diagnosis

Before the beginning of the study, and after ALCAT 5 test, all the patients had followed a period of gluten-free diet (GFD) which was even longer than the 6-week period suggested by the Salerno protocol [5], and all declared a clear improvement of symptom severity. Accordingly, we decided to avoid the repetition of this phase. However, to avoid an effect of gluten ingestion during the days immediately before the beginning of the protocol in sensitized patients, we prescribed a 2-week period of GFD, as a run in period, followed by the 3-week period of the blinded procedure suggested by the Salerno protocol [5], composed of two weeks of capsule ingestion, separated by one week of wash-out period (Fig. 1). After the run in period, the patients continued a strict GFD and were asked to fill in a daily questionnaire to rate the severity of both intestinal and extraintestinal symptoms, considered as gluten- dependent in NCGS (Table 1). Then, in a random order, following a randomization list generated by a computer, the patients were selected to follow a one week period during which gluten (10 capsule/day, 500 mg/capsule) or placebo capsules (rice starch, 10 capsule/day, 500 mg/capsule) were ingested. Rice starch was cho- sen due to its rapid absorption in comparison with other complex carbohydrates. At the end of the first week of capsule ingestion, the patients followed a 1-week wash-out period, without taking cap- sules, but continuing the completion of the daily questionnaires. Then, at the end of the wash-out period, the second period of capsule intake began (Fig. 1).

The daily questionnaire was administered to evaluate the presence and severity of abdominal and extra-intestinal symptoms, indicated in Table 1. The questionnaires used a 100 mm-long visual analogue scale (VAS) and patients were instructed to indicate the severity of each symptom on the line, considering the left end of the line as “absence of symptom” and the right end as “severe symp- tom” [10]. The score of the symptom was calculated by the sum of the score of the single days of each week. In accordance with the Salerno criteria, a mean value of the severity of abdominal pain and bloating during gluten ingestion >30% the value during placebo was considered as indicative of NCGS.

2.2. Statistics

To evaluate agreement between the gold standard test for NCGS and ALCAT 5 we used Cohen’s k. We judged the extent of the agreement according to Landis and Koch [11]. Agreement was considered: poor if k < 0.00; slight if 0.00 k 0.20; fair if 0.21
k 0.40; moderate if 0.41 k 0.60; substantial if 0.61 k 0.80; almost perfect if k > 0.80. Additionally, we calculated the sensi- tivity, specificity, and positive and negative predictive values of ALCAT 5: for these calculations we considered a subject to be positive if she/he displayed a worsening in pain or bloating at the blinded administration of gluten.

3. Results

After blinded administration of gluten, 13 out of 25 patients showed an increase in the severity of abdominal pain >30% than after placebo capsules (52%), and 11 out of 25 showed an increase in the severity of abdominal bloating >30% than after placebo cap- sules (44%).

Alcat Results

Alcat 5 Considering symptom severity after gluten challenge, in the whole group of patients we found a significant correlation between the severity of abdominal pain and abdominal bloating (r 1⁄4 0.64, p < 0.0005) (Fig. 2), and between the modification of abdominal pain and the modification of bloating (r 1⁄4 0.54, p < 0.005) (Fig. 3).

Among the other symptoms we included in the questionnaire, none of the extraintestinal symptoms worsened after gluten chal- lenge (data not shown). Gluten challenge worsened the severity of

flatulence in 11 patients, the severity of nausea in 4 patients, the severity of diarrhoea in 1 patient, and the severity of borborygmus in 1 patient. The number of bowel movements and the Bristol score during gluten challenge were not significantly different than pla- cebo challenge.

ALCAT 5 test proved to be positive in 20 and negative in 5 pa- tients. The comparison between ALCAT 5 and blinded gluten challenge results showed the two tests were concordant in 12 out of 25 patients (48%) when we considered abdominal pain as the main symptom and in 13 out of 25 patients (52%) when we considered bloating as the main symptom. However, the concordance of the two tests improved if the modification of both symptoms was considered: in 16 out of 25 patients (64%) the severity of at least one of the two symptoms during gluten capsule intake was >30% than its severity during placebo capsule intake. In this case, ALCAT 5 and blinded gluten challenge results were concordant in 16 out of 25 patients (64%).

We also evaluated the agreement between the two tests. The agreement between the ALCAT 5 and positive pain symptoms after blinded gluten administration was poor (Cohen’s k 1⁄4 0.06). The agreement between the ALCAT 5 and positive bloating symptoms after blinded gluten administration was slight (Cohen’s k 1⁄4 0.03). Similarly, if we considered positive pain or bloating symptoms after blinded gluten administration, agreement between the two tests was slight (Cohen’s k 1⁄4 0.04).

The ALCAT 5 has a sensitivity of 81% but a poor specificity of 22.2%. The positive predicted value (PPV) of the ALCAT 5 was 65%, while the negative predicted value (NPV) was 40%.

Alcat 5 test

Alcat 5

4. Discussion

Whether symptoms attributed to NCGS are indeed due to gluten intake is still a matter of debate [12]. On pathophysiological grounds, this is a very important topic, as the exact definition of the main actor inducing symptom onset in this condition will allow for a specific treatment. Accordingly, it is important to clarify whether gluten or wheat [13] should be the target of our attention, but also FODMAPs [14] or amylase-trypsin inhibitors [15]. Consequently, the same uncertainty is also present on diagnostic grounds. As far as the role of gluten is concerned, a double-blind, placebo-controlled, gluten challenge was recently proposed [5]: resembling that already adopted in the diagnostic algorithm of food allergy, a blinded administration of gluten or placebo together with a strict symptom occurrence monitoring is today considered an accurate test to diagnose NCGS. We and others [3,4] have recently applied this protocol in patients self-reporting the gluten dependence of their symptoms and some drawbacks should be reported. First of all, the protocol suggests a preliminary 6-week period of GFD to evaluate if symptom improvement may be achieved, to select the subgroup of patients who need a blinded gluten challenge. Since this preliminary phase is unblinded, it is possible that a placebo or nocebo effect may have a role in the improvement of symptoms. The length of this period might be responsible for an excessive extension of the whole protocol and, therefore, it could reduce the patient’s compliance. Second, the organoleptic characteristics of gluten make true blindness very difficult: it must be not visible to the patients and, therefore, it must inevitably be hidden in capsules. Moreover, to avoid excessive capsule size, due to the physical characteristics of gluten, a maximum amount of 500 mg can be contained in one capsule and this causes the need for a high number of capsules to be ingested in a day in order to administer a dose of gluten sufficient to induce symptoms. The ingestion of 10 capsules a day for two weeks, one for the gluten test and the other for the placebo test, i.e. 140 capsules in total, might cause a reduction in patient compliance. Third, daily questionnaires for symptom occurrence monitoring administered over many days might cause a reduction of the patient’s attention, in particular during the last period of the test. Consequently, it is evident that, even if the blinded food challenge is considered the gold standard

in the diagnosis of many adverse reactions to food [16], this test is largely absent in a clinical setting [17].

Accordingly, the availability of alternative tests is advisable. ALCAT 5 is a simple, automated test that could represent a valid option in the diagnosis of NCGS. This is the first evaluation of the accuracy of ALCAT 5 in human disorders: the principle of the test is derived from the ALCAT test, adopted for food intolerance [7], and it was applied to discriminate the role of gluten in self-reported NCGS patients.

Our results show that in a group of patients suffering in particular from functional severe abdominal pain and bloating and without organic diseases, wheat allergy or celiac disease, the prevalence of NCGS is higher than previously reported [3,4], sug- gesting that, in a gastroenterological outpatient clinic, abdominal symptoms are more indicative than extra-intestinal symptoms to guide physicians towards a correct diagnostic algorithm for this condition. All the enrolled patients have previously undergone the ALCAT 5 test, and the comparison between ALCAT 5 and blinded gluten challenge showed that in 64% of cases a concordance was evident. The concordance between the two tests was less accurate when abdominal pain or bloating were considered separately. However, the strict correlation between postechallenge severity of these two symptoms permits the adoption of a combination of them, thus optimizing the performance.

The assessment of the degree of agreement between the double blinded gluten challenge and ALCAT 5 tests showed very low values of Cohen’s k. Considering the single symptoms separately or together, results indicate that the agreement between the tests was no better than would be expected by chance. However, these results are similar to those obtained from a faecal occult blood test and a colonoscopy for the screening of left-sided colon cancer [18,19]. Accordingly, the importance of our results relies on the possibility of using the ALCAT 5 test preliminary to the blinded gluten chal- lenge. Due to the complexity of clinical manifestations of NCGS, frequently characterized by many concomitant symptoms, both intestinal and extraintestinal [2], as often reported by the patients, it may be difficult in many of them to correctly focus on the de- scriptions and the importance of any single symptom during the interpretation of the whole clinical presentation. Therefore, ALCAT 5 might be used to preliminarily screen patients with severe abdominal pain and bloating in order to select, in this subgroup of References patients, which of them should undergo the blinded gluten challenge, at least on the basis of these two main symptoms.

A limit of the study was the absence of a healthy control group, to test for in vitro modification of neutrophils after oral gluten challenge. In a subsequent study, it should be important to test for this modification and to evaluate also the reproducibility of the procedure.

In conclusion, we have compared ALCAT 5 and double-blind, placebo-controlled, gluten challenge results in a group of self- reported NCGS with severe abdominal pain and bloating, to eval- uate if a simplification of diagnostic procedures is possible in these patients. In this subgroup of patients, ALCAT 5 could be used to support the clinical suspicion of the presence of NCGS and to direct these patients to a blinded gluten challenge.

Conflict of interest
The authors have no conflict of interest to declare.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Guarantor of the article

Michele Di Stefano.

Specific author contributions

Michele Di Stefano planned and conducted the study, inter- preted data, drafted the manuscript.

Eugenia Vittoria Pesatori, Giulia Francesca Manfredi, Giacomo Grandi, Alessandro Gabriele, Davide Iozzi conducted the study, collected and interpreted data, drafted the manuscript.

Mara De Amici conducted the study, collected data and drafted the manuscript.

Giuseppe Di Fede planned the study and drafted the manuscript.

All the mentioned Authors approved the final version of the manuscript.

Acknowledgements

None.

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Enhancement of the Efficacy of Chemotherapy with Oxaliplatin Plus 5-Fluorouracil by Pretreatment with IL-2 Subcutaneous Immunotherapy in Metastatic Colorectal Cancer Patients with Lymphocytopenia Prior to Therapy

Research Article (download PDF version)

P. LISSONI1,*, F. BRIVIO2, L. FUMAGALLI2, G. DI FEDE3 and G. BRERA4

1 Division of Radiation Oncology, San Gerardo Hospital, Milan, Italy
2 Division of Surgery, San Gerardo Hospital, Milan, Italy
3 Institute of Biological Medicine, Milan, Italy
4 Ambrosian University, Milan, Italy

*Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italy; Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org

Abstract
The present study was carried out to evaluate the influence of a short-period IL-2 administration on the efficacy of chemotherapy in metastatic colorectal cancer patients with pretreatment lymphocytopenia, which was defined as a lymphocyte count of less than 1500/mm3.

The study included 144 consecutive metastatic colorectal cancer patients, who underwent chemotherapy with oxaliplatin plus 5-fluorouracil. Lymphocytopenia was seen in 41/144 (28%) patients, who were randomized to receive chemotherapy alone or chemotherapy after a prechemoimmunotherapy with IL-2 (3 MIU twice/day for 3 consecutive days), whereas patients with a normal pretreatment lymphocyte count received only chemotherapy.

A normalization of the lymphocyte number was achieved in 12/19 lymphocytopenic patients pretreated with IL-2. The objective tumor regression rate achieved in patients with a normal lymphocyte count prior to chemotherapy was significantly higher compared to that obtained in lymphocytopenic patients treated with chemotherapy alone (54/103 vs. 3/22, p<0.01), whereas no significant difference occurred between patients with normal lymphocyte count and lymphocytopenic patients pretreated with IL-2 (54/103 vs. 8/19).

This study confirms that pretreatment lymphocytopenia is associated with reduced efficacy of chemotherapy in metastatic colorectal cancer patients. Moreover, it suggests that pretreatment with IL-2 before the onset of chemotherapy may enhance the efficacy of chemotherapy in lymphocytopenic patients. Therefore, the administration of IL-2 before the onset of chemotherapy to improve the immune status of cancer patients may be considered as a new chemoimmunotherapeutic combination, which may be recommended in the treatment of advanced cancer patients, particularly in those with cancer-related immune alterations.

(download PDF version)

2006: Lissoni P; Fumagalli L; Brivio F; Rovelli F; Messina G; Di Fede G; Colciago M; Brera G
Division of Radiation Oncology, Milan, Italy

Cancer chemotherapy-induced lymphocytosis: a revolutionary discovery in the medical oncology

Journal of biological regulators and homeostatic agents 2006;20(1-2):29-35
(download PDF version)

The recent advances in the investigation of tumor immunobiology have suggested that cancer chemotherapy, in addition to its well known cytotoxic activity, may play modulatory effects on the endogenous production of cytokines involved in the control of both tumor angiogenesis and antitumor immunity. Cancer chemotherapy constantly acts with inhibitory effects on anti-bacterial, anti-viral and anti- mycotic immune responses, whereas its action on anticancer immunity, which is mainly mediated by lymphocytes, has still to be better investigated and defined.

The present study was carried out to evaluate the influence of chemotherapy on lymphocyte count and its relation to the clinical response in cancer patients suffering from the most commonly frequent tumor histotypes, including lung, colorectal, breast and prostate carcinomas. The study included 144 consecutive metastatic solid tumor patients. Lung cancer patients were treated with cisplatin plus gemcitabine, colorectal cancer patients received oxaliplatin plus 5-fluorouracil, while those affected by breast cancer or prostate carcinoma were treated with taxotere alone.

An objective tumor regression was achieved in 66 out of 144 (46 percent) patients, whereas the remaining 78 patients had only a stable disease (SD)or a progressive disease. Independently of tumor histotype and chemotherapeutic regimen, a lymphocytosis occurred in patients who achieved an objective tumor regression in response to chemotherapy, and lymphocyte mean count observed at the end of the chemotherapeutic treatment was significantly higher with respect to the values seen before the onset of treatment.

On the contrary, lymphocyte mean number decreased on chemotherapy in patients with SD or PD, even though the decline was statistically significant with respect to the pretreatment values in the only patients who had a PD in response to chemotherapy.

This study would suggest that chemotherapy itself may paradoxically act, at least in part, as a cancer immunotherapy by inducing lymphocytosis, as well as previously demonstrated for the only immunotherapy with IL-2, probably by modulating the cytokine network and correcting the altered endogenous production of cytokines, responsible for cancer-related immunodeficiency.

(download PDF version)

Modulation of the anticancer immunity by natural agents: inhibition of T regulatory lymphocyte generation by arabinoxylan in patients with locally limited or metastatic solid tumors

Research Article (download PDF version)

Paolo Lissoni1,*, Giusy Messina1, Fernando Brivio2, Luca Fumagalli2, Luigi Vigoré3, Franco Rovelli3, Luisa Maruelli4, Mauro Miceli4, Paolo Marchiori4, Giorgio Porro1, Michael Held5, Giuseppe di Fede6, Toshi Uchiyamada7

1 Division of Radiation Oncology, San Gerardo Hospital, Milan, Italy
2 Division of Surgery, San Gerardo Hospital, Milan, Italy
3 Laboratory of Immunomicrobiology, San Gerardo Hospital, Milan, Italy
4 Natur-Spiritual, Milan, Italy
5 Biological Medicine Center, Rome, Italy
6 Institute of Biological Medicine, Milan, Italy
7 Daiwa Pharmaceuticals, Tokyo, Japan

dr. Paolo Lissoni

*Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italy; Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org
Key words: Anticancer immunity, arabinoxylan, immunostimulation, T regulatory lymphocytes
Abbreviations: interleukin 10, (IL-10); interleukin 6, (IL-6); interleukin-2, (IL-2); interleukn 12, (IL-12); NK cells, (CD16+CD56+); T cytotoxic lymphocytes, (CD8+); T helper lymphocytes, (TH), (CD4+); T lymphocites, (CD3+); Transforming growth factor beta, (TGF-β) T-regulatory lymphocytes, (T-reg), (CD4+CD25+)

Received: 30 September 2008; Revised: 1 November 2008
Accepted: 17 November 2008; electronically published: December 2008

Summary

In the last years, several immunomodulating antitumor agents have demonstrated in the nature, particularly from Aloe plant and rice bran. However, the major problem concerning the natural antitumor agents is to define their immune mechanisms of action in relation to the more recent advances in tumor immunobiology. At present, the main cause responsible for the lack of an effective antitumor response in advanced cancer patients is belived to be represented by the generation of a subtype of T helper lymphocytes (CD4+) with suppressive activity on anticancer immunity, the so-called T regulatory lymphocytes (T reg), which may be clinically identified as CD4+CD25+ cells. On this basis, a study was planned to evaluate the effect of rice bran extract arabinoxylan on T reg cell count and percentage in solid tumor patients in relation to the various lymphocyte subpopulations. The study included 22 evaluable cancer patients, 16 of whom had an untreatable metastatic solid tumor. Arabinoxylan was given orally at a dose of 2000 mg/day for the first month, followed by a dose of 1000 mg/day for the next month. In each patient we evaluated by monoclonal antibodies the absolute number of lymphocytes, T lymphocytes (CD3+), T helper (TH) lymphocytes (CD4+), T cytotoxic lymphocytes (CD8+), NK cells (CD16+CD56+), T reg lymphocytes (CD4+CD25+) and TH/T reg ratio before and after 2 months of therapy. No substantial change occurred on therapy in the mean number of lymphocytes, CD3+, CD8+ and NK cells. On the other hand, the mean number of TH cells increased, whereas that of T reg cell decreased on treatment, even though none of these differences was statistically significant. On the contrary, TH/T reg mean ratio significantly enhanced after arabinoxylan therapy. In addition to its previously demonstrated stimulatory action on NK function, this study shows that arabinoxylan may inhibit the production of T reg cells, which are responsible for cancer-related immunosuppression, with a following improvement in the anticancer immunity. If further studies will confirm these results, arabinoxylan could be successfully associated with chemotherapy to induce not only a cytotoxic destruction of cancer cells, but also an improvement in the immune status.

I. Introduction
The recent advances in the definition of the mechanisms responsible for tumor progression have suggested the possibility to control cancer growth not only trough chemotherapy-induced cancer cell destruction, but also by stimulating the anticancer immunity. In addiction to the exisence of endogenous antitumor molecules, several agents capable of stimulating the anticancer immunity have alsso isolated from plants. However, the immunomodulatory effects of most natural immunomodulating agents need to be better investigated in an attempt to establish their mechanisms of action in relation to the most recent discoveries concerning the physiopathology of the anticancer immunity. At present, Aloe extracts (Lissoni et al, 1998) and arabinoxylan extract from rice bran (Ghoneum and Jewett, 2000) would represent some of the potential natural agents which could be utilized in the complementary therapy of human neoplasms. Today, it is known that the antitumor immune response is the end-result of several interactions involving cytokines and immune cells, provided by stimulatory or suppressive effects on the anticancer immunity (Atzpodien and Kirchner, 1990; Rosenberg, 1992). Therefore, the lack of an effective anticancer immune response in most cancer patients with advanced disease would simply depend on the prevalence of immunosuppressive mechansisms with respect to the immunostimulatory ones (Atzpodien and Kirchner, 1990). The anticancer immunity is mainly activated by T helper-type 1 lymphocytes by releasing IL-2 (Whittington and Faulds, 1993), and by dentritic cells, which act as antigen-presenting cells producing IL-12 (Banks et al, 1995), T cytotoxic lymphocytes and NK-LAK system, which are involved in the induction of the antigen-dependent and antigen-independent cytotoxicity, respectively (Atzpodien and Kirchner, 1990). Therefore, IL-2 and IL-12 would represent the main anticancer cytokines in humans. On the contrary, the suppression of the anticancer immune response is mediated by several cytokines, namely IL-10 (Moore et al, 1993), IL-6 (Matsuda and Hirano, 1990) and TGF-β (Shevach, 2002). Recently, however, it has been demonstrated that the various endogenous suppressive factors would exert their inhibitory immune effect through a common end-mechanism, consisting of the generation of a subtype of T helper lymphocytes (CD4+cells), provided by a fundamental suppressive activity on the anticancer immunity, the so-called T regulatory lymphocyte (T reg) (Dieckmann et al, 2001), which at present seems to constitute the main mechanism responsible for cancer-related immunosuppressive status. T reg cells may be identified by the simultaneous expression of the alpha-chain of IL-2 receptor (CD25) and CD4 antigen (Dieckmann et al, 2001). Then, T reg cells may be clinically recognized as CD4+CD25+ lymphocytes. Therefore, each eventual natural immunomodulating agent would have to be investigated in relation to its possible effect on T reg generation since, at least from a theoretical point of view, each natural agent capable of counteracting T reg activity could positively influence the prognosis of the neoplastic disease by improving the efficacy of the anticancer immune response. Moreover, our previous preliminary studies have suggested that the percentage of T reg cells with respect to the total number of T helper cells, as expressed as CD4/CD4CD25 ratio, may represent an optimal synthetic immune index to investigate the functional status of the anticancer immunity in the single cancer patient, by representing the synthesis of the actions of the great number of immunostimulating and immunosuppressive factors involved in the modulation of the anticancer immunity (Dieckmann et al, 2001). Within the great number of natural agents derived from plants and potentially usefull to be employed in the complementary therapy of cancer, arabinoxylan would seem to represent one of the potential natural agent, because of its efficacy in improving the clinical status of cancer patients (Ghoneum and Jewett, 2000; Ghoneum and Gollapudi, 2005; Markus et al, 2006; Ghoneum et al, 2007). The immunomodulating properties of this nautral substances extracted from plants have been confirmed by experimental studies, but unfortunately most experiments have been limited to the investigations of they effects on non-specific immune parameters for the anticancer immunity, such as NK cell cytotoxicity. In contrast, since reg cells play a fundamental role in suppressing the generation of the anticancer immunty, each potential antitumor immunomodulatory natural substances, would have to be investigated also in relation to their eventual influence on T reg cell system. On the basis of the recent discoveries in tumor immunobiology (Dieckmann et al, 2001; Shevach, 2002), a study was planned to investigate the possible influence of arabinoxylan on both absolute number of T reg cells and their ratio with respect to the total CD4+ T cells in a group of solid tumor patients, affected by locally limited or metastatic disease.

II. Materials and methods
The study included 24 consecutive patients, 18 of whom had a metastatic solid tumor, which did not respond to the conventional anticancer chemotherapies and for whom no other effective standard treatment was available, while the remaining 6 patients had been surgically treated for a locally limited neoplasm. Patients were followed at Biological Medical Institute of Milan and the protocol was approved by the Director of the Institute. Eligibility criteria were, as follows:histologically proven locally limited or metastatic solid tumor, no double tumor, no chronic therapy with corticosteroids because of their immunosuppressive effects and no concomitant treatment with other immunomodulating agents,such as interferons,interleukins and monoclonal antibodies. At the time of the start of arabinoxylan therapy, patients with untreatable metastatic cancer were under treatment with the only supportive care, consisting of anti-inflammatory agents for pain, anti-dopaminergic drugs for nausea and vomiting and with the pineal hormone melatonin for the therapy of the neoplastic cachexia (Banks et al, 1995). Patients were considered as fully evaluable when they had received arabinoxylan therapy for at least 2 consecutive months. Arabinoxylan was given orally at a dose of 1000 mg twice/day for the first month, followed by a dose of 1000 mg/day for the next month. Arabinoxylan was supplied by DAIWA Pharmaceutical (Tokyo, Japan). It was derived from rice bran treated enzymatically with an extract of the shiitake mushrooms. It is a polysaccharide containing β-1,4-xylopironase hemicellulose, commercially available and known as Biobran. For the immune investigation, venous blood samples were collected in the morning after an overnight fast before the onset of arabinoxylan therapy and after 2 consecutive months of treatment. In each blood sample, we evaluated the absolute number of total lymphocytes, T lymphocytes (CD3+), T helper (TH) lymphocytes (CD4+), T cytotoxic lymphocytes (CD8+), NK cells (CD16+ CD56+ and T regulatory (T reg) lymphocytes (CD4+ CD25+). The different lymphocyte subsets were measured with a flow cytometric assay by using specific monoclonal antibodies supplied by Becton-Dickinson (Milan, Italy). Moreover, because of the importance not only of their absolute number, but also of their percentage with respect to the other lymphocyte subsets, namely to that of CD4+ cells, CD4/CD4CD25 ratio, corresponding to TH/T reg ratio, was also determined before and after therapy. Normal values (95% confidence limits) of T reg number and TH/T reg ratio observed in our laboratory were below 240/mm3 and above 4.0, respectively. Data were reported as mean +/- SE and statistically analyzed by the Student’s t test, the analysis of variance and the chi-square test, as appropriate.

III. Results
Evaluable patients were 22/24, while the remaining 2 patients, both affected by untreatable disseminated liver metastases due to colorectal cancer, rapidly died for disease progression before concluding the two planned months of arabinoxylan therapy. The clinical characteristics of the evaluable patients are reported in Table 1. Figure 1 illustrates changes in the mean number of total lymphocytes, T lymphocytes, T cytotoxic lymphocytes and NK cells occurring after 2 months of arabinoxylan therapy. No substantial variation was found in the mean number of lymphocytes, T lymphocytes, T cytotoxic lymphocytes and NK cells under arabinoxylan treatment. In contrast, as illustrated in Figure 2, TH and T reg mean numbers increased and decreased, respectively, after arabinoxylan therapy, without, however statistically significant differences with respect to the values seen prior to therapy. On the contrary, a statistically significant increase in TH/T reg mean ratio was achieved after arabinoxylan therapy (p<0.025). The increase in TH/T reg ratio under arabinoxylan therapy was more pronounced in patients with an abnormally low ratio prior to therapy with respect to that occurring in those with normal pre-treatment ratio, however without statistically significant differences ( 2.3 +/- 0.4 vs 1.7 +/- 0.5). In more detail,

Figure 1. Changes in the number of lymphocytes, Tlymphocytes (CD3), T cytotoxic lymphocytes (CD8) and NK cells (CD16 CD56) after 2 months of arabinoxylan therapy.

Figure 2. Changes in the mean number of T helper (TH) lymphocytes (CD4) and T regulatory lymphocytes (cd4 cd24) and in TH/T reg mean ratio.

before arabinoxylan therapy, an abnormally low TH/T reg ratio was present in 12/22 (55%) evaluable patients. Arabinoxylan treatment induced a normalization of TH/T reg ratio in 5/12 (42%) patients with an abnormally low ratio prior to therapy. The percentage of arabinoxylan-induced TH/T reg normalization obtained in lymphocytopenic patients was not significantly different from that achieved in patients with normal pre-treatment lymphocyte count ( 3/7(43%) vs 2/5(40%) ). No toxicity was observed under arabinoxylan treatment, which was well tolerated in all patients. Asthenia was present in 8/22 (36%) evaluable patients. An evident relief of asthenia, as assessed by a specific patient report, was obtained under arabinoxylan therapy in 5/8 (63%) patients.

IV. Discussion
Previous experimental studies had already demonstrated some immunomodulating properties of arabinoxylan, in particular consisting of stimulation of NK cytotoxic function (Ghoneum, 1998), whereas NK cell number did not seem to be influenced by arabinoxylan administration. However, it has to be remarked that NK cells were belived to be fundamental in the antitumor immunity until some years ago, before the discovery of the essential role played by the antitumor cytokines, such as IL-2 and IL-12 (Whittington and Faulds, 1993) and dendritic cells, because of their function as antigen-presenting cells (Banks et al, 1995). In fact, it has to be considered that the cytotoxic activity of NK cells is effective only against artificial laboratory cancer cell lines, whose biological malignant properties are different from those presented by fresh human tumor cells (Whittington and Faulds, 1993). In addition, NK cells have been proven to be also able to destroy fresh human cancer cells only after the activation of their cytotoxic function by IL-2 (Atzpodien and Kirchner, 1990). From this point of view, arabinoxylan had been already proven to amplify the stimulatory effect of IL-2 on NK-mediated antitumor cytotoxicity (Ghoneum and Jewett, 2000). In contrast, no study has been performed up to now to evaluate the possible influence of arabinoxylan not only on the mechanisms responsible for the generation of an effective anticancer immune response, but also on those involved in the suppression of anticancer immunity. The results of this preliminary study, carried out to evaluate the influence of arabinoxylan on T reg cells, which represent the most important cells involved in the suppression of the antitumor cytotoxic immune response, demonstrates that arabinoxylan may counteract T reg cell generation by reducing their number and percentage with respect to the total amounts of CD4+ cells and circulating lymphocytes. Since NK cell function is inhibited by T reg activation (Shevach, 2002), the previously demonstrated arabinoxylan-induced stimulation of NK cell cytotoxic function might depend at least in part on its capacity of counteracting T reg generation (Dieckmann et al, 2001). Moreover, this study would suggest that the inhibitory action of arabinoxylan on T reg generation is more pronounced in patients with an abnormally high percentage of T reg cells prior to therapy, with a following pre-treatment abnormally low TH/T reg ratio before therapy, whereas its effect was less evident in patients with a pre-treatment value of TH/T reg ratio within the normal range. Therefore, the influence of arabinoxylan on T reg generation would consist of a modulatory action rather than an inhibitory activity. This finding could explain a potential favourable immunomodulatory effect of arabinoxylan also in patients with autoimmune diseases (Ghoneum, 1998), who in contrast to cancer patients would tend to present abnormally low amounts of T reg cells. In any case, the importance of the inhibition of T reg generation in the induction of an effective anticancer immune response has been recently confirmed by the evidence that the block of T reg activity by specific monoclonal antibodies may induce objective tumor regressions in humans (Yang et al, 2007). Obviously, the major problem is the exact identification of he T reg cell population. Even though T reg cells may express other immune markers, namely FOX-p2 cytoplasmatic antigen, most clinicians are in agreement to identify the CD4+CD25+ cells as T reg lymphocytes (12). In any case, further studies, by evaluating other immune markers, will be required to better identify T reg cells population, namely FOX-p3, even though recently some Authors have shown that FOX-p3 expression by T reg cells is associated with a lower suppressive activity (Dieckmann et al, 2001; Shevach, 2002). Moreover, it has to be remarked that several patients included in the present study were concomitantly under palliative therapy with the anti-cachectic pineal hormone melatonin (Brzezinski, 1997), which may also play immunomodulating effects (Maestroni, 1993). Therefore, further randomized studies with arabinoxylan alone versus arabinoxylan plus melatonin will be required to better define the immunomodulating action of arabinoxylan. If further clinical and experimental studies will confirm the inhibitory action of arabinoxylan on T reg cell system, it could be included in cytokine-based immunotherapies to enhance their efficacy by counteracting T reg cell generation.

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A clinical study of T-regulatory lymphocyte function in cancer patients in relation to tumor histotype, disease extention, lymphocyte subtypes and cortisol secretion

Research Article (download PDF version)

Luigi Vigorè1, Fernando Brivio2, Luca Fumagalli2, Roberto Vezzo1, Giusy Messina6, Franco Rovelli6, Massimo Colciago3, Giovanna D’Amico4, Giuseppe Di Fede5, Paolo Lissoni6

1 Laboratory of Immunomicrobiology, St.Gerardo Hospital, Monza, Milan, Italy
2 Division of Surgery, St.Gerardo Hospital, Monza, Milan, Italy
3 I.N.R.C.A Laboratory of Analyses, Casatenovo, Lecco, Italy
4 Research Center “Fondazione Tettamanti” Clinica Pediatrica Università Milano-Bicocca, Italy
5 Institute of Biological Medicine, Milan, Italy
6 Division of Radiation Oncology, St.Gerardo Hospital, Monza, Milan, Italy

*Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italy; Fax: +390392332284, E-mail: p.lissoni@hsgerardo.org
Key words: Anticancer immunity, immunosuppression, T regulatory lymphocytes
Abbreviations: cytotoxic T lymphocyte-associated antigen-4, (CTLA-4); glucocorticoid-induced TNF-α receptor, (GITR); myeloidderived suppressor cells, (MDSC); NK cells, (CD16CD56); T cytotoxic lymphocytes, (CD8); T helper lymphocytes, (CD4); Tregulatory lymphocytes, (T-reg)

Received: 24 July 2008; Revised: 11 September 2008
Accepted: 12 September 2008; electronically published: October 2008

Summary

Several clinical investigations showed that the immune status is a prognostic variable in cancer patients, even tough the evaluation of the anticancer immunity is not generally considered in the medical oncology. Several immune parameters, including lymphocyte subsets and cytokine blood concentration, had been proposed to quantify the functional status of the anticancer immunity, but recent discoveries would suggest that the end-result of the various immune interactions is represented by a subtype of CD4 lymphocytes capable of suppressing the antitumor immune reaction, the so called T-regulatory lymphocytes (T-reg). This study was performed to detect T-reg count and percentage in solid tumor patients, in relation to tumor histotype, disease extension, lymphocyte sub-populations and cortisol circadian secretion. The study included 114 consecutive cancer patients affected by the most frequent tumor histotypes, 69 of whom showed a metastatic disease. In each patient we evaluated T-reg cells, identified as CD4+CD25+, in relation to T helper (CD4), T cytotoxic (CD8) and NK (CD16CD56) cells. Abnormally high values of T-reg cells were seen in 52/114 (46%) patients, and the percentage of high values of T-reg was significantly higher in metastatic patients than in non-metastatic ones. In contrast, no significant difference was seen in relation to tumor histotype. Patients with increased T-reg count had a significantly lower NK cell number. Finally no significant difference in T-reg number was seen between patients with altered or normal rithm of cortisol. The study confirmed that, irrespectively of tumor histotype the metastatic disease is associated with a progressive and increased T-reg generation, with a following suppression of anticancer immunity.

I. Introduction
At present, there is no doubt about the existence of a sub-type of T lymphocytes, the so-called T regulatory lymphocytes (T-reg), capable of suppressing the cellular immune responses,including the anticancer immunity (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006). However, the exact definition
of T-reg cells in terms of cell surface marker expression still remains controversial, particularly from a clinical point of view. All authors are in agreement to consider Treg lymphocytes as CD4+CD25+ cells, but at present it is still unknown whether the expression of CD4 and CD25 antigens may be sufficient to identify T-reg cells (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006), since several authors retain that the intracytoplasmatic expression of the FOX p3 protein is essential for the differentiation into T-reg cells (Ziegler, 2006; Zou, 2006).Recently, however, some preliminary observations would suggest that the cytoplasmatic expression of FOX p3 by CD4+CD25+ cells may be associated at least in some experimental conditions with a diminished, rather than with an enhanced immunosuppressive activity of T-reg cells (Siddiqui et al, 2007). In contrast, all authors agree that the expression of CD152 antigen, also called cytotoxic T lymphocyteassociated antigen-4 (CTLA-4) (Vasu et al, 2004), is fundamental for the immunosuppressive activity of T-reg cells (Takahashi et al, 2000), since the block of its expression by using anti-CTLA-4 monoclonal antibodies may abolish the suppressive activity of T-reg cells, with a following stimulation of the anticancer immunity in cancer patients (Knutson and Disis, 2007) and an enhanced incidence of autoimmune diseases in the healthy subjects (Lan et al, 2005). Therefore, the addition of a third marker, such as CD152 antigen, may allow to define a more homogeneous cell population provided by a regulatory activity with respect to the simple CD4+CD25+ expression (Dieckmann and Plottner, 2001). In fact, the suppressive regulatory action of CD4+CD25+CD152+ has appeared to be clearly higher than that played by the simple CD4+CD25+ T lymphocytes (Leong et al, 2006).This finding is not surprising, since the simple expression of CD25 marker, corresponding to the !-chain of IL-2 receptor, is not an exclusive characteristic of T-reg lymphocytes, but it is a non-specific property of the overall activated T lymphocytes (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006). At present, preliminary clinical studies would show that the percent of circulating CD4+CD25+ cells may be about 10% of the all CD4+ lymphocytes, and that of CD4+CD25+CD152+ cells may be about 40% of the total CD4+CD25+ cells, then the expected percent of CD4+CD25+CD152+ in the healthy subjects would be less than 5% of the total circulating CD4+ lymphocytes (Jago et al, 2004). Finally, the expression of glucocorticoidinduced TNF-α receptor (GITR) is also associated with an evident suppressive activity by T-reg lymphocytes (Kanamaru et al, 2004), which in fact are stimulated by
cortisol (Sthephens et al, 2004), that in contrast may inhibit the activity of the most other T lymphocytes, namely that of T helper lymphocytes, with a following diminished production of IL-2 (Claman, 1998). As far as the mechanisms responsible for T-reg-induced suppression of the anticancer immunity are concerned, several experimental observations have shown that T-reg cells may suppress the antitumor immune response through the release of immunosuppressive cytokines, namely IL-10 and TGF-β (Dieckmann et al, 2002), even though other authors would suggest that the suppressive activity of Treg cells on CD4+ and CD8+ lymphocyte activation may be relatively independent from the action of cytokines, by mainly requiring cell surface contact (Birebent et al, 2004). IL-2 has been proven to be essential for T-reg generation and some authors consider IL-2 as the main growth factor of T-reg lymphocytes (Antony and Restito, 2005), but more adequate studies have demonstrated that IL-2 may induce both stimulation and inhibition of T-reg generation and activation (Malek and Bayer, 2004). In fact, IL-2 has appeared to induce and promote T-reg differentiation only in the presence of TGF-β (Chen et al, 2003). Therefore, IL-2 would constitute the main human cytokine in influencing the characteristics of the anticancer immunity, since it may be responsible for both activation and suppression of an effective immune response against cancer cell proliferation and dissemination (Wang et al, 2001), namely depending on the whole status of the cytokine network, in particular on the presence or in the absence of adequate concentrations of TGF-β. In the absence of TGF-β, IL-2 stimulates the anticancer immunity, whereas it counteracts the generation of an effective antitumor immunity in the presence of TGF-β. In other words, IL-2 would physiologically control both tolerance and immunity, depending on the presence of TGF-β and other less known factors (Annunziato et al, 2002). In fact, under cancer immunotherapy with IL-2 the percent of T-reg cells has been shown to decrease in responding patients and to enhance in those with disease progression (Cesana et al, 2006). However, the regulation of T-reg functions does not depend only on immune factors, since it is also under a neuroendocrine control (Ji et al, 2004). In particular, cortisol has appeared to stimulate T-reg cell generation (Ji et al, 2004), with a following enhanced release of IL-10, by representing the main mechanism responsible for cortisol-induced immunosuppression. From a clinical oncological point of view, preliminary observations showed an enhanced percent of circulating CD4+CD25+ lymphocytes in cancer patients, namely in those with advanced disease (Sasada et al, 2003). The present study was performed to better establish which is T-reg behaviour in cancer patients in relation to both tumor histotype and disease extension.

II. Materials and methods
The study included 114 consecutive solid tumor patients with locally limited or metastatic disease, whose clinical characteristics are shown in Table 1. Lung cancer and gastrointestinal tumors were the most frequent neoplasms in our patients. For the immune detections, venous blood samples were collected in the morning after an overnight fast. Operable patients and metastatic patients were investigated before the surgical operation and before the onset of chemotherapy, respectively, in an attempt to exclude the possible influence of the various anticancer therapies on the immune status of patients.

In each sample, we have evaluated total lymphocyte count and the various lymphocyte subpopulations by a flow cytometric assay and monoclonal antibodies, including T helper lymphocytes (CD4), T cytotoxic lymphocytes (CD8), NK cells (CD16CD56), and T regulatory (T-reg) lymphocytes (CD4CD25). Normal values (95% confidence limits) of T-reg observed in our laboratory were below 240/mm3. Moreover, because of its importance in regulating lymphocyte functions and proliferation (Claman, 1998; Sthephens et al, 2004), the circadian rhythm of cortisol was also investigated by collecting blood samples at 8.00 A.M. and at 4.00 P.M., and cortisol serum concentrations were measured in duplicate by using the ECLA method. Data were reported as mean +/- SE, and statistically analyzed by the Student’s t test, the analysis of variance and the chi-square test, as appropriate.

III. Results
As reported in Table 2, an abnormally high number of T-reg was seen in 52/114 (46%) patients. Moreover, the percentage of cases with elevated number of T-reg observed in metastatic patients was significantly higher with respect to that found in non-metastatic patients (44/69 (64%) vs 8/45(18%), p < 0.01). Table 3 shows the mean number of T-reg and the mean percentages of T-reg with respect to both total lymphocytes and T helper (CD4+) lymphocytes observed in cancer patients in relation to their disease extension. The mean number of T-reg observed in metastatic patients was higher with respect to that found in patients with locally limited disease, without, however statistically significant differences. In contrast, the mean percentages of T-reg with respect to that of both lymphocytes and CD4 cells were significantly higher in metastatic patients than in the non-metastatic ones (p< 0.05 and p< 0.001,respectively). Moreover, within the metastatic group, patients with a normal lymphocyte count greater than 1500/mm3 showed a significantly higher mean number of T-reg with respect to the non-metastatic patients, whereas no difference was seen between nonmetastatic patients and metastatic patients with lymphocytopenia, consisting of lymphocyte count lower than 1500/mm3. In contrast, the mean percentages of T-reg with respect to total lymphocytes and CD4+ cells observed in both groups of metastatic patients with normal or low total lymphocyte count were significantly higher than in non-metastatic patients (lymphocytes: p< 0.025, CD4+ cells: p< 0.001).

Table 1. Clinical characteristics of 114 solid tumor patients

Table 2. Percentages of abnormally high values of CD4+CD25+ lymphocytes

* P < 0.01 vs non-metastatic patients

Table 3. Mean number of CD4+CD25+ lymphocytes and their mean percentages with respect to total lymphocytes and CD4+ lymphocytes in metastatic and non-metastatic patients

* p<0.05 vs non-metastatic patients; ** p<0.025 vs non-metastatic patients; *** p<0.01 vs non-metastatic patients; **** p<0.001 vs nonmetastatic patients

The mean counts of NK and CD8 cells in relation to that of T-reg are reported in Table 4. As shown, no significant difference in the mean number of CD8 lymphocytes was found between patients with normal or abnormally elevated number of T-reg. On the contrary, patients with elevated number of T-reg showed a significantly lower number of NK cells with respect to that found in those with normal T-reg count. Finally, Table 5 shows the circadian rhythm of cortisol in relation to total lymphocytes, CD4+ cells and T-reg mean number. A normal cortisol rhythm, with morning values greater at least than 50% with respect to the values occurring during the afternoon, was found in 85/114 (75%). Total lymphocyte and CD4+ cell mean numbers observed in patients with altered cortisol rhythm were significantly lower than those found in patients with normal cortisol circadianicity (p<0.01), whereas no significant difference was seen in the mean number of T reg. Figure 1 and Figure 2 illustrate T-reg mean numbers in relation to tumor histoptypes in the overall patients and with respect to their disease extension, respectively. No significant difference was seen in relation to tumor histotype. The highest values of T-reg were observed in pancreatic cancer patients, without however significant differences with respect to the overall other histotypes. The metastatic disease was associated with a higher number of T-reg with respect to the non-metastatic group in all tumor histotypes, even though a statistically significant differences occurred for the only breast cancer (p<0.05) and colorectal cancer (p< 0.01).

Table 4. Mean values of NK cells and CD8+ lymphocytes in cancer patients with normal or abnormally high values of CD4+CD25+ lymphocytes

* p<0.05 vs normal values of CD4+CD25+ lymphocytes

Table 5. Mean numbers of total lymphocytes, T helper (CD4+) lymphocytes and T regulator lymphocytes (CD4+CD25+) in relation to cortisol circadian secretion in cancer patients

* P<0.01 vs patients with altered cortisol rhythm

Figure 1. CD4+CD25+ lymphocyte mean number in relation to tumor histotype

Figure 2. CD4+CD25+ lymphocytes in relation to tumor histotype in metastatic and non-metastatic cancer patients

IV. Discussion
According to previous preliminary clinical investigations (Sasada et al, 2003; Cesana et al, 2006), this study confirms in a greater number of cancer patients that the metastatic disease is characterized by the evidence of an abnormally increased percentage of T-reg lymphocytes with respect to both total circulating lymphocytes and CD4+ lymphocytes. This finding does not seem to represent a specific characteristic of some tumor histotypes, then it could constitute a general alteration occurring during the progression of the neoplastic disease, by representing a fundamental immune parameter of cancer-related immunosuppression.

Several immune molecules have appeared to suppress the anticancer immunity, namely IL-6, IL-10, IL-1, TNF-α and TGF-β, but it seems that the common end result of their mechanisms of action may be represented by the stimulation of T-reg generation, with a consequent inhibition of the activation of an effective anticancer immune reaction. On the same way, several immune cells are able to suppress the anticancer immunity, including macrophages, T helper-2 lymphocytes and some myeloidderived suppressor cells, but also in this case they would act in a suppressive way by promoting the generation of Treg.

Then, the detection of T-reg amounts in terms of both absolute number and percentages with respect to total lymphocytes and CD4+ cells could constitute a simple and adequate clinical immune parameter to quantify the whole status of the anticancer immunity in the single cancer patient. Moreover, future clinical studies will be required to establish the possible prognostic significance of changes in T-reg percentage and number in relation to the anticancer efficacy of the various standard antitumor therapies. Moreover, it has to be remarked that T-reg lymphocytes would not represent the only immune cells involved in the suppression of the anticancer immunity. In fact, there is at least another fundamental immunosuppressive system, consisting of the monocytemacrophage cell lineage (Sica and Bronte, 2007). In more detail, it has been observed that the bone marrow may release myeloid precursors provided by suppressive activity on the antitumor immune response and defined as myeloid-derived suppressor cells (MDSC) (Kusmartsev and Gabrilovich, 2005). These cells have appeared to be characterized by the cell surface expression of GR-1, CD11b and CD80 antigens (Anderson et al, 2002; van Ginderachter et al, 2006). The myeloid suppressor cells would promote the generation and activation of T-reg lymphocytes, which at the other side would stimulate MDSC release from the bone marrow and M2 macrophage differentiation (Terabe et al, 2003; Wie et al, 2006).

Moreover, the myeloid suppressive cells would inhibit the anticancer immunity by promoting macrophage differentiation into the M2 sub-type (Ikemoto et al, 2003), which plays a clear inhibitory effect on the anticancer immunity, namely through the release of IL-6 (Ueno et al, 2000), whereas the M1 macrophage sub-type may either stimulate or suppress the antitumor immunity (Mantovani et al, 2004). M1 and M2 macrophage sub-types have appeared to be characterized by a high production of IL-12 or IL-10, respectively (Ueno et al, 2000).

Then, further studies by concomitantly evaluating T reg and MDSC count, will contribute to better define the immune mechanism responsible for the suppression of the anticancer immunity.

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A phase II study of anastrozole plus the pineal anticancer hormone melatonin in the metastatic breast cancer women with poor clinical status

Research Article (download PDF version)

Paolo Lissoni1*, Giuseppe Di Fede1, Antonio Battista2, Giusy Messina1, Remo Egardi1, Fernando Brivio3, Franco Rovelli1, Massimo Colciago4, Giuseppe Brera5

1 Institute of Biological Medicine, Milan
2 Azienda Sanitaria locale 2, Avellino;
3 Surgery Division, Bassini Hospital, Cinisello,Milan
4 I.N.R.C.A, Casatenovo, Lecco, Italy
5 Ambrosian University, Milan, Italy

dr. Paolo Lissoni

Received: 9 March 2009; Revised 1 April 2009;
Accepted: 13 April 2009; electronically published: 28 May 2009

Summary

The recent advances in the psychoneuroendocrinology have suggested the possibility to modulate tumor hormone dependency through a neuroendocrine approach. In particular, it has been proven that the pineal neurohormone melatonin (MLT) may stimulate estrogen receptor (ER) expression in breast cancer cells and inhibit the aromatase activity. On this basis, a study was planned to evacuate the efficacy of a concomitant treatment with the aromatase inhibitor anastrozole plus MLT in metastatic breast cancer. The study included 14 metastatic breast cancer women of poor clinical conditions with ER positive or unknown. Both anastrozole and MLT were given orally at a dose of 1 mg at noon and of 20 mg in the evening, respectively. The clinical response consisted of complete response in 2 and partial response in 6 patients. Then, an objective tumor regression was achieved in 8/14 (57%) patients, with a median duration of 26 months. No neoplastic cachexia occurred on treatment. This preliminary study shows that a neuroendocrine strategy with anastrozole plus the pineal hormone MLT may represent a new effective and well tolerated regimen in the treatment of metastatic breast cancer women, including those with poor clinical status, with therapeutic results apparently superior to those reported in the literature with the only aromatase inhibitor. Then, these results would justify further randomized studies of aromatase inhibitors with or without a concomitant administration of MLT, in an attempt to establish whether the pineal hormone may enhance the efficacy of the aromatase inibibitors in the treatment of human advanced breast cancer.

I. Introduction
Recent experimental studies have demonstrated that the hormone dependency is at least in part under a psychoneuroendocrine regulation (Cos et al, 2008;Grant et al, 2009). In particular, it has been shown that the pineal hormone melatonin (MLT), whose anticancer properties have been well demonstrated (Bartsch et al, 1981; Maestroni, 1993; Reiter et al, 2002), may in vitro stimulate estrogen receptor (ER) expression on breast cancer cell lines (Molis et al, 1995). Therefore, the hormone dependency of breast cancer cells would not depend only on intrinsic characteristics of cancer cells themselves, but also on host neuroendocrine regulation of tumor cell proliferation and differentiation (Bartsch et al, 2000). Moreover, cancer progression has been proven to be associated with pineal alterations, consisting of a progressive decline in MLT nocturnal production. (Maestroni, 1993). Therefore the advanced cancer would require a substitutive endocrine therapy with MLT (Bartsch et al, 1981; Maestroni, 1993). Previous preliminary clinical studies had already suggested that the concomitant administration of the pineal hormone MLT may apparently increase the efficacy of tamoxifen therapy in the treatment of metastatic breast cancer (Lissoni et al, 1995). Moreover, experimental studies have shown that the activity of aromatase enzyme, which is responsible for the peripheral production of estrogens from testosterone (Bagatell et al, 1994), is under a light/dark circadian rhythm (Bhatnagar et al, 1992). Because of the fundamental role of the pineal hormone MLT in the regulation of the daily photoperiod (Bartsch et al, 1981), it is possible to hypothesize that MLT may be involved in the control of the aromatase activity. In fact, recent studies have demonstrated an inhibitory action of MLT on the aromatase activity (Cos et al, 2005). This finding could reserve a prosiming application in the treatment of both early and advanced breast cancer. This statement is justified by the fact that the aromatase inhibitors represent a new class of agents in the endocrine treatment of breast cancer Plourde et al, 1994), with a potential efficacy superior to that achieved by the previous hormonal therapies with anti-estrogens, such as tamoxifene, even though tumor response rate obtained by the aromatase inhibitors are generally not greater than 40%. On this basis, a phase II study was planned in an attempt to evaluate the efficacy of a neuroendocrinotherapeutic regimen consisting of a concomitant administration of the aromatase inhibitor anastrozole and the pineal hormone MLT in metastatic breast cancer women with poor clinical conditions.

II. Materials and methods
The study included 14 consecutive metastatic breast cancer women (median age: 72 years, range 51-82), who were followed at Biological Medicine Institute in Milan, or at Health Local Unit 2 of Avellino, from Feb. 2002 to Sept. 2003. Eligibility criteria were, as follows: histologically proven metastatic breast cancer, measurable lesions, ER positive or unknown, no ability to tolerate chemotherapy because of age, low performance status (PS), important clinical illnesses other than cancer and/or heavy chemotherapeutic pre-treatments, no previous endocrine therapies for the metastatic disease, no double tumor and life expectancy less than 1 year. Previous heavy chemotherapeutic treatment consisting of at least 3 chemotherapeutic lines was made in 11/14 (79 %) patients. Dominant metastasis sites were, as follows: soft tissues:1; bone:1; lung:7 (neoplastic lymphangitis:2); liver:1; lung + liver:1; bone marrow:3. Time-span since first diagnosis of the primary tumor was 44 months (31-66 months). All patients had an acceptable social conditions. The minimum and median follow-up periods were 60 months and 72 months respectively. In all patients, in the case of disease progression, at least to other endocrine therapeutic lines with other aromatase-inhibitors were planned. The experimental protocol, wich was approved by the Health Direction of Biological Medicine Institute of Milan, was explained to each patient and informed consent was obtained. The treatment consisted of anastrozole at a dose of 1 mg/day orally at noon, plus MLT at 20 mg/day orally in the evening, generally half-hour before sleeping, to correct cancer progression-related decline in MLT night secretion (10). Patients were considered to be evaluable when they were treated for at least 3 consecutive months. The clinical response was evaluated according to WHO criteria. Complete response (CR) was the complete disappearance of all neoplastic lesions for at least 1 month. Partial response (PR) was a reduction greater than 50 % of the sum of all neoplastic lesions, for at least 1 month. Stable disesase (SD) was no increase or decrease greater tha 25 % of tumor volume. Progressive Disease (PD) was an increase in tumor volume greater than 25 % or the appearance of new neoplastic lesions. PS was assessed according to Karnofsky’s score, consisting of the evaluation of the quality of life in relation to patient activity and bed-rest period. ER was positive in 10 and unknown in the remaining 4 patients. The median PS was 80% (range 70-100). Data were statistically evaluated by the chi-square test and the Student’s t test, as appropriate.

Table 1: Clinical characteristics of metastatic breast cancer women and their clinical response (WHO criteria) to a neuroendocrine regimen consisting of anastrozole plus the pineal hormone melatonin.

III. Results
All patients were fully evaluable for the clinical response. The clinical characteristics of patients and their individual clinical response to the treatment are reported in Table 1. As reported, a complete response (CR) was achieved in 2/14 (14%) (soft tissues:1; lung lymphangitis:1). A partial response (PR) was obtained in other 6/14 (43%) (bone:1; lung:3; liver:1; bone marrow:1). Then, an objective tumor response (CR + PR) was reached in 8/14 (57%) patients. The median duration of response was 26 months (range 9-42 months). A stable disease (SD) was seen in other 4/14 (29%), with a median duration of 25 months (range 10-27). Therefore, a disease-control (DC:CR + PR + SD) was achieved in 12/14 (86%) patients, whereas the remaining 2/14 (14%) patients had a progressive disease (PD). No significant difference in tumor response rate was observed between patients with positive or unknown ER ( 6/10(60%) vs 2/4(50%) ). An overall survival at 1 year and at 3 year was achieved in 11/14 (79 %) and in 5/14 (36 %) patients, respectively. Moreover, 3/14 (21%) patients were still alive at 5 years. The treatment was well tolerated in all patients.

Moreover, most patients experienced a relief of asthenia under the treatment and in no patient the neoplastic cachexia occurred. Finally, an evident increase in PS mean values was achieved under treatment, even though it did not reach the statistical significance (86 ±5 vs 93 ± 4, mean ± SE).

IV. Discussion
The results of this preliminary phase II study, by showing a percentage of 1-year survival greater than 70% in patients with live expectancy less than 1 year, would suggest that a neuroendocrine regimen consisting of the aromatase inhibitor anastrozole plus the pineal neurohormone MLT may represent a new effective therapeutic strategy in the treatment of metastatic breast cancer women, also in patients with poor clinical conditions, who would not be able to tolerate the most aggressive therapies. The concomitant administration of the pineal hormone would seem to enhance the efficacy of the aromatase inhibitor in terms of objective tumor regressions with respect to the results commonly reported in the literature with the only aromatase inhibitor (Plourde et al, 1994), which are generally lower than 40%.

The time to progression would seem to be apparently increased by the concomitant treatment with MLT.This finding is not surprising, since MLT could enhance the therapeutic anticancer acitivity of the aromatase inhibitors by either exerting direct antiproliferative antitumor effects (Bartsch et al, 1981; Maestroni, 1993; Reiter et al, 2002), or further inhibiting the aromatase activity by acting on gene and oncogene expression (Molis et al,1995; Cos et al, 2005). In addiction, MLT appeared to stimulate ER expression of breast cancer lines, by transforming ER negative into ER positive breast cancer, as observed in experimental conditions (Danforth et al, 1983).

Since the prognosis of ER positive breast cancers is clearly better than that of ER negative ones, MLT could per se improve the clinical couse of mammary tumors. Finally, because of its interesting therapeutic efficacy as a supportive care (Reiter et al, 2002), MLT would be responsible for the evident improvement in the relief of asthenia and in preventing the occurrence of the neoplastic cachexia. On the other hand, because of the inhibitory effect of MLT (Grant et al, 2009; Reiter et al, 2002) on cancer cell proliferation, the anticancer activity of this polyendocrine regimen would be due not only to an indirect effect, depending on a diminished estrogen production following aromatase enzyme inhibition, but also on a direct inhibition of cancer cell growth, due to MLT itself. Therefore, the results of this preliminary study may justify further clinical randomized investigations with the only aromatase inhibitor versus the concomitant treatment with MLT, in an attempt to confirm the ability of the pineal hormone to enhance the antitumor properties of the aromatase inhibitors in the treatment of metastatic breast cancer women with poor clinical conditions.

References
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Bartsch C, Bartsch H (1981) effect of melatonin on experimental tumors under different photoperiods and times of administration. J Neural Transm 52:269-279.

Bartsch H, Buchberger A, Franz H,Bartsch C,Maidonis I,Mecke D, Bayer E (2000) Effect of melatonin and pineal extracts on human ovarian and mammary tumor cells in a chemosensitivity assay. Life Sci 67:2953-2960.

Bhatnagar AS, Muller P, Schenkel L, Trunet PF, Beh I, Schieweck K. (1992). Inhibition of oestrogen biosynthesis and its consequences on gonadotrophin secretion in the male. J of Steroid Biochemistry and Molecular Biology;41:1021-1027

Cos S, Martinez-Campa C,Mediavilla MD, Sanchez-Barcelo E (2005). Melatonin modulates aromatase activity in MCF-7 human breast cancer cells. J Pineal Res 7:136-142.

Cos S, Gonzales A, Martinez-Campa C, Mediavilla MD, Alonzo-Gonzales C, Sanchez-Barcelo EJ (2008): Melatonin as a selective estrogen enzyme modulator. Curr Cancer Drug Targets. Dec 8(8): 691-702.

Danforth DN, Tamarkin L, Lipmann LE: (1983) Melatonin increase oestrogen receptor binding activity of human breast cancer cells. Nature, 305:323-325.

Grant S.G, Melan MA, Latimer JJ, Witt-Enderby PA (2009) : Melatonin and Breast cancer : cellular mechanism, clinical studies and future perspective. Expert Rev Mol Med 11:e5

Lissoni P, Barni S, Meregalli S, Fossati V, Cazzaniga M, Esposti D, Tancini G (1995) Modulation of cancer endocrine therapy by melatonin:a phase II study of tamoxifen plus melatonin in metastatic breast cancer patients progressing under tamoxifen alone. Br J Cancer 71:854-856.

Maestroni GJM (1993) The immunoneuroendocrine role of melatonin. J Pineal Res 14:1-10.

Molis TM, Spriggs LL,Jupiter Y,Hill SM (1995) Melatonin modulation of estrogen-regulated proteins, growth factors,and protooncogenes in human breast cancer. J Pineal Res 18:93-103.

Plourde PV, Dyroff M, Dukes MD (1994) Arimidex: a potent and selective fourth generation aromatase inhibitor. Breast Cancer Res Treat 30:103-111.

Reiter RJ, Tan DX, Sainz RM, Mayo JC, Lopez-Burillo S. (2002) Melatonin:reducing the toxicity and increasing the efficacy of drugs. Pharm Pharmacol 54:1299-1321

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2009: Lissoni P; Fumagalli L; Brivio F; Rovelli F; Messina G; Di Fede G; Colciago M; Brera G
Division of Radiation Oncology, Milan, Italy

Biotherapy with the pineal hormone melatonin plus aloe and myrrh tincture in untreatable metastatic cancer patients as an essence therapy of cancer

Research Article

P. Lissoni1,*, F. Rovelli1, G. Messina2, F. Brivio3, B. Boniardi1, G. Porro1, L.Vigore4, G. Di Fede1, P. Marchiori1, G. Brera5

1 Institute of Biological Medicine, Milan, Italy
2 Psychiatric Division, Policlinico Hospital, Milan
3 Division of Surgery, Bassini Hospital, Cinisello,Milan;
4 Laboratory of Immunomicrobiology,San Gerardo Hospital, Monza, Milan;
5 Ambrosian University, Milan, Italy.

*Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S. Gerardo, 20052 Monza, Milano, Italia. Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org
Key words: Aloe Vera, Melatonin, Mirrh, and Anticancer Immunity
Abbreviations: Melatonin (MLT), complete response (CR), partial response (PR), stable disease (SD), disease control (DC), progressive disease (PD), T helper lymphocytes (TH, CD4+), T regulatory lymphocytes (T reg, CD4+ CD25+)

Received: 30 July 2009; Revised: 18 October 2009
Accepted: 20 October 2009; electronically published: December 2009

Summary

Background: The recent advances in understanding the immunobiological interactions responsible for cancer progression have allowed us to define the mechanisms of action of some plants, whose antitumor properties were already known by the popular Medicine, in particular Aloe and Myrrha, whose mixture was already therapeutically utilized more than 2000 years ago by the Essence medicine. Moreover, some endogenous natural substances, namely the main hormone produced by the pineal gland melatonin (MLT) may also play anticancer activity. On this basis, a study was performed with a biological regimen consisting of MLT, Aloe and Myrrha in untreatable metastatic cancer patients with life expectancy lower than 1 year. Methods: The study included 35 patients. MLT was given orally at 20 mg/day in the evening and a mixed Aloe and Myrrha tincture was administered at a dose of 5 ml/thrice daily. Results: The clinical response consisted of complete response (CR) in 1, partial response (PR) in 2, stable disease (SD) in 19 patients, whereas the remaining 13 patients had a progressive disease (PD). Thus, a disease control (CR + PR + SD) was achieved in 22/35 (63%)patients. Moreover, a survival longer than 1 year was achieved in 17/35 (49%) patients. Finally, DC was associated with an evident improvement in the immune status, namely consisting of a decrease in the number of T regulatory lymphocytes, which are the main cells responsible for the suppression of the anticancer immunity. Conclusion: This preliminary study shows that a biological anticancer regimen consisting of the pineal hormone MLT in association with Aloe and Myrrha mixture, already known at the times of the Essence medical tradition, may induce a control of the neoplastic disease by stimulating the anticancer immunity, in a relevant percentage metastatic cancer patients, who did not respond to the conventional anticancer treatments and for whom no other standard therapy was available.

References
Davis RH, Parker WL, Sampson RT, Murdoch DP. Isolation of a stimulatory system in an Aloe extract. J Am Pediatr Med Assoc 1991; 81:473-8.

Capasso F, Borrelli F, Capasso R, Di Carlo G, Izzo AA, Pinto L et al. Aloe and its therapeutic use. Phytother Res 1998; 12:124-7.

Vogler BK. Aloe Vera: a systematic review of its clinical effectiveness. B J Gen Pract 1999; 49:823-8.

Claeson P, Zygmunt P, Hogestatt ED. Calcium antagonistic properties of the sesquiterpene T- cadinol. Pharmacol Toxicol 1991; 69:173-7.

Qureshi S, Al-Harbi MM, Ahmed M, Raza M, Giangreco AB, Shah AH. Evaluation of the genotixic, cytotoxic and antitumor properties of Commiphora molmol using normal and Erlich ascites carcinoma cell-bearing Swiss albino mice. Cancer Chemother Pharmacol 1993; 33130-8.

Blazquez C, Casanova ML, Planas A, Del Pulgar TG, Villanueva C, Fernandez-Acenero MJ et al. Inhibition of tumor angiogenesis by cannabinoids. FASEB J 2003; 17:529-31.

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Iguchi H, Kato KI, Ibayashi H. Age-dependent reduction in serum melatonin concentrations in healthy subjects. J Clin Endocrinol Metab 1982; 55:27-9.

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Synchronization of Cortisol Circadian Rhythm by the Pineal Hormone Melatonin in Untreatable Metastatic Solid Tumor Patients and its Possibile Prognostic Significance
on Tumor Progression

Research Article (download PDF version – complete)

FERNANDO BRIVIO1, LUCA FUMAGALLI1, GABRIELE FUMAGALLI2, SIMONETTA PESCIA2, RINALDO BRIVIO2, GIUSEPPE DI FEDE3, FRANCO ROVELLI2 and PAOLO LISSONI3

1 Surgical Division, Bassini Hospital, Cinisello Balsamo, Milan, Italy
2 Department of Clinical Pathology, S.Gerardo Hospital, Monza, Italy
3 Institute of Biological Medicine, Milan, Italy

*Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italy; Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org

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Evaluation of neopterin and ALCAT test in patients with food intolerance

d.ssa Berardi della IRCCS Policlinico San Matteo di Pavia reparto di dermatologia

Background:
Specific foods and food additives can induce adverse gastrointestinal (GI) and cutaneous reactions, including food allergies that involve an abnormal immunologic reaction to food proteins or food intolerance which is not pathogenetically immune-mediated.

Neopterin is synthesized by human monocyte-derived macrophages upon stimulation with interferon-gamma (IFN-gamma). Measurement of neopterin concentrations is useful for monitoring cell-mediated immune activation as there is a positive direct relationship between neopterin levels and severity of immune-mediated disorders. The aim of this study was to evaluate the neopterin serum levels in a group of patients with food intolerance.

Methods:
A group of forty-six patients (40 females, 6 males, median age 33 years) affected by cutaneous disease (27%) or GI symptoms (73%) consequent to food ingestion were tested by ALCAT and neopterin serum levels were measured.

The ALCAT test utilizes electronic haematology instrumentation and computerized data analysis to measure volumetric shifts in peripheral blood cells following incubation with food antigens.
Results are expressed in terms of percent change for cell volume and number. The degree of reactivity was determined by comparing a baseline distribution curve (of WBC) against the distribution curve generated by the analysis of each test agent/blood sample, and calculating the absolute differences between the curves and the standard deviation (SD). Any reactivity under 2 SD was considered non-reactive (negative) and these foods are allowed in the diet.

The Neopterin-MW EIA kit (DRG Instruments GmbH) employed an enzyme immunoassay technique to measure neopterin in a serum sample and was performed according to the Manufacturer’s Instructions and expressed as ng/mL.

Neopterin values were reported as medians with first and third quartiles (interquartile range). ALCAT test data were described as a percentage of non-reactive foodstuffs. Correlation between neopterin serum levels and percentage of non-reactive food was evaluated by means of the Spearman’s correlation coefficient (r).

Results:
The neopterin median value was 2 ng/mL (25°th-75°th: 1.50-2.25 ng/mL). There was a significant positive relationship between neopterin serum levels and percentage of non-reactive foodstuffs (p=0.05, r=0.302).

Conclusion:
This preliminary study confirms that food intolerance is not provoked by immunological mechanisms.

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Psychoneuroendocrine Modulation of Regulatory T Lymphocyte System: In Vivo and In Vitro Effects of the Pineal Immunomodulating Hormone Melatonin

Research Article (download PDF version – complete)

LUIGI VIGORÉ1, GIUSY MESSINA2, FERNANDO BRIVIO3, LUCA FUMAGALLI3, FRANCO ROVELLI4, GIUSEPPE DI FEDE4 and PAOLO LISSONI4

1 Laboratory of Immunomicrobiology, S. Gerardo Hospital, Monza, Milan, Italy
2 Psychiatric Division, Polyclinic Hospital, Milan, Italy
3 Surgical Division, Bassini Hospital, Cinisello, Milan, Italy
4 Institute of Biological Medicine, Milan, Italy

*Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italy; Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org

Abstract

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.

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