Soluble CD30: a biomarker for evaluating the clinical risk versus benefit of IFNbeta1A treatment in multiple sclerosis patients.

Aberrant redox regulation occurs in immune and neurological pathologies, hence targeting the pathways involved in the regulation of the redox system could provide further insights into these diseases and open up new avenues for therapy. Soluble (s) CD30 is of key clinical importance in this respect, as its levels reflect the functionality of the CD30 receptor (CD30R), the specific lymphocyte receptor for thiol disulfide/oxidoreductase thioredoxin 1 (Trx1) which is known to regulate important immune and neurological processes. Increased levels of sCD30 appear to be a common element of oxidative stress, immunological alterations and neurological deficit, therefore these increases could be used as a clinical biomarker and target for therapy. We targeted sCD30 in our study of dendritic cell (DC) regulation of the T helper (Th) cell network in multiple sclerosis (MS) patients, as abnormalities in T regulatory (Treg)/Th1/Th17 pathways contribute to the pathogenesis of this immunological/neurological disease. DC profiles in Treg/Th1/Th2/Th17-types of cytokine production in culture supernatants were used as they determine the type of Th differentiation. Our results show that sCD30 levels increase significantly in MS patients, reflecting the disruption in the regulation of the Treg/Th1/Th17 cell network. A fall in the level of soluble CD30, induced by IFNbeta1a therapy, opposed the increase of neurological deficit through increasing IL10 and TGFbeta levels, thus re-establishing network homeostasis but only when this was accompanied by an increase in IL12p70 levels. Since IL12p70 cytokine production is regulated by Trx1, our results indicate that redox system alterations may be the cause of IFNbeta1a therapeutic inefficacy. We conclude that an increase in the level of IL10, TGFbeta and IL12p70 and a fall in the level of sCD30 represent a means of evaluating the clinical risk/benefit of IFNbeta1a treatment.

Cell cycle control in cellular homeostasis during the immune response: interactions between TH1, TH2 cytokines, and Bcl2 and p53 molecules.

Cytokine regulation of lymphocyte survival may play an important role in the control of the cell cycle during the immune response both in health and disease. Expression of the Bcl2 gene promotes cell survival by countering apoptosis stimuli. The p53 protein has been implicated in the control of the cell cycle, in the synthesis and repair of DNA and in programmed cell death. TH1 and TH2 cytokines exert a mutual cross-regulation on the precursors of TH1- or TH2-type effector cells which are important mediators in directing the immune system towards the appropriate response. TH1 and TH2 cytokines have also been implicated in the modulation of the expression of cell cycle regulator genes. Therefore, the study of the relationships between TH1 and TH2 cytokines and Bcl2 and p53 molecules in healthy subjects could lead to a better understanding of the physiological regulation of the immune response and identify markers for prognostic and diagnostic indices and biotherapeutic treatment. We determined the serum levels of cytokines (IL2, IFN gamma, IL4, IL10, IL5, IL6, IL1 beta, TNF alpha, IL8), soluble receptors (sIL2R, sIL6R), Bcl2-protein and p53-antibody in a group of healthy subjects. Multivariate statistical analyses were used to study the cytokine network relationships with Bcl2-protein and p53-antibody, as they allow a simultaneous evaluation of all variables which reflects the physiological situation. Our overall results suggest that relationships exist between TH1 and TH2 cytokines and the Bcl2-protein and p53-antibody in physiological conditions. This information could now be used in experimental studies to create diagnostic and prognostic indices for the monitoring of health and disease.

Simultaneous measurement of soluble carcinoembryonic antigen and the tissue inhibitor of metalloproteinase TIMP1 serum levels for use as markers of pre-invasive to invasive colorectal cancer.

Matrix metalloproteinases (MMP) are members of a multigene family of zinc-dependent enzymes involved in the degradation of extracellular matrix components. Cancer research suggest that MMP and tissue inhibitors of metalloproteinases (TIMP) may be involved in disease progression; these enzymes could therefore be used as markers in cancer prevention programmes and for clinical monitoring. To establish whether MMP and TIMP can be used effectively as markers we determined serum levels of MMP1 and TIMP1, and studied the relationships between these enzymes and the stage of disease. The potential diagnostic and prognostic value of serum level measurements of MMP1 and TIMP1 was evaluated by comparing them with serum levels of soluble carcinoembryonic antigens (sCEA) and p53 antibodies. Our overall results indicate that simultaneous measurements of serum sCEA and TIMP1 in patients with colorectal cancer could be used as prognostic and diagnostic markers for disease progression from the pre-invasive nodal phase to the invasive phase (stages I, II to III, IV). In addition, serum levels of TIMP1 could be used as a selective marker for metastatic disease (stage III to IV). In fact, the 95% confidence interval of the serum levels of sCEA at stage III (18.4 < or = sCEA < or = 68.6 ng/ml) and TIMP1 at stage IV (1620 < or = TIMP1 < or = 3906 ng/ml) identified statistically significant ranges of values (sCEA P = 0.02, TIMP1 P = 0.02), which may be useful in the monitoring of patients at these disease phases. More specifically, our data suggest that, when the serum level of sCEA is below 18.4 ng/ml and the level of TIMP1 below 1620 ng/ml, there is a 95% probability that the disease is in the pre-invasive nodal phase; when the serum level of sCEA falls between 18.4 ng/ml and 68.6 ng/ml and the level of TIMP1 is below 1620 ng/ml, there is a 95% probability that the disease is in the phase when lymph node infiltration occurs; when the level of sCEA is above 68.6 ng/ml and the level of TIMP1 is at least 1620 ng/ml, there is a 95% probability that the disease is in the metastatic phase.

The TH1 and TH2 cytokine network in healthy subjects: suggestions for experimental studies to create prognostic and diagnostic indices for biotherapeutic treatments.

In vivo and in vitro studies have demonstrated the selective regulatory effect that TH1 and TH2 cytokines reciprocally exert in the regulation of the polarization of precursor cells into TH1 or TH2 types. The study of the network relationships between TH1 and TH2 (TH1/TH2) cytokines in healthy subjects could lead to a better understanding of how the physiological network of cytokines regulates the immune response. Such study could lead to gain suggestions for follow-up experiments to create prognostic and diagnostic indices for biotherapeutic treatments of patients. Hence we determined serum levels (environment network) and PBMC production (cellular network) of IL2, IFN gamma, IL4, IL6 and IL10 in the peripheral blood of healthy subjects; these cytokines made up our networks under basic conditions. Both men and women were studied as hormones can influence the polarization of TH1 and TH2 cells. Cytokines within the physiological network function simultaneously so multivariate statistical methods were used to study TH1/TH2 relationships. The use of mathematical modelling is the only effective way of studying the immune system as a whole. The physiological TH1/TH2 network under activation conditions was evaluated by incorporating: sIL2R and sIL6R into the basic environment network model and the production levels of cytokines by PBMC after PHA stimulus, into the basic cellular network model. The influence of APC was evaluated by adding: serum levels of TNF alpha and IL1 beta to the environment network model, and production levels of IFN gamma, IL10 and IL6, after stimulus with LPS, to the cellular network model. Our results led us to hypothesize that the physiological network of TH1/TH2 cytokines regulates TH polarization by means of specific relationships between TH1 and TH2 cytokines, which may be different in men and women. These relationships could be studied experimentally to create prognostic and diagnostic indices for more efficient prevention programs and biotherapeutic treatments of patients.

Relationships between the activity of MMP1/TIMP1 enzymes and the TH1/TH2 cytokine network.

The evaluation of the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) would appear to be important in cancer patients. Since the activity of these enzymes is regulated at the gene level by cytokines, we studied the serum relationships between MMP1/TIMP1 and the network of TH1/TH2 cytokines in healthy subjects to better understand how the physiological network of cytokines regulates MMP1/TIMP1 activity. Such a study could lead to suggestions for follow-up experiments to create prognostic and diagnostic indices for more efficient disease prevention programs and biotherapeutic treatments of patients. For this purpose, we determined serum levels of MMP1, TIMP1 and interleukin (IL)2, interferon (IFN) gamma, IL4 and IL10 in both healthy men and women (men and women were analyzed separately as hormones are one of the non-cytokine regulatory factors of TH1 or TH2 polarization). These cytokines make up our basic network. Cytokines within the physiological network function simultaneously so mathematical models of multivariate statistical methods were used to study MMP1/TIMP1 and TH1/TH2 network relationships. It has been suggested that mathematical modeling is the only effective way of studying the immune system as a whole. The influence of network activation, antigen presenting cells, antibody response and chemokines on MMP1/TIMP1 balance was also studied. Network activation was evaluated by measuring the levels of soluble IL2 receptors (sIL2R) and sIL6R; the influence of antigen presenting cells was evaluated by measuring serum levels of tumor necrosis factor alpha (TNF alpha) and IL1 beta; antibody response was evaluated by measuring IL5 and IL6 serum levels and the influence of chemokines was evaluated by measuring serum levels of IL8. Our overall results suggest that there are relationships between the activity of MMP1/TIMP1 and the TH1/TH2 network in physiological conditions. These data may be useful in gaining a clearer insight into how the two systems interact and hence regulate the physiological homeostasis. Therefore, this paper provides suggestions for experimental studies on MMP1/TIMp1 enzymes and TH1/TH2 cytokines to create clinical and prognostic markers for patient evaluation.

The significance of an increase in soluble interleukin-2 receptor level in colorectal cancer and its biological regulating role in the physiological switching of the immune response cytokine network from TH1 to TH2 and back.

Current research has still not clarified the biological role of soluble interleukin(IL)-2 receptor (sIL-2R) and the significance of its increase in the serum of colon cancer patients compared to healthy subjects. To address these questions at the immunological level in a group of patients and healthy subjects, we determined the sIL-2R level in the serum and its release from peripheral blood mononuclear cells (PBMC) as a function of tumour necrosis factor (TNF) alpha, IL-1 alpha, IL-1 beta, IL-2, interferon (IFN) gamma, IL-4, IL-6 and IL-10 levels in the serum and PBMC production; and PBMC proliferative responses to IL-2, IL-4 and anti-CD3 monoclonal antibody (CD3), variously combined. The level of sIL-2R in patients' serum was higher than in healthy subjects and correlated with the stage of advancement. Moreover, while in healthy subjects the serum level of sIL-2R was not significantly correlated with other parameters, in patients it was positively related to IL-4, IL-6 and IL-10 serum levels, PBMC IL-4 production and to the PBMC proliferative response to CD3 and CD3 + IL-2; it was negatively correlated to IL-2 serum level and IL-1 beta PBMC release. A negative connection between IFN gamma serum level and the PBMC production of sIL-2R was also found. This suggests that the increase of sIL-2R in the serum of patients, compared to healthy subjects, is involved in the inappropriate expansion of the T helper (TH2) suppressive immune response, which we previously reported. The multivariate statistical method supported the above suggestions and we also found that, in healthy subjects, the up- and down-regulation of sIL-2R in the serum within the physiological ranges seems to have a regulating role in the relationships between TNF alpha, IFN gamma and IL-4, IL-6, contributing to the operation of the cytokine network between TH1 and TH2 cells. However, in patients compared to healthy subjects the increased sIL-2R serum level seems to direct the immune response towards a suppressive type, which may be due to an alteration in the above-mentioned physiological regulating role.

Disease stage prognostic indices in the early clinical screening of colorectal cancer patients.

It is a truism to state that in cancer the extent to which the disease has spread (stage) is probably the most important factor determining patient prognosis and must be given prime consideration in evaluating and comparing different therapeutic regimes. Because of this, clinical screening tests (such as a rectal exam proctoscopy and colonoscopy), if tissue that is not normal is found, should include analyses contributing to the patient disease stage classification. However, the difficulty involved in this, principally due to the absence of reliable early prognostic indices of the disease stage, makes the cancer patients' treatment full of problems and risks. By a retrospective statistical study on pre-surgery peripheral blood immunological parameters of our groups of colorectal cancer patients and healthy subjects, we evaluated our previously suggested possibility of defining stage prognostic indices by parameter blood range values, evaluating their ability in the stage classification compared to the pTNM method. We have investigated the serum levels of various cytokines and cytokine receptors, leukocyte surface markers, peripheral blood mononuclear cell (PBMC) cytokine production and PBMC proliferative response. Statistically significant correlations between a variety of these immunological parameters and the disease stage were found, but as a clinical patient screening of all parameters is quite expensive, to identify the greatest stage weighting parameter and the respective blood range values, we performed a multivariate statistical analysis. We found that the blood ranges of IL-4 serum level and the PBMC proliferative response to anti-CD3 monoclonal antibody (mCD3) stimulus may be reliable prognostic indices which may contribute to an early disease stage classification in colorectal cancer patients, since they seem to be valid as the pTNM method. Moreover, as the immunological prognostic indices could be a useful tool to evaluate the patient immune response, they may also improve the definition of the patient's stage classification for the selection of treatment and restaging procedures for the evaluation of the treatment benefit and recurrent disease.

Bcl-2 and drugs used in the treatment of cancer: new strategies of biotherapy which should not be underestimated.

The theory that an imbalance in the control of the cell cycle contributes to the appearance and progression of neoplastic disease is gaining more ground all the time. This new line of research into tumor disease is a result of the progress made in the comprehension of cell death (apoptosis) and the discovery of alterations in the apoptotic pathway in patients with cancer, which have also been correlated to disease mechanisms. Alterations in the cycle of events that brings about apoptosis can result in tumor cells resistant to chemotherapy. In fact one of the inherent risks of chemotherapy is the generation of new, more aggressive, clonal variants and destruction of healthy cells with deleterious effects on the organism. This review examines the results of studies concerning the identification of the alterations in apoptotic mechanisms in carcinogenesis and the mechanisms governing their regulation. The aim was to evaluate if such data could be of use in identifying drugs able to improve cancer treatment.

The sCEA molecule suppressive role in NK and TH1 cell functions in colorectal cancer.

The soluble form of carcinoembryonic antigen (sCEA), an oncofetal glycoprotein, is frequently produced by human epithelial-tumor cells, particularly of colorectal origin, and evaluated as a prognostic index of tumor progression and patient survival. sCEA molecules are often present at high concentrations in the peripheral blood of colorectal cancer patients, but the function and significance of this are not well understood. Reported data have demonstrated that sCEA can interfere in NK-cell/tumor-cell interaction by drastically reducing the lysis of tumor cells in a dose-dependent manner and can also suppress T and B cell functions. The aim of our study was to evaluate this situation in colorectal cancer by determining peripheral blood immunological parameters in a group of patients and healthy subjects. We evaluated the interleukin (IL)-2, interferon (IFN) gamma, IL-4, sIL-2R and IL-10 levels in the serum and the release of IFN gamma, IL-4 and IL-10 from peripheral blood mononuclear cells (PBMC); the PBMC expression of CD3, CD16 and CD19 phenotypic antigens; the PBMC proliferative responses to IL-2, IL-2 + anti-CD3 monoclonal antibody (mCD3) and mCD3. The statistical evaluation of our overall results strongly indicates that the high level of the sCEA molecules in the patient's serum might act as a suppressive factor for NK and TH1 immunocompetent cells. This may be the cause of sCEA involvement in tumor progression, and indicates the possibility of an improvement in cancer treatment through its manipulation.

IL-10 and sIL-2R serum levels as possible peripheral blood prognostic markers in the passage from adenoma to colorectal cancer.

The pathogenesis of cancer is currently under intense investigation to identify reliable prognostic indices for the early detection of disease. Adenomas have been identified as precursors of colorectal cancer and tumor establishment, and disease progression has been found to reflect a malfunction of the immune system. We previously indicated the investigation of cytokine serum levels in these patients as a useful and non-invasive tool for the study of the disease progression and an imbalance at TH1 and TH2 cell levels was also found. Moreover, the soluble form of interleukin (IL) 2 receptor (sIL-2R) level is an in vivo marker of T cell activation and is used to monitor the activation of the immune system. We therefore performed an immunological study on a group of healthy subjects, subjects with adenomas, and colorectal cancer patients to identify peripheral blood invasiveness markers in the progression from normal mucosa through adenoma to tumor. In this paper we evaluated the relationships between serum levels of interleukin IL-2, sIL-2R, interferon (IFN) gamma, IL-4, IL-6, IL-10 and sICAM-1 and their networks. Our overall data indicate that in the normal mucosa through adenoma to tumor progression, the host immune response proceeded from a TH1 cell-mediated immune response type (healthy subjects) to a type with TH2 suppressive characteristics (adenoma subjects and cancer patients). However, in the adenoma subjects there was no IL-10 or sIL-2R involvement, while these parameters were implicated in the cancer patients' immune responses. Moreover, a concurrent augmentation of sIL-2R and IL-10 levels seems to be prognostic for the passage from adenoma to cancer, and the sIL-2R and sICAM-1 molecules appear to be involved in the invasiveness mechanisms.

Blockade of N-methyl-D-aspartate receptor prevents hypoxic neuronal death and cytokine release.

Neuronal mortality, interleukin-1 beta (IL1 beta) and tumor necrosis factor-alpha (TNF alpha) release were measured in hypoxic hippocampal neuronal cultures. Release of IL 1 beta and TNF alpha was already observed in normoxic cultures, but after hypoxia it was increased approximately 2-fold. Pretreatment with 2-amino-5-phosphonovaleric acid (APV), the N-methyl-D-aspartate (NMDA) receptor antagonist, not only decreased neuronal mortality as expected, but also dramatically lowered cytokine release. However, there was no relationship between the neuronal mortality and the release of each cytokine both in untreated hypoxic cultures and in APV-pretreated ones. We conclude that IL 1 beta and TNF alpha release in hypoxia are dependent on the activation of the NMDA receptor, but that this is not the main mechanism of hypoxia damage in in vitro neuronal cultures.

The role of the soluble CD30 serum level in colorectal cancer: a possible marker for a patient subset which could benefit from IL-2 biotherapy.

On the basis of our previous data suggesting an impairment in host immune response in colorectal cancer caused by an inappropriate switch from TH1 towards TH2 cells, we investigated the role of the soluble CD30 (sCD30) in this disease, as this molecule was found related to immune responses characterized by the activation of a prevalence of TH2 cells. We studied a group of healthy subjects and colorectal cancer patients determining the sCD30 serum level and the following immunological parameters: s interleukin-2 receptor (sIL-2R), IL-2, interferon (IFN) gamma, IL-6, IL-4 and IL-10 levels in the serum and peripheral blood mononuclear cell (PBMC) production; PBMC proliferative responses to IL-2, anti-CD3 monoclonal antibody (CD3) and IL-2 + CD3. Our overall data indicate that in colorectal cancer the sCD30 serum level is also linked to a prevalence of the TH2 immune response activation. However, the Multivariate statistical study underlines that the sCD30 level is principally related to the IL-6 TH2 cytokine. Moreover, it suggests that in colorectal cancer, the sCD30 level might be a marker for identifying a patient subset in which IL-2 biotherapy treatment could contribute to the restoration of the impaired immune system.

Prognostic significance of immunological evaluation in colorectal cancer.

According to the concept that tumour establishment and progression generally reflects a malfunction of the immune system, we have investigated the prognostic significance of immunological parameters in correlation to stage progression in colorectal cancer. In patients and healthy subjects as control group, we determined: serum levels of interleukin (IL)-2, interferon (IFN) gamma, IL-4, IL-6, IL-7, IL-8, tumor necrosis factor (TNF) alpha cytokines and soluble IL-2 receptor (sIL-2R), CD30 (sCD30), ICAM-1 (sICAM-1) molecules, phenotype of peripheral blood mononuclear cells (PBMC); PBMC proliferative response to IL-2, IL-4 and anti-CD3 monoclonal antibody (anti-CD3) variously combined. Our results show that, compared to healthy controls, the group of all patients, but interestingly, also the groups of patients at the various stages of the disease, seem to have different values of these immunological parameters. Since tumour invasion and metastasis are the major causes of cancer treatment failure the early recognition of preinvasive states could lead to an improvement in prognosis. For this purpose our results might be especially useful in making prognostic and diagnostic indices in this neoplasy to identify patients at risk for tumour detention and the patient condition concerning disease progression by a non-invasive method. Moreover, this evaluation which contributes to identify the damage in the patient immune response to tumor could be helpful in identifying the therapeutic substances which might switch this response from being unproductive to productive. Thus, our data leads us to indicate that it might be possible to define reliable prognostic and diagnostic indices in colorectal cancer from the extension of this immunological study by the evaluation of these and other parameters.

Necessity of biotherapeutic treatments inducing TH1 cell functions in colorectal cancer.

Our previous data on colorectal cancer suggest that there are faults at the level of mechanisms of the proliferative responses of patients peripheral blood mononuclear cells (PBMC) to the interleukin (IL)-2 and IL-2 PBMC production, which increase with the stage advancement. The damages in the proliferative response seem to be eliminated by the costimulator effects of the signals produced by the anti-CD3 monoclonal antibody (antiCD3), and the disregulation in TH subsets of CD4+ T cells with a malfunction of TH1 cells and an expansion of TH2, might contribute to this situation. So, by using biotherapeutic treatments to allow the generation of productive immune response in these patients it is essential to identify the defect in their immune system to discover how these mechanisms should be appropriately manipulated in vivo to switch their immune response from a non-productive to a productive one. We have studied this in a group of patients and healthy subjects as the control group, performing their immunological evaluation by determining these parameters: serum levels of IL-2, interferon (IFN) gamma, IL-4, IL-6, IL-7, IL-8, tumour necrosis factor (TNF) alpha, soluble IL-2 receptor (sIL-2R), intercellular adhesion molecule 1 (sICAM-1) and CD30 (sCD30) molecules; PBMC phenotypic antigens expression (CD3, CD4, CD8, CD19, CD16, CD56, CD57, CD25) on peripheral blood mononuclear cells (PBMC); proliferative response of PBMC to IL-2, IL-4 and anti-CD3 monoclonal antibody (antiCD3). Moreover, since mutant c-Ki-ras oncogene is a very frequent finding in colorectal cancers and there are indications which suggest its involvement in tumour progression, the analysis of c-ki-ras codon 12 and 13 were determined and the statistical evaluation of the above immunological parameters were performed by comparing the patient groups with (M+) and without (M-) these mutations with each other, and with the healthy group. The results underline the necessity of biotherapeutic treatments inducing TH1 cell functions in these patients. Moreover in M+ it seems also important to solve the problem of the switch from B to macrophage cells as immune cells which present antigens, and the possible involvement of c-Ki-ras gene mutations in the impairment of T cell receptor activation (TCR).

Cytokine contribution to the repair processes and homeostasis recovery following anoxic insult: a possible IFN-gamma-regulating role in IL-1beta neurotoxic action in physiological or damaged CNS.

The cytokine contribution to the central nervous system (CNS) is currently being investigated, but conflicting results have frequently been found. Since, as stressed in the immune system, the cytokine action can be profoundly influenced by the synergistic and antagonistic interactions of the various cytokines present in the medium, a productive effect of the cytokine network in the repair processes and homeostasis recovery may also be dependent on their balanced response in the CNS. We, therefore, hypothesize that it is necessary to study the cytokine contribution to CNS events, evaluating cytokines both in their network and individually. Recent studies focus on interleukin 1 (IL-1) as a cytokine of primary importance for the outcome of diseases, and it seems to exert this role by regulating the synthesis of new proteins such as gamma interferon (IFN-gamma) cytokine which has been reported as having a very important early role in the balance of interactions in the cytokine network. On this basis, to verify the above hypothesis, we determined the release level of IL-1beta and IFN-gamma cytokines in media of cultured rat hippocampal neurons under physiological and anoxic conditions with and without 2-amino-5-phosphonopentanoate (AP5; an N-methyl-D-aspartate receptor antagonist). Our results show that these cytokines are released in these media, and the anoxic insult seems to determine an increase in IL-1beta and a decrease in IFN-gamma release levels as compared to those under the physiological condition. Moreover, while the IL-1beta and IFN-gamma releases covary positively in the physiological media (because an increase in the IL1-beta level is paralleled by an increase in IFN-gamma), the anoxic insult renders this interaction negative (as the IL-1beta level increase corresponds to a decrease in the IFN-gamma level). These data led us to suggest that IFN-gamma may have a physiological regulating role in the IL-1 neurotoxic action and homeostasis recovery following an insult. In fact, the addition of AP5 before anoxic exposure resulted in a significant increase of cellular survival, but also in a decrease of IL-1beta release and a re-establishment of IL-1beta and IFN-gamma release relations to the initial condition without stress. Our overall results, therefore, sustain our hypothesis on the importance of studying the role of cytokines both in their network and individually.

Immunological directives for biotherapy improvement in the treatment of colorectal cancer.

A major cause of failure in biotherapy in cancer may be the non-existence of predictive indices to individualize the substances which might be helpful to switch the patients' immune response to tumour from an non-productive to a productive one. The defect in the patients' immune system needs to be identified and so the evaluation of their responses to biotherapeutic agents, in correlation to the disease progression is essential. We have addressed this problem in colorectal cancer at systemic level by examining the proliferative response of peripheral blood mononuclear cells (PBMC) to interleukins (IL) IL-2, IL-4, antiCD3 monoclonal antibody (antiCD3), IL-2+CD3, IL-2+IL-4, IL-4+CD3, IL-2+CD3+IL-4; the PBMC expression of phenotypic antigens CD3, CD4, CD8, CD25, DR, CD16, CD56, CD57 and CD19 in the patients and healthy subjects as control group. Analysing our data, it seems that as the disease progresses in these patients the peripheral blood cells change their ability to respond to activation agents which appears to be due to a phenotypic modification of their subsets. Our overall results might give a possible explanation of the variable responses to biotherapy in colorectal cancer patients.

Disregulation in TH1 and TH2 subsets of CD4+ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression.

Recent theories have established that, during an ongoing immune response, the lymphokines produced by TH1 and TH2 subsets of CD4+ T cells are critical to the effectiveness of that response. In vivo and in vitro studies have demonstrated that the type of environmental cytokines plays a determinant role in directing the development of naive T cells into TH1 or TH2 effector cells. Disregulated expansion of one or other subset may contribute to the development of certain diseases. To establish whether a similar situation might exist in the cells of the peripheral blood (PBMC) of colorectal cancer patients, we have performed immunological studies on a group of patients and a group of healthy subjects. We examined the interleukin-2 (IL-2), interferon gamma (IFNgamma), IL-4, IL-6 and tumour necrosis factor alpha levels in serum; the production of IL-4 and IL-2, with and without activating agents, by PBMC, tumour-draining lymph node lymphocytes and tumour cells; and the proliferative response of PBMC to IL-2, IL-4 and anti-CD3 monoclonal antibody (anti-CD3), which were variously combined. The data of the present study lead us to hypothesize that, because of suppressive effects probably due to environmental IL-4, in the peripheral blood of patients there seems to be a disregulation in the functionality of TH1 and TH2 subsets of CD4+ T cells, with an expansion in TH2 and a malfunction in TH1 cells. Moreover it seems that this disregulation increases with as the disease progresses through the stages, suggesting that it can be directly implicated in the mechanisms that allow the tumour to locate and progress in the host.

Progression mechanisms in colon cancer: soluble interleukin-2 (IL-2) receptor, IL-2 plus anti-CD3 proliferative response and tumour stage correlations.

Soluble interleukin-2 receptor (sIL-2R) levels have been found to be elevated in several clinical conditions, including disseminated solid neoplasms, whereas they are generally within the normal range in patients with locally limited neoplastic disease. The aim of the present study was to examine this in our colon cancer patients, and to assess if this situation can affect the in vitro activation of peripheral blood mononuclear cells (PBMC), examining the proliferative response to IL-2 and anti-CD3 monoclonal antibody, the IL-2 serum levels and the PBMC phenotype. The results show that sIL-2R levels were significantly correlated with the stage of the disease, showing an increase from stage I to stage IV; moreover, it is worth noting that the proliferative response to IL-2 plus anti-CD3 is significantly higher than to IL-2 alone in stage IV, without significant alteration in the numerical presence of T and natural killer cells. So it seems that in the peripheral blood of patients, connected with the disease progression, are present cellular populations showing a different response to activation, and that T cells acquire a better response condition than NK. Thus, since the T cellular population includes the tumour-specific cytotoxic precursor cells, this should be helpful for its tumour regressive activity, but it is conceivable that this population cannot perform its functions, owing to a deficiency in responsiveness of the specific ThCD4+ subpopulation.

Identification of factors interacting with a subset of T8+ cells.

In a previously work we have reported that T8+ lymphocytes decrease can be obtained by incubation of lymphocytes from normal blood donors with serum taken from patients in the acute phase of the disease. In this paper is described a partial characterization of the masking serum factor present in the acute phase of the disease. It displays a m.w. of approximately 640,000 d and its effect disappears either after heating 60 min. at 37 degrees C or 30 min. at 56 degrees C, after dialysis against 1M NaCl or lyophilization. Another serum factor, insoluble at 50% ammonium sulphate final saturation, resistant to heating and lyophilization, able to remove (in vitro) the former masking factor from the T8+ cells, is present in the remission phase.

T-lymphocyte subsets modifications in multiple sclerosis: correlation with clinical disease activity.

Serial analyses of peripheral blood T-lymphocyte subsets were performed in 280 samples collected over a 27 months period from 14 multiple sclerosis patients. A significant decrease of T8+ cells was found in 47.1% (47/87) of blood samples collected within four weeks of onset of a relapse opposed to 2.1% (1/47) of samples collected during the four weeks before a relapse and to 3.4% (5/146) of those collected over four weeks after a relapse. Changes of T-cells subsets appear to correlate with disease activity, but normal findings observed before relapses suggest that the decrease of T8+ cells cannot be used as valid predictor of relapse.