Domenico Adorno

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

Abstract 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 γ (IFNγ), IL-4, IL-6 and tumour necrosis factor α 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.

Impact Of Donor-specific Antibodies On Chronic Rejection Occurrence And Graft Loss In Renal Transplantation: Posttransplant Analysis Using Flow Cytometric Techniques1

Background. Improvements in immunosuppressive therapy have greatly reduced acute rejection (ARj) episodes, ensuring better short-term graft outcome, but have not modified long-term survival in renal transplantation. It is now well accepted that chronic rejection (CRj) can be determined by both immune and/or nonimmune mechanisms. The aim of this study was to evaluate the importance of the posttransplant humoral immune response towards mismatched HLA graft antigens in CRj occurrence and graft outcome. Methods. Serum samples from 120 nonpresensitized renal transplant recipients were prospectively screened for 1 year after surgery by means of flow cytometry cross-match (FCXM) and FlowPRA beads (microbeads coated with purified HLA class I and class II antigens) assays. All transplants were followed-up for 2 years or until graft removal. Results. FCXM monitoring identified donor-specific antibodies (DS-Abs) in 29 (24.2%) of 120 transplanted patients. Correlation with clinical data highlighted a higher incidence of ARj in DS-Abs–positive patients compared to negative patients (62% vs. 13%, P <0.00001). Furthermore, graft failure occurred more frequently among FCXM-positive patients than among negative patients (34% vs. 1%, P <0.00001). The deleterious effect of DS-Abs on graft function was confirmed by serum creatinine levels 2 years after transplantation. These were in fact higher in subjects producing DS-Abs than in subjects with only ARj (mean creatinine: 2.5±1.3 mg/dL vs.1.7±0.5 mg/dL, P =0.04). FlowPRA analysis of DS-Ab HLA specificity highlighted the presence of anti-HLA class I antibodies in 85% of FCXM-positive patients, who also presented with a higher incidence of HLA-B mismatches than FCXM-negative patients (1.23±0.66 vs. 0.92±0.59, P =0.02). Conclusions. Flow cytometric techniques are precious tools for investigating the activation of the humoral response against HLA antigens of the graft in renal transplantation. DS-Abs production has a worse impact on organ function and survival than ARj episodes. These findings represent further proof of the threat posed by DS-Abs on long-term graft function and draw attention to the need for a specific immunosuppressive therapy aimed at counteracting the different kinds of immune activation toward graft.

CD30 antigen: not a physiological marker for TH2 cells but an important costimulator molecule in the regulation of the balance between TH1/TH2 response

Understanding the physiological role of CD30 would be an important step forward in transplants because CD30+ T cells can be induced by alloantigens even in the presence of immunosuppressives such as cyclosporine (Csa) and hence can act as regulatory cells in allograft. The results of functional studies on purified T CD30+ cell populations led us to hypothesize that the CD30 costimulator molecule is not a specific marker for TH2 cells in normal conditions, as has been suggested, but rather a marker for an important immunoregulatory subpopulation that regulates the balance between TH1 and TH2 (TH1/TH2) type response. To substantiate this hypothesis we studied the TH1/TH2 cytokine network in peripheral whole blood cultures stimulate with M44 CD30 ligand (CD30L), an agonistic monoclonal antibody (mAb). Four types of whole blood culture were used: the first had been stimulated with anti-CD3 mAb which generates a CD30 cytokine profile similar to alloreactive stimulation; the second with anti-CD3 mAb+M81 (an anti-CD30L mAb) to inhibit CD30/CD30L interaction; the third with anti-CD3+anti-interleukin (IL)4 mAbs to counteract IL4 activity and the fourth with anti-CD3+anti-interferon (IFN)gamma mAbs to counteract IFNgamma activity. Network interactions between soluble CD30 (sCD30, a maker of CD30 expression), sBcl2 (a marker of cell survival) and TH1/TH2 cytokines (IFNgamma, IL2, IL12p70, IL12p40, IL4, IL5 and IL10) were then studied in the supernatants obtained. Our results confirm the hypothesis above by showing that CD30 signals trigger functional mechanisms responsible for changes in levels of production of several important TH1 and TH2 cytokines involved in the regulation of the physiological balance between TH1/TH2 functions. The CD30-stimulated network, in fact, induces IFNgamma production linked to TH1 activity (-->TH1) which is subsequently integrated by IL4 production linked to TH2 activity (-->TH2). This production appears to be regulated, respectively, by IL12p40 (-->TH2) and IL12p70 (-->TH1) production which could maintain the balance between TH1/TH2 type response (TH1<-->TH2). Further CD30 mechanisms are the regulation of the interactions between: IL5-IFNgamma, IL5-IL4, IL2-IL10, IL2-IL12p40 and IL10-IL12p70 production. The immunoregulatory activity of CD30 was confirmed by the lack of production balance between the above-mentioned cytokines observed in cultures in which the interaction between CD30 and its natural ligand (CD30/CD30L) and IL4 or IFNgamma activity had been blocked. We therefore conclude that CD30 may be an important costimulatory molecule and marker for the physiological balance between TH1/TH2 immune response. Consequently, further study of CD30 immunoregulatory mechanisms may allow for the identification of methods for re-establishing equilibrium and hence more effective strategies for the prevention and treatment of immunopathological conditions such as transplant rejection.

Local expression of cytokines in human colorectal carcinoma: evidence of specific interleukin-6 gene expression.

The expression of cytokine mRNAs in tumor tissue, normal mucosa, and peripheral blood mononuclear cells (PBMCs) was studied in 12 patients with colorectal cancer undergoing surgical resection, to characterize local immune conditions. mRNA transcripts for interleukin (IL)-1 beta, IL-2, IL-2-R(p55), IL-4, IL-5, IL-6, and IL-10 were detected using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. IL-6 mRNA was expressed in tumor tissue in 83% of the cases but only in one case in normal mucosa (p < 0.001); serum levels of IL-6 did not show any correlation with IL-6 mRNA; IL-1 beta transcripts were present in all tumor tissue samples; no IL-4 expression was detected; IL-2 mRNA was only present in two tumors; IL-2R(p55) mRNA was found in 58% of tumors but not in normal mucosae (p = 0.005). The expression of IL-10 suggests that it does not play a central role in colorectal cancer immunosuppression, and cytokine expression in PBMCs indicates a different and independent activation. This study suggests a pattern of expression of inflammatory cytokines in the tumor microenvironment, probably produced by infiltrating immune cells. The absence of the specific immune-activating cytokines, IL-2 and IL-4, could indicate an impairment of the anticancer immune response; IL-2R results confirm the dysregulation of the IL-2/IL-2R activation pathway. These findings may lead to a better understanding of the role of cytokines and especially IL-6 at the tumor site and hence their importance in developing an effective immunotherapy.

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.

Abstract 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) α, IL-1α, IL-1β, IL-2, interferon (IFN) γ, 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-1β PBMC release. A negative connection between IFNγ 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 TNFα, IFNγ 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.

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.

Abstract 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 ≤ sCEA ≤ 68.6 ng/ml) and TIMP1 at stage IV (1620 ≤ TIMP1 ≤ 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.

HLA typing does not predict REM sleep behaviour disorder and hallucinations in Parkinson's disease.

HLA‐DR2 haplotype and DQ1 DNA alleles, characterizing 90 to 100% of all narcoleptic patients, were found to be equally distributed in 20 Parkinsons disease (PD) patients with early hallucinations, rapid eye movement (REM) sleep‐related behaviour disturbances (RBD), and sleep onset in REM (SOREM), and in 20 PD patients without hallucinations, despite 10 to 15 years of treatment, and no RBD or SOREM.

Passage from Normal Mucosa to Adenoma and Colon Cancer: Alteration of Normal sCD30 Mechanisms Regulating TH1/TH2 Cell Functions

The pathogenesis of cancer is currently under intensive 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. On the basis of the role of the CD30 molecule in the regulation of TH1/TH2 functions and our previous results, strongly suggesting the validity of serum TH1/TH2 cytokines in the study of tumor progression, we studied network interaction between the production of soluble (s) CD30/sBCl2 in whole blood culture [in basic conditions and after PHA, LPS, and anti-CD3 monoclonal antibody (mAb) stimulation] and levels of TH1/TH2 cytokines (IL2, IFN gamma, IL12, IL4, IL5, IL10). Peripheral blood from a group of healthy subjects, as well as from patients with adenoma and colorectal cancer was used. Our objective was to gain a better insight into the role of the CD30 molecule in the passage from normal mucosa to adenoma and tumor and identify specific disease markers. Our results suggest that the decrease in CD30 expression and the abnormal increase in Bcl2 expression, observed in the peripheral cells of both adenoma and tumor groups determine an imbalance between TH1/TH2 functions. Consequently, changes in sCD30/sBcl2 culture production and TH1/TH2 cytokine serum levels may be reliable markers for tumor progression. In fact, our overall data show that a decrease of sCD30 levels in basic and PHA conditions and an increase of IFN gamma, IL4, IL5, and IL12 serum levels and sBcl2 in all activation condition are indicative of the passage from normal mucosa to adenoma; whilst a decrease of sBcl2 level in basic, LPS and anti-CD3 conditions and of IL2, IFN gamma serum levels, together with an increase of IL5 are indicative of the passage from adenoma to tumor.

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.

CD30 Antigen and Multiple Sclerosis: CD30, an Important Costimulatory Molecule and Marker of a Regulatory Subpopulation of Dendritic Cells, Is Involved in the Maintenance of the Physiological Balance between TH1/TH2 Immune Responses and Tolerance

Objectives: The immunological effect of CD30 on dendritic cells (DCs) was examined in a comparative study of patients with relapsing-remitting multiple sclerosis (RRMS). The patients were divided into two groups on the basis of interferon (IFN)β-1a treatment: IFNβ-1a-treated patients and untreated patients. We have already shown that CD30 is a marker of cells involved in the regulation of the balance between TH1 and TH2 immune responses and so the aim of this study was to confirm this role in DCs and, consequently, to clarify the immunopathological mechanisms of MS and the causes of immunosuppressive drug failure. Methods: We studied network interactions between soluble (s) CD30 and TH1/TH2 cytokines in the supernatants of CD14+-derived immature DC (IDC) and DC cultures from treated and untreated patients. Network interactions between the sCD30 and cytokines in IDC and DC supernatants were also evaluated in relation to TH1/TH2 cytokine serum levels. Results: Our overall results show that CD30 is expressed on IDCs and DCs, indicating an immunological role in resting and activated physiological conditions. This role would appear to be the regulation of the resting and activated physiological balance between the TH1/TH2 immune functions as abnormal increases in sCD30 levels result in impaired regulation. Further studies are undoubtedly required to clarify this situation. IFNβ-1a treatment was found to determine a fall in sCD30 levels, leading to the restoration of the normal functional selection of IDCs from progenitor cells and the regulation of the TH1/TH2 network balance. However, IFNβ-1a treatment may also be responsible for the in vivo suppression of CD30-mediated TH1-DC functions in immune activation. TH1-DC functions are involved in the induction of T-regulatory cells for the physiological deletion of self-aggressive cells. Conclusion: We conclude that CD30 is an important costimulatory molecule and marker of a regulatory subpopulation of DCs which induces and modulates immune cells involved in the maintenance of the physiological balance between TH1/TH2 immune responses and tolerance. Elucidating the mechanisms restoring DC and T-regulatory cell function could lead to more effective therapy and strategies for the prevention and treatment of immunopathological conditions such as autoimmunity, transplant rejection, allergy and tumors.