Granzyme B is elevated in esophageal biopsies from children with eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is an immune-mediated antigen-triggered inflammatory disease of the esophagus. Our aim was to investigate inflammatory responses by an ex vivo biopsy provocation-based method, stimulating biopsies with milk, wheat, and egg extracts. METHODS: An experimental study was conducted on esophageal biopsies from children who underwent esophagogastroduodenoscopy. Supernatants were collected before and after stimulation of the biopsies with food extracts and analyzed for 45 different inflammatory markers. Biopsies were also stained for histological analyzes. RESULTS: Study subjects included 13 controls, 9 active EoE, and 4 EoE in remission, median age 12 years. Of the 45 markers analyzed, three had significant differences between controls and patients with active EoE, Granzyme B, (GzmB), IL-1ra, and CXCL8 (p < .05). Levels of GzmB were higher, and levels of IL-1ra were lower in patients with active EoE compared with controls and EoE in remission both at baseline and after food extract stimulation. CXCL8 increased in active EoE compared with controls only after stimulation. The number of histologically detected GzmB-positive cells were significantly higher in patients with active EoE in contrast to control and EoE remission (p < .05). CONCLUSIONS: The levels of the barrier-damaging protease GzmB were higher in the supernatant both before and after stimulation with food extract ex vivo in patients with active EoE. GzmB was also observed histologically in biopsies from patients with active EoE. The presence of elevated serine protease GzmB in esophageal mucosa of children with active EoE suggests a role in the pathogenesis of this disorder.

Intraarticular nodular fasciitis of the hip.

Intraarticular nodular fasciitis is a rare lesion that has only recently been recognised. We present a case of intraarticular nodular fasciitis of the hip joint in a 25-year-old woman, who presented with a 9-month-history of right groin pain and a decreased range of right hip motion. A polypoid mass, composed of five nodules attached to the synovial membrane of the distal peripheral compartment of the hip was removed arthroscopically, together with two detached nodules. Histopathological examination revealed a myofibroblastic proliferation typical of nodular fasciitis. Complete resolution of symptoms and restoration of function was achieved, without recurrence 2 years after removal of the lesion. To the best of our knowledge, this is the first case of intraarticular nodular fasciitis of the hip, presenting a new indication for arthroscopic treatment.

Investigation of NK cell function and their modulation in different malignancies.

NK cells have become a subject of investigation not only in the field of tumor immunology and infectious diseases, but also within all aspects of immunology, such as transplantation, autoimmunity, and hypersensitivity. Our early studies aside from investigating NK cell activity in experimental animals and humans included studies of perforin expression and modulation in this lymphocyte subset. As NK cell activity is modified by their environment, we showed clinical stage-dependent impairment of their activity and in vitro effect of different sera, Th1 cytokines, and their combination in breast cancer, Hodgkin's disease, and non-Hodgkin's lymphoma patients, especially with respect to metabolic and cell membrane changes of peripheral blood lymphocytes evaluated by spontaneous release of the enzyme lactate dehydrogenase (LDH) that led to the correction of the LDH enzyme release assay for natural cytotoxicity. By long-term immuno-monitoring of patients with malignancies, we also showed the kinetics of NK cell modulation during chemo-immunotherapy. In our more recent studies, we give data of NK function and novel families of NK cell receptor expression in healthy individuals that may be of help in NK cell profiling, by giving referent values of basic and cytokine-induced expression of some NK cell receptors either in evaluation of disease or in immuno-monitoring during cytokine therapy of patients with malignancies. Moreover, we give novel aspects of modulation of NK cell activity by cytokines approved for immunotherapy, IFN and IL-2, in melanoma and other malignancies with respect to alterations in new activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptor characteristics and signaling molecules in CD16- and CD56-defined NK cells and their small immunoregulatory and large cytotoxic subsets in peripheral blood and lymph nodes, as NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling.

Activity of MMP-2 and MMP-9 in sera of breast cancer patients.

Gelatinase A (MMP-2) and gelatinase B (MMP-9) are proteolytic enzymes involved in the process of tumor invasion, and they are considered as possible tumor markers in breast cancer patients. In this study, we examined serum activity of proMMP-2 and proMMP-9 in relation to TNM stage, tumor size, lymph node involvement, grade of differentiation of tumors, as well as steroid and Her2/neu receptor status in breast cancer patients. The activity of gelatinase in the sera of 52 patients was analyzed by SDS-PAGE zymography. The activity of proMMP-2 and proMMP-9 significantly increased with each advancing clinical stage of disease (p=0.02-0.0009) and compared to controls (p=0.015 to p<0.01). We found a positive correlation between the activity of proMMP-2 and proMMP-9 and tumor size (p=0.007; p=0.05). Patients with lymph node-positive cancer have higher proMMP-2 and proMMP-9 activity than those with node-negative cancer. ProMMP-2 and proMMP-9 activity is not associated with the expression of Her2/neu receptors, but patients with Her2/neu overexpression (3+) showed increased proMMP-2 activity. Steroid receptor score is not associated with enhanced gelatinase activity. The relationship between the increase in proMMP-2 and proMMP-9 activity in serum and tumor size and lymph node status suggests the usefulness of these enzymes as staging markers of breast cancer patients.

Effects of dexrazoxane and amifostine on evolution of Doxorubicin cardiomyopathy in vivo.

Doxorubicin is one of the most active drugs in oncology, with cardiotoxicity as a serious side effect of its application. The aim of this study was to investigate dexrazoxane and amifostine impact on the evolution of myocardial changes induced by doxorubicin. BalbC female mice were treated with doxorubicin only (10 mg/kg, single intravenous push), or with dexrazoxane (200 mg/kg, intraperitoneal [ip]) or amifostine (200 mg/kg, ip) 60 mins or 30 mins prior to treatment with doxorubicin, respectively. Blood sampling for determination of conventional serum-marker activity was performed 48 hrs later. The grade of histopathology changes was evaluated by light microscopy 1.5 and 3 months after treatments using the Billingham scoring method. Control groups consisted of nontreated mice. After doxorubicin-only treatment, the grade of heart tissue damage was found to increase in the period between 1.5 and 3 months. A similar but less intense progression was also detected in amifostine-pretreated animals, with significant difference among median Billingham scores between the two time points. The pretreatment with dexrazoxane suspended expansion of tissue lesions in time. Changes in serum enzyme activity revealed two correlations: the greater reduction in alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) leakage is associated with a lower percentage of damaged tissue, and the creatine kinase to alpha-HBDH percent of difference ratio being greater than one is correlated with limited spreading of pathological lesions. Our results indicate that the development of doxorubicin-induced heart failure is based on a slow and persistent expansion of pathological process even long after the completion of the treatment. Dexrazoxane has proved to be successful and superior over amifostine against such an evolution of doxorubicin cardiomyopathy.

Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients.

Natural killer (NK) cells play a role in the innate and adaptive antitumor immune responses. The activity of NK cells is regulated by functionally opposing, activating and inhibitory receptors whose balance ultimately determines whether target cells will be susceptible to NK cell mediated lysis. As melanoma is an immunogenic tumor, the effect of immunomodulating agents is consistently investigated. In this study in 79 metastatic melanoma (MM) patients and 52 controls NK activity, expression of activating NKG2D and CD161 receptors and KIR receptors, CD158a and CD158b, on freshly isolated PBL and NK cells were evaluated. Native NK cell activity of melanoma patients in clinical stage I-III and MM patients was determined against NK sensitive K562, NK resistant Daudi, human melanoma FemX, HeLa and HL 60 target tumor cell lines. In addition, predictive pretherapy immunomodulating effect after 18 h in vitro treatments of PBL of MM patients with rh IL-2, IFN-alpha (IFN), 13-cis retinoic acid (RA) and combination IFN-alpha and RA was evaluated with respect to NK cell lyses against K562 and FemX cell lines. In this study we show for the first time that low expression of CD161 and activating NKG2D receptors, without increased expression of KIR receptors CD158a and CD158b, as well as a decrease in the cytotoxic, CD16(bright) NK cell subset, is associated with a significant impairment in NK cell activity in MM patients. Furthermore, the predictive pretherapy finding that IL-2, IFN, IFN and RA, unlike RA alone, can enhance NK cell activity of MM patients against FemX melanoma tumor cell line can be of help in the design and development of therapeutic regimens, considering that it has recently been shown that low-dose combination of different immunomodulators represents the most promising approach in the therapy of MM.

Sentinel lymph node biopsy may be used to support the decision to perform modified radical neck dissection in differentiated thyroid carcinoma.

BACKGROUND: The prognostic significance of lymph node metastases (LNM) in follicle cell-derived differentiated thyroid carcinoma (DTC) is still controversial. The management of cervical lymph nodes varies from "berry picking" to modified radical neck dissection (MRND). The incidence of LNM in papillary thyroid carcinoma varies from 27% to 80%. The importance of sentinel lymph node (SLN) biopsy for decisions about the surgical management of lymph nodes in DTC has been the subject of several previous studies. PATIENTS AND METHODS: In 40 patients with DTC methylene blue dye was applied peritumorally. Both SLN and non-SLN in the lower third of the jugulo-carotid chain were dissected prior to total thyroidectomy and routine dissection of the central neck compartment and examined by frozen-section and standard histology. MRND was performed in 9 cases of LNM in the lateral neck compartment. RESULTS: The SLN identification rate was 92.5%. Metastases in SLNs were revealed by frozen-section histology in 7 cases, leading to immediate MRND. The findings were confirmed on standard HE examination. In 2 false-negative cases SLN metastases were revealed on standard histology and MRND was performed 1 week later. The specificity of the method was 100%, sensitivity 77.7%, negative predictive value 94%, positive predictive value 100%, with overall accuracy of 95%. CONCLUSION: Our results seem to imply that SLN biopsy in the jugulo-carotid chain using methylene blue dye mapping may be a feasible and valuable method for estimating lymph node status in the lateral neck compartment. It may be helpful in the detection of true-positive but nonpalpable lymph nodes, and in such cases may support the decision to perform MRND in patients with DTC.

Activity of d,l-alpha-tocopherol (vitamin E) against cardiotoxicity induced by doxorubicin and doxorubicin with cyclophosphamide in mice.

The aim of this study was to investigate the cardioprotective activity of vitamin E against doxorubicin alone and doxorubicin in combination with cyclophosphamide in mice. Female BalbC/NIH mice were treated with vitamin E (100 IU/kg, orally) 24 hr before single bolus doses of doxorubicin (10 mg/kg, intravenously), or doxorubicin and cyclophosphamide (150 mg/kg, intraperitoneally). Non-treated animals served as negative controls, while positive control groups received doxorubicin or doxorubicin and cyclophosphamide. For evaluation, serum enzyme activity of aspartate aminotransferase (AST), lactate dehidrogenase (LDH), alpha-hydroxybutirate dehydrogenase (alpha-HBDH), and creatine kinase (CK) at 48 hr and histopathology examination of the heart tissue (Billigham rules) at 1.5 and 3 months followed to treatments were used. In sera of mice treated with vitamin E prior to doxorubicin, the creatine kinase and % alpha-HBDH activity were significantly reduced, compared to positive control. Histopathology changes (scored as 1.5 at 1.5 and 3 months respectively) were not significant compared to negative control at both time points of examination. In animals which received vitamin E before doxorubicin and cyclophosphamide, none of the serum enzymes was significantly reduced compared to positive control, but non-significant increase in AST and creatine kinase activity was detected (3% and 16.57% respectively). The degree of myocardial damage was significantly higher compared to non-treated group (2.0 and 2.5 at 1.5 and 3 months respectively). Current results show that vitamin E in single oral dose failed to inhibit acute cardiotoxic activity of doxorubicin, but suspended further progression of the heart muscle damage over the time. On the contrary, vitamin E did not attain any cardioprotection against doxorubicin and cyclophosphamide in combination.

IL-2-mediated augmentation of NK-cell activity and activation antigen expression on NK- and T-cell subsets in patients with metastatic melanoma treated with interferon-alpha and DTIC.

Considering that well-defined and comprehensive immunological monitoring is the basis for the evaluation of the obtained immunmodulatory effects, we evaluated NK-cell activity, the number of CD3+ CD4+, CD3+ CD8+ T cells and CD16+ CD56+ NK cells, as well as the expression of activation antigens, CD69, CD38 and HLA-DR on CD56+ NK cells, CD8+ and CD3+ T cells, simultaneously with IL-2 and TNF-alpha production, during chemoimmunotherapy with dacarbazine (DTIC) and interferon-alpha (IFN-alpha) in 39 patients with metastatic melanoma. In the first cycle of therapy, there was a significant rise in NK-cell activity, CD4+ T helper cell number, CD4/CD8 T-cell ratio, and the expression of activation antigens CD69 and CD38, on NK and T cells, respectively. However, in the following cycles there was a significant increase only in activation antigens without an increase in the percent or activity of NK cells. The early, but transient, immunopotentiation, present only in the first cycle of combined DTIC and IFN-alpha therapy, suggests that, in spite of increased IL-2 level, associated with augmented NK-cell activity, this therapy has a limited effect probably owing to the adverse effect of persistently high level of TNF-alpha in metastatic disease.

8-Cl-cAMP affects glioma cell-cycle kinetics and selectively induces apoptosis.

8-Chloro-cyclic-adenosine-3',5'-monophosphate (8-Cl-cAMP), a site-selective synthetic cyclic adenosine 3',5'-monophosphate (cAMP) analog exhibits growth inhibition in a broad spectrum of human cancer lines. However, detailed studies on the effects exerted by cAMP analogs on cell-cycle kinetics have been lacking. We have examined and compared the effect of 8-Cl-cAMP on cell-cycle kinetics in two human glioma cell lines, U87MG (p53wt) and U251MG (p53mt). A flow cytometric analysis of cell-cycle distribution as well as apoptosis evaluation were performed by univariate DNA analysis after 24-72 hr of treatment with 10-50 M concentrations of 8-Cl-cAMP. Longer incubation with 8-Cl-cAMP induced dose related accumulation of cells in S phase and a subsequent decrease in the proportion of cells in G0/G1 phase of cell cycle in both cell lines. Time-dependent suppression of cyclin B1 was detected in both glioma cell lines and could be associated with observed G2 delay. However, 8-aCl-cAMP selectively induced apoptotic cell death only in U87MG, but not in U251MG cells. Induction of apoptosis was revealed both by flow cytometry and apoptotic cell morphology. These results provide an insight into the mechanism of 8-aCl-cAMP action, suggesting that the disturbance of cell-cycle kinetics and induction of apoptosis might contribute to its growth-inhibitory effect on cancer cells.