Three-dimensional in vivo imaging of tumors expressing red fluorescent proteins.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

Prognostic significance of periodic acid-Schiff-positive patterns in clear cell renal cell carcinoma.

BACKGROUND: The ability of aggressive tumors to form nonendothelial tumor cell-lined microvascular channels is known as "vasculogenic mimicry" (VM). VM channels are revealed as periodic acid-Schiff (PAS)-positive patterns, and in some tumors their presence predicts clinical outcomes. OBJECTIVE: We aimed to study VM channels in clear cell renal cell carcinoma (cRCC) tumors and explore their prognostic significance and relationship to other suggested prognostic factors such as thymidine phosphorylase (TP) and vascular endothelial growth factor (VEGF) expression. METHODS: We retrospectively studied 45 patients who had undergone radical nephrectomy for clinically confined cRCC (stage T2-T3NOMO) at the Russian Cancer Research Center. The tumor sections were reviewed for disease stage, nuclear grade, perirenal fat invasion, and lymph node involvement, and we performed immunohistochemical staining for VEGF and TP expression, and PAS staining. Disease-free survival probabilities were determined by Kaplan-Meier estimates and prognostic factors were evaluated by univariate analysis. RESULTS: PAS-positive patterns observed in the cRCC tumor included back-to-back closed loops, networks, arcs, and parallel patterns. There was a significant decrease in disease-free survival among patients with PAS-positive networks (p = 0.005), but not among patients with other PAS-positive patterns. TP expression was also a significant predictor of disease-free survival (p = 0.035), but this factor did not correlate with the presence of PAS-positive networks. Notably, in our small sample, the six patients whose tumors were positive for both factors had the highest risk of cancer recurrence. CONCLUSIONS: The presence of PAS-positive networks is an independent and relevant prognostic parameter for disease-free survival in patients with cRCC. Our data suggest that the combination of PAS-positive networks and TP expression may identify patients with the highest risk of cancer recurrence.

Melanoma vasculogenic mimicry is strongly related to reactive oxygen species level.

The concept of 'vasculogenic mimicry' (VM) was introduced to describe the unique ability of highly invasive tumor cells to form capillary-like structures (CLS) and matrix-rich patterned network in three-dimensional culture that mimic embryonic vasculogenic network. Recently, we have shown that CLS formation requires apoptotic cell death through activation of caspase-3-dependent mechanism. In this study, to identify some molecular determinants driving aggressive melanoma cells to express a latent 'angiogenic program' that recapitulates the early events of CLS formation, we focused on the involvement of antioxidants (AOs) in the process of melanoma VM. We have studied the effects of resveratrol, (-)-epigallocathechin gallate, N-acetyl-cysteine (NAC) and Trolox on the ability of melanoma cells to form/destroy CLS. We observed that the formation of CLS was strongly related to reactive oxygen species level. In vivo animal experiments confirmed the involvement of reactive oxygen species level in melanoma VM. To understand the molecular mechanisms of this phenomenon, we specifically looked for induction of apoptosis and vascular endothelial growth factor (VEGF) release. Western blot analysis revealed that the level of VEGF, VEGF receptors (VEGF-Rs) and active caspase-3 dramatically decreased in cells treated with AOs. Here, we also report further experiments designed to determine whether the crosstalk between AOs and apoptosis exists in melanoma VM.

Crosstalk between apoptosis and antioxidants in melanoma vasculogenic mimicry.

The concept of "vasculogenic mimicry" (VM) was introduced to describe the unique ability of highly aggressive tumor cells to form capillary-like structure (CLS) and matrix-rich patterned network in three-dimensional cultures that mimic embryonic vasculogenic network. Here, we provide the experimental evidence that CLS structure formation requires apoptotic cell death through activation of caspase-dependent mechanism. Our results indicate that the formation of CLS is also related to the reactive oxygen species (ROS) levels.

Serum levels of soluble HLA and IL-2R molecules in patients with urogenital chlamydia infection.

Cellular immunity plays a central role in immune response to chlamydial infection, and soluble forms of immune cell membrane antigens take part in the regulation of immune response. Using an immunoenzymatic method, we determined serum levels of soluble HLA molecules (sHLA-I and sHLA-DR) and soluble CD25 molecules (sCD25) in patients with genital chlamydial infection. Specimens from patients with nonspecific inflammation of the urogenital tract were studied and healthy volunteers served as controls. We revealed that serum levels of sHLA-DR and sCD25 increased 3.5- and 2.3-fold, respectively, during chlamydial infection, while the levels of sHLA-I were not changed. Nonspecific inflammation of the urogenital tract was characterized by a 1.5-fold increase in sHLA-I, a 1.6-fold decrease in sCD25, and no changes of sHLA-DR levels in comparison with healthy volunteers. We concluded that Th1 immune responses might dominate during genital chlamydial infection contrary to the state of nonspecific inflammation of urogenital tract.

The involvement of apoptosis in melanoma vasculogenic mimicry.

During development, the formation and remodeling of the primary vascular network occurs by vasculogenesis and angiogenesis. In 1999, the concept of vasculogenic mimicry was introduced to describe the unique ability of highly aggressive tumor cells to form a capillary-like structure and a matrix-rich patterned network in three-dimensional culture that mimic the embryonic vasculogenic network. In this study, we examined the ability of melanoma cells derived from patients with disseminated melanoma to engage in vasculogenic mimicry in order to identify key parameters in the complexity of the formation of capillary-like structure. We showed that disseminated melanoma as well as uveal and cutaneous melanoma adopts a vascular-related phenotype and engages in vasculogenic mimicry: the main geometrical features of capillary-like structure are determined during the first step of the vascular network assembly. We provided experimental evidence that capillary-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism: a broad range caspase inhibitor zVAD-fmk and a caspase-3 inhibitor DEVD blocked capillary-like structure formation. Apoptosis occurs before capillary-like structure formation but not after capillary-like structures have assembled. These observations may provide a better understanding of the mechanisms involved in melanoma vasculogenic mimicry.

Effect of brain injury on immunophenotype of peripheral blood lymphocytes in rats.

Strong immunosuppression occurs after severe traumatic brain injury (TBI) and most likely contributes substantially to the patient morbidity and mortality. However, the mechanisms of this immunosuppression are unknown. For the lowering of stressful factors, severe TBI was induced in anaesthetized rats. The lymphocyte subsets from 60 rats with severe TBI were analyzed using monoclonal antibody by the indirect immunofluorescence method. The blood of 30 rats without TBI was used as a control. When compared to the control group, the rats with TBI showed a remarkable reduction in the relative number of CD4(+), RT-Ia(+), Thy-1(+) and ICO-111(+) lymphocytes during the first 2 h after injury. Further reduction in the number of CD4(+) cells was determined in the rats during all the period of the experimental observation. The number of lymphocytes expressing membrane Thy-1 and ICO-111 antigens was significantly decreased 7 days after the trauma. The relative number of RT-Ia(+) lymphocytes was significantly reduced in rats with TBI during 14 days following the trauma. A significant decrease in the luminescence intensity of all the analyzed antigen-positive cells was also observed in rats with TBI. Between the 7th and the 14th days after the trauma a positive correlation between the number of Thy-1(+) PBLs and the number of RT-Ia(+) lymphocytes was determined. Similar results on lymphocyte immunophenotype were seen in patients with TBI. Thus, the cellular immune response is identical in patients and in animals with TBI. Severe brain traumatic injury leads to a reduced expression of cell surface antigens and causes a decrease in the number of antigen-positive lymphocytes and in the intensity of their luminescence.

Immunophenotype of Peripheral Blood Lymphocytes in Patients with Burns.

Severe immunosuppression occurs after large thermal burns and probably contributes substantially to patient morbidity and mortality. The mechanism of immunosuppression is much unknown. T lymphocyte subsets from 45 severely patients with burns and 35 healthy donors were analyzed using monoclonal antibodies by indirect immunofluorescence method. Compared to healthy donors, patients with burns have shown a profound reduction in relative number of CD3(+) lymphocytes during the 24-h period following injury, which was accompanied by a decrease in CD4 but not CD8 subsets. Activated lymphocytes, while determined by the expression of CD25, CD26 and CD71, were insignificantly increased in patients with burns. The expression HLA DR was insignificantly decreased on peripheral blood lymphocytes from thermally injured patients. Additionally, the significant decrease of CD95 antigen expression was observed in all patients with burns. Also, significant decrease of the luminescence intensity in all analyzed antigens was observed in patients with burns. Numerous positive correlations between CD3(+), CD8(+), CD25(+), CD26(+) and CD71(+) cells were revealed, especially during the first week after thermal trauma. We conclude that the thermal injury produces a profound relative CD3(+) and CD4(+) cell lymphopenia with signs of moderate lymphocyte activation.

Influence of Immune Complexes on Expression of CD54 and CD95 on the Surface of Endothelial Cells of ECV-304 Line.

The etiology of rheumatoid arthritis (RA) remains unknown; however, recent studies have suggested a central role of the vascular endothelium in RA pathogenesis. The immune complex (IC)-mediated vasculitis is typical for RA. The studies reported herein were undertaken to determine 1) whether IC isolated from plasma of patients with RA are capable of inducing expression of ICAM-1/CD54 and Fas antigen/CD95 on the endothelial cell (EC) in vitro, 2) whether the capacity to induce expression of this phenotypic markers of EC activation is determined by the size or by the composition of the IC. The concentrations of IC were chosen to be within the range for IC levels in plasma. We have shown that all IC-containing samples (n = 8) significantly and in a dose-dependent manner increased level of ICAM-1 expression on the EC. In selected experiments EC were incubated with IC in the presence of complement. The presence of serum containing active complement components resulted in further up-regulation of ICAM-1 expression only in 4 of 8 cases, independently on the ability of IC to fix complement. Additionally, we have found that all IC samples significantly and in a dose-dependent manner induced the marked increase in CD95 expression on the EC. Furthermore, the levels of augmented expression of CD95 correlated with the levels of CD54 expression stimulated by the same IC-containing samples. We have demonstrated that these levels of stimulated expression of ICAM-1 as well as levels of Fas antigen expression negatively correlated with percentage amounts of IgM in total protein concentration of IC and directly correlated with IgG content in comparison to IgM in the structure of this IC. Our results show that IC stimulate ECs to express ICAM-1 and CD95, all of which have been implicated in the pathogenesis of rheumatic disease. The studies reported herein provide further information regarding to inflammatory potential of IC in RA.

Immunological Problems of Diagnostics and Therapy in Hematology and Oncology.

Immunology takes reliable place in the system of diagnostics and therapy of oncological and haematological diseases. It should be noted that serodiagnostics of tumors achieved its methodological limit, since all tumor-associated antigens are already known, and the search of new serum immunological markers seems to fail. New markers have attracted attention of investigators: cytoplasmic and surface proteins and glycoproteins, being products of different genes, which control cell viability, such as Pgp170, p53, Bcl-2, CD95 (Fas/APO-1), Her-2/neu and others. All these proteins may be identified by monoclonal antibodies. At present, the identification of these biomarkers by immunohistochemical methods is beginning to use for individualization of therapy. New direction in oncohematology is biotherapy of tumors. Tumor biotherapy means the treatment of oncological patients with vaccines, immunomodulators, cytokines, monoclonal antibodies and so on. Cancer vaccinotherapy took new design due to the achievements in molecular biology and gene engineering. The great success was achieved in the therapy of tumors and leukemias by interferon preparations. Again, the great expectations seem to be in the field of cancer therapy with immunomodulators. Thus, the achievements in the treatment of oncohaematological diseases are as usual related to the achievements in immunology.