Exogenous activated NK cells enhance trafficking of endogenous NK cells to endometriotic lesions

Background: Endometriosis is defined as the presence of endometrial glands and stroma at ectopic locations. Although the prevalence of endometriosis is as high as 35 %-50 %, its pathogenesis remains controversial. An increasing number of studies suggest that changes in immune reactivity may be primarily involved in the development of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of immunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of the innate immune system, have been extensively studied for their ability to defend the organism against infections and malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in lungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells, endometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated the ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions. Methods: As NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by injection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200μl RPMI; Group 2 received i.p dose of 5x106 A-NK in 200μl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2). Results: Our data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the most efficient way for activated NK cells track and infiltrate endometriosis. Conclusion: For the first time, it was shown that both endogenous as exogenous A-NK cells are able to track, migrate and infiltrate endometriosis lesion. This seems to be a promising result, and if confirmed the efficiency of A-NK cells in killing endometriosis lesions, maybe in the future we could use this approach as an alternative treatment for women with endometriosis

Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens

Matrix Metalloproteinases in Cytotoxic Lymphocytes Impact on Tumour Infiltration and Immunomodulation

To efficiently combat solid tumours, endogenously or adoptively transferred cytotoxic T cells and natural killer (NK) cells, need to leave the vasculature, traverse the interstitium and ultimately infiltrate the tumour mass. During this locomotion and migration in the three dimensional environment many obstacles need to be overcome, one of which is the possible impediment of the extracellular matrix. The first and obvious one is the sub-endothelial basement membrane but the infiltrating cells will also meet other, both loose and tight, matrix structures that need to be overridden. Matrix metalloproteinases (MMPs) are believed to be one of the most important endoprotease families, with more than 25 members, which together have function on all known matrix components. This review summarizes what is known on synthesis, expression patterns and regulation of MMPs in cytotoxic lymphocytes and their possible role in the process of tumour infiltration. We also discuss different functions of MMPs as well as the possible use of other lymphocyte proteases for matrix degradation

Local therapy of cancer with free IL-2

This is a position paper about the therapeutic effects of locally applied free IL-2 in the treatment of cancer. Local therapy: IL-2 therapy of cancer was originally introduced as a systemic therapy. This therapy led to about 20% objective responses. Systemic therapy however was very toxic due to the vascular leakage syndrome. Nevertheless, this treatment was a break-through in cancer immunotherapy and stimulated some interesting questions: Supposing that the mechanism of IL-2 treatment is both proliferation and tumoricidal activity of the tumor infiltrating cells, then locally applied IL-2 should result in a much higher local IL-2 concentration than systemic IL-2 application. Consequently a greater beneficial effect could be expected after local IL-2 application (peritumoral = juxtatumoral, intratumoral, intra-arterial, intracavitary, or intratracheal = inhalation). Free IL-2: Many groups have tried to prepare a more effective IL-2 formulation than free IL-2. Examples are slow release systems, insertion of the IL-2 gene into a tumor cell causing prolonged IL-2 release. However, logistically free IL-2 is much easier to apply; hence we concentrated in this review and in most of our experiments on the use of free IL-2. Local therapy with free IL-2 may be effective against transplanted tumors in experimental animals, and against various spontaneous carcinomas, sarcomas, and melanoma in veterinary and human cancer patients. It may induce rejection of very large, metastasized tumor loads, for instance advanced clinical tumors. The effects of even a single IL-2 application may be impressive. Not each tumor or tumor type is sensitive to local IL-2 application. For instance transplanted EL4 lymphoma or TLX9 lymphoma were not sensitive in our hands. Also the extent of sensitivity differs: In Bovine Ocular Squamous Cell Carcinoma (BOSCC) often a complete regression is obtained, whereas with the Bovine Vulval Papilloma and Carcinoma Complex (BVPCC) mainly stable disease is attained. Analysis of the results of local IL-2 therapy in 288 cases of cancer in human patients shows that there were 27% Complete Regressions (CR), 23% Partial Regressions (PR), 18% Stable Disease (SD), and 32% Progressive Disease (PD). In all tumors analyzed, local IL-2 therapy was more effective than systemic IL-2 treatment. Intratumoral IL-2 applications are more effective than peritumoral application or application at a distant site. Tumor regression induced by intratumoral IL-2 application may be a fast process (requiring about a week) in the case of a highly vascular tumor since IL-2 induces vascular leakage/edema and consequently massive tumor necrosis. The latter then stimulates an immune response. In less vascular tumors or less vascular tumor sites, regression may require 9–20 months; this regression is mainly caused by a cytotoxic leukocyte reaction. Hence the disadvantageous vascular leakage syndrome complicating systemic treatment is however advantageous in local treatment, since local edema may initiate tumor necrosis. Thus the therapeutic effect of local IL-2 treatment is not primarily based on tumor immunity, but tumor immunity seems to be useful as a secondary component of the IL-2 induced local processes. If local IL-2 is combined with surgery, radiotherapy or local chemotherapy the therapeutic effect is usually greater than with either therapy alone. Hence local free IL-2 application can be recommended as an addition to standard treatment protocols. Local treatment with free IL-2 is straightforward and can readily be applied even during surgical interventions. Local IL-2 treatment is usually without serious side effects and besides minor complaints it is generally well supported. Only small quantities of IL-2 are required. Hence the therapy is relatively cheap. A single IL-2 application of 4.5 million U IL-2 costs about 70 Euros. Thus combined local treatment may offer an alternative in those circumstances when more expensive forms of treatment are not available, for instance in resource poor countries