Human red blood cells (RBCs) have a remarkable capacity to undergo reversible membrane swelling. Resealed erythrocytes have been proposed as carriers and bioreactors to be used in the treatment of various diseases. This work is aimed at developing a setup allowing the encapsulation of test molecules into erythrocytes by inducing reversible pore formation on the RBC membrane through the application of controlled mechanical shear stresses. The designed setup consists of two reservoirs connected by a glass capillary. Each reservoir is connected to a compressor; during the tests, the reservoirs were in turn pressurized to promote erythrocyte flow through the capillary. The setup was filled with a suspension of erythrocytes, phosphate buffer, and FITC-dextran. Dextran was chosen as the diffusive molecule to check membrane pore dimensions. Samples of the suspension were withdrawn at scheduled times while the setup was operating. Flow cytometry and stereo-optical microscopy analyses were used to evaluate the erythrocyte dextran uptake. The setup was shown to be safe, well controlled, and adjustable. The outcomes of the experimental tests showed significant dextran uptake by RBCs up to 8%. Microscopy observations highlighted the formation of echinocytes in the analyzed samples. Erythrocytes from different donors showed different reactions to mechanical stresses. The experimental outcomes proved the possibility to encapsulate test molecules into erythrocytes by applying controlled mechanical shear stresses on the RBC membrane, encouraging further studies.
IFN-alpha is a promising adjuvant agent in patients with regional melanomatous metastases (stage IIIB). The aim of this study was to verify whether serum evaluation of the interferon induced proteins 2'-5'oligoAS and B2M can predict the clinical response to rIFN-alpha 2A therapy.Forty-two patients who had undergone radical dissection of nodal metastases were evaluated. All patients received adjuvant rIFN-alpha 2A (3 million units s.c. three times weekly) for 3 years or until progression. The patients were followed for a medium period of 43 months (range 19-46). During follow-up 22 of the 42 patients had disease progression. In all patients blood samples were obtained before starting adjuvant therapy and after 1 and 6 months of therapy with rIFN-alpha 2A. 2'-5' oligoAS and B2M were measured by radioimmunoassay.During the first month of adjuvant therapy with rIFN-alpha 2A the serum levels of 2'- 5' oligoAS increased 10-fold, and this trend was maintained in the following 6 months. Similar results were obtained for B2M serum levels. However, we did not find any significant difference in AS and B2M serum levels between responders and non-responders.Our results, therefore, indicate that AS and B2M are markers of the biological activity of administered IFN but that they have little efficacy in predicting the clinical outcome of the treated patients.
221 LAG-3 (CD233) molecule is expressed on activated NK cells and CD4 + and CD8 + T lymphocytes and binds MHC class II molecules with a higher affinity than CD4. Studies in mice have indicated that this molecule has a complex role in controlling T cell functions and that it is directly involved in mediating the activity of murine Treg cells. We studied the expression of CD233 in different human T cell subsets of healthy donors: We found that, although expressed also by CD25-CD4+ T cells, CD233 is strongly up-regulated in naturally occurring CD4+CD25+ T cells and its level of expression is further enhanced in in vitro stimulated CD4+CD25+ T cells. In freshly isolated CD4+CD25+ T cells CD233 is often associated to the expression of CD45RO, HLA-DR, CD71, CD122, CD62L and to the high level of CD25. Moreover, inside the naturally CD4+CD25+ T cells or in in vitro allo-activated CD4+CD25+ T cells, CD233 defined a subset of cells showing high amount of Foxp3 expression. Functional studies using CD4+CD25+CD233+ T cells sorted from in vitro activated CD4+CD25+ showed that this T cell subset displayed potent suppressor activity if compared to the CD233 negative counterpart or to the unsorted population. Stage III-IV metastatic melanoma and colorectal cancer patients did show an increased frequency of CD4+CD25+CD233+ T cells in their PBMC as compared to healthy individuals. Moreover tumor invaded lymph nodes included CD4+ CD25 brigh T cells expressing Foxp3 and CD233. Altogether, our data strongly suggest that, when used in combination with CD25, CD233 could be considered a good marker to define regulatory T cells with enhanced suppressor activity.
Abstract Recent studies increasingly point to a pivotal role of CD4+ T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4+ T cells recognizing the autologous tumor cells in vitro. In fact, the CD4+ T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase κ was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4+ T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient’s peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4+ T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.
AbstractThe identification of genes involved in different biologic functions and in the pathogenesis of diseases has paved the way to the possibility of either interfering with the role of such genes or replacing them in somatic cells in case of loss, which may occur in some genetic diseases or cancer. Such progress has been accomplished thanks to advances in molecular biology and applied technology that allow the transport and insertion of genes into recipient cells by viral or physical vectors as well as the inhibition of gene transcription by antisense oligonucleotides. Methods have also been devised to transfer genes not only in vitro but also in vivo, although this latter approach is still limited owing to poor selectivity and targeting of most vectors when given systemically. Viral and physical vectors have been employed; each of these vectors has distinct advantages and disadvantages, and, therefore, the appropriate vector should be selected according to the therapeutic system involved (1). Retro viral vectors have been used largely for their ability to selectively transfect proliferating cells, a feature that can be advantageous in case one wishes to target only proliferating tumor cells. Owing to the heterogeneous proliferation rate in different parts of a tumor, however, it could be desirable, under some circumstances, to be able to target even the fraction of nonproliferating tumor cells. This can now be obtained by the use of lentivirus (2) or by switching to the use of adenoviruses that can target both dividing and quiescent cells but also induce unwanted inflammmatory reactions from the host.KeywordsGene TherapyMelanoma PatientBystander EffectIntratumoral InjectionAdoptive ImmunotherapyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
