For a full understanding of the properties of the human skin barrier, physical macroscopic parameters of barrier function must be correlated to the structural organization of the barrier on a molecular level. This study was undertaken to relate differences in the relative composition of the three main lipid classes of human stratum corneum, i.e., free fatty acids, cholesterol, and ceramides, to differences in transepidermal water loss, stratum corneum electrical impedance, and corneometer value. A new high performance liquid chromatography/light scattering detection-based analysis method recently developed was used for collection of quantitative lipid data in conjunction with gas chromatography/mass spectrometry/flame ionization detection measurements on the free fatty acid fraction. After subtraction of contaminating lipid fractions we have estimated the molar ratio of the human skin barrier lipid composition to be, respectively, 15% cholesterol esters, 16% saturated long chain free fatty acids, 32% cholesterol, and 37% ceramides. The inter-individual difference in the relative amount of free fatty acids, cholesterol, and ceramides, respectively, can be >100% in the individual case. It was found that the relative amount of ceramides to cholesterol is larger in the wrist area, paralleled by a higher transepidermal water loss and corneometer value as well as different skin electrical impedance values as compared with the upper forearm area. We conclude that the site-dependent differences in the stratum corneum lipid composition are small compared with the large inter-individual variation. Interestingly, in the individual case, no correlation was registered between relative ceramide content and barrier properties.

Ten patients with advanced B-cell lymphoma were treated with a single locoregional injection of CD3CD19 bispecific and costimulating CD28 monospecific antibodies to activate tumor-infiltrating T-lymphocytes. Antibodies were administered at 4 different dose levels (30 g, 270 g, 810 g, 1,600 g of each antibody) either by intratumoral or intralym-phatic injection. Most patients developed responses within different compartments of the immune systems (T cells, NK cells) subsequent to the antibody application. Comparative studies in 2 patients of which treated as well as untreated lymph nodes were available revealed the up-regulation of T-cell activation markers induced by the antibody injection. Additionally, in 1 patient the induction of apoptosis of lym-phoma B cells in the antibody-treated lymph node was observed. Specificity analyses of peripheral blood T cells by means of IFN-ELISpot measurement indicated the recruitment of idiotype-specific T cells, as in 1 out of 3 investigated patients an increased T-cell response toward autologous id-iotype peptides could be demonstrated. We conclude that a single injection of CD3CD19 bispecific antibodies is capable to induce an activation of autologous T lymphocytes if simultaneous costimulatory signaling by CD28 antibodies is provided. Furthermore, our data suggest that at least in some patients lymphoma-specific T cells can be recruited by this immunotherapeutic approach toward B-cell lymphoma. Among the immunotherapeutic approaches toward malignant diseases of B-cell origin, strategies aiming at the activation of T lymphocytes are most promising as these are known to be highly specialized effector cells of the cellular immune system mediating key functions such as cytotoxicity, cytokine production, regulation of other effector cells, and immunological memory. The central role of T lymphocytes in the regulation of the immune system is the rationale for the therapeutic use of professional antigen-presenting cells (APC), namely dendritic cells (DC), that can process and present tumor-associated antigens (TAA) and thus are able to activate tumor-specific T lymphocytes. 1 APC-based approaches for the induction of anti-tumoral T-cell responses are therefore being tested in clinical studies in several human malignancies including B-cell lymphomas and myeloma. 2,3 Tumor-specific T-cell activation by non-cellular immunothera-peutic agents avoids the requirement to generate autologous DC ex vivo for every individual patient. In this view, CD3-based bispe-cific antibodies (CD3anti-TAA) may be used alternatively or in addition to vaccination protocols with DC as they can stimulate T cells in a tumor-specific manner as well. 4 As the application of CD3anti-TAA bispecific antibodies alone will result in an insufficient activation or even apoptosis of the effector cells, 5 the additional stimulation of the effector T cells is an indispensable demand. To address this problem, we used locoregional (i.e., intratumoral) injections of CD3CD19 bispecific antibodies targeting the B-cell anti...

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

Cytomegalovirus (CMV) infection has been suggested to be associated with certain autoimmune phenomena as well as with the development of chronic graft-versus-host disease (cGVHD) following allogeneic bone marrow transplantation. Earlier we found that the CMV-associated host protein CD13 is immunogenic during CMV infection in allogeneic bone marrow transplant patients, resulting in production of CD13-specific antibodies (7). Here we found a close correlation between CD13-specific immunity and later development of cGVHD in 26 of 33 patients who could be evaluated for this disease. Of seven patients with CMV disease, six developed extensive cGVHD, all of whom had CD13 specific antibodies (P=0.0002). All 14 patients who were CD13-immune later developed either limited or extensive cGVHD (P=0.0008). Antibodies in sera from the CD13-immune patients suffering from cGVHD recognized normal structures in cryosectioned skin biopsies from control individuals, producing a staining pattern similar to that of CD13-specific monoclonal antibodies. The antibody binding could be specifically blocked by preincubation of the skin sections with a mixture of monoclonal antibodies against CD13, and was also abolished after preabsorption of sera to mouse cells expressing human CD13. No other common autoantibodies were identified in more than single patients. Furthermore, in vivo binding of IgM antibodies in a CD13-like fashion was preferentially demonstrated in skin and oral mucosa biopsies from the CD13-immune patients suffering from cGVHD. Thus, we suggest that CMV-induced CD13-specific autoimmunity contribute to tissue damage in chronic graft-versus-host reactions.

Early-onset melanoma under the age of 20 years is still a rare disease but has an increasing incidence. The aim of this study was to determine whether CDKN2A germline mutations are present in patients diagnosed with childhood/adolescent melanoma. From the Swedish Cancer Register we identified 60 patients with a diagnosis of cutaneous malignant melanoma before the age of 20 years. A medical history including information on self-reported melanoma heredity was obtained, a physical examination was performed by a dermatologist, and the histopathology slides were reviewed. A blood test was obtained for analysis of germline CDKN2A exon 1 and exon 2 mutations by DNA sequencing. We found only one germline CDKN2A mutation with functional significance, which was an exon 1 missense mutation resulting in a proline-to-leucine substitution in codon 48. This mutation was seen in a patient belonging to a previously reported kindred with hereditary melanoma where this particular germline CDKN2A mutation had been identified. Thus, in the large majority of cutaneous melanoma in childhood/adolescence, any underlying genetic alterations have yet to be identified.