Anti-Galectin-3 IgG autoantibodies in patients with Crohn's disease characterized by means of phage display peptide libraries.

Galectin-3, a member of beta-galactoside-binding lectins, is expressed and secreted by a variety of cell types including human intestinal epithelial cells. The presence of anti-galectin-3 antibody in the sera of patients was analyzed by immunoblotting using recombinant human galectin-3. A substantially higher percentage of sera from Crohn's disease patients contained anti-galectin-3 IgG autoantibodies than from patients with ulcerative colitis, primary biliary cirrhosis, or autoimmune hepatitis and of apparently healthy control volunteers. In Crohn's disease patients the titer of autoantibodies was high and interestingly correlated negatively with disease activity. To characterize and generate artificial epitopes (mimotopes), the anti-galectin-3 monoclonal antibodies A3A12 and B2C10 were used for biopannings of phage display nonapeptide libraries. These mimotopes interfered with the binding of autoantibodies to recombinant and native intestinal epithelial galectin-3. Our data may suggest that galectin-3 mimotopes could be used for the induction of IgG with desired specificity to regulate immune responses in Crohn's disease patients.

Ifosfamide and mitoxantrone in the treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck.

A phase II study was performed to assess the safety and efficacy of ifosfamide and mitoxantrone in recurrent and/or metastatic squamous cell carcinomas of the head and neck. Treatment consisted of ifosfamide 1500 mg/m2 in 1000 ml saline, infused over 60 min and mesna 20% of the total dose of ifosfamide in three doses for 3 days combined with mitoxantrone 12 mg/m2 given as a short infusion on day 1. Treatment courses were repeated every 4 weeks until a total of six cycles. Twenty-two patients entered this trial, 13 of whom had received chemo- and radiation therapy, and nine patients who underwent radiation therapy with or without prior surgery. We observed no objective response, with the exception of two patients who experienced minor response (reduction of tumor size of 25%). The dose-limiting toxicity was myelosuppression with grade 3/4 leukocytopenia in seven patients (32%) and grade 3/4 neutropenia in 15 (68%). Severe organ toxicity except alopecia (91%) was not observed. Ifosfamide combined with mitoxantrone does not improve the therapeutic armentarium in recurrent squamous cell carcinoma of the head and neck.

Vascular endothelial growth factor receptor 2 (VEGFR2) expression in squamous cell carcinomas of the head and neck.

OBJECTIVES: Vascular endothelial growth factor receptor 2 (VEGFR2; Flk-1 [fetal liver kinase]/KDR [kinase insert domain containing receptor]) has been identified as a high affinity receptor for vascular endothelial growth factor (VEGF) on vascular endothelium. Head and neck squamous cell carcinomas (HNSCC) have already been shown to produce substantial amounts of VEGF. VEGFR2 is supposed to play a major role in tumor-neoangiogenesis. METHODS: We investigated 24 tumor specimens and 4 HNSCC cultured tumor cell lines for the incidence and distribution of VEGFR2 by immunohistochemistry using monoclonal antibodies (mAbs) and RT-PCR. RESULTS: Analysis of frozen sections by immunohistochemistry showed that in 90% of tumor specimens VEGFR2-positive cells were found which were associated with vascular endothelium. VEGFR2 was also expressed on tumor cells and vessels, which was confirmed by double immunolabeling of tumor cells with an a-cytokeratin mAb. Furthermore, 2 (JPPA, SCC9) of 4 HNSCC cultured tumor cell lines revealed positive VEGFR2 immunoreactivity. Synthesis of VEGFR2 mRNA on all 4 HNSCC cultured tumor cell lines (JPPA, SCC9, SCC25, and LFFR) and in 6 tumor specimens was confirmed by RT-PCR. In conclusion, our results showed that VEGFR2 is expressed in HNSCCs on tumor cells. VEGFR2 expression is associated with the beginning of vasculogenesis represented by accumulation of VEGFR2-positive cells budding into new vessels ("hot spots"). The focal expression pattern of VEGFR2 on tumor cells suggests an autocrine loop for VEGF in tumor cell growth.

Vascular endothelial growth factor (VEGF) and microvessel density in squamous cell carcinomas of the larynx: an immunohistochemical study.

The distribution of vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, and microvessel density (MVD) were assessed in laryngeal carcinomas by means of immunohistochemistry. Correlation of VEGF with MVD and clinical parameters (T stage, N stage, histological grading, survival, recurrence-free interval) was also examined. VEGF expression was evaluated semi-quantitatively and was observed in varying intensity (i) in tumour cells, (ii) in the stromal department as diffuse, sometimes strong reactivity, especially in close proximity to tumour masses and (iii) in macrophages and endothelial cells. Normal epithelium presented no VEGF reactivity except in the immediate vicinity of tumour transformation. Forty percent of our specimens exhibited substantial VEGF reactivity, whereas 20% showed no staining in tumour cells and stroma. These results could be positively correlated with MVD. Moreover, high-graded carcinomas revealed higher VEGF expression, but there was no association of tumour stage or lymph node status with VEGF or MVD. There was a trend in the survival and recurrence analysis towards a higher risk of disease relapse and shorter survival time for patients with enhanced VEGF expression. Apart from tumour cells, macrophages seem to be a substantial source of VEGF in carcinomas. This observation supports the concept of a pivotal role of these cells in tumour defence--in our case, promoting tumour formation by contributing to neovascularization. VEGF was also found in the connective tissue, where it seems to be bound on collagens and probably builds a reservoir for rapid enzymatic mobilization.

Angiogenic factors in laryngeal carcinomas: do they have prognostic relevance?

Angiogenesis is a cascade-like mechanism essential for tumour growth and metastasis. Recently developed inhibitors of angiogenesis have already proven their benefit in anticancer treatment. Therefore the existence of angiogenic molecules in individual tumours is of major interest. The factors most frequently contributing to angiogenesis are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and some matrix metalloproteinases (MMPs). In order to evaluate the prognostic significance of these molecules we investigated the distribution pattern of VEGF, bFGF, MMP-2 and MMP-9 in 41 laryngeal carcinomas using immunohistochemical methods. The results were correlated with clinicopathological parameters, i.e. T and N stage, histological grading, freedom from disease, overall survival and microvessel density (MVD). Only VEGF revealed a correlation with MVD (p = 0.01) and a weak association with histological grading (p = 0.06). Even though there is controversy about published data on the prognostic relevance of angiogenic factors, our results suggest that the factors examined in this study are not suitable for prognosis in larynx cancer patients.

Distribution of immunoglobulins in squamous cell carcinoma of the head and neck.

The immune response with respect to immunoglobulin production in the tumor was investigated in 23 patients with advanced squamous cell carcinomas of the head and neck. Immunohistochemical staining with monoclonal antibodies against IgG, IgM, IgA, IgD and IgE in the tumor was compared to normal hypopharyngeal mucosa. For IgG, IgA and IgM no significant differences between tumor and control tissues could be found. In contrast, a high number of IgE-positive cells was counted in most squamous cell carcinomas compared to normal mucosa. Most of these cells appeared as plasma cells. Regarding IgD the differences between tumor and control tissues, were less pronounced but also significant.

The expression and cellular distribution of adhesion molecules CD2/LFA-3 and ICAM-1/LFA-1 on mononuclear cells in squamous cell carcinoma of the head and neck.

The expressions and cellular distributions of two pairs of adhesion molecules CD2/LFA-3 (leukocyte function-associated antigen-3) and LFA-1/ICAM-1 (intercellular adhesion molecule-1) were examined in inflammatory cellular infiltrates of advanced squamous cell carcinomas of the head and neck by immunohistochemical techniques including double-staining methods. Thirteen patients were investigated using the following monoclonal antibodies (mAbs): CD2, LFA-3 (CD58), ICAM-1 (CD54), LFA-1 (CD11a), the alpha/beta and gamma/delta T-cell receptor, pan T cells and broadly distributed monocyte/macrophage (m/m phi) [Fc gamma RII (CD32), 25F9, RM3/1]. LFA-3 staining was observed on a high number of cells (968 +/- 112 cells/mm2), correlating to the number of Fc gamma RII (CD32; P < 0.01), 25F9 (P < 0.05) and RM3/1 (P < 0.05) positive m/m phi. Its ligand CD2 was found on 365 +/- 126 cells/mm2, representing about 50% of CD3+ cells (730 +/- 286 cells/mm2). CD2 positivity correlated to CD3 and CD8 (P < 0.01) but not to CD4+ T cells. LFA-1 and ICAM-1 were expressed on lymphocytes as well as on m/m phi. ICAM-1+ cells (902 +/- 205 cells/mm2) correlated to CD3+, CD8+ and RM3/1+ cells (P < 0.01). LFA-1 positivity (803 +/- 255 cells/mm2) showed correlations to nearly all investigated antigens, as well as to CD4+ T cells (P < 0.05). These results show that different m/m phi subsets display distinct patterns of adhesion molecule expressions suggesting different pathways of regulation. The CD3+ lymphocyte population revealed a lack of CD2 expression that was more pronounced in the CD4+ subset and indicated impaired lymphocyte function.

Autotransplanted jejunum in patients with carcinomas of the head and neck: transport of immunosurveillance against tumor cells?

Autologous jejunum, transplanted as a functional replacement immediately after radical dissection of advanced stages of squamous cell carcinomas of the head and neck and subsequently irradiated, was examined by immunohistochemistry (APAAP/PAP-technique). Biopsies from 9 patients were taken at the time of transplantation and up to 24 months thereafter (group 1) and from 5 patients only once after transplantation (group 2). Twenty-six monoclonal antibodies (mAbs) were used as surface markers to give an overview about phenotypical changes with respect to T-, B- and M phi-antigens. 1) B cells: a general increase of CR2+ (CD21, p less than 0.01) could be noticed after transplantation, immunoglobulin positive cells remained unchanged expect for a significant decrease of IgM+ (p less than 0.01) and IgA1+ (p less than 0.01) cells. 2) The number of T cells (CD3+) showed no significant differences although TcR gamma/delta+ cells decreased (p less than 0.01) in the autotransplant. ICAM-1 (CD54) and IL-2R (CD25) were found on a significant (p less than 0.01) higher number of cells after transplantation. 3) Cells with M/M phi morphology showed increased expression of the Fc gamma receptors (CD64, p less than 0.001; CD32, n.s.; CD16, p less than 0.001), of the complement receptors CR1 (CD35, (p less than 0.001) and CR3 (CD11b, p less than 0.02), of HLA-DQ (p less than 0.01), and of the antigens 25F9 (mature M phi; p less than 0.01) and CD4 (p less than 0.02). Correlation analyses of data obtained from the biopsies of the 14 autotransplanted jejunum cases revealed a CD35+ and a 25F9+ subpopulation of M/M phi. Our findings indicate that despite irradiation autotransplanted jejunum contained cells with immunological capacities. Therefore, the replacement of larynx by autologous jejunum may facilitate not only mechanical but also immunological functions.

Expression of leucocyte adhesion molecules at the human blood-brain barrier (BBB).

The expression of leucocyte adhesion molecules was studied on cerebral endothelia by immunocytochemistry. In peritumoral "normal" brain tissue we found low endothelial expression of ICAM1, LFA3, CD44, and CD9, whereas VLA1 was present on vessels in high incidence and density. LFA1, CD2, and CR3 were found on intraluminal and parenchymal leucocytes, but were absent on brain vessels. In brain tumors and inflammatory brain lesions, we observed an up-regulation of endothelial ICAM1 and LFA3 expression, whereas other adhesion molecules on endothelial cells remained unchanged. Within the brain parenchyma, ICAM1 and LFA3 were found on astrocytes and tumor cells; on the contrary, LFA1 was expressed on microglial cells similar to CR3. CD44 and CD9 showed a diffuse neuropil expression in normal and tumoral tissue, whereas VLA1 was not expressed on any parenchymal cells. Our data show that multiple different adhesion molecules are present on blood-brain barrier endothelium (BBB) under normal conditions and some adhesion molecules are up-regulated in brain tumors and under inflammatory conditions. The presence of adhesion molecules in the vessel walls as well as on parenchymal cells like astrocytes and microglia may guide inflammatory cells into and through the brain in the course of immune surveillance and inflammation.

[Histologic study of the effect of postoperative irradiation on free transplanted jejunal segments]. / Histologische Studie über den Einfluss der postoperativen Bestrahlung auf frei transplantierte Jejunalsegmente.

After the reconstruction of defects resulting from the resection of advanced tumors from the upper aerodigestive tract using free microvascular anastomized jejunum, the autotransplant is influenced by local radiotherapy (cumulative dose 50-70 Gray). Biopsies were taken from 15 patients, stained with haemotoxilin-eosin and Giemsa and compared by means of light microscopy with jejunum taken at the time of transplantation. It was possible to observe a widened mucosal and submucosal space two to three months after radiotherapy, similar to the clinical impression of a radiogenic enteritis. The villi were flat and shortened with no or slight epithelial lesions. Additionally, it was possible to observe an inflammatory infiltration consisting mainly of neutrophilic granulocytes, edemas, and telangiectases. The latter were also evident in nonirradiated autotransplanted jejunum and are therefore not only caused by irradiation. One year after radiotherapy the mucosal membrane was atrophic. Fibrosis was to be seen in the lamina propria, accompanied by widened muscularis mucosae. The submucosal space was also widened and fibrotic to a varying degree. It was possible to detect varying stages of alteration in the vascular system up to a complete obliteration. After two years, changes were more pronounced. At no time could any alteration in the nervous system of the plexus submucosus be observed. All of these changes have to be interpreted as a consequence of irradiation. However, lubrication of the mucosal surface and the motility of the transplant are not altered severely by irradiation and therefore the desired functions of the free transplanted jejunal grafts, such as swallowing and phonation, are carried out sufficiently.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic relapsing experimental allergic neuritis induced by repeated transfer of P2-protein reactive T cell lines.

Chronic relapsing experimental allergic neuritis (crEAN) was induced by repeated transfers of P2-protein reactive T lymphocyte lines. Clinically, each intravenous transfer of P2-reactive T cells induced a relapse of the disease with weight loss and flaccid paresis of the hindlimbs followed by recovery. After multiple transfers, recovery from disease was incomplete, leading to increasing neurological deficit during the remissions. The pathology of the lesions during exacerbations was characterized by massive inflammation in the peripheral nervous system, associated with extensive endoneurial oedema, nerve fibre destruction and wallerian degeneration. Selective primary demyelination and remyelination was found in the minority of affected nerve fibres. No onion bulbs were present in chronic lesions. In the central nervous system partial degeneration of the posterior columns reflected the extent of wallerian degeneration in the peripheral nerves and spinal roots. In addition, during stages of active disease some T lymphocytes and upregulation of Ia antigen expression were found in the spinal cord.

Squamous cell carcinoma: infiltrating monocyte/macrophage subpopulations express functional mature phenotype.

Biopsies from 26 patients with advanced stage squamous cell carcinoma of the head and neck were investigated to determine the intensity of the inflammatory cellular infiltrate and the expression of leucocyte antigens. Mononuclear cell infiltration varied considerably between the individual patients and also within the tumour. Tumour-infiltrating cells consisted mainly of T lymphocytes and monocytes (Mo)/macrophages (M phi). Staining procedure with monoclonal antibodies (moabs) against Mo/M phi revealed different clusters of antigen expression: (1) moabs 27E10 and a-CD35 detected a subgroup of Mo/M phi with particular staining of perivasal Mo; (2) moab a-CD1 stained preferentially cells in tumour cell clusters; (3) moabs that reacted with cells of either typical M phi or dendritic morphology throughout the tumour-tissue: a-Fc gamma receptor I-III, a-class II antigens, a-CD4, Rm3/1, a-CD36 and 25F9. Thus, the majority of tumour-infiltrating mononuclear phagocytes were found to possess a rather mature phenotype. The number of Mo/M phi with mature phenotype within the tumours correlated with T lymphocyte infiltration in the tissue.

Monocyte subpopulations in human gliomas: expression of Fc and complement receptors and correlation with tumor proliferation.

Cryostat sections of 12 gliomas and of 3 peritumoral brain tissue samples were investigated for mononuclear cell infiltration by immunohistochemistry, concentrating on cells expressing monocyte/macrophage markers. Only low numbers of T cells were detected in the tumors, whereas in average 20%-30% of all cells present in the samples were recognized by various macrophage markers. These cells carried surface epitopes with known function, like Fc-gamma (Fcg) and complement receptors. Microglial cells, in comparison to typical debris laden macrophages, were only recognized by a restricted panel of macrophages markers (anti-Fcg receptors 1, 2, 3, complement receptor CR3, HLA DR, common leucocyte antigen CD45 and the monocyte marker RM3/1). In peritumoral tissue mainly dendritic, microglia-like cells were present, which revealed decreased expression of antigens CD4, RM3/1 and Fcg receptors in comparison to those in gliomas. A significant positive correlation was found between the number of RM3/1 or CR3 (CD11b)-positive cells and the proliferation rate of the tumors as documented by the number of bromodeoxyuridine-positive or Ki-67+ cells.

[Comparative studies of the effectiveness of specific immunotherapy in house dust mite allergy]. / Vergleichende Untersuchungen zur Wirksamkeit einer spezifischen Immuntherapie bei Hausstaubmilben-Allergie.

In a prospective study, 60 patients with allergic rhinoconjunctivitis and/or asthma due to house dust mites were chosen for hyposensitization treatment with Migen (M) or Pharmalgen (P). Immunotherapy stretched over a whole year and every 3 months clinical results were evaluated by the patient's symptom score, by results of skin prick and conjunctival provocation tests, as well as by RIA and ELISA regarding the total and specific IgE and also specific IgG and IgG4 levels. Out of 30 patients of the M group, 15 were followed up over the whole therapeutic regimen, 4 of whom showed a very good, 7 a good to moderate clinical outcome and 4 showed no improvement at all. In the P group, 17 out of 30 patients were followed up whereby 9 showed a very good and 8 a good to moderate response. In both groups of patients a statistically significant decrease in skin and conjunctival sensitivity to mite allergens was observed after 12 months of therapy. However, there was no correlation between this observation and the failure or success of immunotherapy. Furthermore, in both groups there was significant increase in total and specific IgE (with a slight decrease after 6 to 12 months) and also in specific IgG and IgG4 (especially in the P group), but again these changes in antibody levels gave no indication of a good or bad clinical outcome. Hence, we believe other reasons than the usually presented thesis of inducing "blocking antibodies" by immunotherapy to be responsible for the well-known effects of hyposensitization.

Induction of Fc epsilon R2/CD23 on human epidermal Langerhans cells by human recombinant interleukin 4 and gamma interferon.

Human rIL-4 and human rIFN-gamma are able to induce the expression of the low affinity receptor for IgE (Fc epsilon R2/CD23) on normal human epidermal Langerhans cells, whereas IL-2 and PMA have no effect. A synergistic effect is observed when both cytokines are combined. These receptors are synthesized de novo by the LC since cycloheximide completely inhibits the appearance of Fc epsilon R2/CD23. Fc epsilon R2/CD23+ LC may have a major role in the pathogenesis of atopic eczema, as well as in the regulation of IgE synthesis.