Human basophil chemotaxis and activation are regulated via the histamine H4 receptor.

BACKGROUND: IgE-mediated cross-linking of FcεRI results in the release of mediators stored in basophil granules, such as histamine and proteases, and in the de novo synthesis of sulfidoleukotrienes. OBJECTIVE: In this study, we investigated the role of the histamine receptors, in particular that of the histamine H4 receptor (H4R), in modulating human basophil function. METHODS: The mRNA expression of the histamine receptors was measured by real-time PCR. Migration of basophils was assessed using the modified Boyden chamber technique. The expression levels of CD63 and CD203c on the cell surface and the sulfidoleukotriene release were determined by flow cytometry and ELISA, respectively. RESULTS: We could show that highly purified basophils express the H1R, H2R, and H4R but not the H3R mRNA. Human basophils expressed higher H4R mRNA levels as compared to the expression levels of the H1R (P < 0.01). Histamine and the H4R agonist ST-1006 initiated active migration of basophils (P < 0.001). A significant reduction in FcεRI cross-linking-mediated surface expression of CD63 and CD203c was observed on basophils after pre-incubation with histamine or the specific H4R agonist ST-1006 (P < 0.01). The synthesis and release of sulfidoleukotrienes from basophils after activation with different stimuli, by FcεRI cross-linking or by stimulation with hymenoptera venom allergens, were significantly reduced by histamine or the H4R agonist ST-1006 (P < 0.05-0.001). CONCLUSION: These data imply that the H4R regulates IgE-dependent processes in human basophils and provides a novel function of the H4R preventing an overwhelming immune reaction by engagement of a negative feedback loop.

Brain-derived neurotrophic factor is increased in serum and skin levels of patients with chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria is triggered by many direct and indirect aggravating factors including autoreactive/autoimmune mechanisms, infections, non-allergic and pseudoallergic intolerance reactions. However, the role of neuroimmune mechanisms in chronic spontaneous urticaria so far is unclear. OBJECTIVE: Thus, we wanted to address the regulation of the neurotrophin brain-derived neurotrophic factor (BDNF) in serum and inflammatory skin of patients with chronic spontaneous urticaria in comparison to subjects with healthy skin. METHODS: Fifty adult patients with chronic spontaneous urticaria and 23 skin-healthy subjects were studied. Chronic spontaneous urticaria was defined as recurrent weals for more than 6 weeks. Autologous serum skin test was performed in all patients with chronic spontaneous urticaria and BDNF serum levels were analysed by enzyme immunoassay in all subjects. Furthermore, skin biopsies were taken from weals of eight patients with chronic spontaneous urticaria as well as from healthy skin of eight controls to evaluate the expression of BDNF and its receptors including tyrosine kinase (trk) B and pan-neurotrophin receptor p75(NTR) by immunohistochemistry. RESULTS: BDNF serum levels were detectable in all subjects studied. However, BDNF levels were significantly higher in patients with chronic spontaneous urticaria compared to non-atopic skin-healthy controls (P<0.001). Furthermore, epidermal and dermal expression of BDNF and epidermal expression of p75(NTR) was significantly higher in patients with chronic spontaneous urticaria compared with controls (P<0.05-0.001). There was no difference with regard to the expression of trkB between chronic spontaneous urticaria and controls and no difference in BDNF serum levels between autologous serum skin test-positive (n=23) and -negative (n=27) patients with chronic spontaneous urticaria. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that BDNF is increased in serum and diseased skin of patients with chronic spontaneous urticaria, suggesting a role for neurotrophins in the pathophysiology of this chronic inflammatory skin disease. Further studies are needed to address the functional role of BDNF on key target effector cells in chronic spontaneous urticaria to establish new therapeutic implications.

Specificity of tyrosinase and HMB45 PCR in the detection of melanoma metastases in sentinel lymph node biopsies.

AIMS: Sentinel lymph node biopsy is an increasingly established procedure in the primary staging of high-risk melanoma patients. However, the laboratory evaluation of sentinel lymph node biopsies is a matter of controversy. The aim of this study was to determine the specificity of polymerase chain reaction (PCR) techniques for the evaluation of lymph nodes with regard to melanoma metastases in comparison with histology and immunohistology. METHODS AND RESULTS: Sentinel lymph nodes (n = 41) from 29 melanoma patients and 29 lymph nodes from 27 patients without melanoma were analysed by histology (H&E) and immunohistology (Melan A, HMB45). cDNA of these lymph nodes was subjected to LightCycler PCR amplification using primers specific for tyrosinase and HMB45. Two melanoma sentinel lymph nodes contained naevus cells by histology and immunohistology and were therefore excluded from further evaluation. Eight (20.5%) of the remaining 39 melanoma sentinel lymph nodes were positive by histology and immunohistology and tyrosinase PCR, 15.4% (6/39) were positive only by tyrosinase PCR, 2.6% (1/39) were positive only by histology and immunohistology. HMB45 PCR revealed positive results in 7.7% (3/39) sentinel lymph nodes, which were also positive by tyrosinase PCR and histology and immunohistology. Of non-melanoma lymph nodes 13.8% (4/29) and 14.8% (4/27) of non-melanoma patients were positive by tyrosinase PCR but negative by histology and immunohistology and HMB45 PCR. Thus, tyrosinase PCR had a specificity of only 85.2%. CONCLUSIONS: The specificity of tyrosinase PCR and the sensitivity of HMB45 PCR are too low to recommend these PCR examinations for the guidance of therapy, in particular complete regional lymph node dissection.

Detection of clonal T cell receptor gamma gene rearrangements in cutaneous T cell lymphoma by LightCycler-polymerase chain reaction.

Cutaneous T cell lymphoma is thought to be characterized by a monoclonal T cell infiltrate in the skin that can be detected by polymerase chain reaction-based amplification of T cell receptor gamma gene rearrangements. We sought to establish a new, simple, and fast LightCycler-based real-time polymerase chain reaction assay for the detection of monoclonality in cutaneous T cell lymphoma, which was suitable for routine laboratory application. Monoclonal T cell receptor gamma gene rearrangements were detected by polymerase chain reaction with consensus primers using: (i) a thermocycler followed by polyacrylamide gel electrophoresis; (ii) a Light Cycler followed by melting curve analysis; and (iii) a LightCycler and subsequent polyacrylamide gel electrophoresis. The detection limit of monoclonal Jurkat T cells diluted in polyclonal peripheral blood mononuclear cells was: (i) 1--3% by thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 10% by LightCycler--polymerase chain reaction and melting curve analysis; and (iii) 1% by LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis. In skin biopsies of 22 cutaneous T cell lymphoma patients, a monoclonal or biclonal T cell infiltrate was detected in: (i) 15 of 22 (68%) by thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 13 of 22 (59%) by LightCycler--polymerase chain reaction and melting curve analysis; and (iii) 16 of 22 (72%) by LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis. All three techniques revealed negative results in skin biopsies from 26 patients with benign dermatitis. In conclusion, LightCycler--polymerase chain reaction and melting curve analysis is a fast, simple and specific method to detect monoclonal T cell infiltrates in cutaneous T cell lymphoma. Sensitivity of LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis is slightly higher compared with sensitivity of thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis. Melting curve analysis, however, is less sensitive compared with polyacrylamide gel electrophoresis, and in case of negative results of the melting curve analysis, it is recommended to resolve LightCycler--polymerase chain reaction samples by gel electrophoresis.

Sensitive detection of Borrelia burgdorferi sensu lato DNA and differentiation of Borrelia species by LightCycler PCR.

In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.

Allelic loss at the neurofibromatosis type 1 (NF1) gene locus is frequent in desmoplastic neurotropic melanoma.

Mutations of the tumour-suppressor gene NF1 (neurofibromatosis 1) have been observed in neurofibromas and neurofibrosarcomas of patients with von Recklinghausen's disease and in sporadic nerve sheath tumours. In contrast, melanoma, another tumour type of neuroectodermal origin, rarely shows NF1 alterations. Desmoplastic neurotropic melanoma (DNM) is an uncommon melanoma subtype that shares morphological characteristics with nerve sheath tumours. Therefore, we analysed 15 DNM and 20 melanomas without morphological features of desmoplasia or neuroid differentiation (common melanomas) for loss of heterozygosity (LOH) at the NF1 locus and flanking regions. Allelic loss was detected in 10/15 (67%) DNM but only in 1/20 (5%) common melanomas. LOH was most frequently observed at marker IVS38, located in intron 38 of NF1. These data suggest a role for NF1 in the pathogenesis of DNM and support an earlier hypothesis that exon 37 might encode a functional domain. DNM may represent an interesting tumour model tor the further elucidation of the cellular functions and tumour-suppressive potential of neurofibromin.

11q23 allelic loss is associated with regional lymph node metastasis in melanoma.

Genetic alterations of the long arm of chromosome 11 have been implicated in melanoma pathogenesis, and we recently identified two distinct regions of common allelic loss in chromosomal band 11q23. To establish the point in time of melanoma tumorigenesis at which these two putative tumor suppressor loci become relevant, we investigated allelic loss [loss of heterozygosity (LOH)] in both chromosomal regions in tumors of progressing patients. We analyzed 102 tumor samples from 23 patients for whom at least two (10 patients) or three (13 patients) tumor samples from different clinical progression steps (such as primary tumor and/or in-transit metastasis and/or regional lymph node metastasis and/or distant metastasis) were available. We detected no 11q23 LOH at any stage in 3 of 23 patients and detected LOH at all stages tested in 8 of 23 patients. In 8 of the remaining 12 (67%) patients with 11q23 LOH at some stage during tumor progression, we found this to occur first at regional lymph node metastasis. Two of these patients retained constitutional heterozygosity in several in-transit metastases that developed up to 7 months after lymph node metastases that already had loss. We therefore conclude that 11q23 LOH is associated with regional lymph node metastasis in melanoma. Finally, we detected an allele shift restricted to a histomorphologically distinct part of a primary melanoma and found that the same parental chromosome was affected by allelic loss in a subsequently occurring lymph node metastasis. These findings support our conclusion and give additional evidence for the hypothesis of molecular heterogeneity of early tumor cell populations in melanoma.

Identification of two distinct deletion targets at 11q23 in cutaneous malignant melanoma.

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) are involved in the pathogenesis of malignant melanoma as well as of other malignancies. We have shown previously, by analysis of loss of heterozygosity (LOH), that a tumor-suppressor gene playing an important role in malignant melanoma is likely to be located within a 51-cM region at 11q23. Its loss appeared to be a late event in tumor progression and an indicator of a less favorable clinical outcome. To further test this hypothesis on a larger set of tumors and to refine the region(s) of common allelic loss, we analyzed 21 polymorphic microsatellite repeats on 11q. A PCR-based assay for LOH was used to study normal and tumor tissues from 53 individuals with primary cutaneous malignant melanoma or metastatic disease. Our findings indicate that in cutaneous malignant melanoma there are at least 2 distinct regions of common allelic loss on 11q, one of them centered around marker APOC3 at 11q23.1-q23.2 delineated by markers D11S1347 and D11S4142 and spanning approximately 5 Mb and a second 3-Mb region around marker D11S925 at 11q23.3 delineated by markers D11S528 and D11S1345. Both regions have been described as deletion targets or as being included within larger allelic deletions detected in several other common tumor types. Thus, these 2 putative melanoma-suppressor loci are likely to harbor tumor-suppressor genes relevant to tumorigenesis of melanoma and a number of other common human malignancies.