Fungi can be found throughout the world. They may live as saprophytes, parasites or symbionts of animals and plants in indoor as well as outdoor environment. For decades, fungi belonging to the ascomycota as well as to the basidiomycota have been known to cause a broad panel of human disorders. In contrast to pollen, fungal spores and/or mycelial cells may not only cause type I allergy, the most prevalent disease caused by molds, but also a large number of other illnesses, including allergic bronchopulmonary mycoses, allergic sinusitis, hypersensitivity pneumonitis and atopic dermatitis; and, again in contrast to pollen-derived allergies, fungal allergies are frequently linked with allergic asthma. Sensitization to molds has been reported in up to 80% of asthmatic patients. Although research on fungal allergies dates back to the 19th century, major improvements in the diagnosis and therapy of mold allergy have been hampered by the fact that fungal extracts are highly variable in their protein composition due to strain variabilities, batch-to-batch variations, and by the fact that extracts may be prepared from spores and/or mycelial cells. Nonetheless, about 150 individual fungal allergens from approximately 80 mold genera have been identified in the last 20 years. First clinical studies with recombinant mold allergens have demonstrated their potency in clinical diagnosis. This review aims to give an overview of the biology of molds and diseases caused by molds in humans, as well as a detailed summary of the latest results on recombinant fungal allergens.

Chronic lymphocytic leukemia (CLL) cells require complex microenvironmental and immunologic interactions to survive and proliferate. Such interactions might be best recreated in animal models; however, this needs extensive verification. We therefore investigated the composition of the T-cell compartment in the El-TCL1 transgenic mouse, currently the most widely used murine model for CLL. Immunophenotyping and transplant approaches were used to define T-cell subsets at various stages of CLL. Analogous to human CLL, we observed a skewing of T-cell subsets from naive to antigen-experienced memory T cells that was more pronounced in lymph nodes than in blood. Transplantation of CLL into non-transgenic recipients was feasible without immunosuppression in a pure C57BL/6 background and resulted in the prominent skewing of the T cells of the recipient mice. Both in spontaneously developed CLL and in the transplantation setting, a loss in T-cell receptor diversity was observed, with a relevant number of clonal T-cell populations arising. This suggests that antigen-dependent differentiation toward the T memory pool is initiated by murine CLL cells. In summary, we validate the TCL1 transgenic mouse model for analysis of T-cell phenotypes and suggest a CLL-dependent antigen-driven skewing of T cells in these mice.

IntroductionThe B-cell receptor (BCR) is proposed to generate vital signals in the pathogenesis of chronic lymphocytic leukemia (CLL). Specifically in cases with unmutated BCR sequences, crosslinking of the BCR seems to contribute to the survival and proliferation of the CLL cells. 1 The observed bias in V-gene usage and the nonrandom nature of rearranged CDR3 sequences in CLL also suggest that BCR signaling is involved in the initial selection and development of the malignant clone. 2 Reports have linked the activity of members of the protein kinase C family with the pathogenesis of CLL. [3][4][5][6][7] Protein kinase C (PKC) consists of at least 10 isoenzymes with different activation requirements and substrate specificities. Several of these have been associated with diverse cancer settings. 8 PKC␣ and PKC␦ have been indirectly associated with CLL. 5,7 PKC␤ is involved in the transduction and fine-tuning of BCR signals in normal B cells, 9 and it has recently been implicated in CLL pathogenesis. Studies have shown an overexpression of PKC␤ in human CLL and link the signaling downstream of PKC␤ with other relevant signaling events, such as calcium flux, or PKB/AKT phosphorylation. 3,4 However, to define the role of PKC␤, these studies used inhibitors that are at best only relatively specific for the enzyme, thus warranting genetic evidence for its contribution to CLL pathogenesis. Although PKC␤ is shown to be part of a poor-prognosis gene cluster in CLL linked to the transmission of BCR signals, 10 its role in regulating cell survival is more complex. Depending on overall PKC␤ expression, either enhanced or decreased survival was observed, 3 suggesting that PKC␤ signal strength may determine the outcome. Interestingly, targeted deletion of PKC␤ in a murine model led to a preferential loss of CD5 ϩ B cells, 9 suggesting that in these cells PKC␤ provides an essential developmental signal.We used the TCL1 transgenic mouse model for human unmutated CLL to determine the role of PKC␤ in a clean genetic targeting experiment. 11,12 CLL cells in these mice display unmutated BCR sequences with a bias regarding the V-gene usage, suggesting a role of antigen-dependent selection also in the mouse model. Importantly, the premalignant stage of CD5 ϩ B-cell hyperplasia lends itself to the exploration of initial stages of the disease. 11 We crossed TCL1 transgenic mice with PKC␤ knockout mice to assess the effect of PKC␤ deletion on the development of both the premalignant phenotype and CLL onset. In addition, we used a novel PKC␤ inhibitor, enzastaurin, currently in clinical development, to show the potential of translating our murine findings into the human system (for a review on the substance see Ma and Rosen 13 ). MethodsMice E-TCL1 transgenic mice 11 and PKC␤ knockout mice 9 were provided by C. Croce (Columbus, Ohio) and M. Leitges (Oslo, Norway), respectively. Both strains have been backcrossed for 9 generations to C57BL/6 mice. Genotyping of mice was performed on tail DNA by polymerase chain reaction (PCR) as previousl...

BackgroundVLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions.MethodsVLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined.ResultsAbout 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro.ConclusionsOur data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration,but only a limited role in their protection by stromal cells.

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of ϳ57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and D-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a -ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

No abstract

Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures.

Chronic lymphocytic leukemia (CLL) is the most prevalent lymphoid malignancy in the elderly of the Western world. Although treatment options have improved over the past two decades, 10-15% of patients still have a poor prognosis and are often resistant to therapy. Aberrations in the p53 pathway, such as a deleted (del17p13) or mutated p53 gene, are highly enriched in this class of patients. In an extensive screen for p53-independent apoptosis inducers, actinomycin D was identified from 1496 substances and shown to induce apoptosis in primary CLL cells derived from high-risk patients including those with aberrant p53, revealing a novel p53-independent mechanism of action. Both pro-survival genes BCL2 and MCL1 are targeted by actinomycin D, in contrast to fludarabine the backbone of current treatment schedules. In the well-established TCL1 transgenic mouse model for high-risk CLL, actinomycin D treatment was more effective in reducing tumor load than fludarabine, with no evidence of resistance after three treatment cycles and an overall survival increase of over 300%. Tumor load reduction was coupled to BCL2 downregulation. Our results identify the clinically approved compound actinomycin D as a potentially valuable treatment option for CLL high-risk patients.