A comparison of the allergic responses induced by Penicillium chrysogenum and house dust mite extracts in a mouse model.

UNLABELLED: A report by the Institute of Medicine suggested that more research is needed to better understand mold effects on allergic disease, particularly asthma development. We compared the ability of the fungal Penicillium chrysogenum (PCE) and house dust mite (HDM) extracts to induce allergic responses in BALB/c mice. The extracts were administered by intratracheal aspiration (IA) at several doses (0, 2.5, 5, 10, 20, 40, and 80 µg) four times over a 4-week period. Three days after the last IA exposure, serum and bronchoalveolar lavage fluid (BALF) were collected. The relative allergenicity of the extracts was evaluated based on the lowest dose able to induce a significant response compared to control (0 µg) and the robustness of the response. PCE induced the most robust response at the lowest dose for most endpoints examined: BALF total, macrophage, neutrophil, and eosinophil cell counts, and antigen-specific IgE. Taken together, our data suggest that PCE may induce a more robust allergic and inflammatory response at lower doses than HDM. PRACTICAL IMPLICATIONS: Our data suggest that Penicillium chrysogenum is a robust allergen and may be a more potent allergen source than house dust mite (HDM) in this mouse model. Two critical factors in the development of human allergic disease, exposure levels and sensitization thresholds, are unknown for most allergens including molds/fungi. Human exposure levels are not within the scope of this article. However, the data presented suggest a threshold dose for the induction of allergic responsiveness to P. chrysogenum. Additionally, P. chrysogenum as well as other molds may play an important role in asthma development in our society.

Discrete serum protein signatures discriminate between human retrovirus-associated hematologic and neurologic disease.

The human T-cell leukemia virus type I (HTLV-I) is the causative agent for adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Approximately 5% of infected individuals will develop either disease and currently there are no diagnostic tools for early detection or accurate assessment of disease state. We have employed high-throughput expression profiling of serum proteins using mass spectrometry to identify protein expression patterns that can discern between disease states of HTLV-I-infected individuals. Our study group consisted of 42 ATL, 50 HAM/TSP, and 38 normal controls. Spectral peaks corresponding to peptide ions were generated from MS-TOF data. We applied Classification and Regression Tree analysis to build a decision algorithm, which achieved 77% correct classification rate across the three groups. A second cohort of 10 ATL, 10 HAM and 10 control samples was used to validate this result. Linear discriminate analysis was performed to verify and visualize class separation. Affinity and sizing chromatography coupled with tandem mass spectrometry was used to identify three peaks specifically overexpressed in ATL: an 11.7 kDa fragment of alpha trypsin inhibitor, and two contiguous fragments (19.9 and 11.9 kDa) of haproglobin-2. To the best of our knowledge, this is the first application of protein profiling to distinguish between two disease states resulting from a single infectious agent.

Microfluidic system integration in sample preparation chip-sets - a summary.

An increasing complexity of microfluidic chips and systems used for biochemical assay applications calls for the development of new strategies towards their functionality integration in order to achieve optimum assay performance. Approaches to an integration of microfluidic chips into diagnostic fluidic systems are reviewed with the emphasis on the selection of assay application, integration scheme, interfacing, and fabrication platform. In particular, we discuss a system containing polymer microfluidic chip-sets capable of cell pre-concentration from a complex sample matrix using immunomagnetic separations.

Engineering crystal symmetry and polar order in molecular host frameworks.

A crystal design strategy is described that produces a series of solid-state molecular host frameworks with prescribed lattice metrics and polar crystallographic symmetries. This represents a significant advance in crystal engineering, which is typically limited to manipulation of only gross structural features. The host frameworks, constructed by connecting flexible hydrogen-bonded sheets with banana-shaped pillars, sustain one-dimensional channels that are occupied by guest molecules during crystallization. The polar host frameworks enforce the alignment of these guests into polar arrays, with properly chosen guests affording inclusion compounds that exhibit second harmonic generation because of this alignment. This protocol exemplifies a principal goal of modern organic solid-state chemistry: the precise control of crystal symmetry and structure for the attainment of a specific bulk property.

Parathyroid-produced angiopoietin-2 modulates angiogenic response.

BACKGROUND: Upon explant, parathyroid tissue (PTH) upregulates vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, yet PTH induces a more robust angiogenic response than VEGF alone. This implies that other angiogenic factors are also produced. We tested PTH for production and function of angiopoietin-2 (Ang-2), a protein known to modulate VEGF response. METHODS: With use of reverse transcriptase-polymerase chain reaction and SELDI (Surface Enhanced Laser Desorption/Ionization) (Ciphergen, Freemont, Calif) technology, we tested explanted PTH for Ang-2 production and determined the time sequence of Ang-2 upregulation. With use of an in vitro rat microvessel angiogenesis assay, we determined the angiogenic response to PTH-produced Ang-2. RESULTS: Ang-2 messenger RNA was induced within 1 hour of parathyroid explant, with a maximum level detectable at 24 hours. Ang-2 protein production was maximal at 24 hours, with elimination by 48 hours. Ang-2 supplemented gels appeared to prompt earlier angiogenic induction, whereas sequestration of Ang-2 with soluble Tie2 receptor appeared to delay angiogenic induction. Soluble Tie2 treatment did not significantly decrease cumulative microvessel length, and no significant increase in neovessel length was seen with Ang-2 supplemented gels. CONCLUSIONS: PTH upregulates Ang-2 upon explantation, with peak protein production by 24 hours. Ang-2 appears to functionally enhance initiation of PTH-induced angiogenesis, although the ultimate neovessel length appears to be dependent on other PTH-produced factors.

Selective nucleation and discovery of organic polymorphs through epitaxy with single crystal substrates.

Crystallization of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile (1), previously found to produce six conformational polymorphs from solution, on single-crystal pimelic acid (PA) substrates results in selective and oriented growth of the metastable "YN" (yellow needle) polymorph on the (101)(PA) faces of the substrate. Though the freshly cleaved substrate crystals expose (101)(PA) and (111)(PA) faces, which are both decorated with [101](PA) ledges that could serve as nucleation sites, crystal growth of YN occurs on only (101)(PA). Goniometry measurements performed with an atomic force microscope reveal that the (001)(YN) plane contacts (101)(PA) with a crystal orientation [100](YN)//[010](PA) and [010](YN)//[101](PA). A geometric lattice analysis using a newly developed program dubbed GRACE (geometric real-space analysis of crystal epitaxy) indicates that this interfacial configuration arises from optimal two-dimensional epitaxy and that among the six polymorphs of 1, only the YN polymorph, in the observed orientation, achieves reasonable epitaxial match to (101)(PA). The geometric analysis also reveals that none of the polymorphs, including YN, can achieve comparable epitaxial match with (111)(PA), consistent with the absence of nucleation on this crystal face. In contrast, sublimation of 1 on cleaved succinic acid (SA) substrates, which expose large (010)(SA) faces decorated with steps along [101](SA), affords growth of several polymorphs, each with multiple orientations, as well as oriented crystals of a new metastable polymorph on the (010)(SA) surfaces. The lack of polymorphic selectivity on (010)(SA) can be explained by the geometric lattice analysis, which reveals low-grade epitaxial matches between (010)(SA) and several polymorphs of 1 but no inherent selectivity toward a single polymorph. These observations demonstrate the sensitivity of crystal nucleation to substrate surface structure, the potential of crystalline substrates for selective nucleation and discovery of polymorphs, and the utility of geometric lattice modeling for screening of substrate libraries for controlling polymorphism.

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

The generality of architectural isomerism in designer inclusion frameworks.

We describe herein new structural isomers of a lamellar host system based on organodisulfonate "pillars" that connect opposing hydrogen-bonded sheets, consisting of topologically complementary guanidinium (G) ions and sulfonate (S) groups, to generate inclusion cavities between the sheets. These new isomers-zigzag brick, double brick, V-brick, and crisscross bilayer-expand significantly on our earlier report of architectural isomerism displayed by the discrete bilayer and simple brick forms. We demonstrate here that the discrete bilayer-simple brick isomerism, which was limited to several host-guest combinations based on the G(2)(4,4'-biphenyldisulfonate) host and one pair of compounds based on the G(2)(2,6-naphthalenedisulfonate), can be generalized to other organodisulfonate pillars. Furthermore, in many cases the selectivity toward the different framework isomers reflects a rather systematic templating role of the guest molecules and host-guest recognition during assembly of the lattice. We also describe a convenient approach to identifying and classifying the innumerable possible host architectures based upon the pillar projection topologies for the GS sheets and the intersheet connectivities. The discovery of these new architectures reveals a structural versatility for this class of materials that exceeds initial expectations and observations. Each topology produces different connectivities between the sheets in the third dimension that endows each framework isomer with uniquely shaped and sized inclusion cavities, enabling this host system to conform readily to different guests. The unlimited number of architectures available, combined with the inherent conformational softness and structural tunability of these host lattices, suggests a near universality for the GS system with respect to guest inclusion.

Metric engineering of soft molecular host frameworks.

The self-assembly and solid-state structures of host-guest inclusion compounds with lamellar architectures based on a common building block, a resilient hydrogen-bonded sheet consisting of guanidinium ions and sulfonate moieties of organodisulfonate "pillars", are described. The pillars connect adjacent sheets to generate galleries with molecular-scale cavities occupied by guest molecules. The size, shape, and physicochemical character of the inclusion cavities can be systematically adjusted by interchanging framework components while maintaining the lamellar architecture, enabling prediction and control of crystal lattice metrics with a precision that is unusual for "crystal engineering". The reliability of the lamellar architecture is a direct consequence of conformational flexibility exhibited by these hosts that, unlike rigid systems, enables them to achieve optimal packing with guest molecules. The adaptability of these hosts is further reflected by an architectural isomerism that is driven by guest templating during assembly of the inclusion compounds. Host frameworks constructed with various pillars display metric interdependences among specific structural features that reveal a common mechanism by which these soft frameworks adapt to different guests. This unique feature facilitates structure prediction and provides guidance for the design of inclusion compounds based on these hosts.

Animal models to assess the effects of air pollutants on allergic lung disease.

Animal models provide toxicologists with useful tools for assessing risks associated with respiratory allergy. Both the mouse and BN rat models described exhibit many of the features of human allergic asthma. It is clear that environmental contaminants can exacerbate the expression of these features. Work is under way to explore underlying mechanisms and to develop methods for applying these data to human health risk assessment.

Parathyroid-induced angiogenesis is VEGF-dependent.

BACKGROUND: Autotransplantation of parathyroid tissue after parathyroidectomy is successful at salvaging parathyroid function. The relatively high success of parathyroid transplantation is thought to be due, in part, to the ability of parathyroid tissue to induce angiogenesis and thus recruit a new vasculature. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor produced by a number of tumors and hypoxic tissues. Using a 3-dimensional intact microvessel angiogenesis system, we evaluated the role of VEGF in the stimulation of angiogenesis by human parathyroid cells. METHODS: Freshly isolated rat microvessels embedded in a 3-dimensional collagen I matrix were treated with healthy 1-mm(3) fragments of human parathyroid tissue or isolated parathyroid cells. Other gels were supplemented with VEGF(165) or FLT-1 soluble receptor fusion protein to bind VEGF. After 11 days in culture, the gels were stained with Gs-1 lectin, a marker for rat endothelium, and linear growth of the microvessels was determined by using image analysis. Parathyroid production of VEGF was determined with reverse transcriptase-polymerase chain reaction. RESULTS: A significant increase in microvessel growth was seen in parathyroid coculture (8.4 +/- 1.0 mm) versus VEGF(165) supplemented gels (6.2 +/- 0.3 mm, P <.01). VEGF(165) significantly augmented parathyroid-stimulated angiogenesis (13.7 +/- 2.4 mm, P <.05 vs parathyroid alone). Using quantitative reverse transcriptase-polymerase chain reaction, we identified VEGF messenger RNA (mRNA) induction within 1 hour of parathyroid explant, with a 12-fold increase by 24 hours. Treatment of parathyroid cocultures with 0.2 microg/mL FLT-1 soluble receptor protein completely eliminated the parathyroid induction of angiogenesis. CONCLUSIONS: Parathyroid tissue expresses low levels of VEGF mRNA, which is significantly upregulated on explantation. Furthermore, the increased VEGF expression is essential to drive parathyroid-induced angiogenesis in our model. However, our data suggests that other parathyroid-produced factors are involved in mediating parathyroid-induced angiogenesis.

HER2 regulatory control of angiopoietin-2 in breast cancer.

BACKGROUND: HER2 overexpression is a marker of aggressive breast cancer. Tumors that overexpress HER2 induce endothelial cell retraction and endothelial destabilization. Because angiopoietin-2 (Ang-2) also destabilizes microvessels, we postulated that HER2 signaling upregulates Ang-2 as a mechanism of angioinvasion. METHODS: We tested human breast cancers and breast cancer cell lines for coexpression of HER2 and Ang-2 with Northern blot, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay. Further, we manipulated HER2 signaling with 100 ng/mL MAbHu HER2 (Herceptin; Genentech, San Francisco, Calif) and Heregulin beta1 (100 ng/mL; R&D Systems, Inc, Minneapolis, Minn) to test for HER2 regulation of Ang-2 production. RESULTS: Three of 4 breast cancer cell lines expressed HER2 protein and Ang-2 mRNA. HER cells, a stably transfected cell line that overexpresses HER2 6-fold, showed a 430% increase in Ang-2 mRNA compared to parental MCF-7 cells. Heregulin beta1 stimulation of HER2 signaling in MCF-7 cells increased Ang-2 by 20% (P <.05). HER2 signaling blockade with 100 ng/mL Herceptin reduced Ang-2 mRNA 90% (P <.001). Five of 11 cancers expressed both HER2 and Ang-2; 2 cancers expressed only Ang-2. CONCLUSIONS: We conclude that human breast cancers express Ang-2. HER2 signaling appears to regulate Ang-2 expression, although other signaling pathways may also regulate Ang-2. Ang-2 may be a therapeutic target in these cancers and may define which patients would benefit from Herceptin therapy.

Comparison of respiratory responses to Metarhizium anisopliae extract using two different sensitization protocols.

Metarhizium anisopliae, an entomopathogenic fungus, is a prototypic microbial pesticide licensed for indoor control of cockroaches, a major source of allergens. We have previously demonstrated allergy and asthma-like responses in BALB/c mice intraperitoneally (IP) sensitized in the presence of adjuvant and intratracheally (IT) challenged with the soluble factors from M. anisopliae crude antigen (MACA) (Ward et al., 1998, 2000). This protocol has been used frequently to establish animal models of allergenicity. However, the sensitization protocol is artificial and not representative of an environmental exposure. Concern has been raised that this protocol might produce allergic responses that would not occur under normal environmental exposure conditions. The objective of this study was to compare responses in mice to MACA by two exposure protocols: (1) exclusive respiratory exposures without adjuvant (representative of environmental exposures) and (2) intraperitoneal sensitization in the presence of adjuvant followed by IT challenge (the traditional approach). The intratracheal protocol consisted of four IT exposures of 10 microg MACA in 50 microl HBSS each over a 4-week period. A vehicle control group of mice was exposed IT to HBSS. The intraperitoneal protocol consisted of IP sensitization with 25 microg MACA in 0.2 ml of 1.3% alhydrogel (aluminum hydroxide) followed 14 days later with an IT challenge (10 microg MACA/50 microl HBSS). Airway reactivity responsiveness to methacholine was assessed, serum and bronchoalveolar lavage fluid (BALF) samples were obtained, and the lungs were fixed for histopathology at 1, 3, and 8 days following the last MACA IT challenge. Both groups exhibited immune and pulmonary responses typical of allergic asthma. In general, local responses in the lung, including inflammatory responses (eosinophils, lymphocytes, and macrophages), BALF IgE, and functional responses to methacholine were greater in the IT sensitized group compared to the IP sensitized group, whereas the systemic IgE response was greater in the IP sensitized group. The BALF IL-5 cytokine levels were elevated before and throughout the eosinophil influx. IL-4 was detected in the BALF of IP sensitized, but not IT sensitized mice. Histopathologic changes in the two groups were similar in nature but more severe in the IT mice. The results suggest that the IP sensitization protocol does not induce the level of respiratory responsiveness that results from sensitization by a physiologically relevant route of exposure. Thus total serum IgE levels, which were greater following IP sensitization, may not be the best indicator of allergen potency, at least with respect to respiratory responses.