Intermolecular antigen spreading occurs during the preclinical period of human type 1 diabetes

Intra- and intermolecular spreading of T cell responses to autoantigens has been implicated in the pathogenesis of autoimmune diseases. Therefore, we questioned whether T cell responses from subjects identified as at-risk (positive for autoantibody reactivity to islet proteins) for the development of type 1 diabetes, a cell-mediated autoimmune disease, would demonstrate intermolecular Ag spreading of T cell responses to islet cell proteins. Previously, we have demonstrated that by the time subjects develop type 1 diabetes, they have T cell responses to numerous islet proteins, whereas T cells from normal controls respond to a limited number of islet proteins. Initial testing of PBMC responses from 25 nondiabetic at-risk subjects demonstrated that 16 of the 25 subjects have PBMC responses to islet proteins similar to controls. Fourteen of these 16 subjects were available for follow-up. Eleven of the 14 developed T cell responses to increasing numbers of islet proteins, and 6 of these subjects developed type 1 diabetes. In the nine subjects who already demonstrated T cell Ag spreading at the initial visit, four were available for follow-up. Of these four, two had increases in T cell reactivity to islet proteins, while two maintained their initial levels of T cell reactivity. We also observed Ag spreading in autoantibody reactivity to islet proteins in nine of the 18 at-risk subjects available for follow-up. Our data strongly support the conclusion that intermolecular spreading of T cell and Ab responses to islet proteins occurs during the preclinical period of type 1 diabetes.     

Courtship Behavior of Zaprionus indianus (Gupta) (Diptera: Drosophilidae) from Populations Colonizing South America

We describe for the first time the sexual behavior and the courtship song of males of the African fly Zaprionus indianus (Gupta), a recent invader of South America. The male courtship song is formed by monocyclic pulses and the courtship behavior is simple when compared to that of species of Drosophila. Two interpulse interval (IPI) distributions were observed: pre-mounting and mounting. No significant difference was observed between the pre-mounting IPIs of males that descended from three geographical populations from South America. We also observed the songs produced by females and the homosexual behavior exhibited by males. A sequence of bursts is produced by females as a refusal signal against males, while males emit a characteristic song that identifies sex genus, which differs from the courtship song. The short courtship and mating latencies recorded reveal vigorous males and receptive females, respectively.     

Organization and evolution of the alcohol dehydrogenase gene in Drosophila

The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the structural gene and flanking sequence, was determined for each. These sequences were compared with the Adh region of D. melanogaster to characterize changes that occur in the Drosophila genome during evolution and to identify conserved sequences of functional importance. Drosophila simulans and D. mauritiana Adh are organized in a manner similar to that of D. melanogaster Adh, including the presence of two promoters for the single Adh gene. This study identified conserved flanking elements that, in conjunction with other studies, suggest regions that may be involved in the control of Adh expression. Inter- and intraspecies comparisons revealed differences in the kinds of sequence changes that have accumulated. Sequence divergence in and around the Adh gene was used to assess inter- and intraspecies evolutionary relationships. Finally, there appears to be an unrelated structural gene located directly 3' of the Adh transcribed region.      

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

Dectin-1 and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states

Aspergillus fumigatus is a common cause of invasive and allergic pulmonary disease. Resting conidia of the filamentous fungus are constantly inhaled, but cause infection only after initiating hyphal growth. In this study, we have explored whether macrophages can distinguish between resting spores and the maturing, potentially invasive form of the fungus. Although macrophages bind and ingest A. fumigatus resting conidia efficiently, there is little inflammatory response; NF-kappabeta is not activated, inflammatory cytokines are not induced, and reactive oxygen species are not produced. However, maturing A. fumigatus conidia and germ tubes stimulate NF-kappabeta, secretion of proinflammatory cytokines and production of reactive oxygen by human monocyte-derived macrophages and murine macrophages from multiple anatomical sites. These responses are in part mediated by dectin-1, which binds cell wall beta-glucan that is not present on the surface of dormant conidia, but is present after cellular swelling and loss of the hydrophobic proteinaceous cell wall. Dectin-1 binding to germ tubes augments, but is not required for, TLR2-mediated inflammatory cytokine secretion. Dectin-1 recognition of germ tubes also stimulates TNF-alpha production in the absence of both TLR2 and MyD88 signaling. These data demonstrate one mechanism by which the pulmonary inflammatory response is tailored toward metabolically active cells, thereby avoiding unnecessary tissue damage with frequent inhalation of ubiquitous spores.     

Inhibition of human natural killer cell activity by platelet-derived growth factor. II. Membrane binding studies, effects of recombinant IFN-alpha and IL-2, and lack of effect on T cell and antibody-dependent cellular cytotoxicity

We have previously reported that a 2-h pre-treatment with physiologic (nanogram) quantities of the naturally occurring biologic substance, platelet-derived growth factor (PDGF), significantly inhibits human NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target K-562, as well as the single-cell binding of human NK cells to K-562 targets (i.e., conjugate formation). In this study, we have completed a more detailed and longer time course evaluation of PDGF-mediated human NK cell inhibition, and demonstrate a more marked inhibition of human NK cells at 8 to 20 h as compared to 2 h. We also show that either rIFN-alpha or IL-2 reverses the PDGF-mediated NK cell inhibition; and furthermore, that PDGF does not inhibit the lymphokine augmented NK activity of recombinant alpha-IFN or IL-2-activated human NK cells. In addition, we have further demonstrated that PDGF does not inhibit cytotoxic T cell activity as generated in the MLR, nor does PDGF significantly inhibit antibody-dependent cellular cytotoxicity. Finally, we have demonstrated that human NK cells have roughly 12,000 high-affinity, surface binding sites for PDGF, as determined by the competitive binding of 125I-labeled purified PDGF and unlabeled PDGF to B73.1+ FACS-sorted lymphoid (NK) cells, and Scatchard analysis of these data. The implications of these findings are discussed.