Alterations in the production of hemocytes due to a neoplastic mutation of Drosophila melanogaster

The hemocytes of a genetically induced, temperature-sensitive lethal mutation of Drosophila, Tum¹, were examined both quantitatively and qualitatively during the third larval instar. At the tumor-permissive temperature, 29°C, there was a fourfold increase in the concentration of circulating hemocytes in mutant larvae as compared to control. Additionally, the relative frequency of lamellocytes was 30 times greater in Tum¹ larvae than Basc in the early third instar. However, the severity of this abnormality gradually diminished as Tum¹ approached pupariation; though high frequencies of lamellocytes were always present. At the tumor-restrictive temperature (15°C) the concentration of circulating hemocytes was over twice that found at 29°C for Tum¹ larvae, and did not change during the course of third instar. However, in contrast to 29°C there was no abnormal increase in the frequency of lamellocytes at the tumor-restrictive temperature. Control larvae had equivalent concentrations of hemocytes at both temperatures. In one of two temperature shift experiments, Tum¹ larvae shifted from 15° to 29°C at the beginning of third instar expressed the abnormal hemocyte concentration and differentiation associated with larvae raised continuously at 29°C. In addition, Tum¹ larvae shifted from 29° to 15°C expressed reduced abnormalities of hemocyte differentiation, e.g., with fewer lamellocytes in circulation. The possibility of a temperature-sensitive period for the activation of the Tum¹ gene is discussed.     

Cellular mechanics of germ band retraction in Drosophila

Germ band retraction involves a dramatic rearrangement of the tissues on the surface of the Drosophila embryo. As germ band retraction commences, one tissue, the germ band, wraps around another, the amnioserosa. Through retraction the two tissues move cohesively as the highly elongated cells of the amnioserosa contract and the germ band moves so it is only on one side of the embryo. To understand the mechanical drivers of this process, we designed a series of laser ablations that suggest a mechanical role for the amnioserosa. First, we find that during mid retraction, segments in the curve of the germ band are under anisotropic tension. The largest tensions are in the direction in which the amnioserosa contracts. Second, ablating one lateral flank of the amnioserosa reduces the observed force anisotropy and leads to retraction failures. The other intact flank of amnioserosa is insufficient to drive retraction, but can support some germ band cell elongation and is thus not a full phenocopy of ush mutants. Another ablation-induced failure in retraction can phenocopy mys mutants, and does so by targeting amnioserosa cells in the same region where the mutant fails to adhere to the germ band. We conclude that the amnioserosa must play a key, but assistive, mechanical role that aids uncurling of the germ band.     

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus

In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis.     

Regulation of apoptosis of rbf mutant cells during Drosophila development

Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.     

Dispersal, vaccination and regression of immune defence organs

The thymus in vertebrates and the bursa of Fabricius in birds regress before reproduction, while the immunological information of these organs is maintained as cell memory. Regression at a certain age presupposes that individuals have achieved exposure to a large fraction of parasites in the environment. Here we present a new scenario for regression of immune defence organs, based on optimality reasoning. This scenario links early involution of immune defence organs with (1) effects of exposure to parasites on adaptive immune responses to these parasites, (2) exposure to local parasite communities during natal dispersal and migration as a means of "vaccination" against local parasites, and (3) the function of visits to future breeding sites by juveniles as a means of exposure to local parasites. This scenario provides explanations for why natal dispersal is longer than breeding dispersal, for sex differences in dispersal, and for why the bursa of Fabricius regresses relatively early in life among bird species with delayed start of reproduction.     

Cloning and Characterization of Bombyx mori PP-BP a Gene Induced by Viral Infection

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12₇₈₂) was obtained from the hemolymph RNA pools. Using 5′-RACE with a specially designed primer based on the FDD study, a 1 401 bp cDNA clone was obtained containing a 1 311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.     

Changes in blood leukocytes,bone marrow and lymphoid organs in sheep infected with Haemonchus contortus

Haemonchus contortus infection, which causes a blood loss anaemia in sheep, depleted leukocytes and produced leukopenia and a mild lymphopenia. Thymus atrophy, a decreased size of spleen and enlargement of adrenal glands occurred concomitantly in infected sheep which suggested that they were caused by the stress of infection. The overwhelming change in bone marrow of infected sheep was a 4-fold increase in erythroid series cells.Primary immunization with rat erythrocytes produced similar haemagglutinating antibody responses in infected sheep and in sheep allowed to recover from infection after treatment with thiabendazole. This suggests that extant infection with H. contortus was not associated with a secondary immunodeficiency. Blastogenic responses of blood lymphocytes from infected sheep to larval antigen correlated with faecal egg counts but not with haematocrit or leukocyte values. These results suggest that the unresponsiveness of lymphocytes to worm antigen which occur during infection with H. contortus is more closely related to contact with the parasite than to the pathological effects of infection.     

Morphinane alkaloids in cultured tissues and redifferentiated organs of Papaver somniferum

The capacity of alkaloid synthesis was examined in cultured tissues of Papaver somniferum. Callus, derived meristemoids, redifferentiated roots and     

Cellular defense reactions of the shore crab, Carcinus maenas: in vivo hemocytic and histopathological responses to injected bacteria

The cellular defense reactions of the shore crab, Carcinus maenas, were studied, following injections of the bacteria Bacillus cereus and Moraxella sp., by histological and ultrastructural examination of the gills, heart, and hepatopancreas. The majority of the bacteria were sequestered to the gills, but some were also later evident in the heart and hepatopancreas. The presence of the bacteria in the gills initiated the formation of numerous small cell clumps, composed of both refractile and phagocytic cells, which entrapped many microorganisms. The clumps reached a maximum size 6 hr after inoculation and although some were cleared from the gills others persisted for 7 days, becoming more compact and necrotic during this period. Clump formation appears to occur following recognition of the bacteria as foreign and results in the hemocytes becoming sticky and adherent. The response is very effective in rapidly immobilizing the bacteria, thus restraining the spread of infection. It is proposed that this phenomenon may be a significant component of crustacean cellular host defenses.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Rapid recruitment of innate immunity regulates variation of intracellular pathogen resistance in Drosophila

Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.     

Evaluation of some immune functions in a patient affected by common variable immunodeficiency using luminescent techniques

Common variable immunodeficiency is a primary immunodeficiency characterized by a failure of antibody synthesis, whose fundamental immunologic abnormality is still unknown. In our study, we evaluated some immune functions using chemiluminescence in a 32-year-old woman affected by common variable immunodeficiency. In particular, we showed an impairment of her lymphomonocyte proliferative response which was evaluated using a method based on the bioluminescent measurement of ATP. Besides, we found a reducton of her lymphomonocyte IL2 and IL4 production: the IL4 production was evaluated through an ELISA method, whereas the IL2 activity was determined by its ability to support the IL2-dependent murine T-cell line (CTLL) proliferation which was established through a method based on the bioluminescent measurement of ATP. Finally, we evaluated both yeast-induced and fMLP-induced polymorphonuclear and monocyte oxidative metabolism through a luminol-amplified chemiluminescence; these functions were within normal values. Therefore, in our patient affected by common variable immunodeficiency, we demonstrated an impairment of cellular immunity, which might contribute to the pathogenesis of the disease. © 1997 John Wiley & Sons, Ltd.     

Characteristics of RDGA: Mutants with retinal degeneration in Drosophila

We have characterized mutants of the gene retinal degeneration A (rdgA) in Drosophila using histology, optics, deep pseudopupil techniques, electrophysiology and phototactic testing. Earlier work showed that different mutant alleles differed in whether R7 and R8 (2 receptor types of 8 cells per facet in the compound eye) degenerated. We studied a weakly degenerate allele (without much $\text{R}\text{7}\text{8}$ degeneration), namely rdgA^PC47, and a strongly mutant allele, rdgA^BS12. Our techniques all show that degeneration is more severe in rdgA^BS12, not only for $\text{R}\text{7}\text{8}$ but for R1-6 and ocelli as well. We confirm that $\text{R}\text{7}\text{8}$ degenerates more slowly than R1–6 in rdgA^PC47. Mutants of a different gene, namely rdgB, have been widely used in studies of the visual system. Although retinal degeneration is severe in rdgA, the first synaptic neuropil in rdgA remains much more nearly normal than it does in rdgB.     

Light-dependent development of photosynthetic competence in Scenedesmus mutant no. 8

The heterotrophically grown, $\text{P-700-}\text{free}$ mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or $\text{P-700}$ turn-over could not be detected.When grown mixotrophically, the mutant showed increasing $\text{P-700}$ activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good $\text{P-700}$ turn-over and reasonable rates of NADP reduction.The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of $\text{P-700}$ in linear electron transport.     

The deubiquitinase emperor's thumb is a regulator of apoptosis in Drosophila

We have characterized the gene emperor's thumb (et) and showed that it is required for the regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1. Overexpression of one form of et inhibits apoptosis, consistent with et having an anti-apoptotic function; however, overexpression of a second form of et induces apoptosis, indicating that the two forms of et may have competing functions. et encodes a protein deubiquitinase, suggesting it regulates apoptosis by controlling the stability of apoptotic regulatory proteins.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

95例SARS患者细胞免疫功能抑制的特点及其原因

用ELISA方法测定95例SARS患者急性期、恢复期血清细胞因子水平,用流式细胞仪检测该组患者急性期、恢复期淋巴细胞亚群,并分析细胞因子水平和淋巴细胞亚群变化的关系。结果发现,IL-10和TGF—β在观察期（急性期和恢复期）持续升高,急性期CD4^＋和CD8^＋T细胞显著减少,恢复期患者外周血CD8^＋记忆T细胞减少36．78％。IL-10和TGF-β升高与淋巴细胞变化具有统计学相关。结果提示,SARS病毒感染抑制宿主的细胞免疫功能,引起急性期CD4^＋和CD8^＋T细胞显著降低和恢复期的免疫记忆细胞减少。而IL-10和TGF—β过表达可能在SARS免疫病理中起重要作用。     

Characterization of a dominant-active STAT that promotes tumorigenesis in Drosophila

Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. Here we report the first identification of a dominant-active Stat92E protein, Stat92E^ΔNΔC, which lacks both N- and C-termini. Mis-expression of Stat92E^ΔNΔCin vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y⁷¹¹ and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R^442P substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E^R442P translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R⁴⁴² is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R⁴⁴² may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. We conclude that the N- and C- termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E^R442 has a nuclear function by altering dimer:DNA binding.     

Studies on the in vivo cellular reactions of insects: Clearance of pathogenic and non-pathogenic bacteria in Galleria mellonella larvae

The fate of various doses of bacteria of different pathogenicities injected into Galleria mellonella larvae was monitored over time, from haemocyte and bacterial counts, phagocytic responses and the speed and extent of formation of melanized cell aggregates (nodules). An initial haemocytopenia was recorded in all larvae, probably as a result of wound healing, an increased stickiness of the haemocytes for host tissues and/or cell clump or nodule formation. The results also showed that phagocytosis is the primary cellular defence reaction of this insect for doses of bacteria below ca. 10³ μl^?1 haemolymph while above this level phagocytosis and bacterial clearance are usually rapidly augmented by nodule formation. The extent to which these processes are elicited depends greatly upon the nature of the bacteria injected. In general, the more pathogenic strains produced greater responses than the relatively non-pathogenic forms. This enhanced cellular reactivity was, however, soon overcome by the pathogens which rapidly induced a secondary bacteraemia, a huge drop in haemocyte numbers and death of the larvae. The relative importance of phagocytosis and nodule formation in dealing with various doses of bacteria of differing pathogenicities is discussed.