Bombyxin secretion in the adult silkmoth Bombyx mori: sex-specificity and its correlation with metabolism

Changes in the hemolymph bombyxin titer of the adult silkmoth Bombyx mori were investigated by time-resolved fluoroimmunoassay. Immediately after eclosion, hemolymph bombyxin titers were low in both males and females, and then increased steeply in males to a very high level and this high titer was maintained for at least 3 h, whereas the titer increment in females was small and transient. The difference in the change of bombyxin titer between males and females suggests that bombyxin is responsible for the regulation of physiological changes underlying sexually different activities of the adult moths. However, no evidence was obtained that bombyxin controls adult metabolism as far as the effects of bombyxin on the concentrations of carbohydrates and lipids in the hemolymph were investigated. The change in the hemolymph trehalose concentration was almost the same between sexes, and between intact and neck-ligated moths. Furthermore, bombyxin injection did not affect the hemolymph trehalose concentration nor trehalase activity in the muscle. Although the hemolymph lipid concentration rose after eclosion in males, it was not influenced by bombyxin. These results exhibit striking contrast to the results of our previous study, in which bombyxin showed hypotrehalosemic activity in the larval stage, thus indicating that the action of bombyxin changes during metamorphosis.     

Insulin signaling is involved in hematopoietic regulation in an insect hematopoietic organ

Only a few extracellular hematopoietic factors have been identified in insects. We previously developed an in vitro culture system for the larval hematopoietic organ (HPO) of the silkworm Bombyx mori, and found that cell proliferation is linked to hemocyte discharge from the HPO. In this study, we tested hematopoietic activity of bombyxin, a peptide in the insulin family. When silkworm HPO was cultured with synthetic bombyxin-II, the number of discharged hemocytes increased in a dose-dependent manner, indicating that bombyxin promoted cell proliferation in the HPO. However, a neutralization experiment using anti-bombyxin-II antibody revealed that bombyxin is not the primary effector in larval plasma. Similarly, bovine insulin showed hematopoietic activity. Addition of molting hormone, 20-hydroxyecdysone, circumstantially enhanced the hematopoietic activity of bombyxin and insulin. Bombyxin and insulin induced phosphorylation of different sets of proteins in the HPO, suggesting that their signaling pathways are different.     

Changes in the titer of anti-B virus antibody in captive macaques (Macaca fuscata, M. mulatta, M. fascicularis)

Changes in levels of antibody to B virus (Cercopithecine herpesvirus 1; BV) were examined in BV-positive macaques by ELISA. We observed increases in anti-BV IgG titers in a BV-infected cynomolgus monkey after overseas transportation by air and in a rhesus monkey after transfer from an outdoor group cage to an indoor individual cage. Although shedding of infectious virus was not examined, the increase in antibody titer suggested reactivation of BV. Interestingly, we also found an increase in anti-BV IgG levels during the breeding season in male but not female Japanese macaques kept in an enclosed outdoor colony. Further studies should be performed to investigate whether reactivation of BV led to the observed increase in the anti-BV antibody titer.     

Ecdysteroids in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest

In order to understand the composition and quantitative variation of ecdysteriods in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest, we analyzed the ecdysteroid composition and titers in the adult and egg of this pest. On day 4 after eclosion, the titer of ecdysteriods in the male adult was 0.080 ng/adult, much lower than 5.978 ng/adult in the female adult. During the development of ovaries, the titer of ecdysteriods was low on the first two days, and high in the late period, with the peak (10.48 ng/ovary) appearing on day 3. During the development of eggs, the titer of ecdysteriods was about 0.010 ng/egg from day 1 to day 3, and then decreased to 0.006 ng/egg on day 4. In both adults and eggs, three main components of ecdysteriods were found by identification of HPLC/RIA. They were 20-hydroxyecdysone, 26-hydroxyecdysone, and an unidentified component.     

Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 10(8) dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 10(9) dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10(-5) for NSRO and 3 x 10(-4) for NSRO-A.     

Antibody production in rats vaccinated with inactivated Sendai virus (author's transl)

Adult male and female rats were inoculated with tween 80-ethylether-formalin-ultraviolet inactivated Sendai virus (Sv-V) and examined for the production of hemagglutination inhibition (HI) antibody. There were no significant differences in the antibody titers between males and females, and among the various routes of inoculation except for the intranasal which was not effective. The antibody became detectable 7 days after a single inoculation with 10(5) HAU of Sv-V. The antibody titer, which had its peak 21 days after the inoculation, persisted for 200 days and declined gradually thereafter. The HI antibody titers were correlated with inoculated Sv-V doses and a predominant booster reaction with the vaccine was observed. Maternal antibodies were detected in sucklings born to dams hyperimmunized with the vaccine. The titers were similar to those of the dams until 3 weeks after birth but declined rapidly after weaning at 4-week-old. The titers of fetuses and neonates before suckling were significantly lower than those of the sucklings.     

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.     

Molting hormone titer changes and their significance during development of the colorado beetle, Leptinotarsa decemlineata

Molting hormone (MH) titer in whole animal extracts of Leptinotarsa decemlineata was determined by chemical extraction and the Musca test (1 MU = 3.5 ng ecdysterone) during the developmental span from newly-ecdysed fourth instar larva to an adult 3 days after eclosion. Within the 17-day period, 21 age groups were chosen to estimate the MH titer. Two peaks of MH titer were detected, one in the post-feeding larval stage and the other during the pupal and pharate adult stage. MH activity was first detected in 2-day-old post-feeding larvae, and reached a maximum of 23.5 MU/g tissue on the third day. It began to decline on 3.5 days, and fell to 5.5 MU/g tissue on 4.5 days, the time of larval-pupal ecdysis. In the pupal and pharate adult stage MH rose after the first day and increased to a maximum of 91.5 MU/g tissue on the third day. The titer again declined on the fourth day, and became undetectable one day before adult emergence and in adults 3 days after emergence. MH was demonstrated to be produced by isolated larval abdomens. A peak of 11.5 MU/g tissue was detected in 7-day post-ligation preparations. The titer decreased to 6.9 MU/g tissue in 10-day post-ligation preparations, which was the time of the ecdysis. The finding raises questions concerning the rôle of MH synthesis by other tissues in relation to the function of the prothoracic glands during insect development.     

Population Dynamics of Male-Killing and Non-Male-Killing Spiroplasmas in Drosophila melanogaster

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 × 10⁸ dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 × 10⁹ dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 × 10⁻⁵ for NSRO and 3 × 10⁻⁴ for NSRO-A.     

Assessment in humans of a synthetic peptide-based vaccine against the sporozoite stage of the human malaria parasite, Plasmodium falciparum

Eleven volunteers were injected with an anti-malaria (Plasmodium falciparum) sporozoite vaccine candidate consisting of a synthetic peptide, Ac-Cys-(NANP)3, coupled to tetanus toxoid (TT) and adsorbed to aluminum hydroxide. Two of the volunteers had no previously known exposure to TT. Eight volunteers made detectable antipeptide, anticircumsporozoite protein or antisporozoite antibodies, whose titers increased after multiple injections in four individuals. The maximum antisporozoite titer obtained in an immunofluorescence assay was 1280. In those individuals who produced antipeptide antibody, the overall correlation between IgG anti-Ac-Cys-(NANP)3 antibody in enzyme-linked immunosorbent assay and IgG antisporozoite reactivity in immunofluorescence was highly significant. However, the fine specificity of antibody varied among volunteers with two individuals producing mostly antipeptide antibody. Anti-TT antibody responses increased in all volunteers with the exception of that person who had the highest pretrial anti-TT titer; this individual was one of the two pre-TT-immunized volunteers who failed to produce anti-Ac-Cys-(NANP)3 or sporozoite antibody. For the two non-TT preimmunized volunteers, one produced an antisporozoite fluorescence titer of 320; the other made no detectable antibody against either Ac-Cys-(NANP)3 or sporozoites during a primary response. For the three volunteers monitored, after the first injection, significant T cell proliferative responses to (NANP)3 were observed, which increased up to 4 wk after immunization, when a second injection was given. Responsiveness then declined to background levels and did not reappear after further immunizations. In contrast, a marked TT-specific proliferation was observed for the duration of the study.     

Immunogenicity and cross-reactivity of 2009-2010 inactivated seasonal influenza vaccine in US adults and elderly

The campaign of 2009-2010 Northern Hemisphere seasonal vaccination was concurrent with the 2009 H1N1 pandemic. Using a hemagglutination inhibition (HAI) assay, we evaluated the immunogenicity and cross-reactivity of 2009-2010 inactivated trivalent influenza vaccine (TIV) in US adult and elderly populations. Vaccination of TIV resulted in a robust boost on the antibody response of all subjects to seasonal A/Brisbane/59/2007 (H1N1) and A/Uruguay/716/2007 (H3N2) with over 70% of recipients reaching a seroprotective titer of 40. B/Brisbane/60/2008 was the least immunogenic among the three seasonal vaccine strains with <30% of TIV recipients reaching a seroprotective titer of 40. TIV vaccination also induced a moderate boost on the pandemic specific antibody responses. Twenty-four percent of adults and 36% of elderly reached a seroprotective HAI titer of 40 or more against pandemic A/South Carolina/18/2009 (H1N1) after receiving TIV compared to 4% and 7% at the beginning of vaccination, respectively. In addition, 22% of adults and 34% of elderly showed an increase of 4-fold or more in A/South Carolina/18/2009 specific HAI titers after TIV vaccination. The pandemic specific cross-reactive antibodies strongly correlated with the post-vaccination HAI titers against the seasonal H3N2 vaccine strain in all subjects.     

Anticomplement immunofluorescence for the titration of antibody to varicella-zoster virus

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella-zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type-1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone-fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.     

Presence of antibody against the inducible Hsp71 in patients with acute heat-induced illness

Antibodies against heat shock or stress proteins (Hsps) have been reported in a number of diseases in which they may be involved in the pathogenesis of the disease or may be of use for prognosis. Heat-induced diseases, such as heat cramps, heat exhaustion, or heat stroke, are frequent in hot working or living environments. There are still few investigations on the presence and possible significance of autoantibodies against Hsps in heat-induced illnesses. Using an immunoblotting technique with recombinant human Hsps, we analyzed the presence and titers of antibodies against Hsp60, Hsp71, and Hsp90alpha, and Hsp90beta in a group of 42 young male patients who presented with acute heat-induced illness during training. We also examined the presence of antibody against Hsp71 in a second group of 57 patients with acute heat-induced illness and measured the changes in titers of anti-Hsp71 antibodies in 9 patients hospitalized by emergency physicians. In the first group of young persons exercising in a hot environment, the occurrence of antibodies against Hsp71 and Hsp90alpha was significantly higher among individuals with symptoms of heat-induced illness (P < 0.05) than in the matched group of nonaffected exercising individuals. Moreover titers of antibody against Hsp71 were higher in individuals of the severe and mild heat-induced illness groups, the highest titer being found in the most severe cases. The results from the second group of 57 heat-affected patients exposed to extreme heat were similar. Again, patients with the more severe heat-induced symptoms showed a significantly higher incidence of antibodies to Hsp71 than controls and the titer of anti-Hsp71 was higher in the severely affected group. Finally, in a study of 9 patients, it was observed that the titer of anti-Hsp71 decreased during recovery from severe heat symptoms. These results suggest that measurement of antibodies to Hsps may be useful in assessing how individuals are responding to abnormal stress within their living and working environment and may be used as one biomarker to evaluate their susceptibility to heat-induced diseases.     

Brugia pahangi: circulating antibodies to adult worm antigens in uninfected progeny of homologously infected female jirds

Serum IgG antibody levels to adult Brugia pahangi antigens were measured in uninfected offspring from uninfected and B. pahangi-infected female jirds. Antibody titers to B. pahangi antigens in sera of offspring from infected females mimicked the maternal titer during the suckling period. Neonate titers peaked at 2 weeks of age at levels as high as 1:4100, then decreased to levels well below maternal titers by 8-12 weeks of age. Concurrent maternal and 2-week-old neonate sera recognized identical B. pahangi antigens in Western blots. Spleen cells from 2-week-old filariae-exposed and unexposed offspring failed to produce measurable antibody to B. pahangi in vitro. Progeny of uninfected mothers nursed by B. pahangi-infected females showed circulating IgG antibody titers to adult worm antigens similar to those of homologously reared offspring. Conversely, offspring born to B. pahangi-infected females and nursed by an uninfected female had no serum antibodies to B. pahangi antigens. Blastogenic responses of spleen cells to the mitogens phytohemagglutinin and pokeweed mitogen, and adult B. pahangi antigens, were not different between offspring groups. Mean areas of pulmonary granulomas induced by the intravenous inoculation of B. pahangi antigen-coated beads also did not differ between 4- and 8-week-old progeny of uninfected or infected females. These results suggest that the circulating IgG antibodies to adult B. pahangi antigens demonstrated in offspring of infected female jirds are maternally derived via the milk and do not alter the cellular responses of uninfected offspring to B. pahangi antigens as measured by antigen-stimulated blastogenesis or pulmonary granulomatous inflammatory response.     

Ecdysteroid titers in mated and unmated Drosophila melanogaster females

Radioimmunoassay was used to determine ecdysteroid titers in mated or unmated Drosophila melanogaster females. Whole-body ecdysteroid titers increase after mating and this response is more pronounced after 12-24 hours than it is immediately after mating. In one experiment, females were mated to transgenic males deficient in accessory gland proteins to test whether these peptides mediate the observed increase in female whole-body ecdysteroid titers. Females mated to such transgenic males do not show a pronounced increase in whole-body ecdysteroid titers. The effect of mating on female hemolymph ecdysteroid titers was also investigated. Hemolymph ecdysteroid titers decrease after mating. The ecdysteroid titer change in the hemolymph may result from yolk protein uptake of ecdysteroids into developing vitellogenic oocytes as a consequence of male accessory gland protein stimulation of female oocyte maturation and yolk protein synthesis following mating.     

Glycosphingolipid antigens in neural tumor cell lines and anti-glycosphingolipid antibodies in sera of patients with neural tumors

To characterize biomarkers in neural tumors, we analyzed the acidic lipid fractions of 13 neural tumor cell lines using enzyme-linked immunoabsorbent assay (ELISA) and high-performance thin-layer chromatography (HPTLC) immunostaining. Sulfated glucuronosyl glycosphingolipids (SGGLs) are cell surface molecules that are endowed with the Human Natural Killer-1 (HNK-1) carbohydrate epitope. These glycosphingolipids (GSLs) were expressed in all cell lines with concentrations ranging from 210 to 330 ng per 2 x 10(6) cells. Sulfoglucuronosyl paragloboside (SGPG) was the prominent species with lesser amounts of sulfoglucuronosyl lactosaminyl paragloboside (SGLPG) in these tumor cell lines as assessed by quantitative HPTLC immunostaining. Among the gangliosides surveyed, GD3 and 9-O-acetylated GD3 (OAc-GD3) were expressed in all tumor cell lines. In contrast, fucosyl-GM1 was not found to restrict to small cell lung carcinoma cells. In addition, we have analyzed serum antibody titers against SGPG, GD3, and OAc-GD3 in patients with neural tumors by ELISA and HPTLC immunostaining. All sera had high titers of antibodies of the IgM isotype against SGPG (titers over 1:3,200), especially in tumors such as meningiomas, germinomas, orbital tumors, glioblastomas, medulloblastomas, and subependymomas. Serum in a patient with subependymomas also had a high anti-SGGL antibody titer of the IgG and IgA types (titers over 12,800). The titer of anti-GD3 antibody was also elevated in patients with subependymomas and medulloblastomas; the latter cases also had a high titer of antibody against OAc-GD3. Our data indicate that certain GSL antigens, especially SGGLs, GD3, and OAc-GD3, are expressed in neural tumor cells and may be considered as tumor-associated antigens that represent important biomarkers for neural tumors. Furthermore, antibody titers in sera of patients with these tumors may be of diagnostic value for monitoring the presence of tumor cells and tumor progression.     

Passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen.

The effect of circulating passive antibody on immunity to bovine rotavirus infections in neonatal calves was investigated. In the first experiment, rotavirus antibody titers in the small intestinal lumina of 5- and 10-day-old calves with a wide range of serum rotavirus antibody titers were determined. Neutralizing antibody was present in the small intestinal lumina in titers that correlated with the calves' serum titers (r = +0.84, P less than 0.01). Immunoglobulin G1 was the predominant isotype of intestinal luminal rotavirus antibody. Calves not fed colostrum during the absorptive period lacked rotavirus antibody in circulation and in the intestinal lumen at 7 days of age, even when they were fed large volumes of colostrum with a high rotavirus antibody titer at 48 h after birth. Therefore, rotavirus antibody is not retained in the intestinal lumen for 5 days following a colostrum meal, and the luminal antibody in the 5- and 10-day-old seropositive calves were probably derived from circulating antibody. In a second experiment, calves were passively immunized by subcutaneous injection of colostral whey with a high immunoglobulin G1 rotavirus antibody titer and challenged with virulent bovine rotavirus 48 h later. The passively immunized calves were protected from rotavirus infection and diarrhea compared with calves with comparable serum immunoglobulin concentrations but with lower serum rotavirus with lower serum rotavirus antibody titers. The results of these experiments indicate that circulating immunoglobulin G1 antibody appears in the gastrointestinal tract of neonatal calves and that circulating rotavirus antibody can prevent infection and diarrhea after rotavirus challenge.