Prenatal hypoxia leads to hypertension, renal renin-angiotensin system activation and exacerbates salt-induced pathology in a sex-specific manner.

Prenatal hypoxia is associated with growth restriction and adverse cardiovascular outcomes. Here, we describe renal and cardiovascular outcomes in ageing mouse offspring prenatally exposed to hypoxia (12% O2) from embryonic day 14.5 until birth. At 12 months of age, both male and female offspring exposed to prenatal hypoxia had elevated mean arterial pressure. Glomerular number was reduced by 25% in hypoxia-exposed male, but not female, offspring and this was associated with increased urinary albumin excretion, glomerular hypertrophy and renal fibrosis. Hypoxia-exposed offspring of both sexes were more susceptible to salt-induced cardiac fibrosis, however, renal fibrosis was exacerbated by high salt in males only. In male but not female hypoxia-exposed offspring, renal renin mRNA was increased at weaning. By 12 months, renal renin mRNA expression and concentrations were elevated in both sexes. mRNA expression of At 1a R was also elevated in male hypoxia-exposed offspring at 12 months. These results demonstrate that prenatal hypoxia programs elevated blood pressure and exacerbates salt-induced cardiovascular and renal pathology in a sex specific manner. Given sex differences observed in RAS expression and nephron number, future studies may consider RAS blockade as a therapeutic target in this model.

Prenatal corticosterone exposure programs sex-specific adrenal adaptations in mouse offspring.

Maternal stress can impair foetal development and program sex-specific disease outcomes in offspring through the actions of maternally produced glucocorticoids, predominantly corticosterone (Cort) in rodents. We have demonstrated in mice that male but not female offspring prenatally exposed to Cort (33 µg/kg/h for 60 h beginning at E12.5) develop cardiovascular/renal dysfunction at 12 months. At 6 months of age, renal function was normal but male offspring had increased plasma aldosterone concentrations, suggesting that altered adrenal function may precede disease. This study investigated the long-term impact of prenatal exposure to Cort on adrenal growth, morphology and steroidogenic capacity as well as plasma Cort concentrations in offspring at postnatal day 30 (PN30), 6 months and 12 months of age. Prenatal Cort exposure decreased adrenal volume, particularly of the zona fasciculata, in male offspring at PN30 but increased both relative and absolute adrenal weight at 6 months of age. By 12 months of age, male Cort-exposed offspring had reduced absolute adrenal weight in association with increased adrenal plaque deposition (lipogenic pigmentation). Plasma Cort concentrations were elevated in male 6-month offspring but not at other ages. mRNA expression of Mc2r (ACTH receptor) was increased in males at PN30, and Cyp11a1 expression was decreased at 6 and 12 months of age. There were no changes in the adrenals of female Cort-exposed offspring. This study demonstrates that prenatal Cort exposure induces offspring adrenal gland dysfunction in an age- and sex-specific manner, which may contribute to long-term programmed disease in male offspring after maternal stress.

A case of Murray Valley encephalitis in a 2-year-old Australian Stock Horse in south-east Queensland.

CASE REPORT: This report summarises the findings from a case of naturally-occurring Murray Valley encephalitis in a 2-year-old filly presenting with acute onset of depression and weakness. Serum samples tested at the onset of clinical signs were negative for Hendra and Kunjin virus antibodies, but positive for Murray Valley encephalitis virus (MVEV) using IgM-capture ELISA (1 : 300 dilution). A virus neutralisation assay performed 4 weeks later confirmed a titre of 1 : 160. Sera collected in the weeks preceding neurological signs returned a negative titre for MVEV 2 weeks prior followed by a titre of 1:80 in the week prior to illness. Serological surveillance conducted on 67 co-located horses returned a positive titre of 1 : 20 in one in-contact horse. There was no history of clinical disease in that horse. At 3 months after the onset of clinical signs in the index case, the filly continued to show mild facial paresis and hypermetria; the owners elected euthanasia and gave permission for necropsy. Histopathological analysis of the brain showed a mild meningoencephalitis. CONCLUSION: The progression of a naturally-occurring MVEV infection in a horse has been documented in this case.

Characterization of the Blastocystis-specific faecal IgA immune response in pigs.

Blastocystis is an intestinal protist found in many species including humans and pigs. It has a controversial pathogenesis and has been implicated as a potential cause of irritable bowel syndrome. Our previous studies identified pigs as potential animal models for blastocystosis by demonstrating that they were likely natural hosts of Blastocystis and can harbour subtypes (ST) in common with humans. Furthermore, our finding of a lack of intestinal histopathology associated with Blastocystis infection in pigs is also a consistent finding in examined infected humans. In this study, we aimed to identify and characterize the Blastocystis-specific mucosal IgA response in pigs by immunoblotting, using pig faecal antibodies and Blastocystis antigen. Faeces from 233 pigs representing three age groups (sows/boars, growers/weaners and piglets) and including five dexamethasone-immunosuppressed research pigs were tested. The majority (81·5%) of the pigs had faecal IgA reactivity against Blastocystis proteins of molecular weights of 17·5-120 kDa. Reactivity to a >250 kDa protein was found in 18·5% of pigs. Notably, immunosuppressed pigs and piglets were statistically more likely to have reactivity to this protein compared to growers/weaners and sows/boars, respectively. These results corroborate other findings suggesting that compromised immunity may predispose to blastocystosis and support our contention that pigs are potentially good models for pathogenesis studies.

Mid- to late term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways and nutrient transporters in a sex-specific manner.

Maternal hypoxia is a common perturbation that can disrupt placental and thus fetal development, contributing to neonatal impairments. Recently, evidence has suggested that physiological outcomes are dependent upon the sex of the fetus, with males more susceptible to hypoxic insults than females. This study investigated the effects of maternal hypoxia during mid- to late gestation on fetal growth and placental development and determined if responses were sex specific. CD1 mice were housed under 21% or 12% oxygen from embryonic day (E) 14.5 until tissue collection at E18.5. Fetuses and placentas were weighed before collection for gene and protein expression and morphological analysis. Hypoxia reduced fetal weight in both sexes at E18.5 by 7% but did not affect placental weight. Hypoxia reduced placental mRNA levels of the mineralocorticoid and glucocorticoid receptors and reduced the gene and protein expression of the glucocorticoid metabolizing enzyme HSD11B2. However, placentas of female fetuses responded differently to maternal hypoxia than did placentas of male fetuses. Notably, morphology was significantly altered in placentas from hypoxic female fetuses, with a reduction in placental labyrinth blood spaces. In addition mRNA expression of Glut1, Igf2 and Igf1r were reduced in placentas of female fetuses only. In summary, maternal hypoxia altered placental formation in a sex specific manner through mechanisms involving placental vascular development, growth factor and nutrient transporter expression and placental glucocorticoid signalling. This study provides insight into how sex differences in offspring disease development may be due to sex specific placental adaptations to maternal insults.

Bovine viral diarrhea virus cyclically impairs long bone trabecular modeling in experimental persistently infected fetuses.

Persistent infection (PI) with bovine viral diarrhea virus (BVDV) has been associated with osteopetrosis and other long bone lesions, most commonly characterized as transverse zones of unmodeled metaphyseal trabeculae in fetuses and calves. This study was undertaken to characterize the morphogenesis of fetal long bone lesions. Forty-six BVDV-naïve pregnant Hereford heifers of approximately 18 months of age were inoculated with noncytopathic BVDV type 2 containing media or media alone on day 75 of gestation to produce PI and control fetuses, respectively, which were collected via cesarean section on days 82, 89, 97, 192, and 245 of gestation. Radiographic and histomorphometric abnormalities were first detected on day 192, at which age PI fetal long bone metaphyses contained focal densities (4 of 7 fetuses) and multiple alternating transverse radiodense bands (3 of 7 fetuses). Day 245 fetuses were similarly affected. Histomorphometric analysis of proximal tibial metaphyses from day 192 fetuses revealed transverse zones with increased calcified cartilage core (Cg.V/BV, %) and trabecular bone (BV/TV, %) volumes in regions corresponding to radiodense bands (P < .05). Numbers of tartrate resistant acid phosphatase positive osteoclasts (N.Oc/BS, #/mm(2)) and bone perimeter occupied (Oc.S/BS, %) were both decreased (P < .05). Mineralizing surface (MS/BS, %), a measure of tissue level bone formation activity, was reduced in PI fetuses (P < .05). It is concluded that PI with BVDV induces cyclic abnormal trabecular modeling, which is secondary to reduced numbers of osteoclasts. The factors responsible for these temporal changes are unknown but may be related to the time required for osteoclast differentiation from precursor cells.

Blastocystis subtypes in symptomatic and asymptomatic family members and pets and response to therapy.

BACKGROUND: Blastocystis is a common, enteric parasite. The pathogenicity of the organism is uncertain, but subtypes (ST) 1 and 3 have been reported more likely to cause irritable bowel-like symptoms. AIMS: We treated symptomatic patients positive for Blastocystis with conventional therapy and analysed 16 small-subunit (SSU) rDNA to assess clearance and carriage rates and ST prevalence of the parasite in the asymptomatic household members. METHODS: In a longitudinal, prospective case study, 11 symptomatic patients positive for Blastocystis underwent outpatient clinical assessment to exclude other diagnoses before 14 days of either metronidazole 400 mg three times daily or trimethoprim/sulfamethoxazole 160/800 mg twice-daily therapy. Faecal specimens were collected from patients at baseline, day 15, 28 and 56 after therapy and from 17 family members and eight pets at day 15. Specimens were analysed using faecal smear, culture and polymerase chain reaction analysis of 16SSU rDNA. RESULTS: No patient cleared the organism following therapy. ST 1 (45%), 3 (36%), 4 (36%) and 6 (9%) were found in the symptomatic Blastocystis patients, and ST identified before and after therapy were identical in each individual. All household contacts were positive for Blastocystis and 16/17 (94%) contacts showed identical Blastocystis ST to the symptomatic family member. All pets were positive for Blastocystis with polymerase chain reaction testing, 7/8 (88%) demonstrating ST concordance with the symptomatic Blastocystis patients. CONCLUSIONS: Conventional therapy is ineffective for symptomatic Blastocystis infection. The high prevalence of Blastocystis infection within households suggested transmission between humans and their pets. Subtyping analysis of SSU rDNA alone in Blastocystis does not appear to predict pathogenicity.

Comparative pathology of pulmonary hydatid cysts in macropods and sheep.

The development and appearance of hydatid cysts of Echinococcus granulosus in experimentally infected tammar wallabies (Macropus eugenii) and sheep during the period 9-17 months post-infection (mpi) were studied. Cysts of unknown age were also examined from mature, naturally infected sheep. The cysts grew more rapidly and became fertile within a shorter period in wallabies compared with sheep. Cysts from the wallabies were larger in absolute size and were larger relative to the size of the lungs. Microscopical examination revealed that wallaby hydatid cysts developed in small bronchioles. Hydatid cysts in the wallabies had a thicker germinal membrane, with more nuclei and a thicker laminated layer (LL), than hydatid cysts of similar age found in sheep. In contrast, the adventitial layer was thicker in the ovine cysts, comprising a hyalinized layer of degenerate collagen and necrotic cellular debris surrounded by a layer of granulation tissue that was largely absent from lesions in the wallabies. Multilocular cysts were present in sheep, but not in wallabies. The greater thickness of the germinal membrane in wallaby cysts suggests greater parasite activity, which may explain the more rapid growth rate in this host, whereas the thicker adventitial layer in sheep cysts may be restrictive to growth while simultaneously protecting the hydatid from the host immune response. These differences in the parasite-host relationship between macropods and sheep may reflect the relatively recent introduction of the parasite into Australia.

Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus.

The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques (Macaca fascicularis) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1alpha (HIF-1alpha), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses.

Lung tumor development and spontaneous regression in lambs coinfected with Jaagsiekte sheep retrovirus and ovine lentivirus.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring and experimentally inducible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The first aim of this study was to monitor the development of OPA with minimally invasive, real-time observations of animals experimentally infected with JSRV as well as ovine lentivirus (maedi-visna virus). Worldwide, simultaneous infection of sheep with these 2 retroviruses is a common occurrence, naturally and experimentally; consequently, the lung tumor homogenates used as inocula contained both viruses. Following inoculation, computed tomography was used to detect tumor nodules early, before the onset of clinical signs, and to monitor tumor advancement. However, not only was OPA disease progression observed, but the apparent spontaneous regression of OPA was witnessed. In fact, regression was more common than progression following JSRV inoculation of neonatal lambs. Immune responses were detected, particularly involving CD3(+) T cells and the production of antibodies against JSRV that may mediate the spontaneous regression of JSRV-induced OPA. The second aim of this study was to determine whether OPA tumors harbor genetic alterations similar to those found in human lung adenocarcinoma. No mutations were found in the tyrosine kinase domain of the epidermal growth factor receptor, KRAS codons 12 and 13, or the DNA-binding domain of p53 in tumor DNA from naturally occurring and experimentally-induced OPA cases. Overall, the genetic profile combined with the disease development data provides further important characterization of OPA and describes, for the first time, spontaneous regression of OPA tumors in experimentally infected sheep.

Transplacental infection with non-cytopathic bovine viral diarrhoea virus types 1b and 2: viral spread and molecular neuropathology.

Infection with bovine viral diarrhoea virus (BVDV) represents a reproducible natural animal model in which to study mechanisms of transplacental viral infection. In the present study, BVDV-seronegative heifers were challenged intranasally with non-cytopathic BVDV of genotype 1b or 2. Fetuses were retrieved by caesarean section 7-114 days post-challenge of the dam and subjected to virological, histopathological and immunohistochemistry(IHC) studies. Gross and histopathological changes were only seen in fetuses infected at gestational age 75-85 days and retrieved at gestational age 190 days. Viral antigen could be detected in most tissues from 14 days post-infection, but the primary target organs for histopathological changes were brain, liver and spleen. In the brain, microscopical changes included leucomalacia and macrophage infiltration of meninges and neuropil. Viral antigen was detected in neurons, oligodendrocyte precursors and infiltrating macrophages. IHC revealed normal to slightly increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in the infected fetuses, with evidence of neuronal apoptosis and induction of inducible nitric oxide synthase (iNOS) and phospho-p38alpha mitogen-activated protein kinase (MAPK). These findings suggest that hypoxia may play only a limited role in the pathogenesis of the neural lesions. By contrast, virus-induced cytokine cascades, as part of the fetal innate immune response, and apoptosis of neurons and glial precursor cells may be central to the development of lesions.

Functional genomic and serological analysis of the protective immune response resulting from vaccination of macaques with an NS1-truncated influenza virus.

We are still inadequately prepared for an influenza pandemic due to the lack of a vaccine effective for subtypes to which the majority of the human population has no prior immunity and which could be produced rapidly in sufficient quantities. There is therefore an urgent need to investigate novel vaccination approaches. Using a combination of genomic and traditional tools, this study compares the protective efficacy in macaques of an intrarespiratory live influenza virus vaccine produced by truncating NS1 in the human influenza A/Texas/36/91 (H1N1) virus with that of a conventional vaccine based on formalin-killed whole virus. After homologous challenge, animals in the live-vaccine group had greatly reduced viral replication and pathology in lungs and reduced upper respiratory inflammation. They also had lesser induction of innate immune pathways in lungs and of interferon-sensitive genes in bronchial epithelium. This postchallenge response contrasted with that shortly after vaccination, when more expression of interferon-sensitive genes was observed in bronchial cells from the live-vaccine group. This suggested induction of a strong innate immune response shortly after vaccination with the NS1-truncated virus, followed by greater maturity of the postchallenge immune response, as demonstrated with robust influenza virus-specific CD4+ T-cell proliferation, immunoglobulin G production, and transcriptional induction of T- and B-cell pathways in lung tissue. In conclusion, a single respiratory tract inoculation with an NS1-truncated influenza virus was effective in protecting nonhuman primates from homologous challenge. This protection was achieved in the absence of significant or long-lasting adverse effects and through induction of a robust adaptive immune response.

Integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics.

Recent outbreaks of avian influenza in humans have stressed the need for an improved nonhuman primate model of influenza pathogenesis. In order to further develop a macaque model, we expanded our previous in vivo genomics experiments with influenza virus-infected macaques by focusing on the innate immune response at day 2 postinoculation and on gene expression in affected lung tissue with viral genetic material present. Finally, we sought to identify signature genes for early infection in whole blood. For these purposes, we infected six pigtailed macaques (Macaca nemestrina) with reconstructed influenza A/Texas/36/91 virus and three control animals with a sham inoculate. We sacrificed one control and two experimental animals at days 2, 4, and 7 postinfection. Lung tissue was harvested for pathology, gene expression profiling, and proteomics. Blood was collected for genomics every other day from each animal until the experimental endpoint. Gross and microscopic pathology, immunohistochemistry, viral gene expression by arrays, and/or quantitative real-time reverse transcription-PCR confirmed successful yet mild infections in all experimental animals. Genomic experiments were performed using macaque-specific oligonucleotide arrays, and high-throughput proteomics revealed the host response to infection at the mRNA and protein levels. Our data showed dramatic differences in gene expression within regions in influenza virus-induced lesions based on the presence or absence of viral mRNA. We also identified genes tightly coregulated in peripheral white blood cells and in lung tissue at day 2 postinoculation. This latter finding opens the possibility of using gene expression arrays on whole blood to detect infection after exposure but prior to onset of symptoms or shedding.

Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta).

Multifocal submucosal stromal tumors were diagnosed in a 5.5-year-old rhesus macaque (Macaca mulatta) experimentally infected with simian immunodeficiency virus, strain SIVsmE660, and CD4+ T cell depleted. The animal was negative for simian retroviruses, SRV-1, -2, and -5. Polymerase chain reaction analysis of DNA from tumor and spleen tissue revealed abundant, preferential presence of retroperitoneal fibromatosis herpesvirus, the macaque homologue of the Kaposi sarcoma-associated herpesvirus (human herpesvirus-8), in the tumors. This was corroborated by demonstration of viral latent nuclear antigen-1 in the nuclei of a majority of the spindeloid tumor cells. Low levels of an additional macaque herpesvirus, rhesus rhadinovirus, were also detected in the spleen and tumor tissues. The spindeloid cells labeled positively for vimentin and CD117 but were negative for CD31, CD68, desmin, and smooth muscle cell actin. Collectively, these findings suggest a relation to but not absolute identity with simian mesenchymoproliferative disorders (MPD) or typical gastrointestinal stromal tumors (GISTs).

Greater sensitivity of pigtailed macaques (Macaca nemestrina) than baboons to total body irradiation.

Two juvenile pigtailed macaques (animals 1 and 2) received total body irradiation (TBI) followed by autologous stem cell transplantation, by a procedure known to be well tolerated by baboons. In this procedure, the TBI consisted of treatment on two consecutive days with 255cGy on one side, followed after 1-2 min by a similar dose on the other side. The two pigtailed macaques showed rapid haematopoietic engraftment, but succumbed either to systemic cytomegalovirus (CMV) infection and necrotising colitis or to haemorrhagic cystitis and tubulointerstitial nephritis. For four further pigtailed macaques (animals 3-6) the radiation procedure was changed to four equal doses of 255cGy, given 6-12 h apart. Animals 4-6 all showed engraftment and survived for long periods (>218 days), with no, or only minor treatable, complications. Animal 3 failed to show engraftment and succumbed to radiation-induced vascular lesions and severe multiorgan haemorrhages. The results suggest that pigtailed macaques have a lower tolerance threshold than baboons, rhesus macaques or human beings to TBI, the adverse effects of TBI being indistinguishable from those seen in human patients. The results also suggest that a hyperfractionated radiation procedure can prevent radiation-induced morbidity and mortality in pigtailed macaques.

Studies of the pathogenesis of bovine pestivirus-induced ovarian dysfunction in superovulated dairy cattle.

Two experiments (Experiment I, n=12 Holstein-Friesian heifers; Experiment II, n=8 Jersey cows) were conducted to investigate the pathogenesis of bovine pestivirus-induced ovarian dysfunction in cattle. In both experiments the cattle were superovulated with twice daily injections of a porcine pituitary extract preparation of follicle stimulating hormone (FSH-P), for 4 days commencing on Day 10+/-2 after a presynchronised oestrus. The heifers received a total dose of 30 mg and the cows 32 mg of FSH-P. Prostaglandin F(2alpha) (PGF(2alpha)) was administered 48 h after commencement of superovulation and all cattle were artificially inseminated (AI) between 48 and 66h after PGF(2alpha) treatment. In both experiments bovine pestivirus seronegative cattle (Experiment I, n=6; Experiment II, n=4) were inoculated intranasally with an Australian strain of non-cytopathogenic bovine pestivirus (bovine viral diarrhoea virus Type 1) 9 days prior to AI. Bovine pestivirus infection was confirmed by seroconversion and/or virus isolation in all of the inoculated cattle, consistent with a viremia occurring approximately between Day 5 prior to AI and the day of AI. Ovarian function was monitored in both experiments by daily transrectal ultrasonography and strategic blood sampling to determine progesterone, oestradiol-17beta, luteinising hormone (LH) and cortisol profiles. Non-surgical ova/embryo recovery was performed on Day 7 after AI. In Experiment II half the cattle were slaughtered on Day 2 and the remainder on Day 8 after AI, and the ovaries submitted for gross and histopathological examination including immunohistochemistry to demonstrate the presence of bovine pestivirus antigen. In both studies, comparisons were made between infected and confirmed uninfected (control) animals. Overall the bovine pestivirus infected cattle had significantly lower (P<0.05) ova/embryo recovery rates compared to the control cattle. There was evidence of either an absence (partial or complete) of a preovulatory LH surge or delay in timing of the LH peak in the majority (90%) of infected heifers and cows, and histologically, there was evidence of non-suppurative oophoritis with necrosis of granulosa cells and the oocyte in follicles from the infected cows. By contrast only 20% of the control heifers and cows had evidence of absence of a pre-ovulatory LH surge. These experiments collectively demonstrate that bovine pestivirus infection during the period of final growth of preovulatory follicles may result in varying degrees of necrosis of the granulosa cells with subsequent negative effects on oestradiol-17beta secretion which in turn negatively affects the magnitude and/or timing of the preovulatory LH surge.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy.

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 x 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. At the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Proteolysis of human hemoglobin by schistosome cathepsin D.

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains.

Monocyte macrophages (Mphi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to Mphi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of, various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca(2+)/Mg(2+) enhanced DV binding. This argues against involvement of beta(2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains.

Dengue viral infections; pathogenesis and epidemiology.

Dengue viral infections affect up to 100 million individuals per year. Dengue haemorrhagic fever is a clinical form of disease characterised by intravascular fluid loss. There has been a marked increase in the incidence of this form of the disease over the last few decades, associated with significant mortality, particularly in the paediatric population. A number of theories relating to the pathogenesis of dengue haemorrhagic fever exist that have evolved from the analysis of the epidemiology of this disease. Virological and immunopathological factors are both important but the exact mechanisms for the disease are unknown.