Comparison of microscopic and molecular enumeration methods for insect viruses: Cryptophlebia leucotreta granulovirus as a case study.

Enumeration techniques were compared for quantification of the South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA), used as a biopesticide to control false codling moth (Thaumatotibia leucotreta), an insect pest of various fruits and nuts, including citrus. The routine enumeration method for CrleGV-SA virus particles in experimentation and production of CrleGV-SA biopesticides is dark field microscopy. This method was compared with spectrophotometry, scanning electron microscopy (SEM) and real time quantitative polymerase chain reaction (qPCR). The purpose was to develop an accurate and reliable routine enumeration method for CrleGV-SA occlusion bodies (OBs) and to validate the use of dark field microscopy. Purified and semi-purified CrleGV-SA viral stocks were used. Spectrophotometry was not a suitable or accurate enumeration method. Dark field microscopy and SEM were accurate and statistically comparable (p = 0.064), validating the use of dark field microscopy as an enumeration method for granulovirus (GV). However, SEM has superior resolution and the advantage of easily distinguishing virus particles from debris in semi-purified viral stock preparations. A quantitative PCR technique has been developed based on use of specific oligonucleotide primers for the granulin gene. This has the advantage of not being affected by contamination with non-biological debris or biological material, which impact on the other methods.

Rice sucrose transporter1 (OsSUT1) up-regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles.

The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3-week-old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1- to 10-day infestation time course, using a combination of gene expression and ß-glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide-field fluorescence microscopy was used to confirm the expression of OsSUT1-promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up-regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up-regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid-related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.

Sutherlandia frutescens prevents changes in diabetes-related gene expression in a fructose-induced insulin resistant cell model.

ETHNOPHARMACOLOGICAL RELEVANCE: The African medicinal plant Sutherlandia frutescens (L.) R.Br. (Fabaceae) is traditionally used to treat diabetes and has been shown to have anti-diabetic properties in animal models. The present study investigated the capacity of an aqueous extract of Sutherlandia frutescens to prevent insulin resistance (a precursor of type 2 diabetes) in a human liver cell culture and to identify genes regulated by Sutherlandia frutescens treatment. MATERIALS AND METHODS: A combination of insulin and fructose was used to generate an in vitro model of insulin resistance in human liver cells to compare untreated control, insulin resistant and Sutherlandia frutescens treated insulin resistant cultures. Insulin resistance and its prevention by Sutherlandia frutescens were measured by glucose uptake, gluconeogenesis and lipid accumulation in the cell cultures. Changes in gene expression were quantified using the RT(2)Profiler(TM) PCR Array of 84 diabetes-related genes. RESULTS: The insulin resistant Chang liver cells took up significantly less 2-[(3)H]-deoxyglucose (p<0.05) than controls, released more glucose into the culture medium (p<0.05) and accumulated more intracellular lipid (p<0.05). Simultaneous treatment with Sutherlandia frutescens prevented development of these insulin resistance parameters (p<0.05). A total of 27 potential gene targets of Sutherlandia frutescens were significantly up or down regulated in the Sutherlandia frutescens treated insulin resistant cells. The gene VAMP3, which plays a role in vesicle transport, was down-regulated by insulin resistance, and up-regulated by Sutherlandia frutescens. Twenty six other genes encoding vesicle transporters, receptors, signalling molecules, transcription factors, and metabolic enzymes were significantly regulated by Sutherlandia frutescens. CONCLUSION: These results confirm that Sutherlandia frutescens can prevent insulin resistance in hepatocytes. The identified changes in gene expression indicate several potential mechanisms of anti-diabetic action for Sutherlandia frutescens, reflecting the multiple bioactive compounds previously identified in aqueous extracts of Sutherlandia frutescens.

The Th2 cytokine environment of the placenta.

It is now accepted that local changes to the balance of Th1/Th2-type cytokines occur during pregnancy within the maternal uterus and fetoplacental unit. These changes in cytokine profiles contribute to implantation of the embryo, development of the placenta, and survival of the fetus to term. Overall within the placenta there is a bias in the ratio of Th1:Th2 cytokines towards the Th2-type cytokines. However, there are specific fluctuations in this balance at implantation and during the initiation of parturition. The predominant cytokines at each stage of gestation function both to limit maternal immune rejection of the semi-allogeneic embryo/fetus, especially at the maternofetal interface; and to facilitate the on-going physiological processes within the maternal reproductive tract. These two, at times conflicting, roles are discussed in this review, with key evidence concerning cytokine expression and function from mouse and humans.

A critical review of the role of the major histocompatibility complex in fertilization, preimplantation development and feto-maternal interactions.

From conception to old age, the major histocompatibility complex (MHC) is at the centre of immune responses that aid survival, fitness and adaptation of mammalian species to the environment. Its main function is that of controlling adaptive immunity, particularly T-cell-mediated immunity towards pathogens. In several species, including humans, the MHC is also able to elicit T-cell-mediated immune responses to allogeneic MHC antigens (non-self MHC antigens expressed by another individual from the same species). Although this phenomenon was originally identified in mice by the somewhat unnatural means of tissue transplantation, it was soon realized that it may also play an important role in the natural state, since the mammalian fetus in the maternal uterus is semi-allogeneic, due to the presence of MHC genes inherited from the father. Thus, during normal pregnancy the maternal immune system undergoes changes that lead to tolerance of the fetus. The MHC can play a dual role in the reproduction process: firstly influencing mating choice in some species, affecting the mother-father MHC matching; and secondly influencing the development of the fertilized ovum during the preimplantation period. In this review we examine the role of the MHC at three distinct levels: (i) MHC expression in gametes and its role in fertilization; (ii) MHC expression in placental tissue; and (iii) MHC expression in embryonic tissue. We suggest that the MHC plays a pleiotropic role, both in fitness (survival and reproductive success) and in development, thereby ensuring the survival of the species in future generations.

Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy.

PROBLEM: To investigate the expression of the Th2-type cytokine interleukin (IL)-13 and its receptor in human placenta during gestation. METHOD OF STUDY: Expression of IL-13 and its receptor was analyzed by reverse transcriptase (RT)-polymerase chain reaction (PCR), in situ hybridization and immunohistochemistry using human placental samples. RESULTS: IL-13 mRNA was detected by RT-PCR in placental extracts from all stages of gestation. In situ hybridization revealed IL-13 mRNA in first trimester cytotrophoblast and syncytiotrophoblast. Few positive cells were found within decidual sections from the same pregnancy. Immunohistochemistry revealed a similar pattern to in situ hybridization. This was largely absent in second- and third-trimester placentae. IL-13 receptor alpha chain (IL-13R alpha) was detected by immunofluorescence on the surface of leukocytes in first-trimester villous core and decidua. CONCLUSIONS: The expression of IL-13 is spatially and temporally regulated in the placenta, and associated with the close proximity of a receptor-bearing target leukocyte population.

Regulation of major histocompatibility complex and TAP gene products in preimplantation mouse stage embryos.

PROBLEM: To determine the ontogeny of major histocompatibility complex (MHC) expression and TAP products in mouse embryos. METHOD OF STUDY: mRNAs encoding MHC and associated molecules were identified by reverse transcriptase-polymerase chain reaction, and the protein products were localized by confocal microscopy. RESULTS: mRNAs encoding class Ia (H-2Db) and class Ib (Q7/9) were present in one-cell embryos, whereas beta 2-microglobulin (beta 2-m) transcripts were not detected until the two-cell stage. Transporter TAP1, but not TAP2, transcripts were detected only in blastocysts. H-2 class Ia (classical) protein was detected on the surface of two-cell embryos, H-2 class Ib (nonclassical) protein was detected on one-cell embryos, and beta 2-m transcripts were detected on eight-cell embryos; TAP1 protein was present at low levels in the cytoplasm from the one-cell stage onward, increasing in expression in blastocysts. CONCLUSIONS: In mice, MHC class I mRNAs encoding the heavy chain of H-2- and Q7/9-encoding Qa2 molecules are synthesized soon after conception prior to implantation. Similarly, the nonpolymorphic MHC class I-associated molecule beta 2-m also is expressed before implantation. TAP1, but not TAP2, is first detected at the blastocyst stage, thus preceding the onset of TAP2 in embryonic development.

Decreased levels of the potent regulator of monocyte/macrophage activation, interleukin-13, in the peritoneal fluid of patients with endometriosis.

Endometriosis is characterized by an increase in the number, activation and secretory activity of peritoneal fluid macrophages. Factors regulating the activation of these cells may be important in the pathophysiology of this disease. In this study we measured by enzyme-linked immunosorbent assay the concentrations of the macrophage inhibitory factor interleukin (IL)-13 in the peritoneal fluid of women with and without endometriosis. It was found that women with endometriosis had significantly lower amounts of IL-13 (95 +/- 9.8 pg/ml) in peritoneal fluid, compared with women without endometriosis (115 +/- 30 pg/ml) (P < 0.01). No cycle-specific variation was evident for either group. Another macrophage inhibitory interleukin (IL-10) was also measured, but no differences between women with (16.1 +/- 13.2 pg/ml) or without (10.3 +/- 5.6 pg/ml) endometriosis were seen. The immunolocalization of IL-13 was assessed in eutopic and ectopic endometrium and in isolated peritoneal fluid cells. Glandular epithelial cells and stromal cells in both eutopic and ectopic endometrium were immunopositive for IL-13. No cycle-specific differences in the immunolocalization of IL-13 were seen. In conclusion, the reduced amounts of IL-13 in the peritoneal fluid of women with endometriosis may lead to a lack of suppression of macrophage activation, thereby contributing to the overall pathogenesis of this disease.

Preimplantation mouse embryos express Mhc class I genes before the first cleavage division.

We have used oligonucleotide primers complementary for polymorphic regions of the mouse H-2D gene in a highly sensitive polymerase chain reaction (PCR) assay to detect the transcription of maternal and paternal class I mRNAs in gametes and preimplantation embryos. Using congenic strains of mice differing only at the major histocompatibility loci, class I (H-2D) mRNA of both the maternal and paternal haplotypes was demonstrated in embryos from the one-cell zygote to the late blastocyst stage of development but could not be detected in vas deferens or in vitro capacitated sperm or in ovulated secondary oocytes. These data clearly show that both paternally and maternally inherited Major histocompatibility complex (Mhc) class I genes are transcribed from the earliest stages of embryonic development, and suggest that developmental regulation of expression of their protein products is principally at the post-transcriptional level.

Differentiation-related changes in quantitative binding of immunomagnetic beads.

Experiments have been carried out to demonstrate that cell viability and phenotypic characteristics are not affected by exposure to constant magnetic fields or growth in the presence of Dynabeads. This paper also demonstrates that the changes in the surface concentration of the antigens, SSEA-1 (stage-specific embryonic antigen-1) and histocompatibility antigen (MHC H2-Db) during differentiation as determined by flow cytometric analysis, are mirrored by changes in the numbers of bound immunomagnetic particles (Dynabeads) targeted with monoclonal antibodies to these cell surface antigens. These results clearly indicate that the numbers of beads bound to cells reflect the numbers of specific surface antigens present on the cells.

Down regulation of lamins A and C by murine interferon beta.

Treatment of spontaneously differentiated PSMB embryonal carcinoma cells with murine interferon beta results in a transient decrease in the expression of the nuclear lamins A and C. Reduced levels of mRNAs were observed 4 h after the addition of interferon beta, with reductions in the polypeptides and assembled proteins within the nuclear lamina seen after 8 h of treatment. Expression of the 72-kDa (lamin A) and the 62-kDa (lamin C) polypeptides remained down regulated for 8 h, returning to control levels after 16 h of interferon treatment. The specificity of this response is indicated by the inhibitory action of a neutralizing antibody to interferon.

Lectin-induced abnormalities of mouse blastocyst hatching and outgrowth in vitro.

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.

Interferon beta increases expression of vimentin at the messenger RNA and protein levels in differentiated embryonal carcinoma (PSMB) cells.

Murine interferon beta increases expression of the 58-kDa intermediate filament protein vimentin by differentiated PSMB cells. Enhanced amounts of vimentin mRNA and protein have been detected using Northern hybridization and Western blotting techniques. Immunocytochemical analysis demonstrates the increased assembly of the protein into the intermediate filament network and its relocalization around the cell nucleus. Induction follows a defined time course, with peak protein levels 16 h post interferon addition, followed by a gradual decline over the next 36 h.

Fucosylated glycoconjugates appear on mouse embryos during blastocyst formation.

Mouse embryos were analysed for expression of surface glycoconjugates using a panel of fluorescein-labelled lectins. Morulae and blastocysts but not early cleavage stages stained brightly with fucose binding lectins. By contrast, cleavage stages stained brightly with all the other lectins tested. These findings provide evidence for substantial reorganisation of the cell surface during blastocyst formation and are consistent with a role for fucosylated glycoconjugates in compaction.

The effect of recombinant human tumour necrosis factor on growth and macromolecular synthesis of human epithelial cells.

We have studied the effect of recombinant human tumour necrosis factor (rHuTNF) on growth and macromolecular synthesis in a range of normal and transformed epithelial cell types. Tumour necrosis factor did not affect the growth of normal human mammary epithelial cells, but its growth-inhibitory action on the SV40-transformed human mammary epithelial cell line HBL-100 increased with passage number in association with a progression of malignant phenotype. However, of two lines derived from nude mouse tumours of HBL-100 lines, one, HBLT-12, did not respond to rHuTNF, and the other, HBLT-11 showed some growth stimulation by high dose rHuTNF. Macromolecular synthesis in HBLT-11 was not affected by rHuTNF. The breast cancer cell lines MCF-7 and BT20 were sensitive to the cytotoxic effects of rHuTNF. In MCF-7 a gradual decrease in RNA and DNA synthesis occurred over 48 h, ending with an accumulation of cells in S and G2 phase of the cell cycle and cell death. The addition of alpha- or gamma-interferon increased, but did not accelerate the cytotoxicity of rHuTNF.

DNA fragmentation and cytotoxicity caused by tumor necrosis factor is enhanced by interferon-gamma.

Recombinant human tumor necrosis factor (rhuTNF) induced DNA fragmentation in sensitive cell lines. This fragmentation was similar to that caused by lymphotoxin-containing cytotoxic T cell culture supernatants. The percentage of DNA cleaved correlated with the degree of cell growth inhibition shown by individual cell lines. DNA fragmentation was first seen after 12 h treatment, and increased slowly with time. The presence of 100 microM ZnSO4 inhibited the rhuTNF-induced DNA cleavage in MCF-7 cells. Recombinant interferon-gamma (rhuIFN-gamma) did not induce DNA cleavage, although it reduced the growth of all the cell lines used in this study. However, it interacted with rhuTNF to produce a doubling in the percentage of DNA fragmentation, and increased cytotoxicity in rhuTNF-sensitive cell lines. Pretreatment with rhuIFN-gamma for 1 h prior to rhuTNF treatment also enhanced DNA fragmentation and cell killing.