Paul Masendycz

Hypoxia Prolongs Monocyte/Macrophage Survival and Enhanced Glycolysis Is Associated with Their Maturation under Aerobic Conditions

In chronic inflammatory lesions macrophages are abundant and adapt to the low oxygen concentrations often present there. In low oxygen some cell types die by apoptosis, as reported for macrophage cell lines, while others survive better as they shift their metabolism to anaerobic glycolysis. It was found here that hypoxia prolongs the survival of murine bone marrow-derived macrophages, either in the absence or presence of low CSF-1 (M-CSF) concentrations. Although Akt activity increased in bone marrow-derived macrophages in the low oxygen conditions, the levels of both anti- and proapoptotic Bcl-2 family members decreased. Glycolysis was enhanced as judged by increased glucose uptake, glucose transporter expression, lactate dehydrogenase mRNA expression, and lactate secretion. Human monocytes responded similarly to low oxygen, and a number of genes associated with glycolysis were shown by microarray analysis and quantitative PCR to be up-regulated. Interestingly, human monocyte-derived macrophages showed evidence of enhanced glycolysis even under aerobic conditions. It is proposed that certain monocyte/macrophage populations survive better under conditions of low oxygen, thereby contributing to their increased numbers at sites of chronic inflammation and tumors; it is also proposed that as macrophages differentiate from monocytes they begin to adopt a glycolytic metabolism allowing them to adapt readily when exposed to low oxygen conditions.

Early phase II trial of human rotavirus vaccine candidate RV3

A naturally attenuated, human neonatal strain, rotavirus vaccine candidate RV3, was tested in a limited phase II randomized double-blind controlled trial. Doses of 1 ml, containing placebo or 6.5 x 10(5) fluorescent cell forming units (fcfu) of virus in AGMK cells, were given at 3, 5 and 7 months of age. Limited replication in the small intestine is implied by the lack of virus excretion, and by the occurrence of an immune response in only 46% of the infants. However, those who developed an immune response were partially protected against rotavirus disease during the subsequent winter epidemic (protective efficacy 54%), supporting observations of protection induced by natural infection by this strain. Protection appeared to be heterotypic. Further trials are warranted, employing strategies to increase immunogenicity of this human rotavirus candidate vaccine.

Epidemiological Patterns of Rotaviruses Causing Severe Gastroenteritis in Young Children throughout Australia from 1993 to 1996

ABSTRACT Rotavirus strains that caused severe diarrhea in 4,634 (2,533 male) children aged less than 5 years and admitted to major hospitals in eight centers throughout Australia from 1993 to 1996 were subject to antigenic and genetic analyses. The G serotypes of rotaviruses were identified in 81.9% (3,793 of 4,634) children. They included 67.8% (from 3,143 children) serotype G1 isolates (containing 46 electropherotypes), 11.5% (from 531 children) serotype G2 isolates (27 electropherotypes), 0.8% (from 39 children) serotype G3 isolates (8 electropherotypes), and 1.6% (from 76 children) serotype G4 isolates (9 electropherotypes). G6 (two strains) and G8 (two strains) isolates were identified during the same period. G1 serotypes were predominant in all centers, with intermittent epidemics of G2 serotypes and sporadic detection of G3 and G4 strains. With the exception of two strains (typed as G1P2A[6] and G2P2A[6]) all serotype G1, G3, and G4 strains were P1A[8] and all serotype G2 strains were P1B[4]. Two contrasting epidemiological patterns were identified. In all temperate climates rotavirus incidence peaked during the colder months. The genetic complexity of strains (as judged by electropherotype) was greatest in centers with large populations. Identical electropherotypes appeared each winter in more than one center, apparently indicating the spread of some strains both from west to east and from east to west. Centers caring for children in small aboriginal communities showed unpredictable rotavirus peaks unrelated to climate, with widespread dissemination of a few rotavirus strains over distances of more than 1,000 km. Data from continued comprehensive etiological studies of genetic and antigenic variations in rotaviruses that cause severe disease in young children will serve as baseline data for the study of the effect of vaccination on the incidence of severe rotavirus disease and on the emergence of new strains.

Genetic and Antigenic Characterization of Rotavirus Serotype G9 Strains Isolated in Australia between 1997 and 2001

ABSTRACT Rotavirus serotype G9 is recognized as the most widespread of the emerging serotypes, emerging since 1996 as a frequent cause of severe acute gastroenteritis in children from many countries covering all continents of the world. This study characterized serotype G9 strains collected in three widely separated Australian centers from 1997 to 2001. All G9 strains possessed the VP4 P[8] and VP6 subgroup II genes. The overall prevalence of the G9 strains increased in Australia, from 0.6% of the strains found in 1997 to 29% of the strains found in 2001. The prevalence of G9 relative to all other serotypes varied from year to year and with geographic location. In Melbourne (representing east coast urban centers), G9 made up 11 to 26% of all of the strains found from 1999 to 2001. In Perth (representing west coast urban centers), G9 made up less than 2% of the strains found in 1997 to 2000 but increased to 18.6% of the strains found in 2001. In Alice Springs (representing widely dispersed settlements in northern arid regions), G9 made up 0 to 5% of the strains found from 1997 to 2000 and was the dominant strain in 2001, making up 68.9% of all of the strains found. Three distinct antigenic groups based on reaction with neutralizing monoclonal antibodies (N-MAbs) were identified, including a dominant group (63%) that cross-reacted with the serotype G4 N-MAb. Phylogenetic analysis of the VP7-encoding gene from Australian strains, compared with a worldwide collection of G9 strains, showed that the Australian G9 strains made up a genetic group distinct from other serotype G9 strains identified in the United States and Africa. Future epidemiological studies of the occurrence of G9 strains should combine reverse transcription-PCR and typing with G1 to G4 and G9 N-MAbs to determine the extent of G9 and G4 cross-reactions among rotavirus strains, in order to assess the need to incorporate G9 strains into new candidate vaccines.

A Central Role for the Hsp90·Cdc37 Molecular Chaperone Module in Interleukin-1 Receptor-associated-kinase-dependent Signaling by Toll-like Receptors

Toll-like receptors (TLRs) serve crucial roles in innate immunity by mediating the activation of macrophages by microbial pathogens. The protein kinase interleukin-1 receptor associated kinase (IRAK-1) is a key component of TLR signaling pathways via its interaction with TRAF6, which subsequently leads to the activation of MAP kinases and various transcription factors. IRAK-1 is degraded following TLR activation, and this has been proposed to contribute to tolerance in macrophages by limiting further TLR-mediated signaling. Using a mass spectrometric-based approach, we have identified a cohort of chaperones and co-chaperones including Hsp90 and Cdc37, which bind to IRAK-1 but not IRAK-4 in 293T cells. Pharmacologic inhibition of Hsp90 led to a rapid decline in the expression level of IRAK-1, whereas overexpression of Cdc37 enhanced the activation and oligomerization of IRAK-1 in 293T cells. Significantly, the inhibition of Hsp90 in macrophages resulted in the destabilization and degradation of IRAK-1 but not IRAK-4. Concomitant with the loss of IRAK-1 expression was a reduction in the activation of p38 MAP kinase and Erk1/2 following stimulation with the bacterially derived TLR ligands, lipopolysaccharide and CpG DNA. Moreover, TLR ligand-induced expression of proinflammatory cytokines was also reduced. Thus we conclude that the level of on-going support provided to IRAK-1 by the Hsp90-Cdc37 chaperone module directly influences the magnitude of TLR-mediated macrophage activation. In addition, because further TLR signaling depends on the synthesis of new IRAK-1, the Hsp90-Cdc37 chaperone module could also contribute to tolerance in macrophages by controlling the rate at which nascent IRAK-1 is folded into a functional conformation.

AMINO ACIDS INVOLVED IN DISTINGUISHING BETWEEN MONOTYPES OF ROTAVIRUS G SEROTYPES 2 AND 4

Neutralizing monoclonal antibodies (N-MAbs) to serotype G2 and G4 rotaviruses were used to study intraserotypic variation by selection and characterization of N-MAb-resistant antigenic variants and reaction of N-MAbs with prototype rotavirus strains. Two G2-specific N-MAbs reacted with G2 rotaviruses S2, DS-1, RV-5 and RV-6 but not with 1076. Sequence analysis of the gene encoding VP7 of 1076 virus showed that the differences in amino acid sequence between 1076 virus and the other G2 strains at position 147, 213 and 217 correlated with the loss of N-MAb reactivity. Rotavirus variant mutation mapping data suggested that the amino acid difference at position 213 was likely to be of greatest importance. Rotavirus 1076 was defined as monotype b within G2 strains, whereas S2, DS-1, RV-5 and RV-6 belong to monotype a. The molecular basis for G4 subtypes/monotypes was also studied. The monotype G4b N-MAb 3A3 selected an antigenic variant with an amino acid mutation at position 96, whereas variants of the G4a-reactive N-MAb ST-3:1 showed a mutation at position 94, which produced a new, utilized glycosylation site. Neutralization by N-MAb ST-3:1 was also affected by amino acid changes at position 96. Reactions with these N-MAbs show that serotype G2 viruses can be divided into monotypes and confirm the observation that serotype G4 rotaviruses can be subdivided into subtypes/monotypes a and b. The G2 monotypes relate to differences at particular amino acids within antigenic region C and possibly region B, whereas antigenic region A is most important for G4 monotype differentiation.

Rotavirus strain surveillance—An Australian perspective of strains causing disease in hospitalised children from 1997 to 2007

This study documents rotavirus strains causing severe disease in Australian children during the pre-vaccine era. During the period 1997-2007, rotavirus strains from national multi-centre hospital-based surveillance in Australia were analysed for G and P types. G1P[8] was the dominant genotype identified during the 11-year study, with intermittent peaks associated with genotypes G2P[4], G3P[8] and G9P[8]. The results provide baseline information of the G and P genotypes causing disease in Australian children, and highlight the unpredictable changes in genotype incidence that can occur on both a local and national level. To be optimally effective, rotavirus vaccines must prevent disease caused by all common rotavirus genotypes.

Regulation of the Endosomal SNARE Protein Syntaxin 7 by Colony-Stimulating Factor 1 in Macrophages

ABSTRACT Colony-stimulating factor 1 (CSF-1) is the main growth factor controlling the development of macrophages from myeloid progenitor cells. However, CSF-1 also regulates some of the key effector functions of macrophages (e.g., phagocytosis and cytokine secretion). The endosomal SNARE protein syntaxin 7 (Stx7) regulates vesicle trafficking events involved in phagocytosis and cytokine secretion. Therefore, we investigated the ability of CSF-1 to regulate Stx7. CSF-1 upregulated Stx7 expression in primary mouse macrophages; it also upregulated expression of its SNARE partners Vti1b and VAMP8 but not Stx8. Additionally, CSF-1 induced the rapid serine phosphorylation of Stx7 and enhanced its binding to Vti1b, Stx8, and VAMP8. Bioinformatics analysis and results from experiments with kinase inhibitors suggested the CSF-1-induced phosphorylation of Stx7 was mediated by protein kinase C and Akt in response to phosphatidylinositol 3-kinase activation. Based on mutagenesis studies, CSF-1 appeared to increase the binding of Stx7 to its SNARE partners by inducing the phosphorylation of serine residues in the Habc domain and/or “linker” region of Stx7. Thus, CSF-1 is a key regulator of Stx7 expression and function in macrophages. Furthermore, the effects of CSF-1 on Stx7 may provide a mechanism for the regulation of macrophage effector functions by CSF-1.

Phosphatidylinostitol-3 kinase and phospholipase C enhance CSF-1-dependent macrophage survival by controlling glucose uptake.

Colony stimulating factor-1 (CSF-1)-dependent macrophages play crucial roles in the development and progression of several pathological conditions including atherosclerosis and breast cancer metastasis. Macrophages in both of these pathologies take up increased amounts of glucose. Since we had previously shown that CSF-1 stimulates glucose uptake by macrophages, we have now investigated whether glucose metabolism is required for the survival of CSF-1-dependent macrophages as well as examined the mechanism by which CSF-1 stimulates glucose uptake. Importantly, we found that CSF-1-induced macrophage survival required metabolism of the glucose taken up in response to CSF-1 stimulation. Kinetic studies showed that CSF-1 stimulated an increase in the number of glucose transporters at the plasma membrane, including Glut1. The uptake of glucose induced by CSF-1 required intact PI3K and PLC signalling pathways, as well as the downstream effectors Akt and PKC, together with a dynamic actin cytoskeleton. Expression of constitutively active Akt partially restored glucose uptake and macrophage survival in the absence of CSF-1, suggesting that Akt is necessary but not sufficient for optimal glucose uptake and macrophage survival. Taken together, these results suggest that CSF-1 regulates macrophage survival, in part, by stimulating glucose uptake via Glut1, and PI3K and PLC signalling pathways.

Sequence of the VP7 Gene of an Atypical Human Rotavirus: Evidence for Genetic and Antigenic Drift

The nucleotide sequence of the gene encoding the outer capsid glycoprotein, VP7, isolated from a reassortant human rotavirus, M3014, was determined. The deduced amino acid sequence exhibited significant identity to the VP7 from a standard strain belonging to serotype G4, although the antigenic regions of the M3014 VP7 resembled sequences from both serotype G4 and G9 viruses. However, reactivity with G4 or G9 serotype-specific monoclonal antibodies was not observed. We suggest that the M3014 VP7 was derived from sequential mutation of a G4-like progenitor gene resulting in a protein with novel antigenic properties.