Effect of Chronic Uremia on the Cell Surface Expression of B7 Family Costimulatory Molecules in an HLA-A2 Transgenic Mouse Model of Chronic Kidney Disease

Uremia due to chronic kidney disease (CKD) in humans is associated with immune dysfunction, increased susceptibility to infections, immune-activation–associated inflammation, and poor responses to vaccines. The pathophysiologic basis of these immune defects is hypothesized to be associated with a wide range of immunologic abnormalities, including an inability to sufficiently express the B7 family (B7-1, CD80; B7-2, CD86) of T-cell costimulatory molecules. However, testing the hypothesis that a state of chronic uremia contributes to attenuated expression of CD80 or CD86 has been difficult because few animal models faithfully recapitulate the immune defects observed in human CKD patients. We used a humanized mouse in a model of surgically induced renal failure and secondary chronic uremia to evaluate the effect of uremia on the expression of these markers. In a manner that resembles the changes observed in CKD patients, surgically induced CKD in mice resulted in decreased costimulatory CD86 expression compared with that in sham-operated controls. Immunodeficiency was functionally demonstrated in this mouse model by documenting an attenuated immune response to a cholera-toxin–based hepatitis B vaccine. This model will be useful for investigating the mechanisms involved in chronic uremia-associated immunodeficiency, poor response to vaccination, and problems associated with immunization of CKD patients.Abbreviations: CKD, chronic kidney disease; CT, cholera toxin; HBsAg, hepatitis B surface antigen; HLA, human leukocyte antigen; Th, T-helper cellThe B7 family of costimulatory molecules is critical in regulating adaptive immune responses and includes B7-1 (CD80) and B7-2 (CD86). These proteins are expressed on lymphoid and nonlymphoid cells and are part of a complex signaling network that includes chemokines, cytokines, adhesion molecules, and other immunoglobulin superfamily members. B7-1 and B7-2 deliver costimulatory or inhibitory signals through interaction with T-cell–associated CD28 or CTLA4 (CD152) molecules.^9,11,18,19 In particular, B7–CD28 ligation is necessary for a robust adaptive immune response to antigen that is presented in context with the MHC and T cell receptor complexes. Without B7 costimulation, T cell activation is compromised and the cell may enter an anergic state.¹³ In addition, active binding of B7, expressed by antigen-presenting cells, to CTLA4 that is expressed on responding T cells inhibits T-cell activation.^12,25 Therefore, any disease that alters the expression or function of B7 proteins has the potential to deregulate adaptive immunity.Uremia secondary to chronic kidney disease (CKD) is a disease that has been associated with reduced B7 expression and defects in innate and adaptive immunity in humans.⁹ Innate and adaptive immune dysfunction in CKD patients is associated with hyperinflammatory conditions, infections, atherosclerosis, and cardiovascular disease.^17,23 Some aspects of this dysfunction include impaired activation of T-lymphocytes, an increased T-helper cell (Th) Th1:Th2 ratio, lymphopenia, and impaired function of antigen-presenting cells.¹⁶ The function of antigen-presenting cells appears compromised in uremic patients through defective expression of B7-2 (CD86) on macrophages and dendritic cells.¹⁰ In contrast to that in human CKD patients, this pathology of reduced B7-2 expression has not previously been investigated in an animal model of uremia, and no model has been identified as having a defect in B7-2 expression.³Animal modeling of CKD is imperative to understand the pathogenesis of the disease. Furthermore, modeling will aid in the design and testing of therapeutic approaches. In the current study we examined the effect of surgically induced chronic uremia on the expression level of B7-1 and B7-2 in spleen cells of humanized B6.Cg-Tg(HLA-A/H2-D)2Enge/J transgenic mice (HLA-A2). We hypothesized that surgical induction of CKD and chronic uremia in HLA-A2 mice would result in the subsequent reduction of expression of B7-2 (that is, increased CD80:CD86 ratio) as compared with that of control mice that underwent sham surgery but did not become uremic. We show that the surgically induced chronic uremia protocol that we established results in a pathologic immune phenotype similar to that reported for immunodeficient humans with CKD, thus suggesting the utility of this approach as a new animal model of CKD.     

No evidence for infection of UK prostate cancer patients with XMRV, BK virus, Trichomonas vaginalis or human papilloma viruses

The prevalence of specific infections in UK prostate cancer patients was investigated. Serum from 84 patients and 62 controls was tested for neutralisation of xenotropic murine leukaemia virus-related virus (XMRV) Envelope. No reactivity was found in the patient samples. In addition, a further 100 prostate DNA samples were tested for XMRV, BK virus, Trichomonas vaginalis and human papilloma viruses by nucleic acid detection techniques. Despite demonstrating DNA integrity and assay sensitivity, we failed to detect the presence of any of these agents in DNA samples, bar one sample that was weakly positive for HPV16. Therefore we conclude that these infections are absent in this typical cohort of men with prostate cancer.     

Extractive fermentation-integrated reaction and product recovery

Summary Refinements have been made to a prototype process for the production of ethanol by extractive fermentation. The process is characterized by thein situ extraction of ethanol from a 7 L continuous stirred tank fermenter and the thermal recovery of ethanol from the extracting solvent, which is circulated in a closed loop through the process. Data are provided to show the efficacy and stability of the process under various operating conditions, and the near complete (>96%) continuous conversion of a 300 g/L glucose feed.     

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.     

Molecular characterisation of mouse and human TSSC6: evidence that TSSC6 is a genuine member of the tetraspanin superfamily and is expressed specifically in haematopoietic organs.

Previous analyses of the murine and human TSSC6 (also known as Phemx) proteins were not carried out using the full length sequence. Using 5'-RACE and cDNA library screening, we identified an additional 5' sequence for both the murine Tssc6 cDNA and its human homologue TSSC6. This novel sequence encodes a 5' exon encoding an in frame, upstream start codon, an N-terminal cytoplasmic domain and a transmembrane domain. The deduced, and now full length, murine and human TSSC6 proteins contained four hydrophobic regions together with other features characteristic of the tetraspanin superfamily. Computational analyses of the full length sequences show that TSSC6 is a genuine, albeit relatively divergent member of this superfamily. Using RNA from a number of mouse tissues, we identified seven splice variants of Tssc6. Splice variants of the human gene were also detected. Tssc6 expression was detected early in embryogenesis in primitive blood cells and was confined to haematopoietic organs in the adult mouse. Tssc6 expression was detected in many haematopoietic cell lines and was highest in cell lines of the erythroid lineage.     

Impact of groundwater abstraction on a Banksia woodland, Swan Coastal Plain, Western Australia

Summary The Gnangara Groundwater Mound, centred 38 km north of Perth, Western Australia, is a large, shallow unconfined aquifer that is currently under abstraction as part of the public metropolitan water supply. To investigate the impact of lowering groundwater levels on a Banksia woodland on the Mound, vegetation monitoring near a groundwater abstraction bore (known as P50) began 1 year before becoming operational. In February 1991, 2 years after abstraction commenced, extensive death of the Banksia overstorey was observed within close proximity of the bore, following a short period of high summer temperatures. The site was subsequently revisited and the understorey floristic composition, abundance and vigour of overstorey species resurveyed, and compared with data collected from a site under long‐term monitoring and not currently influenced by abstraction. A lowering of groundwater level by 2.2 m at P50 between the summers of 1990 and 1991, resulting from the cumulative effects of abstraction and below average annual rainfall (low groundwater recharge), coincided with a loss of between 20 and 80% of adults of overstorey species and up to 64% of adults of understorey species within 200 m of the bore. Over a similar time period no significant decreases in the abundance of overstorey or understorey species were recorded in the monitored site not influenced by groundwater abstraction. Of the overstorey species, Holly‐leaf Banksia (Banksia ilicifolia) displayed the greatest susceptibility and lowest net recovery following the abstraction event at P50. The negative impact of groundwater drawdown on Holly‐leaf Banksia populations makes this overstorey species an important indicator of decreasing groundwater levels on the Gnangara Groundwater Mound. Water stress may have been the primary cause of vegetation death in close proximity to the P50 bore, although this would have been exacerbated by extreme summer temperatures (> 45°C) recorded during February 1991. The P50 scenario represents a localized response to an acute drawdown event, in association with other environmental factors, and provides invaluable information on the assessment of groundwater abstraction and poor groundwater recharge events on a Banksia woodland community. However, there are limitations in using the community response at P50 to manage the impact of drawdown events on other plant communities occurring on sandy, shallow aquifers.     

Identification of complexes containing glutathione with As(III), Sb(III), Cd(II), Hg(II), Tl(I), Pb(II) or Bi(III) by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) of mixtures containing glutathione (GSH) and nitrates, oxides or chlorides of the heavy metals, arsenic, antimony, cadmium, mercury, thallium, lead or bismuth allows for definitive identification of complexes in the gas phase. In the positive ion mode, spectra show prominent m/z peaks that are assigned to monocations of general formulae [E(GSH)-xH]+ (E = Cd, Hg, Tl, Pb, As, Sb or Bi; x = 0, 1 or 2), [E(GSH)2-xH]+ (E = Hg, As, Sb, or Bi; x = 1 or 2), [E(GSH)3-xH]+ (E = As, Sb or Bi; x = 2), [E2(GSH)-xH]+ (E = Tl or Pb; x = 1 or 3), [E2(GSH)2-xH]+ (E = Bi; x = 5), [E2(GSH)3-xH]+ (E = Bi; x = 5), and/or [E3(GSH)-xH]+ (E = Tl; x = 2). Spectra obtained in the negative ion mode give m/z peaks observed in assigned to monoanionic species that correspond to some of the monocationic species listed above with two protons removed. The results demonstrate the potential application of ESI-MS as a versatile and efficient approach to study toxic heavy metals in biological systems. In addition, the observations provide a foundation database to understand the chemistry of these heavy metals with bio-molecules.     

Genetic dissection of the human leukocyte antigen region by use of haplotypes of Tasmanians with multiple sclerosis

Association of multiple sclerosis (MS) with the human leukocyte antigen (HLA) class II haplotype DRB1*1501-DQB1*0602 is the most consistently replicated finding of genetic studies of the disease. However, the high level of linkage disequilibrium (LD) in the HLA region has hindered the identification of other loci that single-marker tests for association are unlikely to resolve. In order to address this issue, we generated haplotypes spanning 14.754 Mb (5 cM) across the entire HLA region. The haplotypes, which were inferred by genotyping relatives of 152 patients with MS and 105 unaffected control subjects of Tasmanian ancestry, define a genomic segment from D6S276 to D6S291, including 13 microsatellite markers integrated with allele-typing data for DRB1 and DQB1. Association to the DRB1*1501-DQB1*0602 haplotype was replicated. In addition, we found that the class I/extended class I region, defined by a genomic segment of approximately 400 kb between MOGCA and D6S265, harbors genes that independently increase risk of, or provide protection from, MS. Log-linear modeling analysis of constituent haplotypes that represent genomic regions containing class I (MOGCA-D6S265), class III (TNFa-TNFd-D6S273), and class II (DRB1-DQB1) genes indicated that having class I and class II susceptibility variants on the same haplotype provides an additive effect on risk. Moreover, we found no evidence for a disease locus in the class III region defined by a 150-kb genomic segment containing the TNF locus and 14 other genes. A global overview of LD performed using GOLD identified two discrete blocks of LD in the HLA region that correspond well with previous findings. We propose that the analysis of haplotypes, by use of the types of approaches outlined in the present article, should make it possible to more accurately define the contribution of the HLA to MS.     

Invasion by Ligustrum lucidum (Oleaceae) in NW Argentina: early stage characteristics in different habitat types

Currently biological invasions are considered one of the world's most serious conservation problems. Ligustrum lucidum is the most abundant exotic tree in secondary forest patches of montane forests of NW Argentina. We studied the determinants of success of the early stages of its life cycle in distinct habitat types, with the hope of identifying vulnerabilities that could be exploited to control the invasion. Seed arrival, germination, seedling recruitment and survival, and sapling growth were studied in edges, gaps and forest interior. Seed arrival was also assessed under perches and in open fields. Germination was studied in forest and grassland patches. L. lucidum seedling survival and sapling growth were compared with the most abundant native species survival and growth. Seed arrival was strongly seasonal with a peak in mid-August. Seed rain did not differ significantly among habitat types, however there was a tendency for edges to receive more seeds when only dispersed seeds were considered. Perches strongly enhanced seed arrival; more than 40 times the number of seeds were dispersed beneath citrus plants (i.e. perches) than found in paired open areas. In the forest, seeds in gaps and edges had higher germination rates, but there was no difference in seedling survival. Fruits under closed canopy exhibited the lowest germination. Germination and survival were low in open areas. Neither seedling recruitment nor sapling growth differed between gaps and forest interior. L. lucidum saplings grew significantly more than saplings of the most common native species, and also showed higher seedling survival. L. lucidum is a prolific fruit producer, is capable of germinating and surviving in a broad range of forest environments, it is relatively shade tolerant and has higher survival and faster growth rate in comparison to the most common native species. All these characteristics highlight its potency as a successful invader, and point to few vulnerabilities that could be targets of control measures.