Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin

BACKGROUND: To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 microg/ml; high, 10 microg/ml). METHODS: Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis. RESULTS: Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P < 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater than those of PKH-labeled platelets (P < 0.001). Survival of platelets labeled with biotin did not differ (low, 83.3 h; high, 85.2 h) and was longer than for PKH-labeled platelets (P < 0.01). Labeling methods did not activate platelets (measured by P-selectin expression), nor did they affect platelet responses to adenosine diphosphate (ADP), collagen, or thrombin. CONCLUSIONS: Labeling with two levels of biotin is superior to labeling with PKH dyes, and is useful for measuring concurrently the survival of two differing platelet populations.     

Mutation of BCL-2 Family Proteins in Cancer

Apoptosis is a genetically controlled cell death process that is required for normal development and tissue homeostasis. Suppression of apoptosis can confer a growth advantage to cells and contribute to cancer; many cancers are relatively resistant to apoptosis, including that induced by radiation or chemotherapeutics. Mutations which inactivate pro-apoptotic or activate anti-apoptotic proteins in cancer cells are therefore likely to be responsible for some of these differences. BCL-2 family proteins are key regulators of apoptosis and there is evidence supporting a role for mutation of BCL-2 family proteins in cancer. This includes well established events such as activation of BCL-2 via translocations in follicular lymphoma, as well as more recent observations implicating activation of Bcl-X_L expression and frameshift and missense mutations of BAX and BCL-2 in cancer.     

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.     

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.     

Interaction between smoking and functional polymorphism in the TGFB1 gene is associated with ischaemic heart disease and myocardial infarction in patients with rheumatoid arthritis: a cross-sectional study

Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. Cardiovascular disease (CVD) is common and a major cause of mortality. Studies on cardiovascular morbidity are abundant, whereas mortality studies focusing on cardiovascular outcomes are scarce. The aim of this study was to investigate causes of death and baseline predictors of overall (OM), non-vascular (N-VM), and specifically cardiovascular (CVM) mortality in SLE, and to evaluate systematic coronary risk evaluation (SCORE).

Methods

208 SLE patients were included 1995-1999 and followed up after 12 years. Clinical evaluation, CVD risk factors, and biomarkers were recorded at inclusion. Death certificates and autopsy protocols were collected. Causes of death were divided into CVM (ischemic vascular and general atherosclerotic diseases), N-VM and death due to pulmonary hypertension. Predictors of mortality were investigated using multivariable Cox regression. SCORE and standardized mortality ratio (SMR) were calculated.

Results

During follow-up 42 patients died at mean age of 62 years. SMR 2.4 (CI 1.7-3.0). 48% of deaths were caused by CVM. SCORE underestimated CVM but not to a significant level. Age, high cystatin C levels and established arterial disease were the strongest predictors for all- cause mortality. After adjusting for these in multivariable analyses, only smoking among traditional risk factors, and high soluble vascular cell adhesion molecule-1 (sVCAM-1), high sensitivity C-reactive protein (hsCRP), anti-beta2 glycoprotein-1 (abeta2GP1) and any antiphospholipid antibody (aPL) among biomarkers, remained predictive of CVM.

Conclusion

With the exception of smoking, traditional risk factors do not capture the main underlying risk factors for CVM in SLE. Rather, cystatin C levels, inflammatory and endothelial markers, and antiphospholipid antibodies (aPL) differentiate patients with favorable versus severe cardiovascular prognosis. Our results suggest that these new biomarkers are useful in evaluating the future risk of cardiovascular mortality in SLE patients.     

SELDI-TOF MS Proteomics in Breast Cancer

Background

Proteomic profiling is a rapidly developing technology that may enable early disease screening and diagnosis. Surface-enhanced laser desorption ionization–time of flight mass spectrometry (SELDI-TOF MS) has demonstrated promising results in screening and early detection of many diseases. In particular, it has emerged as a high-throughput tool for detection and differentiation of several cancer types. This review aims to appraise published data on the impact of SELDI-TOF MS in breast cancer.

Methods

A systematic literature search between 1965 and 2009 was conducted using the PubMed, EMBASE, and Cochrane Library databases. Studies covering different aspects of breast cancer proteomic profiling using SELDI-TOF MS technology were critically reviewed by researchers and specialists in the field.

Results

Fourteen key studies involving breast cancer biomarker discovery using SELDI-TOF MS proteomic profiling were identified. The studies differed in their inclusion and exclusion criteria, biologic samples, preparation protocols, arrays used, and analytical settings. Taken together, the numerous studies suggest that SELDI-TOF MS methodology may be used as a fast and robust approach to study the breast cancer proteome and enable the analysis of the correlations between proteomic expression patterns and breast cancer.

Conclusion

SELDI-TOF MS is a promising high-throughput technology with potential applications in breast cancer screening, detection, and prognostication. Further studies are needed to resolve current limitations and facilitate clinical utility.     

Risk factors for Toxoplasma gondii infection in wild rodents from central coastal California and a review of T. gondii prevalence in rodents

Sera from 523 wild rodents were tested for Toxoplasma gondii antibodies using either an indirect fluorescent antibody test (IFAT) (rats and mice, with titer >or=80 considered positive) or a latex agglutination test (LAT) (voles, squirrels, and pocket mice, with titer >or=32 considered positive). Seventeen percent (88/523) of the rodents, including 26% (85/328) of the Peromyscus sp. and 8% (3/37) of Spermophilus beecheyi, were seropositive. Fourteen percent (23/161) of rodents captured in trap sites next to Morro Bay (California) and 15% (16/109) of rodents from sites adjacent to riparian habitats had antibodies to T. gondii, compared to 19% (49/253) of rodents captured in habitats not associated with water; this difference was not statistically significant (P = 0.32). Significantly fewer rodents were captured <200 m from residential housing compared to locations further away (11% vs. 30%, respectively). Factors associated with an increased risk for T. gondii seropositivity in rodents were capture location >or=200 m from residential housing and adult age.     

Nuclear BAG-1 expression inhibits apoptosis in colorectal adenoma-derived epithelial cells

BAG-1 is an anti-apoptotic protein that is frequently deregulated in a variety of malignancies including colorectal cancer. There are three isoforms: BAG-1L is located in the nucleus, BAG-1M and BAG-1S are located both in the nucleus and the cytoplasm. In colon cancer, the expression of nuclear BAG-1 is associated with poorer prognosis and is potentially a useful predictive factor for distant metastasis. However, the function of BAG-1 in colonic epithelial cells has not been studied. Having previously shown a predominant nuclear localisation of BAG-1 in adenoma-derived cell lines,¹ we wanted to determine the function of nuclear BAG-1 in these non-tumourigenic cells, to identify whether nuclear BAG-1 was implicated in tumour progression in the colon. In the current report we established that nuclear BAG-1 inhibits apoptosis in a colorectal adenoma-derived cell line. We demonstrate that apoptosis induced by -radiation or the vitamin D analogue EB1089 in the non-tumourigenic human colorectal adenoma-derived S/RG/C2 cell line, was preceded by a decrease in nuclear and an increase in cytoplasmic BAG-1 expression. This change in subcellular localisation of BAG-1 was due to the redistribution of the BAG-1M isoform. In addition, we have shown that the maintenance of high nuclear BAG-1 through enforced expression of the nuclear localised BAG-1L isoform enhanced cellular survival after -radiation or exposure to EB1089. Furthermore the expression of cytoplasmic BAG-1S isoform fused with a nuclear localisation signal protected against -radiation induced apoptosis. This demonstrates that nuclear localisation of the BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells and that nuclear BAG-1 could potentially be an important survival factor in colorectal carcinogenesis.     

Qualitative evaluation of selective tests for detection of Neospora hughesi antibodies in serum and cerebrospinal fluid of experimentally infected horses

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated. including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but I infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.     

BAG-1 proteins protect cardiac myocytes from simulated ischemia/reperfusion-induced apoptosis via an alternate mechanism of cell survival independent of the proteasome

BAG-1 (Bcl-2-associated athanogene-1) proteins interact with the HSC70 and HSP70 heat shock proteins and have been proposed to promote cell survival by coordinating the function of these chaperones with the proteasome to facilitate protein degradation. Consistent with this proposal, previous analyses in cancer cells have demonstrated that BAG-1 requires protein domains important for HSC70/HSP70 and proteasome binding in order to interfere with the growth inhibition induced by heat shock (Townsend, P. A., Cutress, R. I., Sharp, A., Brimmell, M., and Packham, G. (2003) Cancer Res., 63, 4150-4157). Moreover, cellular stress triggered the relocalization of the cytoplasmic BAG-1S (approximately 36 kDa) isoform to the nucleus, and both BAG-1S and the constitutively nuclear localized BAG-1L (approximately 50 kDa) isoform suppressed heat shock-induced apoptosis to the same extent, suggesting a critical role in the nucleus. Because ischemia (I) and reperfusion (R) are important stress signals in acute and chronic heart disease, we have examined the expression and function of BAG-1 proteins in primary cardiac myocytes (CMs) and the Langendorff-perfused intact heart. The expression of both BAG-1 isoforms, BAG-1S and BAG-1L, was rapidly induced following ischemia in rat CM, and this was maintained during subsequent reperfusion. In control hearts, BAG-1S and BAG-1L were readily detectable in both the nucleus and the cytoplasm. However, BAG-1S did not relocate to the nucleus following simulated I/R. BAG-1 interacted with both RAF-1 and HSC70 in CMs and the whole heart, and binding to HSC70 was increased following I/R. Overexpression of the human BAG-1S and BAG-1 M isoforms significantly reduced CM apoptosis following simulated I/R. By contrast, BAG-1L or BAG-1S fused to a heterologous nuclear localization sequence failed to protect CM. Finally, overexpression of BAG-1 deletion and point mutants unable to bind HSC70/HSP70 failed to offer cardioprotection. Surprisingly, a deletion mutant lacking the N-terminal ubiquitin-like domain, which mediates interaction with the proteasome, still promoted cardioprotection. Therefore, BAG-1 has a novel cardioprotective role, mediated via association with HSC70/HSP70, which is critical upon cytoplasmic localization but independent of the BAG-1 ubiquitin-like domain. Our studies demonstrate that BAG-1 can influence cellular response to stress by multiple mechanisms, potentially influenced by the cell type and nature of the stress signal.