Immune Activation following Irbesartan Treatment in a Colorectal Cancer Patient: A Case Study.

Colorectal cancers are one of the most prevalent tumour types worldwide and, despite the emergence of targeted and biologic therapies, have among the highest mortality rates. The Personalized OncoGenomics (POG) program at BC Cancer performs whole genome and transcriptome analysis (WGTA) to identify specific alterations in an individual's cancer that may be most effectively targeted. Informed using WGTA, a patient with advanced mismatch repair-deficient colorectal cancer was treated with the antihypertensive drug irbesartan and experienced a profound and durable response. We describe the subsequent relapse of this patient and potential mechanisms of response using WGTA and multiplex immunohistochemistry (m-IHC) profiling of biopsies before and after treatment from the same metastatic site of the L3 spine. We did not observe marked differences in the genomic landscape before and after treatment. Analyses revealed an increase in immune signalling and infiltrating immune cells, particularly CD8+ T cells, in the relapsed tumour. These results indicate that the observed anti-tumour response to irbesartan may have been due to an activated immune response. Determining whether there may be other cancer contexts in which irbesartan may be similarly valuable will require additional studies.

Heat shock protein concentration and clarity of porcine lenses incubated at elevated temperatures.

PURPOSE: To quantify the concentration of heat shock proteins in lenses in lens organ culture at elevated temperatures, and to examine the relation between elevated temperature and lens clarity. METHODS: Pig lenses obtained from a local abattoir were dissected aseptically and incubated in medium M199 without serum for 4 days to stabilize, and lenses with protein leakage of less than 10 mg/l were obtained for heat shock exposure. Heat shock was performed by incubation for 1 h in M199 without serum at various temperatures ranging from 37 °C to 55 °C. After incubation for 24 h, cataract blurring of the images was assessed using Scantox™ and Scion Image analysis of the lens photographs. Lens homogenates were subsequently analyzed for Hsp70 and Hsp27 with western blotting. RESULTS: The degree of cataract blurring of the images increased with increasing temperature, but the two functional measures provided different results. Focal length inconsistency, as assessed with the back vertex distance standard error of the mean (BVD SEM; the variability in focal lengths measured at 20 equally spaced locations across the lens, Scantox™), increased nearly linearly with the heat treatment temperature. In contrast, decreased clarity, evident by a fuzzy image with lower contrast, was not markedly altered as the temperature rose until a threshold of approximately 47.5 °C. The inducible isoform of the Hsp70 family (Hsp70) of heat shock proteins was increased at all temperatures above the control except those above 50 °C. Changes in Hsp27 were less clear as the protein content increased only at the incubation temperatures of 39 °C and 48.5 °C. CONCLUSIONS: The porcine lens demonstrates subtle changes in the variability of the focal length, and the variability increases as the incubation temperature rises. In contrast, lens clarity is relatively stable at temperatures up to 47.5 °C, above which dramatic changes, indicative of the formation of cataracts, occur. The lens content of Hsp70 was elevated in lenses exposed to heat shock only up to 50 °C. These data suggest that in a stressful environment, Hsp70 may be associated with protection against loss of clarity. In addition, the functional measures BVD SEM and clarity assess different qualities of the lens, with the former likely more sensitive to subtle changes in the protein structure.

The effects of a 2 week modified high intensity interval training program on the homeostatic model of insulin resistance (HOMA-IR) in adults with type 2 diabetes.

AIM: High intensity interval training (HIIT) induces similar metabolic adaptations to traditional steady state aerobic exercise training. Until recently, most HIIT studies have examined maximum efforts in healthy populations. The current study aimed to examine the effects of a 2 week modified HIIT program on the homeostatic model of insulin resistance (HOMA-IR) in individuals with type 2 diabetes (T2D). It was hypothesized that HIIT would improve HOMA-IR. METHODS: Nine individuals with T2D (age=40.2±9.7 y; BMI=33.9±5.3; fasting plasma glucose [FPG]=8.7±2.9 mmol/L; HbA1C=7.3±1.2%; [mean±SD]) performed 6 individualized training sessions of HIIT (4x30 seconds at 100% of estimated maximum workload followed by 4 minutes of active rest) over 2 weeks. HOMA-IR was calculated from FPG and serum insulin and compared against a prior 2 week baseline period. RESULTS: Blood glucose was reduced immediately after each HIIT session (P<0.05). Anthropometrics, FPG, serum insulin, and HOMA-IR were unchanged after training. However, 6 of the 9 individuals exhibited reduced HOMA-IR values after the training period and there was a significant negative correlation between HOMA-IR value prior to training and change in HOMA-IR after HIIT. CONCLUSION: These observations tend to support the positive health benefits of HITT for individuals with T2D reported in recently published data using a modified HIIT protocol. However, they suggest that the magnitude of the disease should be assessed when examining the effects of exercise interventions in individuals with T2D.

Tumour-infiltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor-negative breast cancer.

BACKGROUND: Regulatory T cells (Tregs) are commonly identified by expression of the transcription factor FOXP3 and are conventionally thought to promote cancer progression by suppressing anti-tumour immune responses. We examined the relationship between FOXP3(+) tumour-infiltrating lymphocytes (TIL) and prognosis in oestrogen receptor (ER)-negative breast cancer, a tumour subtype with poor clinical outcome in which TIL are abundant. METHODS: FOXP3(+) and CD8(+) TIL were assessed by immunohistochemistry in a cohort of 175 ER- breast tumours. Results were confirmed in an independent data set of 78 ER- breast tumours with publically available gene expression data. RESULTS: High FOXP3(+) TIL levels were strongly associated with prolonged recurrence-free survival (HR=0.461, P=0.0002), particularly among basal-like tumours (HR=0.280, P=0.0001), for which FOXP3 status was independent of standard prognostic factors. Over 75% of FOXP3(+) TIL in triple negative breast tumours displayed a conventional CD4(+)CD25(+) Treg phenotype. Importantly, FOXP3(+) TIL were positively correlated with CD8(+) (cytotoxic) T cells (r(s)=0.76, P<0.0001), and were prognostically insignificant in tumours with low levels of CD8(+) TIL. These observations were confirmed in an independent cohort. CONCLUSION: In contrast with current dogma, we show for the first time that FOXP3(+) TIL are associated with robust anti-tumour immunity and favourable prognosis in ER- breast cancer.

Combination antimicrobial susceptibility testing of multidrug-resistant Stenotrophomonas maltophilia from cystic fibrosis patients.

Stenotrophomonas maltophilia is increasingly being isolated from the respiratory tract of individuals with cystic fibrosis, and, because of its multidrug-resistant nature, the selection of suitable treatment regimens can be problematical. Etest methodology was used to facilitate MIC and antimicrobial combination testing on 80 isolates of S. maltophilia cultured from the respiratory tract of Scottish individuals with cystic fibrosis between 2001 and 2010. The overall rate of susceptibility for the 1,410 MIC tests was 23.1%, and resistance was 68.9%. The most active antimicrobials were minocycline, co-trimoxazole, and doxycycline, with 92.4%, 87.3%, and 58.8% of isolates being susceptible, respectively. Of the 517 combinations, 13.2% were synergistic, with the most synergistic being ticarcillin/clavulanate plus aztreonam (91.7% synergistic), ticarcillin/clavulanate plus colistin (40%), and ticarcillin/clavulanate plus levofloxacin (19.4%). Colistin plus tobramycin was the only antagonistic combination (0.2%). By the median susceptible breakpoint index, the most active combinations were minocycline plus co-trimoxazole (median index, 20), minocycline plus piperacillin-tazobactam (median, 20), and co-trimoxazole plus ceftazidime (median, 16.5). The increasing problem of multidrug resistance in organisms recovered from the respiratory tracts of individuals with cystic fibrosis is not going to go away. Current susceptibility testing methods do not address the slow-growing organisms associated with chronic infection, and interpretive standards are based on achievable blood levels of antimicrobials. Addressing these issues specifically for organisms recovered from the respiratory tracts of individuals with cystic fibrosis should lead to better therapeutic outcomes and improved wellbeing of individuals with cystic fibrosis.

Is vancomycin MIC "creep" method dependent? Analysis of methicillin-resistant Staphylococcus aureus susceptibility trends in blood isolates from North East Scotland from 2006 to 2010.

This study investigated "creep" in vancomycin and daptomycin MICs among methicillin-resistant Staphylococcus aureus (MRSA) isolates from blood cultures over a 5-year period in a hospital in the United Kingdom, using different susceptibility testing methods. Trends in vancomycin and daptomycin susceptibility were evaluated by using Etest performed prospectively on isolates in routine clinical practice from December 2007 to December 2010 (n = 102). Comparison was made to results from prospective testing of subcultures at the Scottish MRSA Reference Laboratory, using an automated system (Vitek 2) and retrospective testing (Etest and CLSI reference broth microdilution [BMD] method) of stored isolates from 2006 to 2010 (n = 208). Spearman's rank correlations revealed a significant increase in vancomycin MIC (P = 0.012) and a significant decrease in daptomycin MIC (P = 0.03) by year of study for Etest results from the time of isolation. However, neither trend was replicated in MICs from automated or retrospective testing. The Friedman test revealed a significant difference between vancomycin MICs obtained from the same samples by different testing methods (χ(2) [3 degrees of freedom] = 97; P < 0.001). MICs from automated testing and Etest analysis of stored isolates were significantly lower than those from Etest analysis at the time of isolation for both antibiotics (P < 0.001). Effects of storage on the MIC appeared within the first 6 months of storage. Inconsistent evidence on vancomycin MIC creep and the relevance of the MIC to clinical outcome may arise from differences in susceptibility testing methods, including storage of isolates. There is a need to standardize and streamline susceptibility testing of vancomycin against MRSA.

Combination testing of multidrug-resistant cystic fibrosis isolates of Pseudomonas aeruginosa: use of a new parameter, the susceptible breakpoint index.

OBJECTIVES: The microbiology laboratory at Aberdeen Royal Infirmary operates an extended susceptibility testing service for multidrug-resistant Gram-negative non-fermenting isolates from the sputum of Scottish cystic fibrosis patients. The service aims to provide clinicians with useful treatment options and developed the use of a novel parameter-the susceptible breakpoint index (SBPI), which allows easy ranking of the antimicrobial combinations in order of their possible in vivo effectiveness. METHODS: Three hundred and fifteen isolates of Pseudomonas aeruginosa were submitted for testing. MICs of 14 antimicrobials were determined using the Etest and the results categorized using CLSI guidelines. Usually, six antimicrobial pairs were tested in combination also using the Etest. The results were assessed using the fractional inhibitory concentration index (FICI) and also by a novel parameter, the SBPI. RESULTS: Some 4173 MICs and 1663 combination pairs were performed. The most active individual antimicrobials were colistin, tobramycin and amikacin, with 84%, 69% and 32% of isolates susceptible, respectively. Twenty-eight of 44 antimicrobial combinations were tested >10 times. Of the combinations, 3.6% were synergistic (FICI < or = 0.5) and 0.1% were antagonistic (FICI > 4.0). Amikacin + ceftazidime (17%), ciprofloxacin + ceftazidime (12.9%) and ciprofloxacin + piperacillin/tazobactam (12%) were the most synergistic combinations. By median SBPI, the most effective combinations in vitro were colistin + ticarcillin/clavulanate, colistin + piperacillin/tazobactam and colistin + meropenem. CONCLUSIONS: The Etest is a useful tool for determining MICs and testing antimicrobial combinations. The SBPI is more discriminatory than the FICI, allowing easy ranking of the combinations, and is likely to have clinical relevance.

Conjugated estrogens and breast cancer risk in women.

The relationship between conjugated estrogen(s) (CE) and breast cancer was investigated by the examination of the records of 345 women with newly diagnosed breast cancer and 611 healthy controls belonging to a prepaid health plan. Use of CE was associated with a 40% elevation in risk [relative risk (RR) = 1.4; 95% confidence interval-1.0-2.0]. The RR was 1.3 for menopausal women with intact ovaries and 1.5 for those with ovaries removed. There was statistically significant evidence of a dose-response relationship with the three measures of dose evaluated. RR's rose to about twofold for women with 10 or more CE prescriptions noted in their charts, for those with 5 years or more between their first and last prescription, and for those with a usual daily dose of 1.25 mg or more. The RR associated with having ever used CE and with long-term use was highest among those women with a family history of breast cancer. These data support the hypothesis that long-term use of CE is associated with increased breast cancer risk.

The use of Pseudomonas acyl-CoA synthetase to form acyl-CoAs from dicarboxylic fatty acids.

Pseudomonas acyl-CoA synthetase is shown to act on saturated dicarboxylic acids with a chain length of C10 or greater to produce conjugates containing a single CoA unit. The synthetase can, therefore, be used to generate novel acyl-CoA analogues for studies on proteins that utilise, bind to, or are modulated by acyl-CoAs.

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness.

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein.

Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 A resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding specificity different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identified between the mammalian and parasite ACBPs. These, together with measured binding-specificity differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site.

Transformation of monomorphic Trypanosoma brucei bloodstream form trypomastigotes into procyclic forms at 37 degrees C by removing glucose from the culture medium.

African trypanosomes have been shown previously to undergo efficient transformation from bloodstream forms to procyclic (insect dwelling) forms in vitro by adding citrate and/or cis-aconitate to the culture medium and lowering incubation temperature to 27 degrees C. In this paper, it is shown that strain 427 monomorphic bloodstream forms of Trypanosoma brucei grown in axenic culture at 37 degrees C can be transformed to procyclic forms by simply replacing the glucose carbon source in the culture medium with glycerol. The removal of glucose from the medium results in the loss of the variant surface glycoprotein, the acquisition of cell surface procyclic acidic repetitive protein, the synthesis of procyclic-specific glycosylphosphatidylinositol precursors and the acquisition of substantial resistance to salicyl hydroxamic acid and glycerol within 72 h. A procyclic-specific cytoskeletal protein, known to be a marker of the late stage of transformation, is fully expressed by 96 h but full trans-sialidase activity appears only after 18-30 days. The transformation process described here is slower and less efficient than that previously described for monomorphic trypanosomes, using citrate and/or cis-aconitate and temperature shift as triggers. However, the separation of the transformation process from these stimuli is significant and the effects of glucose deprivation described here may reflect some of the events that occur in vivo in the tsetse fly midgut, where glucose levels are known to be very low.

Behavioral activity during prolonged hypoxemia in fetal sheep.

Experiments were conducted in unanesthetized, chronically catheterized pregnant sheep to determine the fetal behavioral response to prolonged hypoxemia produced by restricting uterine blood flow. Uterine blood flow was reduced by adjusting a vascular occluder placed around the maternal common internal iliac artery to decrease fetal arterial O2 content from 6.1 +/- 0.3 to 4.1 +/- 0.3 ml/dl for 48 h. Associated with the decrease in fetal O2 content, there was a slight increase in fetal arterial PCO2 and decrease in pH, which were both transient. There was an initial inhibition of both fetal breathing movements and eye movements but no change in the pattern of electrocortical activity. After this initial inhibition there was a return to normal incidence of both fetal breathing movements and eye movements by 16 h of the prolonged hypoxemia. These studies indicate that the chronically catheterized sheep fetus is able to adapt behaviorally to a prolonged decrease in arterial O2 content secondary to the restriction of uterine blood flow.

Comparison of methods for assessing synergic antibiotic interactions.

Twenty-five combined microtitre chequerboard/time kill curves were performed on ten isolates from patients with relapsing infection to assess the potential for combination therapy. The isolates were Burkholderia cepacia, Staphylococcus aureus and Klebsiella pneumoniae. No antagonism (FIC index or FBC index >4) was observed with any combination. Synergy by time kill curve (present in 21 combinations) was more often seen at 24 h than 2 or 5 h (P<0.001). On comparing the mean of the FIC and FBC indices, there were significant differences in only four chequerboards (P<0.05). The same checkerboard was repeated on 3 separate days to test reproducibility. There were no significant differences (P>0.05). All combinations showing synergism by FBC index were synergic by FIC index. Synergy by FIC index predicted synergy by FBC index in 67%. All combinations showing synergism by FIC index were synergic by time kill at 24 h but there was poor correlation between synergy at 2 or 5 h and synergy by FIC index or FBC index. In conclusion combining time kill and chequerboard tests gives reproducible results and good correlation between FIC and FBC indices. FIC indices showing synergy were also predictive of synergy in time kill studies. For bactericidal combinations unlikely to be antagonistic, calculation of FIC index may be a good indicator of synergic bactericidal activity.

Prevalence rates and demographic characteristics associated with depression in pregnancy and the postpartum.

Examined the prevalence of depression in a heterogeneous sample of 360 pregnant women. Subjects were assessed with respect to both depressive symptomatology and diagnostic status during pregnancy and after delivery. At both assessments, approximately 25% of the sample reported elevated levels of depressive symptomatology. In contrast, 10% of the women met diagnostic criteria for depression during pregnancy, and 6.8% were depressed postpartum. However, only half of the cases of postpartum depression were new onset (3.4%); the remaining women receiving a diagnosis in the postpartum had also been depressed during pregnancy. Finally, depression during pregnancy was related to different sociodemographic variables than was postpartum depression, suggesting that depression at these two times may be associated with different psychological or etiological factors.

Antibiograms of resistant Gram-negative bacteria from Scottish CF patients.

BACKGROUND: Over a 19-month pilot phase, 93 multiply resistant Gram-negative isolates from Scottish cystic fibrosis patients were sent to a referral laboratory for further investigation. METHODS: In common with the referring diagnostic laboratories, disc diffusion testing was carried out. Antibiotic susceptibility testing was also established by MIC methodology. NCCLS methods were used throughout. Twenty antibiotics were tested. RESULTS: Comparing disc diffusion results against MIC results, there were 167 (14%) major errors. By MIC, Pseudomonas aeruginosa (n = 59), Stenotrophomonas maltophilia (n = 16), Burkholderia cepacia (n = 10) and Alcaligenes xylosoxidans (n = 7) were susceptible to 18%, 11%, 4% and 35% of the antibiotics tested, respectively. Colistin and tobramycin were the most active agents against P. aeruginosa with 60% and 49%, respectively, testing susceptible. Minocycline and gentamicin were most active against S. maltophilia with 58% and 18%, respectively, testing susceptible. B. cepacia were most susceptible to co-trimoxazole (10%) and ciprofloxacin (10%). Five and six of the seven A. xylosoxidans isolates were susceptible to piperacillin and imipenem, respectively. CONCLUSIONS: Improved methods for susceptibility testing of such clinical isolates need to be employed in routine diagnostic laboratories. Levels of resistance in referred isolates were very high and similar to those described in the USA.

Refractory period prolongation by biphasic defibrillator waveforms is associated with enhanced sodium current in a computer model of the ventricular action potential.

Mechanisms through which biphasic waveforms lower defibrillation threshold are unknown. Previous work showed that low-intensity biphasic shocks (BS2), delivered during the refractory period of a control action potential (S1), produced significantly longer responses than monophasic shocks (MS2). To test the hypothesis that longer responses are due to hyperpolarization-induced excitation channel recovery during the first portion of the biphasic waveform, we used the Beeler-Reuter ventricular action potential computer model with the Drouhard-Roberge (BRDR) modification to study refractory period stimulation with MS2 (10 msec) and symmetrical BS2 (10 msec each pulse). At 1.5 times diastolic threshold, BS2 prolonged action potential duration when delivered 50 msec into the S1 refractory period, and produced a maximum BS2 versus MS2 response duration difference of 62 msec. Longer BS2 responses corresponded to enhanced BS2-induced sodium current compared to MS2. Maximum BS2 vs MS2 sodium current difference was 400 uA/cm2. These results show that, in a computer model of the ventricular action potential, hyperpolarization by the first phase of a biphasic waveform enhances S2 sodium current and prolongs duration of refractory-period responses. This effectively shortens the cellular refractory period. Prolonged refractory period responses, produced by biphasic defibrillator waveforms, may underlie enhanced defibrillating efficacy at low shock intensities.

Regulation of prostaglandin synthesis in the human amnion.

An increase in prostaglandin synthesis by intrauterine tissues may be responsible for labour initiation and/or maintenance in humans. In all studies to date, the amnion is the intrauterine tissue whose prostaglandin output consistently increases with the onset of labour. This may be due, in part, to acute activation of the phospholipases A2 and C and to an increase in the specific activity of prostaglandin H synthase (PGHS). A number of factors exist in amniotic fluid, the fetal membranes, the decidua and the placenta that can increase PGHS specific activity. Some of these factors may increase PGHS enzyme activity by gene expression and protein synthesis. Preliminary evidence is presented that suggests the hypothesis that PGHS specific activity increases before the onset of labour rather than as a consequence of labour initiation, and that idiopathic preterm labour may frequently be associated with increased PGHS activity. Hence, activation of PGHS gene expression and/or protein synthesis may be causal for term and preterm labour.