Quantification of neutrophil and monocyte CD64 expression: a predictive biomarker for active tuberculosis.

SETTING: QuantiFERON TB assay (QFT) is used to screen tuberculosis (TB) infection, but it cannot distinguish active TB from latent TB infection (LTBI).OBJECTIVE: To evaluate the quantitative expression of the high-affinity FCgamma receptor I (CD64) on neutrophils (NE) and monocytes (MO) in peripheral blood using flow cytometry, measured in antibody binding capacity (ABC) units as a predictive biomarker of TB.DESIGN: Fifty-two patients were enrolled (45 QFT-positive and 7 QFT-indeterminate). Cultures and molecular analyses were performed.RESULTS: Of the 45 QFT-positive patients, 29 were culture-positive (active TB) and 16 were negative (LTBI). The median NE CD64 ABC and MO CD64 ABC expression was significantly higher (P < 0.001) in culture-positive patients. The NE CD64 and MO CD64 area under the receiver operating characteristic curve values were respectively 0.948 (95%CI 0.838-0.992) and 0.989 (0.901-1.000). By setting the cut-off NE CD64 value at >2400 ABC or MO CD64 value >25 800 the assay sensitivity increased to 95.5% with 100% specificity and 100% positive predictive value. In the QFT-indeterminate group, five culture-positive cases had NE CD64 >2400 ABC or MO CD64 value >25 800; two culture-negative cases had lower values.CONCLUSION: The CD64 quantitative expression on peripheral blood cells may be used as a predictive biomarker for active TB.

Stability of housekeeping genes in human intervertebral disc, endplate and articular cartilage cells in multiple conditions for reliable transcriptional analysis.

Quantitative gene expression analysis is widely used to evaluate the expression of specific tissue markers. To obtain reliable data it is essential to select stable housekeeping genes whose expression is not influenced by the anatomical origin of cells or by the culture conditions. No studies have evaluated housekeeping gene stability in intervertebral disc (IVD) cells and only few studies using cartilaginous endplate (CEP) and articular cartilage (AC) cells are present in the literature. We analysed the stability of four candidate housekeeping genes (GAPDH, TBP, YWHAZ and RPL13A) in human cells isolated from nucleus pulposus (NP) and annulus fibrosus (AF), CEP and AC. Cell isolation, expansion, cryoconservation, and differentiation in 3D pellets were tested. GeNorm, NormFinder, BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability. In each cell population, TBP alone or combined with YWHAZ was identified as the best normaliser in both monolayer and 3D pellets. GAPDH was the best performer only for AC cells in monolayer. In most culture conditions considering groups of two or more cell types, TBP was the most stable and YWHAZ was the second choice. GAPDH was the best performer only in 3D pellets with factors for AC and AF combined with CEP cells. RPL13A was the most stable only for AF with CEP cells at isolation. Our findings will be useful to properly design the experimental set-up of studies involving IVD, CEP or AC cells in different culture conditions, in order to obtain accurate and high quality data from quantitative gene expression analysis.

Identification of cattle carrying alleles associated with resistance and susceptibility to the Bovine Leukemia Virus progression by real-time PCR.

Previous studies have shown a significant association between polymorphisms of the BoLA DRB3 gene and Bovine Leukemia Virus (BLV) infection profile. The presence of allele *1501 has been associated with high proviral load in peripheral blood while allele *0902 has been associated with low proviral load. The purpose of this study was to develop allele-specific real-time PCRs to identify cattle carrying alleles associated with resistance (BoLA DRB3*0902) or susceptibility (BoLA DRB3*1501) to the BLV progression. Specific primers were designed and differential amplification was carried out by real-time PCR and monitored by SYBR® Green dye in DNA samples from peripheral blood. Conditions were also adjusted for traditional PCR amplification (end point amplification). These methods are rapid, simple and suitable for high throughput screening, and could aid in marker-assisted selection of BLV-resistant and susceptible cattle.

Robotic versus laparoscopic total mesorectal excision for rectal cancer: a comparative analysis of oncological safety and short-term outcomes.

BACKGROUND: We assessed feasibility, short-term oncologic safety, and short-term outcomes in robotic total mesorectal excision (R-TME) for rectal cancer compared with laparoscopic TME. METHODS: From March 2008 to June 2009, 50 patients with proven middle/lower rectal adenocarcinoma underwent minimally invasive TME; 25 received R-TME. The groups were balanced (R-TME versus L-TME) in terms of age (median 69 versus 62 years; p = 0.8), disease stage, and body mass index (median 23 versus 26.5 kg/m(2); p = 0.06). There were 37 (74%) anterior resections and 13 (26%) abdominoperineal resections. Twenty-three (46%) patients received preoperative radiochemotherapy. The robot was a four-arm Da Vinci S (Intuitive Surgical, Sunnyvale, CA, USA). RESULTS: Median operating time (R-TME versus L-TME) was 240 versus 237 min (p = 0.2); first bowel movement was 2 versus 3 days (p = 0.5); median hospital stay was 6.5 versus 6 days (p = 0.4). Major complications with reoperation were two in R-TME (one anastomotic leakage, one small bowel perforation) and three in L-TME (one colonic ischemia, two anastomotic leakage). Postoperative complications were 16% versus 24% (p = 0.5). A median of 18 versus 17 (p = 0.7) lymph nodes were retrieved; distal resection margins were disease free in both groups; circumferential margin was involved (<1.0 mm) in one (4%) of L-TME. There were 0 versus 1 (5%) conversions to laparotomy. CONCLUSIONS: R-TME in rectal cancer is feasible, with short-term oncologic and other outcomes similar to those of L-TME. The greater maneuverability and visibility afforded by the robotic approach are attractive. Future studies should more systematically address advantages and costs of R-TME.

Antibody response against three widespread bovine viruses is not impaired in Holstein cattle carrying bovine leukocyte antigen DRB3.2 alleles associated with bovine leukemia virus resistance.

Due to the wide dissemination of bovine leukemia virus (BLV) infection among dairy cattle, control and eradication programs based on serological detection of infected cattle and subsequent culling face a major economic task. In Argentina, genetic selection of cattle carrying alleles of the bovine leukocyte antigen (BoLA) DRB3.2 gene associated with BLV-infection resistance, like *0902, emerges as the best additional tool toward controlling virus spread. A potential risk in expanding or segregating BoLA selected populations of cattle is that it might increase susceptibility to other common viruses. Special concern raises the strong association found between low proviral load and low antibody titer against major BLV structural proteins. This phenomenon might depend on host genetic factors influencing other viruses requiring, unlike BLV, strong and long-lasting humoral immune response to prevent infection. In this study, we demonstrate that there is no association among neutralizing antibody titers against foot and mouth disease virus, bovine viral diarrhea virus, or bovine herpesvirus type 1 and polymorphism of the BoLA DRB3.2 gene. Conversely, there is strong association between BoLA DRB3.2*0902 and low antibody titers against 2 BLV structural proteins--env gp51 and gag p24--to date, the best BLV resistance marker. There is also significant association between low antibody titers against gp51 and p24 and BoLA DRB3.2*1701 and low antibody titers against p24 and BoLA DRB3.2*1101 or 02. Our data suggest that increasing BoLA-selected BLV-resistant cattle or segregating BoLA-associated alleles to BLV susceptibility would not affect the resistance or the predisposition to bovine viral diarrhea virus, bovine herpesvirus type 1, or foot and mouth disease virus infection.

Albendazole enantiomeric metabolism and binding to cytosolic proteins in the liver fluke Fasciola hepatica.

Fascioliasis causes important economic losses in ruminant species all over the world. Its control is largely based on the use of the flukicidal compound triclabendazole (TCBZ). However, its chemically related benzimidazole anthelmintic albendazole (ABZ) is being successfully used to control TCBZ-resistance flukes. This research gains some insights into the comparative molecular behaviour of both anthelmintics within the target fluke. The goals of the current work were: (i) to assess the competitive binding of ABZ and TCBZ to cytosolic proteins of F. hepatica, and (ii) to evaluate the enantioselective biotransformation of ABZ in microsomal fractions obtained from TCBZ-susceptible and TCBZ-resistant strains of the liver fluke. Cytosolic proteins from fluke specimens bound TCBZ with greater affinity (83%) than ABZ (44%) and the fraction of TCBZ bound to cytosolic proteins was not displaced by ABZ. The microsomes from both -susceptible and resistant flukes sulphoxidized ABZ into ABZ sulphoxide (ABZSO). However, this oxidative activity was 49% higher in microsomes from TCBZ-resistant flukes (P < 0.001) with a predominant production of the (+) ABZSO enantiomer. As earlier shown for TCBZ, the results reported here confirm an enhanced ability for ABZ oxidation in TCBZ-resistant flukes. While this enhanced oxidative metabolism of ABZ may cooperate to the resistance phenomenon, other pharmacodynamic-based mechanisms may be involved, which would explain why, although being chemically-related, ABZ remains efficacious against TCBZ resistant flukes under field conditions.

Association of BLV infection profiles with alleles of the BoLA-DRB3.2 gene.

Bovine leukaemia virus (BLV) causes lymphosarcoma and persistent lymphocytosis (PL). Some MHC class II gene polymorphisms have been associated with resistance and susceptibility to the development of lymphosarcoma and PL, as well as with a reduced number of circulating BLV-infected lymphocytes. Previously, 230 BLV-infected Holstein cattle were classified into two infection profiles characterized by low and high proviral loads (LPL and HPL respectively). Here, the influence of the polymorphism at the BoLA-DRB3.2* gene of these animals was examined. After genotyping, the association between the BoLA-DRB3.2* alleles and the BLV infection profile was determined as the odds ratio (OR). Two subtypes of allele *11 were identified (ISAG*0901 and *0902). Allele ISAG*0902 showed a stronger association with the LPL profile (OR = 8.24; P < 0.0001) than allele *11 itself (OR = 5.82; P < 0.0001). Allele ISAG*1701 (*12) also showed significant association with the LPL profile (OR = 3.46; P < 0.0055). Only one allele, ISAG*1501 or 03 (*16), showed significant association with HPL (OR = 0.36; P < 0.0005). The DRB3.2* alleles were assigned to three categories: resistant (R), susceptible (S) and neutral (N). Based on their DRB3 genotypes, cattle were classified as homozygous or heterozygous. The RR and RN genotypes were associated with the LPL profile, while the SS and NS genotypes were associated with the HPL profile. The RS genotype could not be associated with any particular profile. Our results show that allele ISAG*0902 appears to be the best BLV resistance marker in Holstein cattle.

Chicken antibodies: a useful tool for antigen capture ELISA to detect bovine leukaemia virus without cross-reaction with other mammalian antibodies.

The 24 kDa protein from the gag of the bovine leukaemia virus was cloned and expressed as a fusion protein GST-p24. This recombinant protein was then used to immunize a Leghorn chicken. The partially purified chicken anti-GST IgY was used to develop a solid-phase assay by binding the IgY to an ELISA plate. When the fusion protein contacts the antibody, it binds it by its N-terminal, leaving the C-terminal, which carries the sequence that acts as a capture antigen in solution maximally exposed, reducing the risk of epitope masking. The conditions of the fusion protein on the solid phase maximize the presentation of the antigens' epitopes in solution. For the first time, a system has been developed with a non-mammalian coating antibody. Besides optimizing the recognition of low-molecular-weight antigens synthesized as fusion proteins, it avoids cross-reactions with commonly used secondary antibodies, mostly raised in mammalian hosts.

Partial characterization of enterocin MR99 from a corn silage isolate of Enterococcus faecalis.

AIMS: To assess the inhibitory activity on Gram-positive and Gram-negative bacteria of several species of enterococci recovered from a natural corn silage. METHODS AND RESULTS: The inhibitory activity of strains of Enterococcus faecalis (58), Enterococcus faecium (35), Enterococcus gallinarum (3) and Enterococcus casseliflavus (4) were studied employing indicator strains from various sources (clinical, food and ATCC). Enterococcus faecalis MR99, the only strain with inhibitory activity, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromone-responsive plasmid. The partially chromatography-purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6.2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4.0, 5.0, 6.0, 7.0 and 8.0 and was resistant to heat. Inactivation was at 15 min at 121 degrees C. Enterocin MR99 was bactericidal on strains of Listeria monocytogenes, Staph. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram-negative bacilli, PCR studies demonstrated a lack of relationship between them. CONCLUSIONS: The active component had a protein nature, was resistant to heat and presented a wide inhibitory spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The biological properties of Ent. faecalis MR99 suggest that this strain merits further investigations so it can be applied in human and veterinary health programmes.

Damaged microtubules can inactivate BCL-2 by means of the mTOR kinase.

Rapamycin, a specific inhibitor of the serine/threonine mTOR kinase, markedly inhibited both cell growth and apoptosis in human B-cell lines. Besides arresting cells in G(1) by increasing p27(kip1), rapamycin tripled the cellular level of the BCL-2 protein. The activity was dose-dependent and specific for the p27(kip1) and BCL-2 proteins. Rapamycin did not affect bcl-2 mRNA although it increased cellular BCL-2 concentration by inhibiting phosphorylation, a mechanism initiating the decay process. To add new insight, we combined rapamycin treatment with treatment by taxol, which, by damaging microtubules, can phosphorylate BCL-2 and activate apoptosis. It was found that the mTOR kinase was activated in cells treated with taxol or with nocodazole although it was inhibited in cells pre-treated with rapamycin. BCL-2 phosphorylation, apoptosis and hyperdiploidy were also inhibited by rapamycin. In contrast, taxol-induced microtubule stabilization or metaphase synchronization were not inhibited by rapamycin. Taken together, these findings indicate that mTOR belongs to the enzymatic cascade that, starting from damaged microtubules, phosphorylates BCL-2. By regulating apoptosis, in addition to the control of a multitude of growth-related pathways, mTOR plays a nodal role in signaling G(1) and G(2)-M events.

Sensitivity of trypanosomatid protozoa to DFMO and metabolic turnover of ornithine decarboxylase.

alpha-Difluoromethylornithine (DFMO), the specific and irreversible inhibitor of ornithine decarboxylase (ODC), was able to induce the arrest of proliferation in Leishmania mexicana and ODC-transformed Trypanosoma cruzi cultures grown in a semi-defined medium essentially free of polyamines. Conversely, Crithidia fasciculata and Phytomonas 274 were not affected by the inhibitor. The drug-resistance of Crithidia and Phytomonas was neither caused by an impairment of DFMO uptake nor by a decrease of the enzyme affinity for the inhibitor. We were also able to rule out the possibility of ODC overexpression in the drug-tolerant parasites. The measurements of ODC metabolic turnover indicated that the enzymes from Crithidia and Phytomonas have a short half-life of 20-40 min, while ODC from Leishmania and transgenic Trypanosoma cruzi are rather stable with a half-life longer than 6 hours. Analyses of polyamine internal pools under different growth conditions have shown that DFMO was able to markedly decrease the levels of putrescine and spermidine in all parasites, but the depletion of spermidine was higher in trypanosomatids containing an ODC with slow turnover. Our results suggest that in these parasites cultivated in the presence of the drug, spermidine might decrease below critical levels needed to maintain trypanothione concentrations or other conditions essential for normal proliferation.

The complete genomic sequence of a BLV strain from a Holstein cow from Argentina.

DNA was extracted from the peripheral blood of a seropositive, PCR-positive, BLV-infected Holstein cow (No. 38) from Argentina. The DNA was amplified via PCR with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 38 DNA was cloned, sequenced, and compared phylogenetically to three other full-length BLV sequences. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Endoscopic laser ablation of nondysplastic Barrett's epithelium: is it worthwhile?

The clinical value of endoscopic ablation of nondysplastic Barrett's epithelium is controversial. It has been stated that ablation, combined with acid suppression or antireflux surgery, may reduce the risk of adenocarcinoma, thereby obviating the need for endoscopic surveillance in these patients. Eighteen symptomatic patients were enrolled in a prospective study of Nd:YAG laser ablation of Barrett's esophagus followed by treatment with proton pump inhibitors or antireflux surgery. All patients had intestinal metaplasia and no associated dysplasia or carcinoma. Laser treatment was performed with noncontact fibers and a power output of 60 watts. The mean number of treatment sessions was three (range 1 to 5), and the mean energy delivered during each session was 2800 joules (range 600 to 4800 joules). All patients were given a standard dose of omeprazole (40 mg/day) throughout the study period. In two patients a mild distal esophageal stricture occurred and required a single dilatation. Macroscopic and histologic eradication of the specialized columnar epithelium was documented in 8 of 12 patients with tongues of Barrett's metaplasia, in one of four patients with circumferential Barrett's metaplasia, and in two of two patients with short-segment Barrett's esophagus. In five patients (28%) only a partial ablation could be achieved despite repeated laser treatment. Two patients (11%), one with tongues and the other with circumferential Barrett's metaplasia, were considered nonresponders. Adenocarcinoma undermining regenerated squamous epithelium was found, 6 months after eradication, in one patient who underwent esophagogastric resection. Twelve patients agreed to undergo antireflux surgery. Over a mean follow-up period of 14 months (range 4 to 32 months), two patients presented with recurrent Barrett's metaplasia: one at 8 months after successful Nissen fundoplication and the other after 1 year of continuous omeprazole treatment. Progression of Barrett's metaplasia was found in two other patients receiving pharmacologic therapy in whom a partial response to laser treatment had been obtained. In conclusion, Nd:YAG laser therapy of nondysplastic Barrett's esophagus, performed in conjunction with omeprazole treatment and followed by antireflux surgery, allows a partial regression of specialized columnar epithelium in most patients. However, this is a time-consuming procedure that produced only temporary eradication, did not prove effective in reducing cancer risk, and did not obviate the need for endoscopic surveillance.

Endoscopic palliation of oesophageal cancer: results of a prospective comparison of Nd:YAG laser and ethanol injection.

OBJECTIVE: To evaluate the effectiveness of intratumoral alcohol injection compared with Nd:YAG laser in the treatment of unresectable fungating cancers of the oesophagus. DESIGN: Prospective, randomised clinical study. SETTING: University hospital, Italy. SUBJECTS AND INTERVENTIONS: 47 consecutive patients were randomly allocated to have endoscopic Nd:YAG laser treatment (n = 24), or intratumoural injection of 98% alcohol (n = 23). MAIN OUTCOME MEASURES: Morbidity, mortality, dysphagia score, survival. RESULTS: One patient in the laser group needed analgesic support during and after the treatment, whereas 18 (78%) of those treated with alcohol experienced mild pain and most of them required analgesics. An improvement of at least 2 points in the dysphagia score was noted in 21 patients (88%) in the laser group and in 18 in the alcohol group (78%). The mean dysphagia-free intervals between each treatment were 30 and 37 days, respectively. The median survival was 6 months in each group. There were no significant differences in the mean dysphagia scores of patients still alive. There were no complications in the laser group, but one oesophageal perforation occurred during the preliminary dilatation before the second session of alcohol injection. There were no procedure-related deaths. CONCLUSION: The two techniques allowed similar palliation of dysphagia and improvement of quality of life. Intratumoral injection of alcohol is an effective and inexpensive therapeutic option in the palliation of fungating oesophageal lesions.

Cloning of a trypanosomatid gene coding for an ornithine decarboxylase that is metabolically unstable even though it lacks the C-terminal degradation domain.

Mammalian ornithine decarboxylase (ODC) is among the most labile of cellular proteins, with a half-life of usually less than an hour. Like other short-lived proteins ODC is degraded by the 26S proteasome. Its degradation is not triggered by ubiquitination, but is stimulated by the binding of an inducible protein, antizyme. Truncations and mutations in the C terminus of mammalian ODC have been shown to prevent the rapid turnover of the enzyme, demonstrating the presence of a degradation signal in this region. Moreover, ODCs from the trypanosomatid parasites Trypanosoma brucei and Leishmania donovani, which lack this C-terminal domain, are metabolically stable, and recombination of T. brucei ODC with the C terminus of mammalian ODC confers a short half-life to the fusion protein when expressed in mammalian cells. In the present study we have cloned and sequenced the ODC gene from the trypanosomatid Crithidia fasciculata. To our knowledge, this is the first protozoan shown to have an ODC with a rapid turnover. The sequence analysis revealed a high homology between C. fasciculata ODC and L. donovani ODC, despite the difference in stability. We demonstrate that C. fasciculata ODC has a very rapid turnover even when expressed in mammalian cells. Moreover, ODC from C. fasciculata is shown to lack the C-terminal degradation domain of mammalian ODC. Our findings indicate that C. fasciculata ODC contains unique signals, targeting the enzyme for rapid degradation not only in the parasite but also in mammalian cells.

Improving results of esophageal stenting: a study on 160 consecutive unselected patients.

BACKGROUND AND STUDY AIMS: Esophageal stenting is a valuable treatment in the management of malignant dysphagia. Recently, self-expanding stents have proved effective in reducing morbidity and mortality. The aim of this study was to evaluate the early and late results of esophageal stenting in a series of 160 consecutive unselected patients with unresectable esophageal and cardial carcinoma treated between November 1992 and December 1996. PATIENTS AND METHODS: The procedure was successful in 159 patients (99.4%). A traditional tube was employed in 84 patients (52.5%). Metallic self-expanding stents have been available since June 1993 and were used in 75 selected patients (46.9%). The tumor was located in the cervical, upper, middle and lower thoracic esophagus, and at the gastric cardia in 16, 16, 56, 22 and 34 patients, respectively. In the remaining 15 patients an esophagovisceral anastomosis was involved. Preoperative chemo- and/or radiotherapy were performed in 82 patients (51.3%). RESULTS: Overall hospital morbidity was 11.3% (18/159) and included four dislodgments, four incomplete expansions of a self-expanding stent, two perforations, two incomplete sealings of a malignant respiratory tract fistula, two hemorrhages, two persistent foreign body sensations, one arrhythmia and one aspiration pneumonia. Hospital mortality was 1.3% (2/159) and was recorded in patients who underwent traditional intubation. At discharge, dysphagia was improved at least 2 degrees in 152 patients (96.8%). The overall long-term morbidity was 23.5% (37/157). Mean survival after the procedure was 4.7 months. CONCLUSIONS: Intubation is a safe palliative procedure which can be performed with low morbidity and mortality rates. Self-expanding metallic stents have enhanced the indications and the outcomes of the procedure, resulting in the treatment of strictures where the placement of a traditional tube is difficult or technically impossible.

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.

Ornithine decarboxylase from Crithidia fasciculata is metabolically unstable and resistant to polyamine down-regulation.

Ornithine decarboxylase (ODC) of Crithidia fasciculata extracts shows maximal activity during exponential growth of the parasite and decreases markedly in the stationary phase. The inhibition of protein synthesis by cycloheximide evoked a rapid loss of enzyme activity with a half-life of about 30 min. Upon removal of DFMO from Crithidia cultures treated with the drug for 24 h, the ODC activity increased at the same rate as total protein synthesis. The addition of putrescine at high concentrations to parasites cultivated in a synthetic medium showed that Crithidia ODC levels were not reduced by polyamines.