One-step purification and concentration of DNA in porous membranes for point-of-care applications.

The emergence of rapid, user-friendly, point-of-care (POC) diagnostic systems is paving the way for better disease diagnosis and control. Lately, there has been a strong emphasis on developing molecular-based diagnostics due to their potential for greatly increased sensitivity and specificity. One of the most critical steps in developing practical diagnostic systems is the ability to perform sample preparation, especially the purification of nucleic acids (NA), at the POC. As such, we have developed a simple-to-use, inexpensive, and disposable sample preparation system for in-membrane purification and concentration of NAs. This system couples lateral flow in a porous membrane with chitosan, a linear polysaccharide that captures NAs via anion exchange chromatography. The system can also substantially concentrate the NAs. The combination of these capabilities can be used on a wide range of sample types, which are prepared for use in downstream processes, such as qPCR, without further purification.

An individual reproduction model sensitive to milk yield and body condition in Holstein dairy cows.

To simulate the consequences of management in dairy herds, the use of individual-based herd models is very useful and has become common. Reproduction is a key driver of milk production and herd dynamics, whose influence has been magnified by the decrease in reproductive performance over the last decades. Moreover, feeding management influences milk yield (MY) and body reserves, which in turn influence reproductive performance. Therefore, our objective was to build an up-to-date animal reproduction model sensitive to both MY and body condition score (BCS). A dynamic and stochastic individual reproduction model was built mainly from data of a single recent long-term experiment. This model covers the whole reproductive process and is composed of a succession of discrete stochastic events, mainly calving, ovulations, conception and embryonic loss. Each reproductive step is sensitive to MY or BCS levels or changes. The model takes into account recent evolutions of reproductive performance, particularly concerning calving-to-first ovulation interval, cyclicity (normal cycle length, prevalence of prolonged luteal phase), oestrus expression and pregnancy (conception, early and late embryonic loss). A sensitivity analysis of the model to MY and BCS at calving was performed. The simulated performance was compared with observed data from the database used to build the model and from the bibliography to validate the model. Despite comprising a whole series of reproductive steps, the model made it possible to simulate realistic global reproduction outputs. It was able to well simulate the overall reproductive performance observed in farms in terms of both success rate (recalving rate) and reproduction delays (calving interval). This model has the purpose to be integrated in herd simulation models to usefully test the impact of management strategies on herd reproductive performance, and thus on calving patterns and culling rates.

Advances in predicting nutrient partitioning in the dairy cow: recognizing the central role of genotype and its expression through time.

In recent years, it has become increasingly clear that understanding nutrient partitioning is central to a much broader range of issues than just being able to predict productive outputs. The extent to which nutrients are partitioned to other functions such as health and reproduction is clearly important, as are the efficiency consequences of nutrient partitioning. Further, with increasing environmental variability, there is a greater need to be able to predict the ability of an animal to respond to the nutritional limitations that arise from the environment in which it is placed. How the animal partitions its nutrients when resources are limited, or imbalanced, is a major component of its ability to cope, that is, its robustness. There is mounting evidence that reliance on body reserves is increased and that robustness of dairy cows is reduced by selection for increased milk production. A key element for predicting the partition of nutrients in this wider context is to incorporate the priorities of the animal, that is, an explicit recognition of the role of both the cow's genotype (genetic make-up), and the expression of this genotype through time on nutrient partitioning. Accordingly, there has been a growing recognition of the need to incorporate in nutritional models these innate driving forces that alter nutrient partitioning according to physiological state, the genetically driven trajectories. This paper summarizes some of the work carried out to extend nutritional models to incorporate these trajectories, the genetic effects on them, as well as how these factors affect the homeostatic capacity of the animal. At present, there are models capable of predicting the partition of nutrients throughout lactation for cows of differing milk production potentials. Information concerning genotype and stage of lactation effects on homeostatic capacity has not yet been explicitly included in metabolic models that predict nutrient partition, although recent results suggest that this is achievable. These developments have greatly extended the generality of nutrient partitioning models with respect to the type of animal and its physiological state. However, these models remain very largely focussed on predicting partition between productive outputs and body reserves and, for the most part, remain research models, although substantial progress has been made towards developing models that can be applied in the field. The challenge of linking prediction of nutrient partitioning to its consequences on health, reproduction and longevity, although widely recognized, is only now beginning to be addressed. This is an important perspective for future work on nutrient partitioning.

Predicting energy x protein interaction on milk yield and milk composition in dairy cows.

Feed management is one of the principal levers by which the production and composition of milk by dairy cows can be modulated in the short term. The response of milk yield and milk composition to variations in either energy or protein supplies is well known. However, in practice, dietary supplies of energy and protein vary simultaneously, and their interaction is still not well understood. The objective of this trial was to determine whether energy and protein interacted in their effects on milk production and milk composition and whether the response to changes in the diets depended on the parity and potential production of cows. From the results, a model was built to predict the response of milk yield and milk composition to simultaneous variations in energy and protein supplies relative to requirements of cows. Nine treatments, defined by their energy and protein supplies, were applied to 48 cows divided into 4 homogeneous groups (primiparous or multiparous x high or low milk potential) over three 4-wk periods. The control treatment was calculated to cover the predicted requirements of the group of cows in the middle of the trial and was applied to each cow. The other 8 treatments corresponded to fixed supplies of energy and protein, higher or lower than those of the control treatment. The results highlighted a significant energy x protein interaction not only on milk yield but also on protein content and yield. The response of milk yield to energy supply was zero with a negative protein balance and increased with protein supply equal to or higher than requirements. The response of milk yield to changes in the diet was greater for cows with high production potential than for those with low production potential, and the response of milk protein content was higher for primiparous cows than for multiparous cows. The model for the response of milk yield, protein yield, and protein content obtained in this trial made it possible to predict more accurately the variations in production and composition of milk relative to the potential of the cow because of changes in diet composition. In addition, the interaction obtained was in line with a response corresponding to the more limiting of 2 factors: energy or protein.

(+)-Totarol from Chamaecyparis nootkatensis and activity against Mycobacterium tuberculosis.

The isolation of (+)-totarol as active compound against Mycobacterium tuberculosis is reported from Chamaecyparis nootkatensis outerbark.

Increasing intervention implementation in general education following consultation: a comparison of two follow-up strategies.

This study examined two strategies for increasing the accuracy with which general education teachers implemented a peer tutoring intervention for reading comprehension. The intervention was implemented for 5 elementary school students who had been referred for consultation services. Initial implementation of the intervention by the teachers was variable, and the data exhibited a downward trend. When consultants held brief daily meetings with the teachers to discuss the intervention, implementation improved for 2 of 5 participants. Four of the teachers implemented the intervention at levels substantially above baseline during the performance feedback condition, whereas implementation for 1 teacher increased following discussion of an upcoming follow-up meeting with the principal. Student reading comprehension scores improved markedly during the peer tutoring intervention. Three students maintained these gains 4 weeks after the intervention ended. The implications of these findings for the maintenance of accurate treatment implementation in applied settings are discussed.

Effects of contingent reward and instruction on oral reading performance at differing levels of passage difficulty.

This study examined the effects of reinforcement contingencies designed to increase the performance of existing reading skills as well as the effects of instruction--modeling and practice--designed to increase skill level for oral reading fluency across three levels of reading materials. Results showed that a combination of contingencies, modeling, and practice was effective in producing substantial increases in reading fluency for all participants at their assigned grade levels. These results demonstrate one strategy for experimentally determining those instructional components that are required to increase oral reading rate.

Nosocomial pneumonia and tracheitis in a pediatric intensive care unit: a prospective study.

We conducted a prospective study in the multidisciplinary pediatric intensive care unit (pediatric ICU) of a tertiary-care university hospital in order to determine the incidence, risk markers, risk factors, and complications related to bacterial nosocomial pneumonia (BNP) and tracheitis (BNT) in children. A cohort of 1,114 consecutive admissions to the pediatric ICU was enrolled over a 56-wk period; 154 cases were excluded mostly (75%) because they already had a respiratory infection at entry. The final sample included 960 admissions (831 patients). Diagnosis of BNP or BNT was based on Centers for Disease Control of Atlanta criteria using a consensus method involving three experts, who also attributed complications to BNP and BNT. A total of 29 BNP and BNT (3.0%; 95% CI: 1.1 to 4.1%) were diagnosed (BNP: 1.2%, 95% CI: 0.7 to 1.9%; BNT: 1.8%, 95% CI: 0.8 to 2.6%). Three factors were retained by multivariate analysis as independent risk factors or markers for BNP (immunodeficiency, immunosuppression, and neuromuscular blockade), and two for BNT (head trauma and respiratory failure). Gram-negative bacteria and Staphylococcus aureus were the microorganisms most frequently found in the tracheal aspirates. Prescription of antibiotics was commonly attributable to BNP (75%) and BNT (59%). Death, as well as multiple organ system failure, resulted from BNP in 8% of cases, but never from BNT. In BNT, the reintubation rate was 24%. Nosocomial bacterial respiratory infections are rare in critically ill children. However, BNP causes significant complications, and more attention should be focused on BNT in the critically ill child.

[Clinical value of the study of proliferation and cell activation antigens with flow cytometry in bladder cancer]. / Intérêt clinique de l'étude d'antigènes de prolifération et d'activation cellulaire par cytométrie en flux dans le cancer de la vessie.

The proliferative rate of tumor cells is frequently proportional to their degree of aggressiveness. The objective of the present study was to determine the correlation between the expression of three antigens associated with proliferation and the stage and ploidy of bladder cancers. The reactivity of antibodies against the nuclear antigen Ki-67 and membrane antigens T43 and the epidermal growth factor receptor (EGFR) was studied by flow cytometry on a series of 35 clinical samples of superficial and infiltrating bladder cancer and as well as on 5 specimens of normal urothelial cells. A preferential expression of the T43 antigen was observed on invasive cancer. EGFR was less frequently expressed; however, seven of the nine positive samples were from invasive cancers. Ki67 on the other hand did not show any selective expression according to tumor stage. When comparing Ki-67 and T43 expression on individual tumor samples, a negative correlation was found in that no tumor strongly expressed both markers simultaneously. These results suggest that markers of proliferation and activation do not all measure the same tumor parameters. Together, they may provide prognostic information that may be useful in the follow-up of patients with bladder cancer.

Toxicity of ribosome-inactivating proteins-containing immunotoxins to a human bladder carcinoma cell line.

Immunotoxins were prepared by linking the type 1 ribosome-inactivating proteins (RIP) momordin I, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6 to the 48-127 monoclonal antibody (MAb) recognising a glycoprotein (gp54) expressed on all human bladder tumours tested and on human bladder carcinoma cell lines, in particular on the T24 cell line. T24 cells required a 2 hr contact with immunotoxins to ensure binding and endocytosis. A time course of exposure, followed by further incubation without the immunotoxins, showed that maximum inhibition of protein synthesis by T24 cells was reached after 2 hr of contact followed by 3 days without the immunotoxins. Under optimal conditions, 48-127/RIP immunotoxins at nanomolar concentrations inhibited by 50% protein synthesis of target T24 cells. No toxicity was observed if (i) target cells were treated with non-conjugated RIP, (ii) target cells were treated with momordin I- or PAP-S-containing immunotoxins made with an irrelevant antibody and (iii) a non-target cell line was treated with the same 2 RIP conjugated to 48-127 antibody. The in vitro selective toxicity of these immunotoxins encourages further studies in view of a possible use in clinical trials for the local therapy of human bladder carcinomas.

Immune response to verotoxin 1 and 2 in children with Escherichia coli O157:H7 hemorrhagic colitis and classic hemolytic uremic syndrome.

OBJECTIVES: To compare neutralizing antibody titres against verotoxin (vt)-1 and vt-2 between children with uncomplicated hemorrhagic colitis (hc) and those with classic hemolytic uremic syndrome (hus). vt antibody titres were also compared in children with hc who received trimethoprim-sulfamethoxazole with those who did not. DESIGN: Prospective study. SETTING: Tertiary pediatric hospital. POPULATION STUDIED: Children with hc (n=41) or classic hus (n=12). INTERVENTIONS: Serum antibodies against vt-1 and vt-2 were determined by quantitative neutralization. MAIN RESULTS: Antibodies were detected in 40% (21 of 53) of serum samples for vt-1 and in 100% (53 of 53) of samples for vt-2. A positive immune response, defined as a fourfold increase in vt antibody titres or as a single titre of 1/64 or greater, was found in 0% (0 of 12) of patients with hus compared with 7% (three of 41) of those with hc for vt-1 (P=0.4); and in 17% (two of 12) of patients with hus compared with 22% (nine of 41) of those with hc for vt-2 (P=0.3). The rate of seroconversion against either vt-1 or vt-2 was comparable in treated and untreated patients with uncomplicated hc. CONCLUSIONS: There was no evidence that neutralizing antibody levels against vt-1 or vt-2 in classic hus or after antibiotic therapy are substantially different from those in patients with uncomplicated hc.

Hyperammonemia-hyperornithinemia-homocitrullinuria syndrome: neurologic, ophthalmologic, and neuropsychologic examination of six patients.

We report the clinical, electrophysiologic, ophthalmologic, and neuropsychologic features of six patients with hyperammonemia-hyperornithinemia-homocitrullinuria syndrome, an inborn error of ornithine metabolism. Pyramidal signs, decreased vibration sense, bucco-facio-lingual dyspraxia, and learning difficulties or subnormal intelligence were found in the majority. Anomalies of peripheral nerve conduction velocity and of evoked potentials were common, and one patient had markedly abnormal white matter images on cranial magnetic resonance imaging. One patient had retinal depigmentation and chorioretinal thinning. The clinical severity varied greatly among patients; in general, the three younger patients had less neurologic and intellectual impairment than did the three older patients. Only two of our patients have had episodes of symptomatic hyperammonemia. We conclude that hyperammonemia-hyperornithinemia-homocitrullinuria syndrome can be associated with widespread manifestations in the central and peripheral nervous systems. Although the control of hyperammonemia is an essential element in the treatment of these patients, the relationship of hyperammonemia to the chronic neuropsychologic problems of these patients is unclear.

Randomized, controlled trial of antibiotic therapy for Escherichia coli O157:H7 enteritis.

We undertook a prospective, controlled study to evaluate the effect of trimethoprim-sulfamethoxazole in children with proven Escherichia coli O157:H7 enteritis on the duration fo symptoms, on fecal excretion of pathogen, and on the risk of progression to hemolytic-uremic syndrome. There was no statistically significant effect of treatment on progression of symptoms, fecal pathogen excretion, or the incidence of HUS (2/22 vs 4/25; p = 0.67). Our results suggest that a multicentric trial using rapid diagnostic methods to permit early randomization should be carried out.

Strategies of chemoprevention based on antigenic and molecular markers of early and premalignant lesions of the bladder.

Using monoclonal antibodies, we have identified a series of tumor-associated antigens selectively expressed on tumor subtypes with distinct clinical behaviours. The mucinous antigen M344 and the gp200 surface antigen 19A211 are preferentially expressed on papillary superficial tumors and carcinoma in situ lesions of the bladder. The combination of these two antigenic markers in immunocytology and flow cytometry studies of exfoliated cells has improved the sensitivity of detection for bladder tumors. Moreover, the detection of M344- and 19A211-positive exfoliated cells from previously treated but currently tumor-free patients appears to be predictive of tumor recurrence on follow-up. These results, as well as results of bladder mapping studies in tumor patients, suggest that these antigenic changes occur in a premalignant stage and may provide tools to monitor the efficacy of chemopreventive measures. Other markers, such as the surface antigen T138 and the soluble molecules autocrine motility factor (AMF) and tumor collagenase stimulating factor (TCSF), are produced by primary or recurrent tumors with a higher metastatic potential. They may be useful in identifying high risk patients for distant failure. The highly restricted antigen 19A211 is also expressed on cervix condylomas and carcinoma. This observation led us to investigate a possible viral etiology of some bladder cancers. Using PCR techniques, we detected the presence of human papillomavirus (HPV) 16 DNA sequences in a significant proportion of bladder tumors. HPV positivity was inversely correlated with the presence of p53 mutations in exons 5-9 of the same tumors as measured by PCR-SSCP technique. This combination of markers may provide a basis for chemoprevention strategies targeted to distinct etiological events.

Arterial catheter-related infections in children. A 1-year cohort analysis.

To determine the incidence of infection secondary to arterial catheterization in children as well as the risk markers, we prospectively evaluated, during a 1-year period, all arterial catheters installed in children admitted to the pediatric intensive care unit. A total of 340 cannulas were placed in 310 children aged 80 +/- 4 months (mean +/- SEM) for a period of 64 +/- 4 hours. Most catheters were inserted percutaneously (99%) in the radial artery (86.5%). Ninety-two percent (313/340) of the catheters were sterile (group 1), 5% (17/340) were contaminated (less than 10 colony-forming units on semiquantitative culture) (group 2), and 3% (10/340) were considered either locally infected (ie, greater than or equal to 10 colony-forming units) (eight of 10) or associated with a possible catheter-related sepsis (two of 10) (group 3, or infected group). The incidence of local inflammation at the insertion site was higher in group 2 than in group 1 (18% vs 2.9%) but not statistically different between groups 3 and 1 (10% vs 2.9%). The duration of arterial catheterization was longer in group 3 than in group 1 (125 +/- 31 vs 61 +/- 4 hours). The risk of infection was nonexistent in the first 48 hours of catheterization. Thereafter it was calculated as being 6.2% (10/161), but it correlated poorly with the duration of arterial catheterization. These results confirm the very low incidence of infection related to arterial catheterization in children. Thus, routine catheter reinsertion is, in our opinion, unjustified.

[Sterilization and quality control]. / Stérilisation et contrôles de qualité.

The dental profession has always considered the sterilization of surgical instruments as an important element in the prevention of infection. During the last decade the concern with blood and to a lesser degree with saliva in the transmission of infection, due primarily to the hepatitis and HIV viruses, has resulted in many corporate organizations releasing recommendations to reinforce the use of sterilization techniques and disinfection techniques when sterilization is not applicable or justifiable. As a result of the fact that the two terms are often intermingled, the authors review the fact that each one pursues different objectives and put the emphasis on sterilization by briefly reviewing the principal advantages and disadvantages of each method of sterilization presently available for use in dental offices. Subsequently, they stress the importance of introducing quality control in any infection prevention program, in order to assure that the sterilization process is not at the mercy of human error or mechanical failure. The pharmaceutical industry, food chains and hospital centres have for more than 40 years realized the importance of using chemical indicators and especially biological testing. Only biological testing can put one's mind at ease, for the professional in a dental office and the patient receiving care, that the instruments or objects that one comes in contact with are truly sterile. The dental profession did not wait for the discovery of the hepatitis B and HIV viruses to become preoccupied with the spread of disease and have sterilized their surgical instruments for many years.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel lymphocyte activation antigen present on allospecific primed lymphocytes and defined by the monoclonal antibody VG01.

This study describes VG01, a monoclonal antibody to a novel lymphocyte activation antigen. The cells reacting with VG01 were first characterized by indirect immunofluorescence and fluorescence-activated cell sorter analysis. The antigen defined by VG01 was expressed on circulating T lymphocytes (23.8 +/- 9.0) and on the majority of large granular lymphocytes and monocytes (greater than 90%), while granulocytes stained weakly. All other cells tested so far--thymocytes, hematological malignancies and several cell lines--were negative. Upon activation with mitogens or allogeneic cells more than 90% of the transformed lymphocytes became VG01-positive. The kinetic studies demonstrated that the antigen defined by VG01 lags the expression of the IL-2 receptor by 48 hr and remains present for up to 18 days after stimulation in culture. Thus, this antigen does not seem to be involved in the initial steps of T cell activation and its expression continues after cell division has stopped. Assays with purified sorted populations showed that lymphocyte proliferation to mitogens and in mixed lymphocyte culture came primarily from the VG01- population. The responding cells became VG01+ during activation--and when alloantigen-primed lymphocytes were sorted and restimulated with the original stimulators, all the in vitro memory response came from the VG01+ population. Thus, VG01 antibody is efficient in selecting for alloreactive T lymphocytes in vitro and has potential for selective modulation of immune responses. Its reactivity with activated T cells and NK cells may also help define a common functional program for these two cell types, adding to the understanding of their mechanism of action and/or their origin.

Incidence of infection related to arterial catheterization in children: a prospective study.

The incidence of infection related to arterial catheterization has not been studied in critically ill children, using systematic catheter cultures. We studied prospectively 68 children in whom 70 arterial catheters were inserted. After the aseptic catheterization procedure, no component of the system was changed. The insertion site was inspected daily for signs of inflammation. Upon removal, catheters were cultured using a semiquantitative method. Blood and infusion fluid specimens were also cultured if septicemia was clinically suspected. Mean duration of catheterization was 59 +/- 6 (SE) h. In our series, all catheter and infusion fluid cultures were negative. Local inflammation was not predictive of catheter tip infection and correlated poorly with duration of catheterization (r = 0.2). In our experience, the incidence of infection related to arterial catheterization is low. Routine change of infusion fluid, tubing, dressing and insertion site as well as systematic catheter culture in the absence of fever appears unwarranted.