Hormonal and metabolic changes following severe head injury or noncranial injury.

In order to evaluate the effect of head injury in severely traumatized patients on the response of plasma cortisol, glucagon, insulin, glucose, and FFA as well as urinary N and catecholamines excretions, 36 patients were prospectively studied over 5 consecutive days following injury. They were divided into three groups: group I, severe isolated head injury (n = 14); group II, multiple injury combined with severe head injury (n = 12); group III multiple injury without head injury (n = 10). The results demonstrate similar hormonal and metabolic changes between these three groups of patients, characterized by elevated urinary adrenaline, noradrenaline excretion, increased cortisol, glucagon, insulin plasma levels throughout the study and elevated N urinary excretion with strongly negative N balances during the first 5 days postinjury. A significant correlation was observed between N intake and 5 day cumulated N balance (r = 0.63, p less than 0.001). In addition, N balance was negatively correlated with urinary excretion of adrenaline (r = -0.47, p less than 0.01) and noradrenaline (r = -0.44, p less than 0.05) as well as plasma levels of glucagon (r = -0.44, p less than 0.05). Isolated severe head injury seems to induce a full response in the secretion of the catabolic counterregulatory hormones comparable to that encountered in patients with multiple injury and associated with a marked increase in protein catabolism; additional noncranial major injury does not seem to enhance these responses.

Task-shifting in the delivery of hormonal contraceptive methods: Validation of a questionnaire and preliminary results.

Guilbert ER, Morin D, Guilbert AC, Gagnon H, Robitaille J, Richardson M. International Journal of Nursing Practice 2011; 17: 315-321 Task-shifting in the delivery of hormonal contraceptive methods: Validation of a questionnaire and preliminary results In order to palliate the access problem to effective contraceptive methods in Quebec, Canada, as well as to legitimate nurses' practices in family planning, a collaborative agreement was developed that allow nurses, in conjunction with pharmacists, to give hormonal contraceptives to healthy women of reproductive age for a 6 month period. Training in hormonal contraception was offered to targeted nurses before they could begin this practice. A questionnaire, based on Rogers's theory of diffusion of innovations, was elaborated and validated to specifically evaluate this phenomenon. Preliminary results show that the translation of training into practice might be suboptimal. The validated questionnaire can now be used to fully understand the set of factors influencing this new practice.

Detection of new cross-reacting carcinoembryonic antigen(s) on cultured tumor cells by mixed hemadsorption assay.

Antisera highly specific for carcinoembryonic antigen (CEA) from New Zealand White rabbits and a goat reacted strongly in antibody binding tests with cultured tumor cell lines, irrespective of the ability of the cell lines to produce CEA. The most reactive were colon carcinoma and melanoma cell lines, the former known to produce CEA and the latter not associated with CEA production. The reactivity was not diminished by absorption with perchloric acid extracts of normal lung or spleen, whereas absoprtion with purified CEA preparations abolished the reactivity. Quantitative absorption studies indicated that reactivity against CEA-producing cell lines could be totally removed by absorption with other CEA-producing lines but not with melanoma cell lines. Reactivity against melanoma cell lines could be completely removed by colon carcinoma cells as well as by melanoma cells. Antisera raised against purified CEA, after absorption with extracts of normal lung, still contained two populations of antibodies, one that binds a newly described antigen cross-reacting with CEA which is present on melanoma cells.

Validation of a Low-Cost Human Papillomavirus Genotyping Assay Based on PGMY PCR and Reverse Blotting Hybridization with Reusable Membranes.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

Development of an aggregation assay to screen FimH antagonists.

Alpha-D-mannopyranosides are potent FimH antagonists, which inhibit the adhesion of Escherichia coli to highly mannosylated uroplakin Ia on the urothelium and therefore offer an efficient therapeutic opportunity for the treatment and prevention of urinary tract infection. For the evaluation of the therapeutic potential of FimH antagonists, their effect on the disaggregation of E. coli from Candida albicans and guinea pig erythrocytes (GPE) was studied. The mannose-specific binding of E. coli to yeast cells and erythrocytes is mediated by type 1 pili and can be monitored by aggregometry. Maximal aggregation of C. albicans or GPE to E. coli is reached after 600 s. Then the FimH antagonist was added and disaggregation determined by light transmission over a period of 1400 s. A FimH-deleted mutant of E. coli, which does not induce any aggregation, was used in a control experiment. The activities of FimH antagonists are expressed as IC(50)s, the half maximal inhibitory concentration of the disaggregation potential. n-Heptyl alpha-D-mannopyranoside (1) was used as a reference compound and exhibits an IC(50) of 77.14 microM , whereas methyl alpha-D-mannopyranoside (2) does not lead to any disaggregation at concentrations up to 800 microM. o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl alpha-D-mannopyranoside (3) shows a 90-fold and 2-chloro-4-nitrophenyl alpha-D-mannopyranoside (4) a 6-fold increased affinity compared to 1. Finally, 4-nitrophenyl alpha-D-mannopyranoside (5) exhibits an activity similar to 1. As negative control, D-galactose (6) was used. The standardized aggregation assay generates concentration-dependent, reproducible data allowing the evaluation of FimH antagonists according to their potency to inhibit E. coli adherence and can therefore be employed to select candidates for experimental and clinical studies for treatment and prevention of urinary tract infections.

Female thyroid cancer : the role of reproductive and hormonal factors in Switzerland

We conducted a study on 91 women with thyroid cancer and 306 controls in hospital for acute nonneoplastic, non-hormone-related disorders in order to investigate the role of reproductive and hormonal factors in the etiology of epithelial thyroid cancer in the Canton of Vaud, Switzerland. Non-significant increases in cancer risk with an increasing number of full-term pregnancies (odds ratio, OR, after allowance for age and previous benign thyroid disease = 1.6, for > or = 3 vs. 0 full-term pregnancies, 95% confidence interval, CI: 0.7-3.6) and spontaneous abortions (OR = 2.0 for > or = 2 vs. 0 spontaneous abortions, 95% CI: 0.7-5.2) were seen. A significantly elevated OR (2.8, 95% CI: 1.1-7.2) was found in those women whose first pregnancy ended with an abortion. Whereas most other reproductive, menstrual and hormonal factors examined did not seem to affect the risk of thyroid cancer significantly, a clue emerged of an association between thyroid cancer and artificial menopause (OR = 6.3, for women who underwent artificial menopause vs. premenopausal women, 95% CI: 1.7-23.2). Although not necessarily causal, the relationship between the risk of epithelial thyroid cancer and the occurrence of spontaneous abortions and artificial menopause deserves attention in future studies, in the light of the high incidence of thyroid cancer in young and middle-aged women.

Estradiol levels in men with congenital hypogonadotropic hypogonadism and the effects of different modalities of hormonal treatment.

Objective: To evaluate the degree of E-2 deficiency in male congenital hypogonadotropic hypogonadism (CHH), and its response to different hormonal treatments.Design: Retrospective and prospective studies.Setting: Academic institution.Patient(s): Untreated or treated CHH, healthy men, untreated men with Klinefelter syndrome (KS). Intervention(s): Serum sex hormone-binding globulin (SHBG) and total E-2 (TE2) as well as bioavailable (BE2) and free (FE2) levels were measured and determined.Main Outcome Measure(s): Total, bioavailable, and free testosterone, TE2, BE2, FE2 were compared in normal men, untreated and treated CHH and in untreated KS.Result(s): TE2, BE2, and FE2 levels were very significantly lower in untreated patients with CHH (n = 91) than in controls (n = 63) and in patients with KS (n = 45). The TE2 correlated positively with serum total T in patients with CHH. The TE2 also correlated very positively with serum LH in the combined population of patients with CHH and healthy men, suggesting that low E-2 levels in CHH are due to severe LH-driven T deficiency. All fractions of circulating E-2 were very significantly higher in patients with CHH receiving T enanthate (n = 101) or the FSH-hCG combination (n = 88) than in untreated patients with CHH. Contrary to dihydrotestosterone (DHT), both T enanthate and combined FSH-hCGtherapy significantly and prospectively increased TE2 levels in patients with CHH.Conclusion(s): Contrary to KS, the male hypogonadism observed in CHH is associated with profound E-2 deficiency, which can be overcome by aromatizable androgen or combined gonadotropin therapy. (Fertil Steril (R) 2011; 95: 2324-29. (C) 2011 by American Society for Reproductive Medicine.)

Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers.

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented. Copyright © 2011 John Wiley & Sons, Ltd.

Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant.

Since new technologies based on solid phase assays (SPA) have been routinely incorporated in the transplant immunology laboratory, the presence of pretransplantation donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules has generally been considered as a risk factor for acute rejection (AR) and, in particular, for acute humoral rejection (AHR). We retrospectively studied 113 kidney transplant recipients who had negative prospective T-cell and B-cell complement-dependent cytotoxicity (CDC) crossmatches at the time of transplant. Pretransplantation sera were screened for the presence of circulating anti-HLA antibody and DSA by using highly sensitive and HLA-specific Luminex assay, and the results were correlated with AR and AHR posttransplantation. We found that approximately half of our patient population (55/113, 48.7%) had circulating anti-HLA antibody pretransplantation. Of 113 patients, 11 (9.7%) had HLA-DSA. Of 11 rejection episodes post-transplant, only two patients had pretransplantation DSA, of whom one had a severe AHR (C4d positive). One-year allograft survival was similar between the pretransplantation DSA-positive and -negative groups. Number, class, and intensity of pretransplantation DSA, as well as presensitizing events, could not predict AR. We conclude that, based on the presence of pretransplantation DSA, post-transplantation acute rejections episodes could not have been predicted. The only AHR episode occurred in a recipient with pretransplantation DSA. More work should be performed to better delineate the precise clinical significance of detecting low titers of DSA before transplantation.

A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine.

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 microl) is performed by protein precipitation using 600 microl of acetonitrile, after the addition of 100 microl darunavir-d(9) (DRV-d(9)) at 1000 ng/ml in MeOH/H(2)O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 microl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 microl is injected onto a 2.1 x 50 mm Waters Atlantis-dC18 3 microm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1-9.8%, and accurate (range of inter-day deviation from nominal values -3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir-glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.

Label-free cytotoxicity screening assay by digital holographic microscopy.

Abstract We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.