Examination of the kinetics of herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using surface plasmon resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (KD) of 3.2 x 10(-6) M and gD2(306t) had a KD of 1.5 x 10(-6) M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Delta290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 3.3 x 10(-8) M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Delta290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

Hematological malignancies with a deletion of 11q23: cytogenetic and clinical aspects. European 11q23 Workshop participants

OBJECTIVE: Using receiver-operating characteristic (ROC) curves, we tried to determine the diagnostic threshold of amniotic fluid index (AFI) that will identify abnormal fetal size (birth weights under 2500 g or at least 4000 g) at 37 weeks or beyond. METHODS: We analyzed prospectively over 2 years all parturients with intact membranes and known AFI in early labor. Patients with the following conditions were excluded: pregestational or gestational diabetes, known anomalies, and preterm labor. Two ROC curves were constructed, and the areas (+/- standard error of the mean [SE]) under the curves were calculated. P < .05 was considered significant. RESULTS: Of the 1038 subjects meeting study criteria, 3.6% and 11.5% gave birth to infants who were small for gestational age (SGA) or macrosomic, respectively. Overall, 28.7% had oligohydramnios (AFI at most 5.0 cm) and 3.6% had hydramnios (AFI at least 24.0 cm). Small for gestational age was more common in patients with AFI at most 5.0 cm (6.4%) than in those with adequate fluid (AFI 5.1-23.9; 2.5%), or hydramnios (2.7%; P = .012). Macrosomic newborns were less likely to be born to women with oligohydramnios (7.7%) than to those with adequate amniotic fluid (13.1%) or hydramnios (13.5%). Areas under ROC curves are not significantly different from the area under the nondiagnostic line, indicating that AFI (0-34 cm) cannot differentiate between newborns under 2500 g and at or over 2500 g or under 4000 and at or more 4000 g. CONCLUSION: Intraparterium AFI appears to be a poor screening test to identify risk for delivery of SGA or macrosomic fetus.     

Prognosis of intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer by immunological urinary measurements: statistically weighted syndrome analysis

PURPOSE: The goal of this research was to discover new biological indicators in urine which could be used for short-term prognosis of local Bacillus Calmette-Guerin (BCG) therapy outcome in patients with superficial bladder cancer. PATIENTS AND METHODS: We measured and statistically evaluated soluble immunological molecules in urine from bladder cancer patients (n = 34) receiving BCG intravesically. Urine was collected following each of 6 weekly treatments, processed and assayed. The data base included measurements of interleukin-1 (IL-2, IL-4, IL-6, IL-10, IL-12, soluble intercellular adhesion molecule-1 (sICAM-1), tumour necrosis factor-alpha (TNF alpha), soluble CD14 (sCD14), interferon-gamma (IFN gamma), GM-CSF, volume of urine and its pH. The clinical response was evaluated by urine histology and random quadrant biopsy 3 months after the start of therapy. Patients were divided into 2 groups, with good and poor therapeutic effect. The initial complete response rate was 62% (21/34). The data base was analyzed using traditional multivariate statistical methods and a pattern recognition method which deals with combinatorial-statistical analysis (statistically weighted syndromes (SWS) method) of the gradated features. The SWS method is capable of identifying robust patterns in small "fuzzy" sets with high dimensional objects and some missing values. RESULTS: Only one parameter gave significant differences at p < 0.05, GM-CSF at instillation 6. Repeated measurement analysis of variance, backward stepwise multiple logistic regression and linear discriminant analysis failed to show any significance. However, significant differences in the structure of correlation between features in the groups with and without therapeutic effect were observed and four highly informative variables (the masses of sICAM-1, TNF alpha, sCD14 and pH) relating to 5th-6th installations were selected by SWS. These features provided accurate individual prediction of therapeutic outcome for all our patients. Cross-validation analysis and computer simulation showed the statistically significant stability of the prediction. CONCLUSION: We have selected a set of urinary variables that could be considered as a perspective combination of indicators (syndromes) of outcome of pre-operation BCG therapy of patients with superficial bladder cancer. A larger patient database will provide testing and evaluation of the biological and clinical significance of selected features. The computational syndrome-disease approach should be applicable for the solution of decision-making problems for management of cancer.     

The correct formulation of clinical questions for the practice of evidence based medicine

Evidence-based medicine is the conscientious, explicit, and judicious use of the best evidence available to make decisions about the care of individual patients. The practice of EBM means integrating individual clinical expertise with the best available external clinical evidence from systematic research. The good practice of EBM begins with a well formulated clinical question, meaning that it should be clear, directly relevant to the problem at hand and answerable by searching in medical literature. This paper begins by presenting the central tasks of clinical work from which clinical questions arise. The components of well formulated clinical questions are then presented. Finally, some of the steps for the correct formulation of questions are discussed.     

Relapsing cutaneous polyarteritis nodosa associated with streptococcal infections

BACKGROUND: Polyarteritis nodosa is an aggressive, often fatal form of vasculitis associated with multi-organ involvement. Cutaneous polyarteritis nodosa is purported to be a more benign form of this disorder with involvement limited to the skin. METHODS: The identification of a female patient from childhood to adulthood documenting repeated episodes of cutaneous polyarteritis nodosa following bouts of recurrent streptococcal pharyngitis. RESULTS: Repeated bouts of streptococcal pharyngitis at ages 11, 28, and 33 years were followed by episodes of cutaneous polyarteritis nodosa, documented by histopathologic skin changes and clinical presentation, and confirmed by therapeutic management. CONCLUSIONS: Various infectious and non-infectious conditions have been linked both to the initiation and relapse of this disease. We describe a patient with recurrent episodes of cutaneous polyarteritis nodosa spanning a period of over 20 years with each episode appearing to be linked to a prior streptococcal infection.     

Analysis of the immunologic heterogeneity of HIV-1: I. "Arginine-glutamine trigger" in the fourth position of the central tetrapeptide of the V3 loop of gp120

This article reviews the existing knowledge base concerning the biology of spinal fusion, with the understanding that the focus is weighted toward posterolateral lumbar spinal fusion because of a relative paucity of biologic information on healing of other types of fusions. The discussion focuses first on the basic science of spinal fusion healing from the standpoint of animal modeling. Next, the discussion centers on the multitude of local factors that can affect fusion healing. Finally, the numerous systemic factors known to affect fusion healing are discussed.     

Intravascular ultrasound predictors of restenosis after balloon angioplasty of the femoropopliteal artery

The skepticism surrounding the potential benefits of resistance exercise training prevalent just decades ago has evolved over the years to an understanding of the integral nature muscular overload plays in the training programs for athletes. The science of training elite athletes is progressing rapidly, as insights into the physiological adaptations resulting from varying program configurations become available. Resistance training impacts several body systems, including muscular, endocrine, skeletal, metabolic, immune, neural, and respiratory. An understanding and appreciation of basic scientific principles related to resistance training is necessary in order to optimize training responses. Careful selection of the acute program variables in a workout to simulate sports-specific movements is required for optimal transfer of gains made in training to competition. Thus, whether athletes require predominantly eccentric, isometric, slow-velocity, or high-velocity strength or power in their athletic event will dictate the time commitment to each component and form the basis for designing individual workouts. Program variation over a training period is essential to maximize gains and prevent overtraining.     

Evaluation of fine needle capillary sampling in superficial and deep-seated lesions. An analysis of 670 cases

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.