Evaluation of antibody-based and nucleic acid-based assays for diagnosis of hepatitis E virus infection in a rhesus monkey model

We have evaluated four hepatitis E virus (HEV) specific antibody assays, using sequential samples taken from 86 rhesus monkeys at intervals for up to 86 weeks after they had been infected with different doses of HEV. The animals are a common experimental model of hepatitis E. The large collection of sequential samples used avoids uncertainties encountered in previous studies regarding the precise infection status of study subjects and minimizes bias due to the individuality of response to infection. One assay (YES IgG) was produced with synthetic peptides; the others (E2 IgM, E2 IgG, and GL IgG) were produced with recombinant antigens. The results were compared with the viral RNA contents of the serum and stool samples and the occurrence of these virological and immunological markers in the course of the infection was temporally related to the development of hepatitis. Diagnostic utility of the markers was assessed according to their response rates and prevalence at different times in the course of infection. All the animals produced E2 IgG and developed viremia and all but one also produced E2 IgM and excreted the virus in stool, whereas response rates for the other antibodies were lower and decreased with virus dose. Hepatitis occurred over a period of 4 weeks between 3 and 7 weeks after infection. Virological activity occurred mainly during the incubation period and the prevalence of viral markers declined rapidly after the onset of hepatitis. Production of the E2 antibodies immediately preceded the onset of hepatitis, and this was followed about one week later by production of the other antibodies. Seroprevalence E2 IgM reached a peak value 3 weeks after the onset of hepatitis, whereas seroprevalence of GL IgG and YES IgG peaked after the disease had subsided. E2 IgG persisted in all animals for the entire duration of the experiment of up to 86 weeks and possibly beyond and, thus, can serve as a useful epidemiological marker of HEV infection.     

Rapid Intradermal Delivery of Liquid Formulations Using a Hollow Microstructured Array

Purpose

The purpose of this work is to demonstrate rapid intradermal delivery of up to 1.5 mL of formulation using a hollow microneedle delivery device designed for self-application.

Methods

3M??s hollow Microstructured Transdermal System (hMTS) was applied to domestic swine to demonstrate delivery of a variety of formulations including small molecule salts and proteins. Blood samples were collected after delivery and analyzed via HPLC or ELISA to provide a PK profile for the delivered drug. Site evaluations were conducted post delivery to determine skin tolerability.

Results

Up to 1.5 mL of formulation was infused into swine at a max rate of approximately 0.25 mL/min. A red blotch, the size of the hMTS array, was observed immediately after patch removal, but had faded so as to be almost indistinguishable 10 min post-patch removal. One-mL deliveries of commercial formulations of naloxone hydrochloride and human growth hormone and a formulation of equine anti-tetanus toxin were completed in swine. With few notable differences, the resulting PK profiles were similar to those achieved following subcutaneous injection of these formulations.

Conclusions

3M??s hMTS can provide rapid, intradermal delivery of 300?C1,500 µL of liquid formulations of small molecules salts and proteins, compounds not typically compatible with passive transdermal delivery.     

The fatty acid-binding protein-2 A54T polymorphism is associated with renal disease in patients with type 2 diabetes

The intestinal fatty-acid binding protein-2 (FABP2) gene codes a protein responsible for the absorption of long-chain fatty acids. To test whether FABP2 is a candidate gene for renal disease in patients with type 2 diabetes, a functional A54T polymorphism was genotyped in 1,042 Brazilians with type 2 diabetes. Patients were classified as having normoalbuminuria (urinary albumin excretion [UAE] <20 microg/min; n = 529), microalbuminuria (UAE 20-199 microg/min; n = 217), or proteinuria (UAE >199 microg/min; n = 160). Patients with end-stage renal disease (ESRD) (n = 136) were also included. The prevalence of the TT genotype was higher in patients with renal involvement compared with those with normoalbuminuria (odds ratio [95% CI] 2.4 [1.1-5.4]) following adjustment for type 2 diabetes duration, BMI, hypertension, A1C, and cholesterol levels. The risk was similar considering different stages of renal involvement. In a second independent patient sample (483 type 2 diabetic Caucasians residing in Massachusetts), a significant association was also observed between the TT genotype and proteinuria or ESRD (2.7 [1.0-7.3]; P = 0.048). This study thus provides evidence that FABP2 confers susceptibility to renal disease in type 2 diabetic patients.     

MR imaging of the knee: pseudotear of the lateral meniscus caused by the meniscofemoral ligament

Sagittal MR images of the knee often show a linear band of increased signal in the medial aspect of the posterior horn of the lateral meniscus that can be confused with a meniscal tear. This pseudotear is due to the meniscal insertion of the meniscofemoral ligament. To study the normal appearance of the medial aspect of the posterior horn of the lateral meniscus, we analyzed 109 MR examinations and correlated the findings with the results of arthroscopy. The meniscofemoral ligament was visualized in 54 cases (50%), and in 42 cases (39%) it caused the appearance of a pseudotear on sagittal images. The pseudotear had one of two orientations. The most common (35/42) was an oblique orientation coursing from the superior surface posteriorly and inferiorly. The other (7/42) was a more vertical orientation parallel to the base of the meniscus. Knowledge of the characteristic location and orientation of the meniscofemoral ligament should help to distinguish it from a true meniscal tear on MR images.     

Randomized controlled study of colloid preload before spinal anaesthesia for caesarean section

We randomized women having elective Caesarean section to receiveeither no preload (control group, n=33) or 4% gelatin solution(Gelofusine) 15 ml kg^–1 (colloid group, n=35)i.v. before spinal anaesthesia. Intravenous metaraminol wastitrated at 0.25–0.75 mg min^–1 to maintainsystolic arterial pressure (SAP) in the target range 90–100%of baseline after the spinal injection. The control group requiredmore vasopressor in the first 10 min [median 1.7 (range 0–2.9)mg vs 1.4 (0–2.8), P=0.02] at a greater maximum infusionrate [0.5 (0–0.75) vs 0.25 (0–0.5) mg min^–1,P=0.0005] and had a lower minimum SAP [90 (51–109) vs101 (75–127) mm Hg, P=0.006] than the colloid group. Nauseawas less frequent in the colloid group (6 vs 24%) but neonataloutcome was similar in the two groups. Colloid preload improvedhaemodynamic stability but did not affect neonatal outcome whenarterial pressure was maintained with an infusion of metaraminolduring spinal anaesthesia for Caesarean section. Br J Anaesth 2001; 87: 772–4     

A genome-wide allelotype study of primary and corresponding recurrent glioblastoma multiforme in one patient

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain tumor. Although comprehensive therapeutic measures are available, recurrence is very frequent and the prognosis of GBM remains dismal. To date, little is known about the molecular pathogenesis associated with GBM recurrence. According to Knudson ' s two-hit hypothesis of tumor suppressor gene (TSG) inactivation,1 deletion of a chromosomal region, as revealed by loss of heterozygosity (LOH), is often indicative of the presence of a potential TSG. Allelotype studies involving a comprehensive LOH analysis of the whole genome can provide more detailed and thorough information for detecting genetic anomalies than traditional LOH analysis. The present study is designed to conduct a genome-wide allelotype analysis of one patient ' s primary and corresponding recurrent GBM tumors in an effort to reveal molecular genetic alterations associated with the recurrence of this malignancy.     

Application of tumor markers CEA, TPA, and SCC-Ag in patients with low-risk FIGO stage IB and IIA squamous cell carcinoma of the uterine cervix

OBJECTIVE: The aim of this study was to determine the potential clinical utility of tumor markers carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), and squamous cell carcinoma antigen (SCC-Ag) in patients with FIGO stage IB and IIA squamous cell carcinoma of the uterine cervix with low-risk clinicopathologic factors (negative lymph node metastasis, no lymphovascular space involvement, no bulky tumor size, no parametrial invasion, no deep stromal invasion, and well-differentiated cellular histology). METHODS: A retrospective study was performed on 558 patients with FIGO stage IB-IIA and pathology-proven invasive squamous cell carcinoma of the uterine cervix, treated at the Veterans General Hospital, Taipei, between December 1986 and November 1990. Serum specimens were drawn before operation. A total of 140 assessable patients were enrolled into the study (including 109 stage IB patients and 31 stage IIA patients; all patients had no clinicopathologic risk factors and had at least one tumor marker datum). Survival curves were constructed according to the Kaplan-Meier method and survival curves were compared using the log-rank test. RESULTS: In univariate analysis of survival, CEA, TPA, and SCC-Ag all have roles in the prediction of prognosis. In Cox proportional hazards model using CEA, TPA, and SCC-Ag as covariates, TPA demonstrated the most significant risk factor (P = 0.031). CONCLUSIONS: We concluded that preoperative evaluation of serum TPA might be of great value in the prediction of survival of patients without any clinicopathologic risk factors and this special group of patients should be paid much attention in the follow-up period. From this study, preoperative elevation of TPA defines a group of otherwise low-risk invasive cervical cancer patients who are at high risk for recurrence. Adjuvant therapy might be necessary for this special subset of patients. A prospective study with a larger sample should be conducted to prove this particular finding.     

Angiotensin-I converting enzyme insertion/deletion polymorphism and its association with diabetic nephropathy: a meta-analysis of studies reported between 1994 and 2004 and comprising 14,727 subjects

Aims/hypothesis The ACE insertion/deletion polymorphism has been examined for association with diabetic nephropathy over the past decade with conflicting results. To clarify this situation, we conducted a comprehensive meta-analysis encompassing all relevant studies that were published between 1994 and 2004 and investigated this potential genetic association.Methods A total of 14,727 subjects from 47 studies was included in this meta-analysis. Cases (n=8,663) were type 1 or 2 diabetic subjects with incipient (microalbuminuria) or advanced diabetic nephropathy (proteinuria, chronic renal failure, end-stage renal disease). Control subjects (n=6,064) were predominantly normoalbuminuric.Results No obvious publication bias was detected. Using a minimal-case definition based on incipient diabetic nephropathy, subjects with the II genotype had a 22% lower risk of diabetic nephropathy than carriers of the D allele (pooled odds ratio [OR]=0.78, 95% CI=0.69–0.88). While there was a reduced risk of diabetic nephropathy associated with the II genotype among Caucasians with either type 1 or type 2 diabetes, the association was most marked among type 2 diabetic Asians (Chinese, Japanese, Koreans) (OR=0.65, 95% CI=0. 51–0.83). This OR is significantly different from the OR of 0.90 (95% CI= 0.78–1.04) that was obtained for type 2 diabetic Caucasians (p=0.019). Using a stricter case definition based on advanced diabetic nephropathy, a comparable risk reduction of 24–32% was observed among the three subgroups, although statistical significance was reached only among Asians.Conclusions/interpretation The results of our meta-analysis support a genetic association of the ACE Ins/Del polymorphism with diabetic nephropathy. These findings may have implications for the management of diabetic nephropathy using ACE inhibitors especially among type 2 diabetic Asians.     

In Vitro and In Vivo Characterization of MEMS Microneedles

Transdermal drug delivery TDD systems have many advantages but are conventionally limited by the low permeability of skin. The idea of using microneedles to painlessly penetrate the topmost impermeable stratum corneum has previously been put forward. In this paper, the fabrication of solid and hollow silicon microneedles with straight side-walls and with the following dimensions: 20–100 m in diameter and 100–150 m in length is described. In vitro tests demonstrate that with prior solid microneedle application, transdermal drug transport is significantly increased by 10–20 times, with the degree of enhancement being related to needle diameter. In vivo tests in diabetic animals, however, were unable to demonstrate any delivery of insulin through the hollow microneedles. It is proposed that two factors, microneedle length and tip sharpness, have to be improved for systemic drug delivery to be seen in vivo.     

Protein kinetic signatures of the remodeling heart following isoproterenol stimulation

Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (²H₂O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of β-adrenergic–induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.