Deborah A. Payne

Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.

Background Worldwide, vaginal transmission now accounts for more than half of newly acquired HIV-1 infections. Despite the urgency to develop and implement novel approaches capable of preventing HIV transmission, this process has been hindered by the lack of adequate small animal models for preclinical efficacy and safety testing. Given the importance of this route of transmission, we investigated the susceptibility of humanized mice to intravaginal HIV-1 infection. Methods and Findings We show that the female reproductive tract of humanized bone marrow–liver–thymus (BLT) mice is reconstituted with human CD4+ T and other relevant human cells, rendering these humanized mice susceptible to intravaginal infection by HIV-1. Effects of HIV-1 infection include CD4+ T cell depletion in gut-associated lymphoid tissue (GALT) that closely mimics what is observed in HIV-1–infected humans. We also show that pre-exposure prophylaxis with antiretroviral drugs is a highly effective method for preventing vaginal HIV-1 transmission. Whereas 88% (7/8) of BLT mice inoculated vaginally with HIV-1 became infected, none of the animals (0/5) given pre-exposure prophylaxis of emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) showed evidence of infection (Chi square = 7.5, df = 1, p = 0.006). Conclusions The fact that humanized BLT mice are susceptible to intravaginal infection makes this system an excellent candidate for preclinical evaluation of both microbicides and pre-exposure prophylactic regimens. The utility of humanized mice to study intravaginal HIV-1 transmission is particularly highlighted by the demonstration that pre-exposure prophylaxis can prevent intravaginal HIV-1 transmission in the BLT mouse model.

Systemic Administration of Antiretrovirals Prior to Exposure Prevents Rectal and Intravenous HIV-1 Transmission in Humanized BLT Mice

Successful antiretroviral pre-exposure prophylaxis (PrEP) for mucosal and intravenous HIV-1 transmission could reduce new infections among targeted high-risk populations including discordant couples, injection drug users, high-risk women and men who have sex with men. Targeted antiretroviral PrEP could be particularly effective at slowing the spread of HIV-1 if a single antiretroviral combination were found to be broadly protective across multiple routes of transmission. Therefore, we designed our in vivo preclinical study to systematically investigate whether rectal and intravenous HIV-1 transmission can be blocked by antiretrovirals administered systemically prior to HIV-1 exposure. We performed these studies using a highly relevant in vivo model of mucosal HIV-1 transmission, humanized Bone marrow/Liver/Thymus mice (BLT). BLT mice are susceptible to HIV-1 infection via three major physiological routes of viral transmission: vaginal, rectal and intravenous. Our results show that BLT mice given systemic antiretroviral PrEP are efficiently protected from HIV-1 infection regardless of the route of exposure. Specifically, systemic antiretroviral PrEP with emtricitabine and tenofovir disoproxil fumarate prevented both rectal (Chi square = 8.6, df = 1, p = 0.003) and intravenous (Chi square = 13, df = 1, p = 0.0003) HIV-1 transmission. Our results indicate that antiretroviral PrEP has the potential to be broadly effective at preventing new rectal or intravenous HIV transmissions in targeted high risk individuals. These in vivo preclinical findings provide strong experimental evidence supporting the potential clinical implementation of antiretroviral based pre-exposure prophylactic measures to prevent the spread of HIV/AIDS.

One Percent Tenofovir Applied Topically to Humanized BLT Mice and Used According to the CAPRISA 004 Experimental Design Demonstrates Partial Protection from Vaginal HIV Infection, Validating the BLT Model for Evaluation of New Microbicide Candidates

ABSTRACT Recent iPrEx clinical trial results provided evidence that systemic preexposure prophylaxis (PrEP) with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) can partially prevent rectal HIV transmission in humans. Similarly, we have previously demonstrated that systemic administration of the same FTC-TDF combination efficiently prevented rectal transmission in humanized bone marrow/liver/thymus (BLT) mice. The CAPRISA 004 trial recently demonstrated that topical application of the tenofovir could partially prevent vaginal HIV-1 transmission in humans. To further validate the usefulness of the BLT mouse model for testing HIV prevention strategies, we evaluated the topical administration of tenofovir as used in CAPRISA 004 to prevent vaginal HIV transmission in BLT mice. Our results demonstrate that vaginally administered 1% tenofovir significantly reduced HIV transmission in BLT mice (P = 0.002). Together with the results obtained after systemic antiretroviral PrEP, these topical inhibitor data serve to validate the use of humanized BLT mice to evaluate both systemic and topical inhibitors of HIV transmission. Based on these observations, we tested six additional microbicide candidates for their ability to prevent vaginal HIV transmission: a C-peptide fusion inhibitor (C52L), a membrane-disrupting amphipathic peptide inhibitor (C5A), a trimeric d-peptide fusion inhibitor (PIE12-Trimer), a combination of reverse transcriptase inhibitors (FTC-TDF), a thioester zinc finger inhibitor (TC247), and a small-molecule Rac inhibitor (NSC23766). No protection was seen with the Rac inhibitor NSC23766. The thioester compound TC247 offered partial protection. Significant protection was afforded by FTC-TDF, and complete protection was offered by three different peptide inhibitors tested. Our results demonstrate that these effective topical inhibitors have excellent potential to prevent vaginal HIV transmission in humans.

Capillary electrophoresis and its application in the clinical laboratory.

Over the past 10 years, capillary electrophoresis (CE) is an analytical tool that has shown great promise in replacing many conventional clinical laboratory methods, especially electrophoresis and high performance liquid chromatography (HPLC). The main attraction of CE was that it was fast, used small amounts of sample and reagents, and was extremely versatile, being able to separate large and small analytes, both neutral and charged. Because of this versatility, numerous methods for clinically relevant analytes have been developed. However, with the exception of the molecular diagnostic and forensic laboratories CE has not had a major impact. A possible reason is that CE is still perceived as requiring above-average technical expertise, precluding its use in a laboratory workforce that is less technically adept. With the introduction of multicapillary instruments that are more automated, less technique-dependent, in addition to the availability of commercial and cost effective test kit methods, CE may yet be accepted as a instrument routinely used in the clinical laboratories. Thus, this review will focus on the areas where CE shows the most potential to have the greatest impact on the clinical laboratory. These include analysis of proteins found in serum, urine, CSF and body fluids, immunosubstraction electrophoresis, hemoglobin variants, lipoproteins, carbohydrate-deficient transferrin (CDT), forensic and therapeutic drug screening, and molecular diagnostics.

miRNA : The new gene silencer

MicroRNAs (miRNAs) can be defined as small, noncoding sets of 19 to 24 nucleotides that have been associated with messenger RNA expression. miRNAs are members of a class of small regulatory RNAs that includes small interfering RNAs (siRNAs). miRNAs regulate the expression of downstream gene targets, including transcription factors, oncogenes, and tumor suppressor genes. Transcriptional profiling using genomic microarrays and beads has enabled the discovery of numerous miRNAs that are differentially expressed in normal tissues vs tumors and associated with cancer development, diagnosis, and prognosis. miRNA signatures can be used to detect and classify cancer and predict the severity of disease, with certain profiles of miRNA expression linked to aggressive cancers with advanced disease present at diagnosis. miRNAs have also become targets of novel anticancer gene therapy with antisense molecules that can inhibit miRNA activity currently being tested for their efficacy in a strategy of reducing miRNA activity on reporter genes bearing miRNA-binding sites. In the future, sophisticated genomic and proteomic techniques combined with complex bioinformatics data analyses will be required to translate these recent basic science discoveries to clinically useful diagnostic tests.

Porphyria cutanea tarda: multiplicity of risk factors including HFE mutations, hepatitis C, and inherited uroporphyrinogen decarboxylase deficiency.

The coexistence of factors considered to contribute to development of porphyria cutanea tarda was studied in 39 consecutive patients. Highly prevalent factors were alcohol intake in 79%, smoking in 86%, hepatitis C virus infection in 74%, estrogen use in 73% of 11 females, and at least one mutation in the HFE (hereditary hemochromatosis) gene in 65%. The C282Y mutation was found in 29%, H63D in 47%, and S65C in 0%. HFE genotypes included C282Y/C282Y in 9%, H63D/H63D in 9%, C282Y/H63D in 12%, C282Y/wild type in 9%, and H63D/wild type in 26%. Less prevalent were HIV infection in 15% (or 25% of those tested, N = 24) and erythrocyte uroporphyrinogen decarboxylase deficiency, which distinguishes familial (type 2) from “sporadic” (type 1) porphyria cutanea tarda, in 19%. Multiple contributing factors coexisted in both types 1 and 2, with 92% of all patients having three or more factors. These observations indicate that this porphyria is multifactorial in the individual patient, and therefore is seldom attributable to a single identifiable cause. Profiling for all potentially contributing factors is important for individualizing management.

Monitoring of BK Viral Load in Renal Allograft Recipients by Real-Time PCR Assays

BK virus (BKV) is a nonenveloped, double-stranded DNA virus of the polyomavirus family that primarily affects immunocompromised people. BKV may cause nephropathy in renal transplant recipients receiving immunosuppressive therapy, resulting in renal dysfunction and, possibly, graft loss. Monitoring of BK viral load in urine and blood has been used as a surrogate marker of BKV nephropathy (BKVN). Although real-time polymerase chain reaction (PCR) is the method of choice, currently there is no US Food and Drug Administration-approved or standardized BK viral load assay. Different PCR assays vary significantly in sample types, DNA extraction method, PCR primers and probes, and reference materials used to generate a standard curve. These differences can affect the accuracy, specificity, and dynamic ranges of various real-time PCR assays. These analytic differences cause difficulty in comparing test results, making it impossible to establish universal standardized cutoff values that correlate with clinical manifestations of BKVN. In this review, we summarize real-time PCR assays used for managing BKVN.

Molecular Events in the Progression of Recurrent Respiratory Papillomatosis to Carcinoma

CONTEXT Identification of the type of human papillomavirus (HPV) by polymerase chain reaction and sequencing to determine coinfection or superinfection (by more than 1 HPV type) and other molecular events have not been reported in a series of patients exhibiting the morphologic spectrum of recurrent respiratory papillomatosis progressing to carcinoma. DESIGN Four cases of juvenile-onset recurrent respiratory papillomatosis progressing to carcinoma (no history of smoking or irradiation in 2 cases) were studied. Morphologically distinct foci (squamous papilloma, pulmonary papillomatosis, squamous dysplasia subjacent to carcinoma, and squamous carcinoma) were subjected to laser capture microdissection and polymerase chain reaction amplification using general primers in addition to type-specific primers for HPV types 16 and 18. Direct sequencing of polymerase chain reaction products identified the type of HPV. The tissue sections were immunostained using antibodies to p53, pRb, p21(WAF1), and p16 proteins with a semiquantitative assessment. RESULTS Human papillomavirus 11 was the only type of HPV identified in all lesions of all cases associated with recurrent respiratory papillomatosis. There was a marked increase in p53 protein expression in foci of dysplasia and carcinoma as compared to squamous papilloma and pulmonary papillomatosis. An inverse correlation between p53 and p21(WAF1) protein expression was noted in all lesions. pRb protein expression increased from the benign to the malignant end of the spectrum. p16 protein was expressed in all lesions. CONCLUSIONS Infection by HPV-11 may be an early event associated with progression of recurrent respiratory papillomatosis to carcinoma. Increased expression of p53 and pRb proteins and a reduced expression of p21(WAF1) protein appear to be significant subsequent events.

Developing a sustainable process to provide quality control materials for genetic testing

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community.Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps.Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step.Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Prevalence of herpesviruses in cranial nerve ganglia.

A number of cranial nerve disorders are known to result from viral infection or reactivation, including Bells palsy, Ramsay Hunt syndrome and herpetic laryngitis. The consequences of these diseases are well established although the patient population at risk is not. Prevalence studies in the general population are an initial step toward defining individuals at risk. The aim of this study was to determine the prevalence of herpesvirus DNA in cranial nerve ganglia in a random population sample. Qualitative molecular biologic analysis using polymerase chain reaction assay of the trigeminal, geniculate, vestibular, spiral and vagal ganglia was used in 18 randomly selected fresh cadaver heads. Herpes simplex virus (HSV) DNA was detected in 42% of all ganglia surveyed. Varicella zoster virus (VZV) DNA was detected in 44% of all ganglia. The difference in the prevalence rate between viruses was not significant ( p = 0.63). At least 1 of the 2 viruses was found in 65% of all ganglia. Both HSV and VZV can commonly be recovered from cranial nerve ganglia. In order to confirm a viral etiology for various cranial nerve disorders, demonstration of a significant difference in prevalence of the viruses in specimens from afflicted individuals will be necessary.A number of cranial nerve disorders are known to result from viral infection or reactivation, including Bells palsy, Ramsay Hunt syndrome and herpetic laryngitis. The consequences of these diseases are well established although the patient population at risk is not. Prevalence studies in the general population are an initial step toward defining individuals at risk. The aim of this study was to determine the prevalence of herpesvirus DNA in cranial nerve ganglia in a random population sample. Qualitative molecular biologic analysis using polymerase chain reaction assay of the trigeminal, geniculate, vestibular, spiral and vagal ganglia was used in 18 randomly selected fresh cadaver heads. Herpes simplex virus (HSV) DNA was detected in 42% of all ganglia surveyed. Varicella zoster virus (VZV) DNA was detected in 44% of all ganglia. The difference in the prevalence rate between viruses was not significant (p = 0.63). At least 1 of the 2 viruses was found in 65% of all ganglia. Both HSV and VZV can commonly be recovered from cranial nerve ganglia. In order to confirm a viral etiology for various cranial nerve disorders, demonstration of a significant difference in prevalence of the viruses in specimens from afflicted individuals will be necessary.