Evaluation of a collaborative model for successful implementation of a National CD4 enumeration EQA program in Cameroon.

Participation in an EQA program is critical to the quality assurance process. Reliable and precise CD4 T-cells enumeration are essential to improve the clinical management of patients by evaluating the disease progression and by monitoring the effectiveness of ART in HIV-patients. The CIRCB, CD4 reference laboratory, in collaboration with the Canadian QASI-program, recruited sites, distributed and analyzed CD4-panels in 61 sites across Cameroon. A trend and performance analysis in the pre-analytical, analytical and post-analytical phases was performed. Continuous training and corrective actions carried out from 2014 to 2018 increased the number of participating sites from 15 to 61 sites, the number of unacceptable results decreased from 50 to 10%. Specific challenges included errors in pre analytic (17.5%), analytic (77.0%) and post-analytic (5.5%) phases. This EQA requires the application of good laboratory practices, fluidic communication between all the stakeholders, continuous training, application of specific on-site corrective measures, and timely equipment maintenance in order to avoid repetitive errors and to increase laboratory performance. It could be extended to other HIV-1 testing like viral load and EID point-of-care. Partnership with QASI serve as a model for implementation of a successful EQA model for resource limited countries wanting to implement EQA for HIV testing and monitoring in alignment with 90-90-90 targets.

Pre-exposure prophylaxis differentially alters circulating and mucosal immune cell activation in herpes simplex virus type 2 seropositive women.

OBJECTIVE: Oral tenofovir-based pre-exposure prophylaxis (PrEP) is an important tool for prevention of new HIV infections, which also reduces subclinical herpes simplex virus type 2 (HSV-2) shedding and symptomatic lesions in HIV-negative, HSV-2-seropositive individuals. However, the impact of PrEP on mucosal immunity has not been examined in detail. DESIGN: Here we evaluate paired genital tissue and systemic immune profiles to characterize the immunological effects of PrEP in HIV-negative, HSV-2-seropositive African women sexually exposed to HIV. METHODS: We compared local and systemic innate and T-cell characteristics in samples collected during PrEP usage and 2 months after PrEP discontinuation. RESULTS: We found that frequencies of cervical CCR5CD4 cells, regulatory T cells, and tissue macrophages were significantly reduced during PrEP use compared with after PrEP discontinuation. In contrast, peripheral blood CD4 and CD8 T cells expressing markers of activation and trafficking were increased during PrEP usage. CONCLUSION: Together, our data are consistent with PrEP altering immunity differentially in the female genital tract compared with circulation in HSV-2+ women. Further study including comparison with HSV-2 negative women is needed to define the overall impact and mechanisms underlying these effects. These results point to the critical need to study the human mucosal compartment to characterize immune responses to mucosal infections.

Compatibility of stabilized whole blood products with CD4 technologies and their suitability for quality assessment programs.

BACKGROUND: CD4 T cell enumeration is the most widely used prognostic marker for management of HIV disease. Internal quality control and external quality assessment (EQA) programs are critical to ensure reliability of clinical measurements. The utility of stabilized whole blood products (SWBP) as a test reagent for EQA programs such as Quality Assessment and Standardization for Immunological measures relevant to HIV/AIDS (QASI) program have been demonstrated previously. Since then, several new commercial SWBPs and alternative CD4 enumeration technologies have become available. Seven SWBPs were evaluated on seven different enumeration platforms to determine which product(s) are most suitable for EQA programs that support multiple analytical technologies. METHOD: Assessment of SWBPs was based on two criteria: (1) accuracy of CD4 T cell measurements and; (2) stability under sub optimal storage conditions. RESULTS: Three SWBPs (Multi-Check, StatusFlow and CD4 Count) showed accurate CD4 T-cell absolute count and percentage values across six of the enumeration platforms. All products retain stability up to 18 days at 21-23°C with the exception of Multi-Check-high on FacsCount and Multi-Check-Low and StatusFlow-Low on Pima. One of the products (CD4 Count) retained stability for three days on all platforms tested when stored at 37°C. CONCLUSION: This study demonstrated that the characteristics of commercially available SWBPs vary across multiple CD4 platforms. The compatibility of testing panels for EQA programs with multiple analytical platforms needs to be carefully considered, especially in large multiplatform CD4 EQA programs. The selection of a suitable cross-platform SWBP is an increasing challenge as more reagents and platforms are introduced for CD4 T-cell enumeration.

Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

Factors affecting the accuracy of urine-based biomarkers of BSE.

BACKGROUND: Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD. OBJECTIVE: Urine is well suited as the matrix for an ante-mortem test for TSE diseases because it would permit non-invasive and repeated sampling. In this study urine samples collected from BSE infected and age matched control cattle were screened for the presence of individual proteins that exhibited disease specific changes in abundance in response to BSE infection that might form the basis of such an ante-mortem test. RESULTS: Two-dimensional differential gel electrophoresis (2D-DIGE) was used to identify proteins exhibiting differential abundance in two sets of cattle. The known set consisted of BSE infected steers and age matched controls throughout the course of the disease. The blinded unknown set was composed of BSE infected and control samples of both genders, a wide range of ages and two different breeds. Multivariate analyses of individual protein abundance data generated classifiers comprised of the proteins best able to discriminate between the samples based on disease state, breed, age and gender. CONCLUSION: Despite the presence of confounding factors, the disease specific changes in abundance exhibited by a panel of urine proteins permitted the creation of classifiers able to discriminate between control and infected cattle with a high degree of accuracy.

Analysis of clusterin glycoforms in the urine of BSE-infected Fleckvieh-Simmental cows.

Currently approved tests for bovine spongiform encephalopathy (BSE) monitoring in cattle are based on the detection of the disease-related isoform of the prion protein in brain tissue and consequently are only suitable for postmortem diagnosis. Previously, to meet the demand for an antemortem test based on a matrix that would permit easy access and repeated sampling, two-dimensional differential gel electrophoresis (2D-DIGE) was used to perform an unbiased screen of bovine urine. Data demonstrated the altered abundance of particular isoforms of the multifunctional glycoprotein clusterin in urine samples obtained from BSE-infected and age-matched Fleckvieh-Simmental cattle. Unfortunately, the use of particular isoforms of a relatively abundant urine protein such as clusterin for diagnosis faces many of the same challenges encountered in tests based on PrP(d) detection. In both instances the specific detection of the marker protein is complicated by the high background levels of proteins with identical amino acid sequences, but lacking the disease-specific posttranslational modifications or configuration. The goal of the current study was to define the distinguishing characteristics of the clusterin isoforms observed. Biochemical and mass spectrometry analyses in combination with the generation of bovine clusterin subunit-specific antibodies enabled us to demonstrate that it was ß-subunits of clusterin possessing N-linked glycans of complex structure that exhibited differential abundance in response to BSE infection. The charateristic highly glycosylated clusterin ß-subunit was detectable as early as 16 mo post infection (mpi) by one-dimensional (1D) Western blot analysis of urine obtained from BSE-infected cattle.

A radiation-induced adaptive response prolongs the survival of prion-infected mice.

Previously, it has been demonstrated that an "adaptive response" that includes the prevention, repair, and removal of oxidative damage can be evoked by radiation at dose rates substantially lower than those at which risks have been observed. The exact pathogenic mechanism of prion diseases is unknown, but circumstantial evidence suggests that oxidative stress plays a central role. Exposure of prion-infected mice to four 500 mGy/fraction doses of (60)Co γ-radiation administered every other day at a low dose rate (0.5 mGy/min) starting at 2 days before infection, 7 days postinfection (dpi), or 50 dpi significantly prolonged the survival of infected mice. The 500-mGy radiation treatments started at 50 dpi also significantly prolonged the symptom-free period of the disease and caused a significant delay in the rise of the 8-hydroxydeoxyguanosine concentration observed in the urine of nonirradiated infected mice at 98 dpi. The duration of the reduction in oxidative stress achieved by the radiation treatments was similar in length to the prolonged survival of the irradiated mice. This suggests that the adaptive response induced by low-dose whole-body radiation treatments prolongs the survival of prion-infected mice by reducing oxidative stress.

Efficient knockdown of human prnp mRNA expression levels using hybrid hammerhead ribozymes.

Prion diseases are invariably fatal infectious diseases of the central nervous system. The prion protein has been identified as the underlying causative agent as PrP knockout mice (prnp(0/0)) are resistant to infection. This suggests that a significant reduction in the expression levels of PrP(c) should interrupt disease progression. Accomplishing this in vivo, upon presentation of symptoms, requires a mechanism that significantly reduces prnp mRNA levels while lacking potential side effects that may be cytotoxic or lethal to the host. Hybrid hammerhead ribozymes (HyHamRzs) include both a helicase recruitment signal and a tRNA(Val) promoter. HyHamRzs offer a means of highly specific and significant mRNA cleavage. In this study, data demonstrate increased activity granted to HamRzs by the addition of the helicase recruitment signal. Results show that three different HyHamRzs, targeting different locations along the full length prnp mRNA, reduced expression levels by greater than 95% relative to the control. It is postulated that HyHamRzs, modified to enhance serum stability and delivered intravenously to neurons by forming a complex with the modified rabies virus G protein (RVG), may offer a potential gene therapy strategy.

The identification of disease-induced biomarkers in the urine of BSE infected cattle.

BACKGROUND: The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob Disease (vCJD) have led to profound changes in the production and trade of agricultural goods. The rapid tests currently approved for BSE monitoring in slaughtered cattle are all based on the detection of the disease related isoform of the prion protein, PrPd, in brain tissue and consequently are only suitable for post-mortem diagnosis. OBJECTIVES: In instances such as assessing the health of breeding stock for export purposes where post-mortem testing is not an option, there is a demand for an ante-mortem test based on a matrix or body fluid that would permit easy access and repeated sampling. Urine and urine based analyses would meet these requirements. RESULTS: Two dimensional differential gel eletrophoresis (2D-DIGE) and mass spectrometry analyses were used to identify proteins exhibiting differential abundance in the urine of BSE infected cattle and age matched controls over the course of the disease. Multivariate analyses of protein expression data identified a single protein able to discriminate, with 100% accuracy, control from infected samples. In addition, a subset of proteins were able to predict with 85% +/- 13.2 accuracy the time post infection that the samples were collected. CONCLUSION: These results suggest that in principle it is possible to identify biomarkers in urine useful in the diagnosis, prognosis and monitoring of disease progression of transmissible spongiform encephalopathy diseases (TSEs).

Severe subacute GM2 gangliosidosis caused by an apparently silent HEXA mutation (V324V) that results in aberrant splicing and reduced HEXA mRNA.

We have characterized the molecular basis of beta-hexosaminidase A (HEX A) deficiency in a patient ascertained through an ophthalmologic examination that revealed cherry red spots on his retina. The absence of neurological deficit in this child until 3 3/4 years of age indicated residual HEX A must be present. Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele, and 1A > T (M1L) on the paternal allele were identified. The effects of the amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as expected, no HEX A activity was associated with the M1L mutation but surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed by the wild type cDNA. The effect of the S4P change was much less than that of another HEXA mutation, G269S, associated with an adult onset form of G(M2) gangliosidosis. This indicated that the S4P change was not the cause of disease and suggested that one of the mutations on the maternal allele, 10T > C or 972T > A, had its effect at the mRNA level. This was confirmed by Northern blot analysis that showed only 7% of the normal level of HEXA mRNA in proband fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed normal transcripts from both the maternal and paternal allele, as well as a low abundance aberrant transcript from the maternal allele. Sequencing of this aberrant transcript revealed a new exon 8 donor site created by the 972T > A mutation that resulted in a 17 bp deletion and destabilization of the resulting abnormal transcript. The remaining normal mRNA produced from the 972T > A allele must account for the delayed onset of clinical symptoms in this child.

Identification of a direct Dlx homeodomain target in the developing mouse forebrain and retina by optimization of chromatin immunoprecipitation.

Understanding homeobox gene specificity and function has been hampered by the lack of proven direct transcriptional targets during development. Dlx genes are expressed in the developing forebrain, retina, craniofacial structures and limbs. Dlx1/Dlx2 double knockout mice die at birth with multiple defects including abnormal forebrain development and decreased Dlx5 and Dlx6 expression. We have successfully applied chromatin immunoprecipitation (ChIP) to identify a direct transcriptional target of DLX homeoproteins from embryonic tissues in vivo. We optimized cross-linking conditions to enrich for protein-DNA complexes, then using specific high affinity DLX antibodies captured immunoenriched DLX genomic DNA transcriptional targets. DLX homeobox proteins bind differentially to the Dlx5/Dlx6 intergenic enhancer in newborn retina (DLX2) and embryonic striatum (DLX1, DLX2) in situ. Reporter gene assays demonstrated the functional significance of the binding of DLX proteins to this regulatory element, confirmed in vitro by electrophoretic mobility shift assays, using tissue extracts or recombinant DLX proteins. ChIP provides the best approach to identify direct Dlx homeoprotein targets from developing tissues in situ. The use of this technology will advance our understanding of Dlx gene function in the vertebrate in vivo and can be applied to examine targets of other homeobox genes and other classes of transcription factors.