Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses.

Encouraging protection results from current mRNA-based SARS-CoV-2 vaccine platforms are primarily due to the induction of SARS- CoV-2- specific B cell antibody and CD4 + T cell. Even though, current mRNA vaccine platforms are adept in inducing SARS-CoV2-specific CD8 + T cell, much less is known about CD8 T cells contribution to the overall vaccine protection. Our allogeneic cellular vaccine, based on a secreted form of the heat-shock protein gp96-Ig, achieves high frequencies of polyclonal CD8 + T cell responses to tumor and infectious antigens through antigen cross-priming in vivo. We and others have shown that gp96-Ig, in addition to antigen-specific CD8 + T cell anti-tumor and anti-pathogen immunity, primes antibody responses as well. Here, we generated a cell-based vaccine that expresses SARS-Cov-2 Spike (S) protein and simultaneously secretes gp96-Ig and OX40L-Fc fusion proteins. We show that co-secretion of gp96-Ig-S peptide complexes and the OX40L-Fc costimulatory fusion protein in allogeneic cell lines results in enhanced activation of S protein-specific IgG antibody responses. These findings were further strengthened by the observation that this vaccine platform induces T follicular helper cells (TFH) and protein-S -specific CD8 + T cells. Thus, a cell-based gp96-Ig vaccine/OX40-L fusion protein regimen provides encouraging translational data that this vaccine platform induces pathogen-specific CD8+, CD4 + T and B cell responses, and may cohesively work as a booster for FDA-approved vaccines. Our vaccine platform can be rapidly engineered and customized based on other current and future pathogen sequences.

The induction of a mesenchymal phenotype by platelet cloaking of cancer cells is a universal phenomenon.

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

Platelets, immune cells and the coagulation cascade; friend or foe of the circulating tumour cell?

Cancer cells that transit from primary tumours into the circulatory system are known as circulating tumour cells (CTCs). These cancer cells have unique phenotypic and genotypic characteristics which allow them to survive within the circulation, subsequently extravasate and metastasise. CTCs have emerged as a useful diagnostic tool using "liquid biopsies" to report on the metastatic potential of cancers. However, CTCs by their nature interact with components of the blood circulatory system on a constant basis, influencing both their physical and morphological characteristics as well as metastatic capabilities. These properties and the associated molecular profile may provide critical diagnostic and prognostic capabilities in the clinic. Platelets interact with CTCs within minutes of their dissemination and are crucial in the formation of the initial metastatic niche. Platelets and coagulation proteins also alter the fate of a CTC by influencing EMT, promoting pro-survival signalling and aiding in evading immune cell destruction. CTCs have the capacity to directly hijack immune cells and utilise them to aid in CTC metastatic seeding processes. The disruption of CTC clusters may also offer a strategy for the treatment of advance staged cancers. Therapeutic disruption of these heterotypical interactions as well as direct CTC targeting hold great promise, especially with the advent of new immunotherapies and personalised medicines. Understanding the molecular role that platelets, immune cells and the coagulation cascade play in CTC biology will allow us to identify and characterise the most clinically relevant CTCs from patients. This will subsequently advance the clinical utility of CTCs in cancer diagnosis/prognosis.

An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood.

Circulating tumor cells (CTCs) carry valuable biological information. While enumeration of CTCs in peripheral blood is an FDA-approved prognostic indicator of survival in metastatic prostate and other cancers, analysis of CTC phenotypic and genomic markers is needed to identify cancer origin and elucidate pathways that can guide therapeutic selection for personalized medicine. Given the emergence of single-cell mRNA sequencing technologies, a method is needed to isolate CTCs with high sensitivity and specificity as well as compatibility with downstream genomic analysis. Flow cytometry is a powerful tool to analyze and sort single cells, but pre-enrichment is required prior to flow sorting for efficient isolation of CTCs due to the extreme low frequency of CTCs in blood (one in billions of blood cells). While current enrichment technologies often require many steps and result in poor recovery, we demonstrate a magnetic separator and acoustic microfluidic focusing chip integrated system that enriches rare cells in-line with FACS™ (fluorescent activated cell sorting) and single-cell sequencing. This system analyzes, isolates, and index sorts single cells directly into 96-well plates containing reagents for Molecular Indexing (MI) and transcriptional profiling of single cells. With an optimized workflow using the integrated enrichment-FACS system, we performed a proof-of-concept experiment with spiked prostate cancer cells in peripheral blood and achieved: (i) a rapid one-step process to isolate rare cancer cells from lysed whole blood; (ii) an average of 92% post-enrichment cancer cell recovery (R2 = 0.9998) as compared with 55% recovery for a traditional benchtop workflow; and (iii) detection of differentially expressed genes at a single cell level that are consistent with reported cell-type dependent expression signatures for prostate cancer cells. These model system results lay the groundwork for applying our approach to human blood samples from prostate and other cancer patients, and support the enrichment-FACS system as a flexible solution for isolation and characterization of CTCs for cancer diagnosis. © 2018 International Society for Advancement of Cytometry.

An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters.

Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200µm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing.

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue.

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.

Development of the Sensory Hypersensitivity Scale (SHS): a self-report tool for assessing sensitivity to sensory stimuli.

Sensory hypersensitivity is one manifestation of the central sensitization that may underlie conditions such as fibromyalgia and chronic fatigue syndrome. We conducted five studies designed to develop and validate the Sensory Hypersensitive Scale (SHS); a 25-item self-report measure of sensory hypersensitivity. The SHS assesses both general sensitivity and modality-specific sensitivity (e.g. touch, taste, and hearing). 1202 participants (157 individuals with chronic pain) completed the SHS, which demonstrated an adequate overall internal reliability (Cronbach's alpha) of 0.81, suggesting the tool can be used as a cross-modality assessment of sensitivity. SHS scores demonstrated only modest correlations (Pearson's r) with depressive symptoms (0.19) and anxiety (0.28), suggesting a low level of overlap with psychiatric complaints. Overall SHS scores showed significant but relatively modest correlations (Pearson's r) with three measures of sensory testing: cold pain tolerance (-0.34); heat pain tolerance (-0.285); heat pain threshold (-0.271). Women reported significantly higher scores on the SHS than did men, although gender-based differences were small. In a chronic pain sample, individuals with fibromyalgia syndrome demonstrated significantly higher SHS scores than did individuals with osteoarthritis or back pain. The SHS appears suitable as a screening measure for sensory hypersensitivity, though additional research is warranted to determine its suitability as a proxy for central sensitization.

Contributions of physical function and satisfaction with social roles to emotional distress in chronic pain: a Collaborative Health Outcomes Information Registry (CHOIR) study.

Individuals with chronic pain show greater vulnerability to depression or anger than those without chronic pain, and also show greater interpersonal difficulties and physical disability. The present study examined data from 675 individuals with chronic pain during their initial visits to a tertiary care pain clinic using assessments from Stanford University's Collaborative Health Outcomes Information Registry (CHOIR). Using a path modeling analysis, the mediating roles of Patient-Reported Outcomes Measurement Information Systems (PROMIS) Physical Function and PROMIS Satisfaction with Social Roles and Activities were tested between pain intensity and PROMIS Depression and Anger. Pain intensity significantly predicted both depression and anger, and both physical function and satisfaction with social roles mediated these relationships when modeled in separate 1-mediator models. Notably, however, when modeled together, ratings of satisfaction with social roles mediated the relationship between physical function and both anger and depression. Our results suggest that the process by which chronic pain disrupts emotional well-being involves both physical function and disrupted social functioning. However, the more salient factor in determining pain-related emotional distress seems to be disruption of social relationships, than global physical impairment. These results highlight the particular importance of social factors to pain-related distress, and highlight social functioning as an important target for clinical intervention in chronic pain.

Analytical performance of RNA isolated from BD SurePath™ cervical cytology specimens by the PreTect™ HPV-Proofer assay.

Several commercial HPV ancillary tests are available for detection of E6/E7 RNA. It is not clear how storage of a cervical Pap affects the analytical and clinical performance of the PreTect™ HPV-Proofer assay. To investigate the qualitative performance of RNA extracted from BD SurePath™ liquid-based cytology (LBC) specimens for the detection of human papillomavirus (HPV) E6/E7 mRNA using the PreTect™ HPV-Proofer assay, studies including stability, reproducibility, residual specimen analysis, and storage medium comparison assays were performed. Cervical cytology specimens were collected and stored in BD SurePath™ LBC preservative fluid and/or PreTect™ Transport Media. RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit and RNA integrity was evaluated in the PreTect™ HPV-Proofer assay. The performance of RNA isolated from cervical cells collected and stored in BD SurePath™ LBC preservative fluid or PreTect™ Transport Media was also evaluated through a storage medium comparison study. The RNA was found to be stable for a minimum of 21 days when stored at ambient temperature and displayed high reproducibility with the mean percentage reproducibility ranging from 90.5% to 100% for the HPV types detected by the PreTect™ HPV-Proofer assay. The prevalence rate of HPV types in this study cohort was consistent with published reports. A 93.7% first pass acceptance rate was demonstrated across all cytology grades. The positive human U1 snRNP specific A protein (U1A) and HPV rate for BD SurePath™ LBC and PreTect™ Transport Media specimens was statistically equivalent for both normal and abnormal specimens. This data support the use of RNA isolated from BD SurePath™ LBC for ancillary HPV testing and demonstrates the feasibility of using BD SurePath™ preservative fluid as a specimen type with the PreTect™ HPV-Proofer assay.

Generation of monoclonal antibodies to the AML1-ETO fusion protein: strategies for overcoming high homology.

The chromosomal translocation t(8;21) often found in acute myeloid leukemia generates an oncogenic fusion protein AML1-ETO. This chimeric oncoprotein disrupts wild-type AML1 function and dysregulates genes important for normal myelopoiesis. Monoclonal antibodies that can capture and detect the AML1-ETO fusion protein would help with early diagnosis and treatment prognosis of acute myeloid leukemia. We report the development of murine monoclonal antibodies (MAbs) that specifically bind epitopes encoded by either AML1 or ETO. Since alignment to the human ETO protein indicated almost 100% homology to the mouse ortholog, a strategy was needed to instruct humoral immunity in mice to focus and respond to self-epitopes. Our strategy to develop capture/detector reagents involved producing MAbs that would bind to epitopes within the non-fused myelopic protein (i.e., either AML1 or ETO). This included a process to select antibodies for their ability to also recognize the translocated chromosomal AML1-ETO fusion protein and to identify complementary capture/detector antibody pairs. Construction of a peptide hapten-carrier complex and use of a rapid immunization protocol resulted in IgM-IgG ETO specific MAbs. These MAbs bound specifically to a recombinant form of AML1-ETO fusion protein expressed in HEK and to an endogenous AML1-ETO form of the fusion protein expressed in Kasumi-1. We report the development of murine hybridoma MAbs derived from immunizations with a peptide "self-epitope." Our findings provide a potential strategy to instruct humoral immunity in mice to focus and respond to self-epitopes. This strategy has been validated with the oncogenic fusion protein AML1-ETO involved in acute myeloid leukemia.

The selection and characterization of antibodies to minichromosome maintenance proteins that highlight cervical dysplasia.

BACKGROUND: Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. METHODS: Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. RESULTS: Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10(-9) M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. CONCLUSION: In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples.

Recovery of DNA from BD SurePath cytology specimens and compatibility with the Roche AMPLICOR Human Papillomavirus (HPV) Test.

BACKGROUND: The AMPLICOR HPV Test has been validated for use with cervical cells collected in liquid-based preservative fluids, such as BD SurePath. It is currently recommended, however, that residual BD SurePath samples be stored at 4 degrees C prior to testing. OBJECTIVES: The aim of this study was to demonstrate that DNA isolated from SurePath cervical cytology specimens and stored at ambient temperature was also compatible with the AMPLICOR HPV Test. STUDY DESIGN: DNA was extracted using the AmpliLute Media Sample Preparation Kit. Amplification and detection of HPV was performed both as directed by the manufacturer and with minor protocol modifications. RESULTS: Cervical specimens collected in SurePath preservative fluid remained stable for testing with the AMPLICOR HPV Test for at least 21 days. The performance of DNA extracted from specimens stored at room temperature was equivalent to DNA extracted from specimens stored at 4 degrees C. The beta-globin internal control was detected in all of the 146 residual SurePath cervical cytology specimens tested using the AMPLICOR HPV Test, and high-risk HPV was detected in 46.2% (18/39) of ASCUS cases, in 63.3% (19/30) of LSIL cytology specimens, and 92.3% (24/26) of HSIL cases. Concordance of AMPLICOR HPV Test results with Hybrid Capture II was 83.9%. CONCLUSIONS: The AMPLICOR HPV Test can be successfully and reproducibly performed from DNA isolated from residual SurePath cervical cytology specimens stored at ambient temperature for at least 21 days. This provides clinical laboratories flexible storage conditions for residual SurePath cytology specimens.

Isolation of RNA from cell lines and cervical cytology specimens stored in BD SurePath preservative fluid and downstream detection of housekeeping gene and HPV E6 expression using real time RT-PCR.

This study was performed to demonstrate that RNA isolated from cell lines and cervical cytology specimens stored in SurePath preservative fluid would be functional in real-time RT-PCR assays. RNA was isolated from cervical cell lines or cytology samples stored in SurePath preservative at room temperature for 2-5 weeks using five commercially available RNA purification kits, three of which contain proteinases. The quality of the RNA was assessed by real time RT-PCR amplification of GAPDH, GUSB, U1A, HPV 16 and 18 E6 mRNAs. RNA was isolated successfully from cells that were stored in SurePath preservative fluid with only the three protocols that contained proteinases. GAPDH was amplified in 98-100% of the samples, GUSB in 90-98%, and the least abundant transcript, U1A, was amplified in 81-96% of the samples. HPV 16 and 18 E6 transcripts were detected in 56% of high grade, 39% of low grade and 2% of normal samples, with a concordance between DNA genotype and E6 mRNA expression of 97%. We demonstrated that RNA can be extracted from cervical cell lines and cytology specimens stored in BD SurePath preservative fluid with three different procedures that all contain proteinases. This RNA is suitable for real-time RT-PCR applications.

Hamartoma torácico congénito: a propóstio de un caso / Congenital thoracic hamartoma: A case report

A pesar de su poca frecuencia las tumoraciones de la cavidad torácica fetal constituyen casos realmente curiosos descritos en la literatura mundial. Dentro de estos los hamartomas, los linfangiomas, tumores fibrosos y teratomas mediastinales han sido reportados según hallazgos clínicos o de necropsia, pues no obstante su histología inequívocamente benigna puede acompañarse de otras malformaciones como el secuestro pulmonar, la hipoplasia pulmonar, pulmones quísticos, hidrops fetalis e hidrotórax. En este trabajo presentamos un hallazgo de la necropsia realizada a un feto masculino de 1360 g y 27 semanas de edad gestacional procedente de una interrupción de embarazo por diagnóstico prenatal de hernia diafragmática derecha, observándose durante la necropsia integridad del músculo diafragmático y la presencia de un tumor sólido y pediculado en la cavidad torácica derecha con estudio histológico concluyente de un hamartoma, acompañado de una hipoplasia moderada del pulmón derecho(AU)

In spite of its scarce frequency tumors of the fetal thoracic cavity constitute weird cases described in the world literature. Among them hamartomas, lymphangiomas, fibrosis tumors and mediastinal teratomas have been reported according to clinical or necropsy findings because no matter the undoubtedly benign histology, they may be together with some other anomalies, such as, the pulmonary sequestrum, pulmonary hypoplasia, cystic lungs, hydrops fetalis and hydrothorax. In this work it is presented the necropsy finding of a male fetus of 1360 grams and 27 weeks of gestation interrupted by the prenatal diagnosis of right diaphragmatic hernia, noticing during the necropsy, the integrity of the diaphragmatic muscle and the presence of a solid and pediculated tumor in the right thoracic cavity with a final histological research of harmatoma joined by moderated right lung hypoplasia(EU)

Toracópago: presentación de un caso / Thoracopagus: Presentation of a case

Con una incidencia variable el Thoracópagus o Toracópago es el tipo más común de gemelos unidos, representando el 75 por ciento de los casos que se reportan y son una condición rara en la cual existe predominio del sexo femenino. Se presenta el caso de gemelos unidos del tipo Toracópagos, con una fusión amplia, procedentes de la interrupción por malformaciones congénitas complejas de un embarazo múltiple en una gestante de edad avanzada, que se estudió anatómica y morfológicamente en el acto de necropsia. Aunque no existe acuerdo sobre la embriogénesis de esta malformación, la teoría más aceptada es la propuesta de que esta alteración resulta de la unión secundaria de dos discos embrionarios separados originalmente(AU)

With a variable incidence the thoracopagus is the most common type of conjoined twins representing the 75 percent of the reported cases and they show a weird condition in which females prevailed. It is presented the case of thoracopagus conjoined twins with a wide fusion coming from the interruption for complex congenital malformation of a multiple pregnancy in an aged pregnant who was anatomically and morphologically studied in the necropsy. Although there is no resolution about embryogenesis of this malformation the most accepted theory is proposal that this alteration is the result of a secondary joined of two embrionary disks initially separated(EU)

Isolation of RNA from residual BD SurePath liquid-based cytology specimens and detection of HPV E6/E7 mRNA using the PreTectt HPV-Proofer assay.

Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.

Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein.

Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.

In vivo expression of a GST-fusion protein mediates the rapid generation of affinity matured monoclonal antibodies using DNA-based immunizations.

Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.

Comparison of two ultrasonic instruments for post removal.

The relative performance of two different ultrasonic units commonly used clinically for post removal was evaluated using tips designed specifically for post vibration. Twenty-four extracted maxillary and mandibular cuspids with crowns removed at the labial cementoenamel junction were treated endodontically. Post spaces were made 10 mm into the roots before cementing a 16 mm #5 (0.050-inch) Para-Post with zinc phosphate cement. The teeth were divided into three similar groups of eight. Post retention was assessed in group 1. Ultrasonic vibration was applied to groups 2 and 3 until post removal. The average force required to dislodge the posts from the teeth in group 1 (control group, no ultrasound) was 40.5 kg (SD = 12.3 kg). The average time for post removal in group 2 (Spartan) was 4:52 min (SD = 2:26). The average time for post removal in group 3 (Enac) was 1:31 min (SD = 0:34). The difference between groups 2 and 3 was statistically significant (p < 0.005). Use of ultrasonic tips designed for post vibration and maximization of audible sound level during ultrasonic treatment of posts seem to play an important role in the effectiveness and efficiency of post removal. The results obtained indicate that both the Enac ultrasonic unit with the ST-09 vibration tip and the Spartan ultrasonic unit with the Analytic VT-S tip were effective. Nevertheless, the Enac ultrasonic unit with the ST-09 vibration tip was clearly more efficient under these study conditions, resulting in typical post removal times of <2 min.