Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults.

BACKGROUND: Cytomegalovirus (CMV) infection leads to effector memory CD8+ T cell expansion and is associated with immune dysfunction in older adults. However, the molecular alterations of CMV-specific CD8+ T cells in CMV infected healthy young and middle-aged adults has not been fully characterized. RESULTS: We compared CD8+ T cells specific for a CMV epitope (pp65495-503, NLV) and an influenza A virus (IAV) epitope (M158-66, GIL) from the same young and middle-aged healthy adults with serum positive for anti-CMV IgG. Compared to the IAV-specific CD8+ T cells, CMV-specific CD8+ T cells contained more differentiated effector memory (TEM and TEMRA) cells. Isolated CMV-specific central memory (TCM) but not naïve (TN) cells had a significant reduced activation-induced expansion in vitro compared to their IAV-specific counterparts. Furthermore, we found that CD70 expression was reduced in CMV-specific CD28+CD8+ TCM and that CD70+ TCM had better expansion in vitro than did CD70- TCM. Mechanistically, we showed that CD70 directly enhanced MAPK phosphorylation and CMV-specific CD8+ TCM cells had a reduced MAPK signaling upon activation. Lastly, we showed that age did not exacerbate reduced CD70 expression in CMV- specific CD8+ TCM cells. CONCLUSION: Our findings showed that CMV infection causes mild expansion of CMV-NLV-specific CD8+ T cells, reduced CD70 expression and signaling, and proliferation of CMV-NLV-specific CD8+ TCM cells in young and middle-aged healthy adults and revealed an age-independent and CMV infection-specific impact on CD8+ memory T cells.

Amplification and thrifty single-molecule sequencing of recurrent somatic structural variations.

Deletion of tumor-suppressor genes as well as other genomic rearrangements pervade cancer genomes across numerous types of solid tumor and hematologic malignancies. However, even for a specific rearrangement, the breakpoints may vary between individuals, such as the recurrent CDKN2A deletion. Characterizing the exact breakpoints for structural variants (SVs) is useful for designating patient-specific tumor biomarkers. We propose AmBre (Amplification of Breakpoints), a method to target SV breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, AmBre validates SVs called by whole-exome/genome sequencing and hybridization arrays. AmBre involves a PCR-based approach to amplify the DNA segment containing an SV's breakpoint and then confirms breakpoints using sequencing by Pacific Biosciences RS. To amplify breakpoints with PCR, primers tiling specified target regions are carefully selected with a simulated annealing algorithm to minimize off-target amplification and maximize efficiency at capturing all possible breakpoints within the target regions. To confirm correct amplification and obtain breakpoints, PCR amplicons are combined without barcoding and simultaneously long-read sequenced using a single SMRT cell. Our algorithm efficiently separates reads based on breakpoints. Each read group supporting the same breakpoint corresponds with an amplicon and a consensus amplicon sequence is called. AmBre was used to discover CDKN2A deletion breakpoints in cancer cell lines: A549, CEM, Detroit562, MOLT4, MCF7, and T98G. Also, we successfully assayed RUNX1-RUNX1T1 reciprocal translocations by finding both breakpoints in the Kasumi-1 cell line. AmBre successfully targets SVs where DNA harboring the breakpoints are present in 1:1000 mixtures.

An innovative system for 3D clinical photography in the resource-limited settings.

BACKGROUND: Kaposi's sarcoma (KS) is the most frequently occurring cancer in Mozambique among men and the second most frequently occurring cancer among women. Effective therapeutic treatments for KS are poorly understood in this area. There is an unmet need to develop a simple but accurate tool for improved monitoring and diagnosis in a resource-limited setting. Standardized clinical photographs have been considered to be an essential part of the evaluation. METHODS: When a therapeutic response is achieved, nodular KS often exhibits a reduction of the thickness without a change in the base area of the lesion. To evaluate the vertical space along with other characters of a KS lesion, we have created an innovative imaging system with a consumer light-field camera attached to a miniature "photography studio" adaptor. The image file can be further processed by computational methods for quantification. RESULTS: With this novel imaging system, each high-quality 3D image was consistently obtained with a single camera shot at bedside by minimally trained personnel. After computational processing, all-focused photos and measurable 3D parameters were obtained. More than 80 KS image sets were processed in a semi-automated fashion. CONCLUSIONS: In this proof-of-concept study, the feasibility to use a simple, low-cost and user-friendly system has been established for future clinical study to monitor KS therapeutic response. This 3D imaging system can be also applied to obtain standardized clinical photographs for other diseases.

Binding and isolation of tumor cells in biological media with perfluorocarbon microbubbles.

With the emerging interest in personalized medicine, there is strong demand for new technologies for clinical sample interrogation. Exfoliated tumor cells in variety of pathological samples (e.g., blood, bone marrow, urine) could provide invaluable information for diagnosis and prognosis of cancers. Here we describe a detailed method for capture and isolation of tumor cells in medium, blood, or large issue buffy coat using EpCAM-targeted buoyant microbubbles (MBs). Perflorohexane gas lipid shell MBs were prepared with emulsification method and conjugated with antibody as described by us before [25]. The binding of EpCAM-targeted MBs to A549 (human lung carcinoma) and 4T1 (mouse breast carcinoma) cells spiked into BSA/PBS or blood was more than 90%, which was comparable with commercial anti-EpCAM immunomagnetic beads (DynaBeads). Anti-EpCAM MBs efficiently (75-82%) isolated BxPC3 pancreatic tumor cells spiked into medium, blood or a buffy coat, within 15-30 min of incubation. We discuss MB parameters and experimental conditions critical to achieve efficient cells binding and isolation. In conclusion, MB-assisted cell isolation is a promising method for rapid enrichment of cells and biomarkers from biological samples.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-kappaB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IkappaBalpha degradation and activation of the NF-kappaB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-kappaB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

Laboratory-Scale Production of Sterile Targeted Microbubbles.

Perfluorocarbon gas-filled microbubbles are clinically used as ultrasound contrast agents. We have been developing targeted microbubbles based BACS (buoyancy activated cell sorting) or BUBLES (buoyancy enabled separation) for ex vivo cell isolation from bloods for circulating tumor cell (CTC) detection and hematopoietic cell isolation. Recently, we further applied targeted microbubbles for multimarker cell sorting, and as artificial antigen presenting cells (aAPC) for T cell activation and expansion by taking advantage of a number of interesting properties of lipid-shelled microbubbles. This chapter will describe the process of manufacturing sterile targeted microbubbles for research applications.

John Cunningham virus T-antigen expression in anal carcinoma.

BACKGROUND: Anal carcinoma is thought to be driven by human papillomavirus (HPV) infection through interrupting function of cell regulatory proteins such as p53 and pRb. John Cunningham virus (JCV) expresses a T-antigen that causes malignant transformation through development of aneuploidy and interaction with some of the same regulatory proteins as HPV. JCV T-antigen is present in brain, gastric, and colon malignancies, but has not been evaluated in anal cancers. The authors examined a cohort of anal cancers for JCV T-antigen and correlated this with clinicopathologic data. METHODS: Archived anal carcinomas were analyzed for JCV T-antigen expression. DNA from tumor and normal tissue was sequenced for JCV with viral copies determined by quantitative polymerase chain reaction and Southern blotting. HPV and microsatellite instability (MSI) status was correlated with JCV T-antigen expression. RESULTS: Of 21 cases of anal cancer (mean age 49 years, 38% female), 12 (57%) were in human immunodeficiency virus (HIV)-positive individuals. All 21 cancers expressed JCV T-antigen, including 9 HPV-negative specimens. More JCV copies were present in cancer versus surrounding normal tissue (mean 32.54 copies/µg DNA vs 2.98 copies/µg DNA, P = .0267). There was no correlation between disease stage and viral copies, nor between viral copies and HIV-positive or -negative status (28.7 vs 36.34 copies/µg DNA, respectively, P = .7804). In subset analysis, no association was found between JCV T-antigen expression and HPV or MSI status. CONCLUSIONS: Anal carcinomas uniformly express JCV T-antigen and contain more viral copies compared with surrounding normal tissue. JCV and its T-antigen oncogenic protein, presumably through interruption of cell regulatory proteins, may play a role in anal cancer pathogenesis.

The essentiality of alpha-2-macroglobulin in human salivary innate immunity against new H1N1 swine origin influenza A virus.

A novel strain of influenza A H1N1 emerged in the spring of 2009 and has spread rapidly throughout the world. Although vaccines have recently been developed that are expected to be protective, their availability was delayed until well into the influenza season. Although anti-influenza drugs such as neuraminidase inhibitors can be effective, resistance to these drugs has already been reported. Although human saliva was known to inhibit viral infection and may thus prevent viral transmission, the components responsible for this activity on influenza virus, in particular, influenza A swine origin influenza A virus (S-OIV), have not yet been defined. By using a proteomic approach in conjunction with beads that bind alpha-2,6-sialylated glycoprotein, we determined that an alpha-2-macroglobulin (A2M) and an A2M-like protein are essential components in salivary innate immunity against hemagglutination mediated by a clinical isolate of S-OIV (San Diego/01/09 S-OIV). A model of an A2M-based "double-edged sword" on competition of alpha-2,6-sialylated glycoprotein receptors and inactivation of host proteases is proposed. We emphasize that endogenous A2M in human innate immunity functions as a natural inhibitor against S-OIV.

A sensitive array-based assay for identifying multiple TMPRSS2:ERG fusion gene variants.

Studies of gene fusions in solid tumors are not as extensive as in hematological malignancies due to several technical and analytical problems associated with tumor heterogeneity. Nevertheless, there is a growing interest in the role of fusion genes in common epithelial tumors after the discovery of recurrent TMPRSS2:ETS fusions in prostate cancer. Among all of the reported fusion partners in the ETS gene family, TMPRSS2:ERG is the most prevalent one. Here, we present a simple and sensitive microarray-based assay that is able to simultaneously determine multiple fusion variants with a single RT-PCR in impure RNA specimens. The assay detected TMPRSS2:ERG fusion transcripts with a detection sensitivity of <10 cells in the presence of more than 3000 times excess normal RNA, and in primary prostate tumors having no >1% of cancer cells. The ability to detect multiple transcript variants in a single assay is critically dependent on both the primer and probe designs. The assay should facilitate clinical and basic studies for fusion gene screening in clinical specimens, as it can be readily adapted to include multiple gene loci.

Lipid Microbubble-Conjugated Anti-CD3 and Anti-CD28 Antibodies (Microbubble-Based Human T Cell Activator) Offer Superior Long-Term Expansion of Human Naive T Cells In Vitro.

Stimulation of human primary T cells with immobilized anti-CD3 and anti-CD28 Abs in vitro provide a system to study T cell activation and proliferation and an avenue for expanding T cells for immunotherapy. Magnetic beads conjugated with anti-CD3 and anti-CD28 Abs (Dynabeads Human T-Activator [D-TCA]) have been a golden standard for stimulating human primary T cells in vitro. In this study, we report that an application using anti-CD3 and anti-CD28 Abs conjugated on lipid microbubbles (microbubble-based human T cell activator [MB-TCA]) to stimulate primary human naive T cells resulted in expansion superior to D-TCA. In 56-d cultures with three repeated stimulation cycles (14 d per stimulation), we found that 1) MB-TCA induced significantly better expansion (20- and 10-fold increase) of naive CD4+ and CD8+ T cells than did D-TCA; 2) MB-TCA- and D-TCA-stimulated T cells had a similar number of initial cell divisions, but MB-TCA had significantly lower activation-induced cell death than D-TCA; 3) MB-TCA-stimulated T cells produced less TNF-α than did D-TCA; and 4) blocking TNF-α action via adding an Ab against TNF-αR (TNFRSF1A) significantly improved expansion of T cells activated by D-TCA in vitro. Together, we demonstrated that the MB-TCA induces a better expansion of human naive T cells in vitro and offers advantages in both basic and clinical applications in which the outcome depends on the number of T cells.

p16 Expression Correlates with Invasive Ocular Surface Squamous Neoplasms in HIV-Infected Mozambicans.

BACKGROUND: p16 immunohistochemistry is widely used to diagnose human papillomavirus (HPV)-related squamous neoplasms of cervix, anogenital, head, and neck tissues. The incidence of these HPV-related squamous neoplasms is markedly increased in the HIV-infected population. Ocular surface squamous neoplasia (OSSN) is also more common in HIV-infected patients. However, the expression pattern of p16 in OSSN among HIV-infected patients is unclear. Here, we examined the expression of p16 in OSSN surgical excisions collected from a large HIV-infected cohort from -Mozambique. METHODS: OSSN surgical tissue specimens were collected from 75 Mozambican patients. Formalin-fixed, paraffin-embedded tissue blocks from these OSSNs were sectioned, stained with hematoxylin and eosin (H&E), and p16 expression by immunohistochemistry. H&E slides were reviewed to determine if OSSNs were noninvasive conjunctival intraepithelial neoplasms or invasive squamous cell carcinomas (SCC). Cases were classified as p16 positive or negative based on diffuse nuclear and cytoplasmic expression of p16 in neoplastic cells. RESULTS: p16 positivity was found in a minority of OSSN cases (14/75). p16 positivity was significantly associated with the invasive SCC type of OSSN in HIV-infected patients (p value of 0.026). CONCLUSIONS: The majority of OSSNs in our HIV-infected cohort do not express p16. However, those cases that are p16-positive are significantly more likely to be the invasive SCC form of OSSN. We propose that p16 expression may identify more aggressive OSSNs in HIV-infected populations.

Hepatocellular carcinoma: Clinical-pathological features and HIV infection in Mozambican patients.

BACKGROUND AND AIMS: Mozambique had been ranked among the countries with the highest global incidence of HCC with chronic hepatitis B infection and high exposure to aflatoxin-B1 (AFB1) being major risk factors. Indeed, HCC remains one of the most frequent cancer in Maputo. On the other hand, Mozambique has a high prevalence of infection with Human Immunodeficiency virus (HIV). Our study aims to describe the epidemiology, clinicopathological and serological features of patients with HCC in Maputo Central Hospital and its relationship with HIV. METHODS: A series of 206 patients, diagnosed with HCC via fine needle aspiration, were consecutively included in the study. Patient data was collected using a questionnaire and all patients were tested for HBV, HCV, HIV. RESULTS: Median age was 49 years old and the M: F sex ratio was 2.4. A total of 114 (56.2%) of the patients were HBsAg positive. Hepatitis C antibodies were present in 8.9% of cases, and co-infection with HBV and HCV (HBsAg/anti-HCV) was observed in 4 (2.0%) cases. The remainder, 36.3%, were neither hepatitis B- nor C-related. HIV was detected in 34 cases (18.0%) cases. HIV-HBV or HIV-HCV co-infections were observed in 22 (68.8%) and 2 (6.2%) cases. Overall, positivity for HIV was associated with younger age, and especially in patients with HBsAg+/anti-HCV+. CONCLUSIONS: Our data emphasize the need for a reinforcement of secondary prevention measures in Mozambique. Serological screening for HBV in people born before universal anti-hepatitis B immunization (2001), effective screening, and specific management in HIV(+) patients are urgently needed.

Silica cloaking of adenovirus enhances gene delivery while reducing immunogenicity.

Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the robustness of viral gene expression and the versatility of nanoparticle technology. Here, we describe a method for cloaking adenovirus (Ad) in silica (SiAd) as a nanoparticle formulation that significantly enhances transduction. Intratumoral injections in human glioma xenografts revealed SiAd expressing luciferase improved tumor transduction while reducing liver uptake. In immune-competent mice SiAd induced no inflammatory cytokines and reduced production of neutralizing antibodies. Finally, SiAd expressing TNF-related apoptosis-inducing ligand inhibited tumor growth of glioma xenografts. These results reveal that silica cloaking of Ad can enhance viral gene delivery while reducing immunogenicity.

Optimization of primer design for the detection of variable genomic lesions in cancer.

Primer approximation multiplex PCR (PAMP) is a new experimental protocol for efficiently assaying structural variation in genomes. PAMP is particularly suited to cancer genomes where the precise breakpoints of alterations such as deletions or translocations vary between patients. The design of PCR primer sets for PAMP is challenging because a large number of primer pairs are required to detect alterations in the hundreds of kilobases range that can occur in cancer. These sets of primers must achieve high coverage of the region of interest, while avoiding primer dimers and satisfying the physico-chemical constraints of good PCR primers. We describe a natural formulation of these constraints as a combinatorial optimization problem. We show that the PAMP primer design problem is NP-hard, and design algorithms based on simulated annealing and integer programming, that provide good solutions to this problem in practice. The algorithms are applied to a test region around the known CDKN2A deletion, which show excellent results even in a 1:49 mixture of mutated:wild-type cells. We use these test results to help set design parameters for larger problems. We can achieve near-optimal designs for regions close to 1 Mb.

Network-based classification of breast cancer metastasis.

Mapping the pathways that give rise to metastasis is one of the key challenges of breast cancer research. Recently, several large-scale studies have shed light on this problem through analysis of gene expression profiles to identify markers correlated with metastasis. Here, we apply a protein-network-based approach that identifies markers not as individual genes but as subnetworks extracted from protein interaction databases. The resulting subnetworks provide novel hypotheses for pathways involved in tumor progression. Although genes with known breast cancer mutations are typically not detected through analysis of differential expression, they play a central role in the protein network by interconnecting many differentially expressed genes. We find that the subnetwork markers are more reproducible than individual marker genes selected without network information, and that they achieve higher accuracy in the classification of metastatic versus non-metastatic tumors.

Development of Human-Derived Cell Culture Lines for Recurrent Respiratory Papillomatosis.

Recurrent respiratory papillomatosis (RRP) is mainly caused by human papillomavirus (HPV) 6 and 11. While various adjuvant therapies have been reported, no effective therapy has been documented to universally "cure" this disease. In the era of precision medicine, it would be valuable to identify effective intervention based on drug sensitivity testing and/or molecular analysis. It is essential to be able to successfully carry out in vitro culture and expand tumor cells directly from patients to accomplish this goal. Here we report the result of successful culture of HPV-infected cell lines (success rate 70%, 9/13) that express the E6/E7 RNA transcript, using pathologic tissue biopsies from patients treated at our institution. The availability of such a system would enable ex vivo therapeutic testing and disease modeling.

Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles.

Circulating tumor cells (CTCs) are extremely rare cells found in blood of metastatic cancer patients. There is a need for inexpensive technologies for fast enrichment of CTCs from large blood volumes. Previous data showed that antibody-conjugated lipid shell immuno-microbubbles (MBs) bind and isolate cells from biological fluids by flotation. Here, blood-stable MBs targeted to several surface markers for isolation of breast tumor cells were developed. MBs coated with anti-human EpCAM antibodies showed efficient binding of EpCAM+ breast cancer cell lines SKBR-3, MCF-7, and MDA-MB-453, whereas anti-human EGFR MBs showed binding of EpCAMLOW/NEGATIVE cell lines MDA-MB-231 and BT-549. Multitargeted anti-human EpCAM/EGFR MBs bound all cell lines with over 95% efficiency. Highly concentrated MB-bound tumor cells were collected in a microliter volume via an inverted vacuum-assisted harvesting setup. Using anti-EpCAM and/or anti-EpCAM/EGFR MBs, an efficient (70-90%) recovery and fast (30min) isolation of the above-mentioned cells and cell clusters was achieved from 7.5mL of spiked human blood. Using anti-EpCAM MBs and anti-EpCAM/EGFR MBs, cytokeratin-positive, CD45-negative CTCs were detected in 62.5% (10/16) of patients with metastatic breast cancer and CTC clusters were detected in 41.7% (5/12) of CTC-positive samples. Moreover, in some samples MBs isolated cytokeratin positive, CD45 negative tumor-derived microparticles. None of these structures were detected in blood from non-epithelial malignancies. The fast and inexpensive multitargeted platform for batch isolation of CTCs can promote research and clinical applications involving primary tumors and metastases.

In vivo sampling of extracellular beta-thymosin by ultrafiltration probes.

In vivo detection and monitoring of extracellular beta-thymosin will facilitate the understanding of their biological function and association with disease progression. A novel technique using capillary ultrafiltration (CUF) probes linked to mass spectrometry is capable of sensing extracellular thymosin beta-4 and/or thymosin beta-10 in vivo in wounded skin and other tissue microenvironments. In this review, we highlight the association of extracellular beta-thymosin with skin wound healing and the potential adjuvant effects on vaccination. The fabrication and biological application of CUF probes are also described. Data from CUF probe-captured beta-thymosin may guide future exploration of extracellular beta-thymosin.

Viral discovery and sequence recovery using DNA microarrays.

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.