Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

Use of multiple PCR primer sets for optimal detection of human papillomavirus.

Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/Inex at Telelab (Skien, Norway) and group Ineg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp(5+)-Gp6+ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp+, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp+, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.     

Correlation of optic nerve sheath diameter measurements by computed tomography and magnetic resonance imaging

BackgroundTraditionally, intracranial pressure is measured by direct ventriculostomy, which is invasive. Noninvasive measures such as bedside ultrasound and magnetic resonance imaging have been advocated and utilized recently to assess the intracranial pressure. The role of this study is to determine the degree of agreement between measurements of the optic nerve sheath diameter by computed tomography (CT) and magnetic resonance imaging (MRI).Materials and MethodsRetrospective chart review of 100 consecutive patients who had both MRI and CT scan of the head from January 1, 2011, until March 31, 2013, at our center was performed. A discrepancy of 0.2 mm between the 2 measurements was set as acceptable difference. The measurements of optic nerve sheath diameter (ONSD) were compared for agreement between the 2 modalities using the method by Bland and Altman.ResultsA total of 100 patients with both MRI and CT scan of the head were selected. Of these 100 patients, 24 were male and 76 were female. The average age was 63 years. No ONSD abnormality was detected in any of the patients. The discrepancy in measurements of the ONSD between CT and MRI in transverse plane was less than the predetermined cut-off value of 0.2 mm. Within-subject variance was estimated at 0.0058 for both CT and MRI.ConclusionComparable results without significant discrepancy as predetermined by the study groups were obtained from CT scan. Measurement of ONSD by CT scan can be used to indirectly asses the intracranial pressure in addition to clinical assessment and other signs of increased intracranial pressure on CT scan.     

Real time optic nerve sheath diameter measurement during lumbar puncture

Measurement of optic nerve sheath diameter (ONSD) using point of care ultrasound has been used to indirectly assess the intracranial pressure (ICP) particularly in conditions where it is raised. Direct pressure measurements using probes reaching the ventricle system correlated with ONSD using ultrasound. Attempts were made to measure the ONSD pre and post lumbar puncture (LP) after draining cerebrospinal fluid (CSF) as well as post ventricular shunt placement. We report ONSD measurement and demonstrate dynamic changes during LP in a patient with known idiopathic intracranial hypertension (IIH).     

DCLK1, a promising colorectal cancer stem cell marker,regulates tumor progression and invasion through miR-137 and miR-15a dependent manner

Cancer stem cells (CSCs) are thought to be a major player in tumor initiation, progression, and metastasis. Targeting CSCs for elimination presents a promising therapeutic strategy; however, this approach will require a stronger understanding of CSC biology and identification of CSC-specific markers. The present study was conducted to examine the correlation between DCLK1 and miR-137 and miR-15a levels in colorectal cancer. A total of 222 samples, including 181 colorectal cancer specimens, 24 adenomatosis, and 17 non-adenomatosis colonic polyps, were stained for DCLK1 expression using immunohistochemistry. Also, expression of miR-137 and miR-15a was assessed in colorectal cancer with high and low DCLK1 expression levels. Most colorectal cancer specimens (76%) showed strong expression of DCLK1, whereas only 21% of adenomatous and none of non-adenomatous colonic polyps showed strong DCLK1 expression. A significant difference in DCLK1 expression was found between colorectal cancer, adenomatous, and non-adenomatous colonic polyps (P < 0.001). Higher expression of DCLK1 was more frequently detected in colorectal cases with larger tumor size (P = 0.03), poor differentiation (P = 0.03), and lymph node involvement (P = 0.04). Comparison of miR-137 and miR-15a in colorectal cancer cases revealed a significant inverse correlation with DCLK1 expression (P = 0.03 and P = 0.04, respectively). DCLK1 may act as a candidate marker for colorectal cancer stem cells. The critical role of DCLK1 in colorectal cancer suggests that it may represent an early diagnostic marker and therapeutic target; however, further investigation is warranted.

     

Ultrasound Modulates the Splenic Neuroimmune Axis in Attenuating AKI

We showed previously that prior exposure to a modified ultrasound regimen prevents kidney ischemia-reperfusion injury (IRI) likely via the splenic cholinergic anti-inflammatory pathway (CAP) and α7 nicotinic acetylcholine receptors (α7nAChR). However, it is unclear how ultrasound stimulates the splenic CAP. Further investigating the role of the spleen in ischemic injury, we found that prior splenectomy (–7d) or chemical sympathectomy of the spleen with 6-hydroxydopamine (6OHDA; –14d) exacerbated injury after subthreshold (24-minute ischemia) IRI. 6-OHDA–induced splenic denervation also prevented ultrasound-induced protection of kidneys from moderate (26-minute ischemia) IRI. Ultrasound-induced protection required hematopoietic but not parenchymal α7nAChRs, as shown by experiments in bone marrow chimeras generated with wild-type and α7nAChR^–/– mice. Ultrasound protection was associated with reduced expression of circulating and kidney-derived cytokines. However, splenocytes isolated from mice 24 hours after ultrasound treatment released more IL-6 ex vivo in response to LPS than splenocytes from sham mice. Adoptive transfer of splenocytes from ultrasound-treated (but not sham) mice to naïve mice was sufficient to protect kidneys of recipient mice from IRI. Ultrasound treatment 24 hours before cecal ligation puncture–induced sepsis was effective in reducing plasma creatinine in this model of AKI. Thus, splenocytes of ultrasound-treated mice are capable of modulating IRI in vivo, supporting our ongoing hypothesis that a modified ultrasound regimen has therapeutic potential for AKI and other inflammatory conditions.     

Tolerogenic dendritic cells produced by lentiviral‐mediated CD40‐ and interleukin‐23p19‐specific shRNA can ameliorate experimental autoimmune encephalomyelitis by suppressing T helper type 17 cells

Down‐regulation of soluble or membrane‐bound co‐stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow‐derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV‐DCs) and/or p19 subunit of interleukin (IL)‐23 (p19LV‐DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In‐vitro studies showed that double‐transduced BMDCs (CD40⁺p19LV‐DCs) resemble tolerogenic DCs due to profound down‐regulation of CD40, lower expression of proinflammatory cytokines (IL‐6 and IL‐12), increased IL‐10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)_35–55‐specific T cells for production of IL‐10 compared with CD40LV‐DCs, p19LV‐DCs and BMDCs transduced with control lentiviral vector (CoLV‐DCs). Moreover, injection of transduced CD40⁺p19LV‐ BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL‐17 or increased production of IL‐10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV‐DCs‐treated EAE mice. In conclusion, simultaneous knock‐down of CD40 and IL‐23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL‐17‐dependent autoimmune diseases, including multiple sclerosis.