Skin mediated human papillomavirus infection in breast: A report of four cases

To address the ambiguity of different modes of human papillomavirus (HPV) transmission in breast, the immunohistochemical expression of two oncoproteins E6/E7 of HPV16 was analyzed in primary breast cancer (BC) and adjacent normal skin of 4 samples. The patients were of 35–55 years old having no previous history of cancer. The E6/E7 expressions were evident in both skin and BC. In skin, high/moderate cytoplasmic expressions of E6/E7 proteins were seen in all samples, whereas in BC, high/moderate cytoplasmic expressions of the proteins were observed in 2–3 samples. Thus, it seems that HPV infection in the breast may occur through the skin

Inhibition of Autotaxin Ameliorates LPA-Mediated Neuroinflammation and Alleviates Neurological Dysfunction in Acute Hepatic Encephalopathy

Growing evidence suggests an essential role of neuroinflammation in behavioral abnormalities associated with hepatic encephalopathy (HE). Here, we report the involvement of autotaxin–lysophosphatidic acid (LPA) signaling in HE’s pathogenesis. We demonstrate that the autotaxin (ATX) inhibitor PF-8380 attenuates neuroinflammation and improves neurological dysfunction in the mouse model of HE. In the thioacetamide (TAA)-induced model of HE, we found a twofold increase in the levels of ammonia in the brain and in plasma along with a significant change in HE-related behavioral parameters. Mice with HE show an increased brain weight, increased levels of tumor necrosis factor-α (TNF-α), IL-1β (interleukin-1β), interleukin-6 (IL-6), and LPA 18:0 in the cerebral cortex and hippocampus, and increased levels of LPA 18:0 in plasma. Treatment with the autotaxin inhibitor (ATXi) did not affect liver injury, as we observed no change in liver function markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) and no change in ammonia levels in the brain and plasma. However, ATXi treatment significantly ameliorated the neuroinflammation, reduced the levels of LPA 18:0 in the cerebral cortex and hippocampus in the brain and plasma, and reduced brain edema and the levels of IL1β, IL-6, and TNF-α. The neurobehavioral symptoms for HE such as the cognitive and motor function deficit and overall clinical grading score were significantly improved in ATXi-treated mice. Mouse astrocytes and microglia stimulated with NH4CL with or without ATXi showed significant attenuation of oxidative stress and the neuroinflammatory effect of NH4CL in ATXi-treated cells

Study of association and molecular analysis of human papillomavirus in breast cancer of Indian patients: Clinical and prognostic implication

<div><p>Objectives</p><p>Human papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile.</p><p>Methods</p><p>HPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done.</p><p>Results</p><p>High prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20–45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants.</p><p>Conclusion</p><p>Thus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.</p></div

Comparative HPV infection in adjacent normal breast, fibro adenomas, benign phyllode and breast tumors.

<p>Comparative HPV infection in adjacent normal breast, fibro adenomas, benign phyllode and breast tumors.</p

Analysis of HPV16 physical status in breast tumor samples and MCF7 breast cancer cell line.

<p>Representative agarose gel of physical status of HPV16 genome at <b>(a)</b> E2A region. <b>(b)</b> E2B region and <b>(c)</b> E2C region <b>(d)</b> Histogram represent significant high frequency of integrated viral genome in both pre-therapeutic and NACT samples (p≤0.01). Frequency of integration at three regions of the E2 gene in <b>(e)</b> pre-therapeutic cases and <b>(f)</b> NACT samples. [Here M: 100bp marker, NC represent Negative control with no DNA, +Ve represent Episomal control where HPV16 plasmid was used. SiHa is the HPV16 positive Cervical cancer cell line used as Integration control for E2A and E2B region while Episomal control for E2C region]</p

Determination of HPV prevalence in primary breast tumors and breast cancer cell line MCF7.

<p><b>(a)</b> Representative agarose gel showing positive HPV infection in pre-therapeutic sample 2766T and MCF7 cell line while, neo-adjuvant chemotherapy treated (NACT)sample 4242T is HPV negative. +Ve means Positive control having HPV16 plasmid. <b>(b)</b> Frequency of HPV infection in BC samples. Samples were identified as positive when PCR bands were seen with respective subtype specific primers. Representative agarose gel of <b>(c)</b> HPV16 <b>(d)</b> HPV18 <b>(e)</b> HPV33 detection PCR. [M: 100bp marker, NC represent Negative control with no DNA, +Ve represent Positive control having HPV16, HPV18 and HPV33 plasmid in their respective subtype detection gels] <b>(f)</b> Distribution of different genotypes of HPV in different type of BC samples.</p

Kaplan–Meier 5-year survival probability curves with cumulative survival of breast cancer patients (BC) based on hrHPV status.

<p>Survival probability of BC patients in <b>(a)</b> pre therapy group (p = 0.04) and <b>(b)</b> neo-adjuvant chemotherapy treated (NACT) group (p = 0.13). [N: total number of samples included in this study. hrHPV(+) indicates infection by either HPV16, HPV18 or HPV33].</p

Sequence variation of LCR, E6 and E7 region and methylation status of LCR of HPV16 in pre-therapeutic breast tumor cases.

<p>Schematic representation of identified sequence variations in <b>(a)</b> the LCR region and <b>(b)</b> the E6 and E7 gene. Here, bold lettered variants were novel ones. Upper head arrow thickness indicate the frequency of the given variation where “N” represent number of samples. In LCR, lower head arrows indicates binding of the transcription factors while in E6 and E7 gene it highlighted different protein domains. Methylation sites in the LCR were also marked (Bold triangle). ‘*’ indicates transcription factors predicted by Alibaba 2.1 TF Binding Prediction software. Representative agarose gel showing methylation status at <b>(c)</b> the enhancer region <b>(d)</b> the P97 promoter region. <b>(e)</b> Histogram showed significant high frequency methylation in P97 promoter than enhancer region (p = 0.004). [M: 1000bp marker, NC represent Negative control with no DNA, K1 used as digestion control, K2 used as DNA integrity control, Mock: mock digestion without enzyme, HhaI: DNA digested with HhaI enzyme, HapII: DNA digested with HpaII enzyme].</p

Schemetric diagram represent summerised results of the current study.

<p>Here, upper head arrow indicates high, lower head arrow indicates low. PT represent pre-therapeutic BC. gDNA; Genomic DNA of patients.</p

Detection and quantification of E6 and E7 mRNA of HPV16 in pre-therapeutic breast tumor samples and MCF7 breast cancer cell line.

<p><b>(a)</b> The dot plot showed wide variation of dCt value of E6, E7 and full length E6/E7 mRNA expression. High dCt value indicates low expression and vice versa. Here, SiHa cell line was used as positive control for E6 and E7 expression. Bold horizontal line represent average dCt value. <b>(b)</b> Representative agarose gel of post real time PCR of E6 showed different splice forms of E6. <b>(c)</b> Agarose gel represents post real time PCR of E7. <b>(d)</b> Representative agarose gel of post real time PCR showed full lenth E6/E7 mRNA with its dfferent splice forms. Bold lettered represent novel splice form. <b>(e)</b> Representative gel showed post real time PCR of β2-microglobulin used as endogenous control gene. <b>(f)</b> Schematic diagram of different splice forms of E6 and E6-E7 transcripts. Bold line represent exon and V- shaped thin line represents intron. Nucleotide position in HPV16 genome was also indicated. Number of left and right arm represent splicing donar and acceptor sites respectively. [Here M: 1000bp marker; NC represent Negative control with no cDNA].</p