Decoding the Genetic and Structural Features of HPV16 E5 Oncogene in Cervical Cancer Isolates from Pakistan: A Pilot Study.

Background: Many anogenital cancers are caused by high-risk HPV. The most common subtype is HPV16, which is prevalent in the world, including Pakistan. Various amino acid residues in HPV16 E5 are associated with high cell cycle progression and proliferation. Lack of studies on HPV16E5 in Pakistan prompted the current study. This is the first report on the occurrence of pathogenic E5 variant of HPV16 in tissue sections obtained from invasive cervical cancerous patients in Pakistan. Methods: A subset of 11 samples from HPV-positive biopsies were subjected to E5 gene amplification using PCR and analyzed using bioinformatics programs. The bioinformatics analysis detected mutations causing structural variations, which potentially contribute to the oncogenic properties of proteins. Results: The two-point mutations, C3979A and G4042A, observed in isolate 11 caused the substitution of isoleucine for leucine and valine at positions 44 and 65 in E5 protein. The rest of the isolates had Leu44Val65 amino acids. Intratypic variations and phylogenetic analysis revealed that the majority of the isolates were closely clustered with European-Asian lineage. Moreover, C3979A and G4042A contributed to higher degree of interactions with host receptors, i.e. EGFR. Conclusion: This is the first study reporting HPV16 variants in a Pakistani population based on variations in the E5 region. Our findings indicate that isolate 11 has a strong interaction with the intracellular domain of EGFR, which may enhance the generation of downstream signals. Since this was a pilot study to explore E5 gene mutation, future studies with large samples are absolutely needed.

Molecular Pharming for low and middle income countries.

Interest in applications and benefits that Molecular Pharming might offer to Low and Middle Income Countries has always been a potent driver for the research discipline, and a major reason why many scientists entered the field. Although enthusiasm remains high, the reality is that such a game-changing innovation would always take longer than traditional uptake of new technology in developed countries, and be complicated by external factors beyond technical feasibility. Excitingly, signs of increasing interest by LMICS in Molecular Pharming are now emerging. Here, three case studies from Thailand, South Africa and Brazil are used to identify some of the key issues when a new investment into Molecular Pharming manufacturing capacity is under consideration. At present, academic research is not necessarily addressing these issues. Only by understanding the concerns, can members of the academic community contribute to helping the development of Molecular Pharming for LMICs by focusing their research efforts appropriately.

Apoptosis of Leukemia Cells by Ocimum basilicum Fractions Following TNF alpha Induced Activation of JNK and Caspase 3.

PURPOSE: Leukemia, one of the major cancers, affects a large proportion of people around the world. Better treatment options for leukemia are required due to a large number of side effects associated with current therapeutic regimens. In the present study, we sought to determine the pathway of triggering apoptosis of leukemic cells by Ocimum basilicum (O. basilicum) plant extract. MATERIALS/METHODS: Methanolic extract of the O. basilicum plant material was prepared. The crude extract was fractionated into several fractions through column chromatography using ethyl acetate and n-hexane as eluting solvents. Cell viability of leukemic cells was assessed via Cell titer GLO assay and apoptosis was measured through Annexin V/PI staining. Two apoptotic molecules JNK and caspases were analyzed through western blotting while pro-inflammatory cytokines TNFα, CCL2 and CXCL8 using qPCR. Fractions were characterized through LC-MS. RESULTS: The most potent with lowest IC50 values among the fractions were BF2 (2:8 n-hexane:ethyl acetate) and BF3 (3:7 n-hexane:ethyl acetate). Cytotoxicity was associated with apoptosis. Apoptosis was found caspasedependent and P-JNK activation was detected sustained. A significant increase in the level of TNF α and a decrease in the level of CXCL8 were observed in BF2 and BF3 treated cells. CONCLUSION: The fractions of O. basilicum extract were found to kill cells following JNK pathway activation. Excellent results were obtained with BF2 and BF3 probably due to predominant Epicatechin and Cinnamic acid derivatives in these fractions.

The Role of Serotonin during Skin Healing in Post-Thermal Injury.

Post-burn trauma significantly raises tissue serotonin concentration at the initial stages of injury, which leads us to investigate its possible role in post burn wound healing. Therefore, we planned this study to examine the role of serotonin in wound healing through in vitro and in vivo models of burn injuries. Results from in vitro analysis revealed that serotonin decreased apoptosis and increased cell survival significantly in human fibroblasts and neonatal keratinocytes. Cellular proliferation also increased significantly in both cell types. Moreover, serotonin stimulation significantly accelerated the cell migration, resulting in narrowing of the scratch zone in human neonatal keratinocytes and fibroblasts cultures. Whereas, fluoxetine (a selective serotonin reuptake inhibitor) and ketanserin (serotonin receptor 2A inhibitor) reversed these effects. Scald burn mice model (20% total body surface area) showed that endogenous serotonin improved wound healing process in control group, whereas fluoxetine and ketanserin treatments (disruptors of endogenous serotonin stimulation), resulted in poor reepithelization, bigger wound size and high alpha smooth muscle actin (α-SMA) count. All of these signs refer a prolonged differentiation state, which ultimately exhibits poor wound healing outcomes. Collectively, data showed that the endogenous serotonin pathway contributes to regulating the skin wound healing process. Hence, the results of this study signify the importance of serotonin as a potential therapeutic candidate for enhancing skin healing in burn patients.

Identification of IgE- binding pollen protein from Cannabis sativa in pollen-hypersensitive patients from north Pakistan.

Cannabis sativa (C.sativa) is well-known for its medicinal, industrial and recreational use. However, allergies in relation to Cannabis sativa (C.sativa) are rarely reported. C. sativa is one of the common weeds found in Pakistan and its pollen grains are common in spring and fall season. Although categorized as an aeroallergen, there are limited number of reports regarding allergenic potential in C. sativa. Therefore, the current study is aimed at exploring the IgE- binding potential among the C. sativa pollen in local pollen allergic patients. Initial screening of C. sativa sensitized individuals was carried out by dot blot from the sera of pollen allergic patients. Proteins from the pollen grains were extracted and resolved on 10% gel. Eight bands were visible on gel however only one protein fragment i.e. of 14KDa size was found to bind to IgE as analyzed through protein gel blot analysis. Strong IgE affinity of a 14 kDa protein fragment from C. sativa pollen extract suggests its allergenic potential. Further study is required to find the exact nature of this protein fragment.

Detection of human papillomavirus-16 DNA in archived clinical samples of breast and lung cancer patients from North Pakistan.

PURPOSE: Over the past few decades, human papillomavirus (HPV) has been recorded as a key player in the development of various genital cancers, most notably cervical cancer. It has also been associated with some non-genital cancers. A subset of oropharyngeal cancers are known to be caused by HPV. Its aetiological involvement has been suggested for breast and lung cancer as well. However, reports regarding the HPV DNA detection vary widely from different parts of the world. Due to scarcity of local data in this regard, the current study aimed at retrospective detection of HPV presence in the archival samples of breast and lung cancer patients from north part of the country. METHODS: A total of 55 formalin-fixed paraffin-embedded tissue sections of invasive ductal carcinoma of breast (n = 46) and lung (n = 9) were collected for this study. Genotyping for HPV16 and 18 was carried out through PCR. RESULTS: HPV16 DNA was found in both breast and lung carcinoma samples with the prevalence rate of 17 and 11 %, respectively. An interesting association was found between ER/PR (Oestrogen/Progesterone receptor) and HER2/Neu (Human epidermal growth factor receptor-2) positivity with HPV occurrence in breast tumours. CONCLUSION: Current study shows the presence of HPV16 DNA in archived clinical biopsy sections from breast and lung cancers (17, 11 %), respectively. A positive correlation of HPV16 presence was found with ER/PR and HER2-positive breast cancers. These initial findings warrant further investigation in order to determine HPV prevalence and aetiological role in local cancers, especially in ER/PR/HER2-positive breast cancers on a larger scale.

Retrospective analysis of HPV 16/18-related disease burden using archival clinical samples.

PURPOSE: Estimation of HPV-related disease burden lies at the core of effective disease management. HPV testing is heavily reliant on its retrospective detection in archival clinical cancer samples, especially in parts of the world where HPV screening is not routinely practiced. During the last decade, valuable insights were gained through regional reports based on occasional screening of cervical smears or biopsy sections for the presence of high-risk HPV. HPV 16 and 18 were found to be predominant high-risk HPV subtypes with some regional differences and incidences of co-infections, detected mostly through PCR-based methods. In cases of multiple infections, the presence of viral DNA may not signify its etiologic involvement. The current study, therefore, combines PCR-based detection method with the immunohistochemical (IHC) detection of early viral protein E6 expression, in order to obtain a reliable read out for the disease causing viral subtype, especially in cases of co-infections with oncogenic subtypes other than HPV 16 and 18. Immunohistochemistry (IHC) and PCR-based methods are routinely used laboratory techniques in local hospitals. The concordance between IHC and PCR-based analyses may be useful for determining effective method for the retrospective testing of HPV 16 and 18 disease-related burden. METHODS: A total of 49 paraffin-embedded cervical cancer biopsy sections representing patients from the northwest region of the country were collected from the tertiary care hospital for this study. Genotyping for HPV 16 and 18 was carried out through PCR. The HPV 16/18 E6 protein expression was evaluated by IHC and was compared with the clinicopathological features of cervical cancer. RESULTS: Molecular analysis of 33 (67 %), E6-expressing paraffin-embedded cervical cancer biopsy sections revealed the presence of HPV 16 (n = 23; 47 %), HPV 18 (n = 6; 12 %) and co-infection (n = 4; 8 %) in 49 tumors through PCR. Despite the PCR-based detection of viral DNA in 37 cervical cancer samples, IHC analysis of E6 expression revealed the etiological involvement of HPV 16/18 in 33 out of 37 cervical cancer samples. Overall, there was 85 % concordance in the results of the two techniques. CONCLUSION: IHC analysis provides more conclusive evidence regarding the etiological involvement of the viral subtypes, especially in the presence of multiple infections. About two-thirds (67 %) of cervical cancer samples were found to be caused due to HPV 16/18. Latent occurrence of HPV 16 and 18 is suggested in less than 10 % cervical cancer samples which were found to harbor viral DNA without E6 expression. Furthermore, E6 expression was found to be significantly correlated with the tumor grade.

Clinical role of HER2 gene amplification and chromosome 17: a study on 154 IHC-equivocal cases of invasive breast carcinoma patients.

Accurate evaluation of human epidermal growth factor receptor 2 (HER2) status is quite crucial for invasive breast tumor patients in order to select anti-HER2 therapy for effective clinical outcomes. Immunohistochemistry (IHC) assay is routinely used to evaluate the HER2 oncoprotein overexpression but is unable to explain the chromosomal and genetic alterations and has been considered as a hot issue in IHC-equivocal cases. We investigated these molecular aberrations in correlation with prognostic factors. A cohort of 154 IHC-equivocal (+2) cases was selected and retrospectively analyzed by dual-probe fluorescence in situ hybridization (FISH) assay by using locus-specific HER2 and centromere enumeration probes (CEP17) for the identification of HER2 proto-oncogene amplification and chromosomal copy number per cell, respectively. The data were analyzed by SPSS 16.0 version using chi-square test (p < 0.05). We identified 36 out of 154 cases (23.4 %) showing HER2 gene amplification (average HER2 gene copies per cell >4 or <4 with HER2/CEP17 ratio >2) in concordance with HER2 oncoprotein overexpression, and significant correlation was observed with prognostic parameters including histological type, tumor grade II to III, histology and pathological type, lymphatic invasion, ductal carcinoma in situ (DCIS), and estrogen-positive and progesterone-negative receptors. Of the 154 cases, 18 cases (11.7 %) showed polysomy 17 with CEP17 probe signals per cell ≥3 and 22 cases (14.3 %) presented monosomy 17 (CEP17 probe signals per cell ≤1). Our data indicate that the use of anti-HER2 therapy should not be suggested unless true evaluation of HER2 protein expression is made regarding gene amplification essentially in IHC-ambiguous invasive breast tumors.

Assessment of the activity of directly acting antivirals and other products against different genotypes of hepatitis C virus prevalent in resource-poor countries.

Certain food additives and drugs used for other indications have been shown to inhibit in vitro replication of HCV and have been proposed as cheap options for the treatment of HCV infections in resource-poor countries. We here report that the in vitro anti-HCV (genotypes 1a, 1b, 2a and 4b) activity of nitazoxanide, silymarin, silibinin and the green tea extract EGCG is very weak when compared to directly acting antivirals. HCV-infected patients in resource-poor countries should receive the best possible treatment (if possible via expanded access programs); it is therefore advisable not to plan clinical studies with drugs/compounds with weak anti-HCV activity.

Transcriptional up-regulation of BMP-4 and BMPR-II genes in the peripheral blood of breast cancer patients: A pilot study.

BACKGROUND: Bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta (TGF-ß) super family, which are primarily known for their inherent role in osteogenesis and ontogenesis. Accumulating evidence suggests the regulatory role of BMP-4 in cellular proliferation, apoptosis, differentiation and thus a possible oncogenic role. OBJECTIVE: Variable cellular expression and in vitro functional assays are indicative of the involvement of BMP related signaling in Breast cancer (BC). The differential expression of BMP-4 in the peripheral blood of BC patients may therefore be considered as a potential biomarker. Therefore, this study aimed to evaluate transcriptional expression of BMP-4 and its cognate receptor BMPR-II in the peripheral blood from the BC patients in relation to the healthy individuals. METHODS: The expression pattern of BMP-4 and BMPR-II was analyzed in the blood of breast cancer patients (n = 22) and healthy controls (n = 22) through Semi Quantitative Reverse transcription Polymerase chain reaction. RESULTS: An up-regulated expression of BMP-4 and BMPR-II was observed in the peripheral blood of breast cancer patients especially in the advanced-stage of the disease. Moreover, BMP-4 and BMPR-II expressions were found to be correlated. CONCLUSION: The current preliminary results based on the transcriptional analysis suggest the prospective use of BMP4 as a biomarker, however further validation is required.

Knowledge and awareness about breast cancer and its early symptoms among medical and non-medical students of Southern Punjab, Pakistan.

Breast cancer is the leading cause of morbidity and mortality globally but has an even more significant impact in developing countries. Pakistan has the highest prevalence among Asian countries. A general lack of public awareness regarding the disease often results in late diagnosis and poor treatment outcomes. The literacy rate of the Southern Punjab (Pakistan) is low compared to its Northern part. It is therefore vital that university students and especially medical students develop a sound knowledge about the disease so that they can spread awareness to others who may be less educated. This study therefore considers current knowledge and understanding about the early signs of breast cancer amongst a study group of medical and non-medical university students of the Southern Punjab, Pakistan. A cross-sectional descriptive analysis of the university students was carried out using a self-administered questionnaire to assess their awareness of breast cancer from March to May 2014. A total of 566 students participated in this study, out of which 326 were non-medical and 240 were from a medical discipline. Statistical analysis was carried out using Graph Pad Prism Version 5 with a significance level set at p<0.05. The mean age of the non medical and medical participants was 23 (SD 2.1) and 22 (SD 1.3) years, respectively. Less than 35% students were aware of the early warning signs of the breast cancer development. Knowledge of medical students about risk factors was significantly better than the non medical ones, but on the whole was insufficient. Our study indicated that knowledge regarding breast cancer was generally insufficient amongst the majority of the university students (75% non-medical and 55% medical) of Southern Punjab, Pakistan. This study highlights the need to formulate an awareness campaign and to organize conferences to promote breast cancer awareness among students in this region.

Prevalence of High risk Human Papillomavirus in cervical dysplasia and cancer samples from twin cities in Pakistan.

INTRODUCTION: Human Papilloma Virus (HPV) is small DNA virus mostly infecting mucosa and cutaneous keratinocytes. So far, more than 200 Human papillomaviruses are known. HPV have been divided into high- and low-risk on the basis of their oncogenic potential. High risk HPV is considered to be the main etiological cause for cervical cancer. OBJECTIVE: The current study was designed to screen the local cervical cancer patients from the twin cities of Pakistan for the occurance of high risk HPV. METHODOLOGY: A total of 67 formalin fixed paraffin-embedded samples of cervical cancer biopsies were obtained from the government hospitals in Islamabad and Rawalpindi. Cervical cancer biopsies were examined for the presence of HPV DNA. Polymerase chain reaction (PCR) was used for the amplification of a region in the HPV-L1 gene for the general detection of the Papilloma virus and for the genotype specific detection of high risk HPV 16 and 18 using the GP5/GP6 primers and genotype specific primers, respectively. RESULTS: HPV DNA was detected in 59 out of 67 samples analyzed. 30 samples showed the presence of HPV16 while 22 samples were positive for HPV18. HPV subtype could not be determined in 7 samples. CONCLUSION: Our results show a strong association between HPV infection and cervical cancer among women in twin cities of Pakistan. One way to minimize the disease burden in relation to HPV infection in Pakistani population is the use of prophylactic vaccines and routine screening. An early diagnosis of HPV infection will allow better health management to reduce the risk of developing cervical cancer.

Indoleamine 2,3-dioxygenase expression and activity in patients with hepatitis C virus-induced liver cirrhosis.

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. It plays a key role in various malignancies, infection and autoimmune diseases. IDO induces immunosuppression through the depletion of tryptophan and its downstream metabolites. Hepatitis C virus (HCV) has infected more than 12 million individuals in Pakistan. The aim of the present study was to assess the expression and activity of IDO in HCV-infected patients. The functional enzymatic activity of IDO was measured by colorimetric assay. Serum samples from 100 HCV-infected patients were taken to examine IDO activity and samples from 100 healthy volunteers were used as controls. Liver sections from patients with HCV (n=35) and healthy controls (n=5) were used for immunohistochemical studies. Immunohistochemical analysis revealed that IDO was overexpressed in 28 of 35 (80%) cirrhotic liver samples, whereas 5 of 35 (14.2%) cases presented moderate and 2 of 35 (5.7%) cases presented mild expression of IDO. The enzymatic activity of IDO was significantly higher in the serum samples of HCV-infected patients as compared with those in the control. These data indicate that the expression of IDO correlated with the pathogenesis of disease. In summary, it is suggested that the high expression of IDO in the progressively cirrhotic livers of HCV-infected patients might contribute to the development of hepatocellular carcinoma. IDO may characterize a novel therapeutic target against HCV.

Roles for human papillomavirus type 16 l1 cysteine residues 161, 229, and 379 in genome encapsidation and capsid stability.

Human papillomavirus (HPV) capsids are formed through a network of inter- and intra-pentameric hydrophobic interactions and disulfide bonds. 72 pentamers of the major capsid protein, L1, and an unknown amount of the minor capsid protein, L2, form the structure of the capsid. There are 12 conserved L1 cysteine residues in HPV16. While C175, C185, and C428 have been implicated in the formation of a critical inter-pentameric disulfide bond, no structural or functional roles have been firmly attributed to any of the other conserved cysteine residues. Here, we show that substitution of cysteine residues C161, C229, and C379 for serine hinders the accumulation of endonuclease-resistant genomes as virions mature within stratifying and differentiating human epithelial tissue. C229S mutant virions form, but are non-infectious. These studies add detail to the differentiation-dependent assembly and maturation that occur during the HPV16 life cycle in human tissue.

Significant correlation of TLR4 expression with the clinicopathological features of invasive ductal carcinoma of the breast.

Toll-like receptor 4 belongs to the family of pattern recognition receptors and is therefore an important part of innate immunity. Since the last decade, Toll-like receptors (TLRs) have gained much interest in cancer research due to their role in tumor progression. However, there is scarcity of data evaluating TLR4 expression in different cell populations of tumor in relation to clinicopathological parameters of invasive ductal carcinoma of the breast. Therefore, we investigated TLR4 expression in 50 primary invasive ductal carcinoma and 17 lymph node sections through immunohistochemistry. Significant association of TLR4 expression with local metastasis and absence of necrosis was observed. The incidence of lymph node metastasis was higher in patients with high TLR4 expression in the tumor cells. We also observed an interesting inverse relationship of TLR4 with necrosis. In endothelial and lymphocytic population constituting breast tumors, a significant association of TLR4 expression was seen with the tumor grade. Therefore, we conclude that TLR4 expression is of biological interest both as a prognostic marker as well as a possible therapeutic target.