A regulatory approach on serum bilirubin level in subjects with impaired glucose tolerance.

The present study was undertaken to find the role of dietary intervention and physical exercise on serum bilirubin level in IGT subjects. Thirty three newly detected otherwise healthy subjects with IGT, aged 35-63 years, were randomly selected to participate in a 12 weeks diet and exercise program. Nine participants were within 35-40 years while majority fifteen participants aged 41-50 years and rest six participants were above 50 (51-63) years. A male preponderance was observed among the study participants where 53.3% of the total participants were male (n=16) and 46.7% were female (n=14). Mean bilirubin (mg/dl) level was recorded 0.68 ± 0.29 at base line and with follow-up, the value was 0.66 ± 0.26 mg/dl. For men (n=16), serum bilirubin were 0.77 ± 0.39 and 0.75 ± 0.36 mg/dl at base line and follow-up while for women (n=14), the values were 0.67 ± 0.33 and 0.59 ± 0.28 mg respectively. The 35-40 years group (n=9) showed bilirubin from 0.66 ± 0.23 at base line to 0.73 ± 0.19 mg/dl at follow-up while 41-50 years group (n=15) had 0.70 ± 0.34 and 0.58 ± 0.26 mg/dl and for 51-63 years group (n=6), the values were 0.65 ± 0.29 and 0.73 ± 0.33 mg/dl respectively. Participants with BMI 20-25 had bilirubin 0.62 ± 0.29 mg/dl at base line and 0.71 ± 0.21 mg/dl at follow-up while with BMI >25 (n=20) had 0.71 ± 0.30 and 0.63 ± 0.2 8 mg/dl respectively. No significant changes in serum bilirubin were observed among the groups and therefore, the dietary intervention and physical exercise during the period did not have a significant role in this respect.

Antibacterial Effect of Curcuma longa (Turmeric) Against Staphylococcus aureus and Escherichia coli.

This observational study was conducted during the period from July 2010 to June 2011 in the Department of Pharmacology in the collaboration of Department of Microbiology, Mymensingh Medical College, Mymensingh to determine the profile of antibacterial effect of Crude Turmeric paste aqueous turmeric extract, and standard antibiotic Amikacin against Staphylococcus aureus and Escherichia coli. Three separate experiments were done e.g. (Expt- I) Inhibitory effect of Crude Turmeric paste incorporated into nutrient agar (NA) media, (Expt- II) Minimum inhibitory concentration of (a) Aqueous Turmeric extract and (b) Amikacin by broth dilution technique and (Expt-III) their subculture study in nutrient agar (NA) media for confirmation of respective results of previous experiments. Inhibitory effects were observed against the growth of Staph Aureus and Esch coli at 10% and 30% respectively of Crude Turmeric paste incorporated into NA media. The broth dilution technique was followed to determine the MIC of Aqueous Turmeric extract and Amikacin. The MIC of Aqueous Turmeric extract was 800 µg/ml against Staph aureus and that against Esch coli was 2000 µg/ml and the MIC of Amikacin was 10 µg/ml for both the bacteria. The MIC of Amikacin was the lowest in comparison to MIC of Aqueous Turmeric extract for complete inhibition of growth of Staph aureus and Esch coli. The subculture study showed similar results with that of previous experiments in terms of inhibitory effects of Crude Turmeric paste and MIC of Aqueous Turmeric extract and Amikacin against all of the organisms studied.

Intermittent prophylaxis of recurrent febrile seizures with clobazam versus diazepam.

Febrile seizures are the most common type of seizure among children that can be prevented by using prophylactic drugs like Clobazam and Diazepam. The present prospective study was conducted in the Department of Pediatrics, Mymensingh Medical College Hospital and Community Based Medical College Hospital, Bangladesh over a period of 1 year from July 2012 to June 2013 to compare the effectiveness of intermittent Clobazam versus Diazepam therapy in preventing the recurrence of febrile seizures and assessed adverse effects of each drug. A total of 65 patients (32 children administered Clobazam and rest 33 children received Diazepam) of simple and complex febrile seizures aged 6 months to 5 years of both sexes were the study population. Data were collected by interview of the patients, clinical examination and laboratory investigations using the research instrument. Data were analyzed by using Chi-square (χ2) Test, Student's 't' Test and Fisher's Exact Test. For all analytical tests, the level of significance was set at 0.05 and p<0.05 was considered significant. The proportion of patients was higher between age 12-36 months and male was predominant in the both Clobazam and Diazepam groups. Over 31% of patients in Clobazam group who experienced episode of fever within 3 months, 40.6% within 6 months and 9.4% within 9 months compared to 36.4% in Diazepam group within 3 months, 45.5% within 6 months & 12.1% within 9 months after discharge from the hospital. Three (9.4%) patients in Clobazam group and 7(21.3%) in Diazepam group who experienced febrile convulsion during the follow up period. From the data adverse effects within 3 and 6 months experienced by the patient's drowsiness, sedation and ataxia were higher in Diazepam group than those in Clobazam group. However, within 9 months lethargy and irritability were somewhat higher in Clobazam group than those in Diazepam group. The mean duration of hospitalization was significantly higher in Diazepam group compared to Clobazam group (6.0±1.0 vs. 4.6±0.08 days, P<0.001). Seven (21.2%) out of 33 children with febrile seizures in Diazepam group had a history of recurrent seizures, whereas 3(9.4%) of 32 children in the Clobazam group. The risks of recurrent febrile seizure in the Diazepam group was 2.6 times greater compared to those in the Clobazam group (P=0.186). The result indicates that Clobazam is safe, efficacious, requires less frequent dosing and has less adverse effects such as drowsiness, sedation, ataxia and irritability as compared to Diazepam. So, Clobazam may be an alternative to Diazepam given intermittently for prevention of recurrent febrile seizures.

Current Trends of Using Antimicrobials in Acute Watery Diarrhoea in Children Below 5 Years of Age at Paediatrics Outpatient Department of a Tertiary Care Hospital in Bangladesh.

Childhood diarrhoea is a major public health problem in developing countries like Bangladesh which is commonly caused by intestinal infection, mainly viral. Diarrhoea is causing second leading mortality in children below 5 years, where about 2 billion cases occur globally in each year. This study was proposed to evaluate the antimicrobials utilization pattern prescribed for AWD in children below 5 years of age, in a tertiary care hospital in Bangladesh. This record based, cross-sectional, descriptive type of observational study carried out at pharmacology department of Mymensingh Medical College. A total of 205 diarrhoeal patient's up to 5 years of age, attending the paediatric OPD from January 2021 to December 2021, were enrolled in the study. Out of 205 patients 182(88.8%) were prescribed antimicrobials. Azithromycin was the most frequently prescribed antimicrobial (60.0%) and among the antiprotozoals metronidazole was the prescribed most (24.9%). ORS and Zinc were prescribed in all patients (100%). Empirical excessive use of antibiotics was observed in this study. Emphasis on educational and training programs may help in a better and judicious use of drugs in children.

Antibacterial Activities of Methanolic Seeds Extract of Black pepper (Piper nigrum L.) against Gram Positive Staphylococcus aureus & Gram-Negative Escherichia coli.

Evaluation of the in vitro antibacterial activity of Methanolic extracts isolated from Black pepper seeds (Piper nigrum L.) against two infection causing pathogens, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Between July 2022 and June 2023, this experimental study was conducted at the Mymensingh Medical College's Department of Pharmacology and Therapeutics in conjunction with the Department of Microbiology. Using the disc diffusion and broth dilution methods, the antibacterial activity of methanolic extract of black pepper seeds (MBPE) was evaluated at various doses. The solvents Methanol and 10.0% Di Methyl Sulfoxide (DMSO) were used to make the extract. Using the broth dilution procedure, the conventional antibiotic Ciprofloxacin was utilized and the outcome was contrasted with that of Methanol extracts. Methanolic extract of black pepper seeds (MBPE) at seven distinct concentrations (100, 80, 60, 40, 20, 10 and 5 mg/ml) were utilized, then later in chosen concentrations as needed to confirm the extracts' more precise margin of antimicrobial sensitivity. At 80 mg/ml and above doses of the MBPE, it had an inhibitory impact against the aforementioned microorganisms. For Staphylococcus aureus and Escherichia coli the MIC were 60 and 75 mg/ml in MBPE respectively. As of the MIC of Ciprofloxacin was 1µg/ml against Staphylococcus aureus and Escherichia coli. In comparison to MICs of MBPE for the test organisms, the MIC of Ciprofloxacin was the lowest. This study clearly shows that Staphylococcus aureus and Escherichia coli are sensitive to the methanolic extract of black pepper seeds' antibacterial properties.

Anti-angiogenic therapy increases intratumoral adenovirus distribution by inducing collagen degradation.

Conditionally replicating adenoviruses (CRAd) are a promising class of gene therapy agents that can overcome already known glioblastoma (GBM) resistance mechanisms but have limited distribution upon direct intratumoral (i.t.) injection. Collagen bundles in the extracellular matrix (ECM) have an important role in inhibiting virus distribution. In fact, ECM pre-treatment with collagenases improves virus distributions to tumor cells. Matrix metalloproteinases (MMPs) are an endogenous class of collagenases secreted by tumor cells whose function can be altered by different drugs including anti-angiogenic agents, such as bevacizumab. In this study we hypothesized that upregulation of MMP activity during anti-angiogenic therapy can improve CRAd-S-pk7 distribution in GBM. We find that MMP-2 activity in human U251 GBM xenografts increases (*P=0.03) and collagen IV content decreases (*P=0.01) during vascular endothelial growth factor (VEGF-A) antibody neutralization. After proving that collagen IV inhibits CRAd-S-pk7 distribution in U251 xenografts (Spearman rho=-0.38; **P=0.003), we show that VEGF-blocking antibody treatment followed by CRAd-S-pk7 i.t. injection reduces U251 tumor growth more than each individual agent alone (***P<0.0001). Our data propose a novel approach to improve virus distribution in tumors by relying on the early effects of anti-angiogenic therapy.

Antibacterial Effects of Henna (Lawsonia inermis) Leaf Extracts (Chloroform) Against Two Food Borne and Nosocomial Infection Causing Pathogens: Staphylococcus aureus and Escherichia coli.

Evaluation of the in vitro antibacterial activity of Chloroform extracts isolated from Henna (Lawsonia inermis) leaf against two food born & nosocomial infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Escherichia coli. This interventional study was carried out during the period of January 2021 to December 2021 in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. The antibacterial activity was tested at different concentrations of Chloroform Henna leaf extracts by using disc diffusion and broth dilution method. The extract was prepared by using solvents chloroform and 0.1% DMSO. The test microorganisms were also tested for their activity against a standard antibiotic Ciprofloxacin by broth dilution method and the result was compared with that of Chloroform extracts. Chloroform Henna Extracts (CHE) were used initially in nine different concentrations (2.5, 5, 10, 20, 50, 100, 200, 500 and 1000mg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the CHE, 100mg/ml and above concentrations showed inhibitory effect against Staphylococcus aureus and 300mg/ml and above concentrations showed inhibitory effect against Escherichia coli. The MIC for Staphylococcus aureus and Escherichia coli were 100 and 350mg/ml in CHE respectively. The MIC of Ciprofloxacin was 1µg/ml against both Staphylococcus aureus and Escherichia coli. The MIC of Ciprofloxacin was the lowest in comparison to MICs of CHE for the test organisms. The present study showed that Chloroform Henna extracts demonstrated antibacterial effects against food borne pathogens. From this study, it is clearly observed that there is definite antibacterial effect of the chloroform extract of Henna leaves against Staphylococcus aureus and Escherichia coli.

Pattern of Prescribing Drugs in Chronic Obstructive Pulmonary Disease in a Tertiary Care Hospital of Bangladesh.

Chronic obstructive pulmonary disease (COPD) is a chronic obstructive disease of the airways. It is one of the most common and important chronic respiratory conditions in terms of years lived with disability. Incidence is increasing in Bangladesh like other developing countries. To evaluate drug prescription pattern for COPD, this cross-sectional, observational study was conducted from January to December in 2020 at the Department of Pharmacology in collaboration with the Department of Medicine in Mymensingh Medical College, Bangladesh. A total of 168 patients were selected for the study by non-random purposive sampling technique. Age distribution indicates that 31.5% of patients were in the 50-59 years age group and males were 93.5%. The majority (82.1%) of the participants were smokers. In this study, majority (34.12%) of the drugs were used as oral form and second most common dosage form was nebulization (26.75%). Bronchodilators were mostly prescribed 652(57.19%), followed by corticosteroids 222(19.47%) and antibiotics 165(14.47%) among drugs used for COPD. Beta sympathomimetics 322(45.49%) were mostly prescribed, followed by anticholinergics 186(28.52%) and methylxanthines 144(22.08%) as bronchodilators. Out of 1140 drugs for COPD, 53.06% and 34.12% were delivered as inhalation and oral forms, respectively. Inhalation route was the most (60.37%) preferred one over oral route (37.63%) for steroid use. The most of the patients [152 (90.48%)] were treated with combination therapy. Mostly (39.6%) used Fixed Dose Combination (FDC) therapy was salbutamol and ipratropium bromide followed by salmeteroal and Fluticasone (30.83%). Both FDC were prescribed in 57.7% of study population. Considering nomenclature, trade name was used in 24.4% of prescription.

Antibacterial Activities of Mint (Mentha piperita) Leaf Extracts (Aqueous) Against Two Food Borne infection causing pathogens: Staphylococcus aureus and Escherichia coli.

Evaluation of the in vitro antibacterial activity of Aqueous extracts isolated from Mint (Mentha piperita) leaf against two food born infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Escherichia coli. This interventional study was carried out in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Bangladesh from January 2021 to December 2021. The antibacterial activity was tested at different concentrations of Aqueous Mint leaf extracts by using disc diffusion & broth dilution method. The extract was prepared by using solvents Aqueous. The test microorganisms were also tested for their activity against a standard antibiotic Gentamicin by broth dilution method and the result was compared with that of Aqueous extracts. Aqueous extract of Mint leaves (AMLE) were used initially in eight different concentrations (25, 50, 100, 200, 400, 600, 800 and 1000µg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the AMLE, 200µg/ml and above concentrations showed inhibitory effect against Staphylococcus aureus and 400µg/ml and above concentrations showed inhibitory effect against Escherichia coli. Minimum inhibitory concentration (MIC) for Staphylococcus aureus and Escherichia coli were 200 and 400µg/ml in AMLE respectively. The MIC of Gentamicin was 1µg/ml against Staphylococcus aureus and 1.5µg/ml against Escherichia coli. The MIC of Gentamicin was the lowest in comparison to MICs of AMLE for the test organisms. This study showed that Aqueous Mint extracts demonstrated antibacterial effects against food borne pathogens. It is clearly observed that there is definite antibacterial effect of the aqueous extract of Mint leaves against Staphylococcus aureus and Escherichia coli.

Maxillary sinusitis and fungal infection.

This cross sectional study was done to identify the fungal etiology of maxillary sinusitis. This study was done in the department of Otolaryngology & Head-Neck Surgery, Shahid Suhrawardy Medical College Hospital, Dhaka, Bangladesh. The study period was 5 years (January 2003 to December 2007). Total 63 patients who were diagnosed clinically and radiologically as a maxillary sinusitis were enrolled in this study. All the patients were included randomly. Among 63 patients 8(12.69%) patients had laboratory proved fungal maxillary sinusitis. Collection of the laboratory specimen was done from antral wash out and biopsy for histopathology was taken by endoscopic surgery. By histopathological and fungal stain revealed noninvasive type of fungal infection in all cases. Post nasal drip (100%), Headache (100%), Nasal obstruction (65% cases) were the main presenting symptoms in case of fungal maxillary sinusitis. Anti fungal treatment along with systemic antibiotic was given in case of proven maxillary sinusitis and 100% cure rate was observed after treatment.

Comparison between the effects of alcohol and hexane extract of spirulina in arsenic removal from isolated tissues.

Chronic arsenic poisoning (arsenicosis) is a major public health problem in Bangladesh. People are consuming high concentration of arsenic (>10 ppb) through their drinking water. But still now, there is no specific treatment of it. Spirulina, natural bluish-green microalgae, is found to be effective in the treatment of arsenicosis recently. Keeping this fact in mind the present study was conducted in the Department of Pharmacology, BSMMU to compare the effectiveness of alcohol & Hexane extract of Spirulina in arsenic removal from isolated tissues (rat liver). The experiment was performed in two phases-in phase I, liver tissues incubated with arsenic at 37 degree centigrade at different incubation period & accumulation of arsenic was measured. In phase II, arsenic-loaded liver tissues were incubated at 37 degree centigrade in presence and absence of alcohol extract & hexane extract of spirulina. Arsenic removal (%) from liver tissues by alcohol extract and hexane extract of spirulina was estimated by Atomic Absorption Spectrophotometer. This study showed that there is time dependent accumulation of arsenic in isolated liver tissue and highest accumulation found was 0.69 microg/g tissues after 45 minutes incubation, which was highly significant. Removal of arsenic (%) from arsenic loaded liver tissues by alcohol extract & hexane extracts were 33.8% & 83.0% respectively. Between the two extracts of spirulina the hexane extract causes more percentage removal of arsenic which is highly significant (p<0.001). So, the present study suggests hexane extract of spirulina is more effective in removal of arsenic from rat liver tissues than alcohol extract.

Demography and findings of reported rape cases.

Six hundred and ninety nine cases of alleged rape were studied by the authors during the period from 2007-2008 at the Mymensingh Medical College, Mymensingh. Of these cases, 122 had positive findings of recent sexual intercourse; 250 cases had the positive findings of habituated sexual intercourse, and 327 cases had no findings of sexual intercourse but they complained of forcible sexual intercourse and found no sign of sexual intercourse. Most of the alleged victims of rape were nulliparous 87.12% and parous was only 12.87%. 430 (61.51%) cases of reported victims who were students of schools and colleges were not considered as rape cases considering their victim's history of love affairs, leaving home secretly with their lovers, living with them for many days. Gang rape was not so common (4.29% of raped cases) in our study. Age groups, their occupations, living areas, time of arrival for medico-legal examination have been studied. Most of the cases were students (61.51%). A few numbers of victims were subjected to gang rape. Examination and reporting the cases have been discussed.

Post mortem outcome of organophosphorus compound poisoning cases at Mymensingh Medical College.

Post mortem studies were carried out on victims of Organophosphorus Compounds (OPC) poisoning cases at Mymensingh Medical College Morgue, Mymensingh from January 2007-December 2008. Data were collected from Post mortem department of Forensic Medicine, Mymensingh Medical College, Mymensingh, Bangladesh. Out of 1862 autopsy cases 692(37.16%) were identified as OPC poisoning cases. The main cause of ingestion of OPC was to commit suicide but accidental and homicidal cases were very few. Of the OPC cases, 401(57.8%) were males while the remainder 291(42.2%) were females. The study reveals that among the total OPC cases, 657(94.94%) were suicidal, 03(0.43%) were homicidal, and in 05(0.72%) cases the motive of ingestion was something else. The study further reveals that the incidence of OPC cases was highest in 21 to 30 years age group and it was 265 cases (38.29%).

Hypoglycemic effect of Catharanthus roseus in normal and streptozotocin-induced diabetic rats.

The effects of crude juice (at 0.5 and 1 ml/kg b.w.) and aqueous extract (at 0.30 and 0.45 gm/kg b.w.) of leaves of Catharanthus roseus on serum glucose level in streptozotocin induced diabetic rats were examined at 8 hours, 12 hours and 24 hours following single oral administration. The administration of crude juice at 1 ml/kg b.w. continued for another 9 doses (total 10 single morning doses given) and its effect was examined on the 4th and 11th day. The rats were made diabetic by single intraperitoneal injection of streptozotocin at 45 mg/kg b.w. Glibenclamide was used in the study for comparison. The crude leaf juice at 0.5 and 1 ml/kg b.w. reduced the serum glucose level in streptozotocin induced diabetic rats throughout the 24-hour period significantly (P varies between 0.05 and 0.001 at different times). The aqueous extract at 0.30 and 0.45 gm/kg reduced the serum glucose level in streptozotocin diabetic rats at 8 and 12 hour significantly (P varies between 0.05 to 0.01 at different times) but not at the 24 hour. Glibenclamide, at 500 mug/kg, also reduced the serum glucose level in streptozotocin induced diabetic rats throughout the 24-hour period (P<0.001). The crude leaf juice at 1 ml/kg also significantly reduced the serum glucose level in the streptozotocin induced diabetic rats on the 4th and 11th day (P<0.001 on both occasions). The effect of crude leaf juice at 1 ml/kg b.w administered daily orally over a 10 day period was also examined on a group of normal rats at different times. The study showed significant reduction at 8 hr (P<0.05), 12 hr, 24 hr and on the 4th day (P<0.01 on these 3 occasions) and also on the 11th day (P<0.001).

Dysregulation of Aromatase in Breast, Endometrial, and Ovarian Cancers: An Overview of Therapeutic Strategies.

Aromatase is the rate-limiting enzyme in the biosynthesis of estrogens, which play crucial roles on a spectrum of developmental and physiological processes. The biological actions of estrogens are classically mediated by binding to two estrogen receptors (ERs), ERα and ERß. Encoded by the cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) gene, aromatase is expressed in a wide variety of tissues, as well as benign and malignant tumors, and is regulated in a pathway- and tissue-specific manner. Overexpression of aromatase, leading to elevated systemic levels of estrogen, is unequivocally linked to the pathogenesis and growth of a number malignancies, including breast, endometrium, and ovarian cancers. Aromatase inhibitors (AIs) are routinely used to treat estrogen-dependent breast cancers in postmenopausal women; however, their roles in endometrial and ovarian cancers remain obscure. While AI therapy is effective in hormone sensitive cancers, they diminish estrogen production throughout the body and, thus, generate undesirable side effects. Despite the effectiveness of AI therapy, resistance to endocrine therapy remains a major concern and is the leading cause of cancer death. Considerable advances, toward mitigating these issues, have evolved in conjunction with a number of histone deacetylase (HDAC) inhibitors for countering an assortment of diseases and cancers, including the aforesaid malignancies. HDACs are a family of enzymes that are frequently dysregulated in human tumors. This chapter will discuss the current understanding of aberrant regulation and expression of aromatase in breast, endometrial, and ovarian cancers, and potential therapeutic strategies for prevention and treatment of these life-threatening diseases.

Preoperative discrimination of benign and malignant ovarian tumors using color Doppler sonography and its correlation with histopathology.

A total of forty clinically diagnosed ovarian tumors were studied over a period of one year. The study aimed at evaluating the efficacy of trans-vaginal color Doppler sonography (TV-CDS) in preoperative discrimination of benign and malignant ovarian tumors and to correlate the imaging findings with postoperative histopathological findings. The pulsatility Index (PI) and Resistive Index (RI) were studied as primary effecacy variables of TV-CDS. In the present study, using TV-CDS, 72.5% of the ovarian tumors were found benign and 22.8% were malignant which were significantly correlated with postoperative histopathological findings. The average PI for benign and malignant tumors were 1.98 and 0.77 respectively and RI for benign and malignant tumors were 0.62 and 0.41 respectively. The low PI and RI values in malignant tumors as compared to benign ones were statistically highly significant (p < 0.001). The validity tests for TV-CDS were found to be 90% sensitive, 100% specific and 97% accurate. The study concludes that TV-CDS is a useful imaging diagnostic modality in preoperative discrimination of benign and malignant ovarian tumors due to its excellent characterization of tumors neo-vascularization.

Analysis of glioblastoma tumor coverage by oncolytic virus-loaded neural stem cells using MRI-based tracking and histological reconstruction.

In preclinical studies, neural stem cell (NSC)-based delivery of oncolytic virus has shown great promise in the treatment of malignant glioma. Ensuring the success of this therapy will require critical evaluation of the spatial distribution of virus after NSC transplantation. In this study, the patient-derived GBM43 human glioma line was established in the brain of athymic nude mice, followed by the administration of NSCs loaded with conditionally replicating oncolytic adenovirus (NSC-CRAd-S-pk7). We determined the tumor coverage potential of oncolytic adenovirus by examining NSC distribution using magnetic resonance (MR) imaging and by three-dimensional reconstruction from ex vivo tissue specimens. We demonstrate that unmodified NSCs and NSC-CRAd-S-pk7 exhibit a similar distribution pattern with most prominent localization occurring at the tumor margins. We were further able to visualize the accumulation of these cells at tumor sites via T2-weighted MR imaging as well as the spread of viral particles using immunofluorescence. Our analyses reveal that a single administration of oncolytic virus-loaded NSCs allows for up to 31% coverage of intracranial tumors. Such results provide valuable insights into the therapeutic potential of this novel viral delivery platform.

Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy.

Glioblastoma multiforme patients have a poor prognosis due to therapeutic resistance and tumor relapse. It has been suggested that gliomas are driven by a rare subset of tumor cells known as glioma stem cells (GSCs). This hypothesis states that only a few GSCs are able to divide, differentiate, and initiate a new tumor. It has also been shown that this subpopulation is more resistant to conventional therapies than its differentiated counterpart. In order to understand glioma recurrence post therapy, we investigated the behavior of GSCs after primary chemotherapy. We first show that exposure of patient-derived as well as established glioma cell lines to therapeutic doses of temozolomide (TMZ), the most commonly used antiglioma chemotherapy, consistently increases the GSC pool over time both in vitro and in vivo. Secondly, lineage-tracing analysis of the expanded GSC pool suggests that such amplification is a result of a phenotypic shift in the non-GSC population to a GSC-like state in the presence of TMZ. The newly converted GSC population expresses markers associated with pluripotency and stemness, such as CD133, SOX2, Oct4, and Nestin. Furthermore, we show that intracranial implantation of the newly converted GSCs in nude mice results in a more efficient grafting and invasive phenotype. Taken together, these findings provide the first evidence that glioma cells exposed to chemotherapeutic agents are able to interconvert between non-GSCs and GSCs, thereby replenishing the original tumor population, leading to a more infiltrative phenotype and enhanced chemoresistance. This may represent a potential mechanism for therapeutic relapse.

Depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer a survival advantage in experimental malignant glioma.

Myeloid-derived suppressor cells (MDSCs) accumulate in the glioma microenvironment during tumor progression and promote immunosuppression. Interleukin-12 (IL-12) immunogene therapy can alter MDSCs toward an antigen-presenting cell phenotype and these mature cells can have a central role in antigen presentation. It remains unclear, however, how MDSC depletion can affect glioma immunotherapy. In this study, we generated a replication-deficient adenoviral vector, Ad.5/3.cRGD-mIL12p70, that transduces the GL261-based murine glioma cell line, resulting in the induction of biologically active, murine IL12p70 expression. Ex vivo, IL-12 expressed by GL261 cells induced interferon-γ synthesis in CD8(+) T cells (P<0.001), CD4(+) T cells (P=0.009) and natural killer cells (P=0.036). When injected 1 week after tumor implantation, Ad.5/3.cRGD-mIL12p70 successfully prolonged the survival of glioma-bearing mice. Sixty percent of animals treated with IL-12 immunotherapy were long-term survivors over 175 days, whereas all the control group animals expired by 40 days after tumor implantation (P=0.026). Mice receiving Ad.5/3.cRGD-mIL12p70 also accumulated 50% less MDSCs in the brain than the control group (P=0.007). Moreover, in the IL-12 group, MDSCs significantly overexpressed CD80 and major histocompatibility complex class II molecules (P=0.041). Depletion of MDSCs with Gr1(+) antibody had no survival benefit induced by IL-12-mediated immunotherapy. Of note, IL-12 therapy increased the presence of myeloid dendritic cells (mDCs) in the glioma microenvironment (P=0.0069). Ultimately, the data show that in the context of IL-12 immunogene therapy, MDSCs are dispensable and mDCs may provide the majority of antigen presentation in the brain.

Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: targeted delivery of the therapeutic payload in an orthotopic brain tumor model.

Oncolytic virotherapy is a promising novel therapy for glioblastoma that needs to be optimized before introduced to clinic. The targeting of conditionally replicating adenoviruses (CRAds) can be improved by relying on the tumor-tropic properties of neural stem cells (NSCs). Here, we report the characterization of an FDA approved NSC, HB1.F3-CD, as a cell carrier for CRAd-S-pk7, a glioma-tropic oncolytic adenovirus. We show that NSCs replicate and release infectious CRAd-S-pk7 progeny capable of lysing glioma cell lines. Moreover, ex-vivo-loaded NSCs, injected intracranially in nude mice bearing human glioma xenografts (i) retained their tumor tropism, (ii) continued to replicate CRAd-S-pk7 for more than a week after reaching the tumor site and (iii) successfully handed off CRAd-S-pk7 to glioma cells in vivo. Delivery via carrier cells reduced non-specific adenovirus distribution in the mouse brain. Moreover, we assessed biodistribution of loaded NSCs after intracranial injection in animal models semi-permissive to adenovirus replication, the Syrian hamster and cotton rat. NSCs did not migrate to distant organs and high levels of CRAd-S-pk7 DNA were observed only in the injected hemisphere. In conclusion, this optimized carrier system, with high efficiency of adenovirus delivery and minimal systemic toxicity, poses considerable advantages for anti-glioma oncolytic virotherapy.