Qatar Foundation Annual Research Forum Volume 2011 Issue 1

T-cells genetically modified to express a chimeric antigen receptor (CAR) against a tumor associated antigen are attractive anti-cancer therapeutic agents. The 5T4 cell surface oncofoetal antigen is an attractive target antigen for cancer immunotherapy as it is expressed by a wide spectrum of cancers including gastric, ovarian and colorectal while showing limited expression in normal adult tissues. Previously it was shown that human and murine T-cells engineered to express human 5T4 specific CAR can specifically lyse human 5T4-expressing tumor targets in vitro and in vivo, respectively. This study aimed to isolate single chain variable fragments (scFvs) specific for murine 5T4 (m5T4) and to examine their efficacy in the context of CAR in a fully autologous model. Screening four novel hybridoma cell-lines producing anti-m5T4 monoclonal antibodies to clone the scFv yielded one functional m5T4-specific scFv from the hybridoma cell-line P1C9. The P1C9 scFv expressed as a fusion protein with the Fc domain of human IgG significantly labels m5T4-expressing targets. Murine T-cells modified to express the P1C9 scFv fused to the CD3? molecule can specifically lyse target cells in vitro and result in IFN-? cytokine release, while T-cells expressing the non-signalling CAR derivative; P1C9 scFv fused to the murine MHC-I transmembrane domain, were unable to lyse m5T4-expressing tumor targets. On-going experiments aim to test the efficacy and assess the toxicity of m5T4-specific CAR against m5T4-expressing tumor models in vivo. This model will allow further understanding of how gene-modified T-cells function in an autologous setting with the aim to improve human T-cell based cancer immunotherapy.

Background: Caring for an autistic child places significant stress on the lives of family members. To date, no study has evaluated how looking after a child with autism affects quality of life (QoL) of caregivers in Qatar or the Gulf country region. Our study is aimed at quantifying the health-related QoL of caregivers of children with autism in Qatar.

Methods: We recruited two groups of caregivers of children between 3 to17 years old. The first group was formed of caregivers of children with autism from two developmental paediatric rehabilitation clinics, and the second group was formed of caregivers of typically-growing children and who were visiting a family clinic for routine medical check-up. Demographic information of all caregivers and children were collected. The Arabic version of the Short Form-36 (SF-36), which is a generic type questionnaire, was administered to caregivers in both groups to assess QoL.

Results: A total of 98 participants consented to take part in the study. 56 were caregivers of children with autism, and 42 were caregivers of a typically-growing children. There was no statistically significant difference in QoL domains between both groups, but caregivers of autistic children rated their health as poor and likely to get worse (p=0.003). Mental health components were consistently poorer than the physical components in the autistic group, and female caregivers had poorer mental health than males in this cohort of participants (p<0.05).

Conclusion: This study provided evidence for the impact of caring for a child with autism on the life of the caregiver. However, there seems to be a need to develop condition-specific QoL questionnaires that can specifically measure QoL in caregivers of autistic children more accurately than generic questionnaires. The findings should still help health policy-makers provide more focused support to the children with autism and their families.

Background: Cigarette smoking is one of the preventable causes of ill health in Qatar. Qatar community pharmacists are in an ideal position to play an important role in smoking cessation. This role necessitates adequate smoking cessation knowledge and education. The study objectives were to assess Qatar community pharmacists' smoking cessation knowledge and to gauge their perceptions of which aspects of smoking-related education would be most interesting.

Methods: A pretested survey was used to solicit community pharmacists' anonymous responses. The survey was designed after reviewing relevant smoking cessation literature. A phone call was made to all community pharmacists in Qatar to request their participation. Interested pharmacists were sent the survey link by email or by fax. Data was descriptively analyzed using the Statistical Package of Social Sciences software version 18.

Results: Over 20 weeks, we collected 112 surveys (35% response rate). Smoking cessation knowledge was evaluated using 8 true or false questions. Thirty seven percent of respondents scored less than 60% and 13% scored more than 80%. The mean score was 61% with a standard deviation of 17%. Eighty-nine percent of respondents indicated that they have not received before any smoking cessation education. Nevertheless, at least 70% indicated that they were interested in receiving additional smoking cessation education. Respondents were mostly interested in receiving education on motivating smokers to quit and on counseling on behavioral techniques (89% and 86% respectively). Sixty nine percent indicated a preference for mailings of printed materials as method of information delivery.

Conclusion: Despite their low smoking cessation knowledge, Qatar community pharmacists are interested in receiving additional smoking cessation education. A smoking cessation education program should be offered to these pharmacists to give them the knowledge they need to be competent smoking cessation counselors.

Introduction: Controlled drug release from implantable silicone elastomers was reported to occur mainly via the osmotic rupture release mechanism. In this work, we are attempting to determine the effects of drug volumetric loading, particle size, device shape, and dissolution media on the release rate of the water-soluble osmotically active drug, Papaverine Hydrochloride (PH) from silicon elastomers.

Materials and Methods: Medical grade silicone (Sylgard kit) was purchased from Dow Corning. All other chemicals were purchased from Sigma-Aldrich and used as received. The release profiles from devices of three geometries (cylinders, cubes and tablets), of different drug particle sizes (< 45μm, 63–125μm or 125–250μm) in different dissolution media (phosphate buffered saline, distilled water, and 3% sodium chloride solution) were investigated. The effect of degree of elastomer crosslinking and the percentage of volumetric loading of the osmotic excipient and pore forming agent, Trehalose, on the release rate were also investigated. The drug fraction released of PH was analyzed using ultraviolet spectrophotometric analysis. Data obtained was then plotted as cumulative percentage release against time and then further analyzed.

Results and Discussion: Contrary to previous reports, devices formulated with the same volumetric loading and smaller drug particles sizes released drug faster than those devices with a larger particle size. The drug release from tabular implants was of faster rate when compared to cylindrical and cubic devices. Contrary to the expected, higher drug release rate was also observed from cubic elastomeric devices with higher crosslinking density. In addition, the release profiles demonstrated that osmotic release was the predominant mechanism governing the release of PH from silicone elastomers.

Conclusion: Our results show that osmotic rupture is the predominant mechanism of release of PH from silicone elastomer. Further studies must be conducted to confirm the effect of the elastomer crosslinking density on drug release rate.

Acknowledgements: This report was made possible by a UREP award [UREP 07-120-3-027] to HM Younes from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the authors.

Background: Fetal movements are clinically correlated to fetal wellbeing. Ultrasounds are the most accurate measurements of the fetal movement but expensive and intrusive. To avoid these constraints, Fetal activity is captured through data acquired using low cost and nonintrusive accelerometer . Time-frequency distributions (TFDs) are often used to represent the energy, temporal and spectral characteristics of non-stationary signals in the time and frequency plane.

Many quadratic TFDs were proposed in the literature such as Wigner-Ville distribution, Spectrogram, B-Distribution, Choi-Williams , etc. The drawbacks of the majority of these techniques is that only a few parameters can be modified in the kernel, generally the lag and Doppler parameters, so that they cannot be easily adapted to the data.

Objectives: This work aims at designing a new kernel with several parameters that leads to a higher resolution time frequency representation (TFR) of the signal, therefore improving the characteristic of the fetal movement.

Methods: The proposed TFD can be considered as an extension of the Gaussian TFD (also called Choi-Williams distribution). The kernel function of the proposed kernel is given by the sum of the weighted derivative Gaussian TFD (refer to the equation).

Results and Conclusion: The weighted parameters in the above formula can be estimated by maximizing the concentration of the instantaneous frequency. The resulting TFD is compared with other methods and applied to the analysis and classification of the fetal movement data recorded by the accelerometers. The results obtained indicate that the proposed time-frequency methodology allows the detection of fetal movement data recorded by accelerometers.

Introduction: Autism spectrum disorder (ASD) affects children at around the age of 3 years old and last throughout the person's lifetime. ASD imposes great burden on the family, and forces family members to considerably modify their daily lives to suit their reality with autistic child(ren). To our knowledge, no previous research assessed the burden of ASD on the lives of parents/caregivers of autistic children in Qatar or the Gulf region.

Methods: Caregivers of a child with autism between 3–17 years were recruited from children rehabilitation clinics. The control group was represented by caregivers of a typically-growing child visiting a primary health care facility for a routine medical examination. Data collected from both groups included demographic information of caregivers and children and quality of life information. The Lebanese Arabic version of the Short Form-36 (SF-36) was used to assess quality of life.

Results: Children in the AG spent more time indoors, watching TV, or sleeping than children in the control group (p←0.05). Around 40% of caregivers in the AG said they would encourage their child to get married and become parent when s/he grows up. Half of the sample in the AG utilizes special education classes and other facilities, and the remaining half has access problems. There was no statistically significant difference between quality of life domains between the two groups of caregivers, but caregivers of autistic children rated their health as poor and likely to get worse (p=0.003). However, mental health components were consistently poorer in the AG compared to the physical components, and female caregivers also had poorer mental health than males in this cohort of participants (p<0.05).

Conclusion: This study provided evidence for the impact of caring for a child with autism on the life of the caregiver. The findings should help health policy-makers provide more focused support to the children with autism and their families.

Background: Anterior cruciate ligament (ACL) tears are common injuries occurring upwards of 250,000 times annually only in the U.S. These injuries create an annual billion dollar expense. It is important to understand these injury mechanisms as these injuries continue to be one of the largest problems in orthopedic sports medicine. Improved understanding of the injury mechanisms may improve prevention, rehabilitation and surgical procedures.

Objective: This study used a 3-Dimensional finite element (FE) knee joint model to investigate the combinations of movement, which cause ACL injury.

Method: Digital bone structures were created from magnetic resonance images (MRI). Ligament bundles were modeled based on the origins and insertion sites determined from MRI. Bone was modeled as rigid, and a transversely isotropic Mooney-Rivlin material was applied to the ligament structures. This study incorporates a novel approach for developing bundle specific prestrain within 3-D ligament structures. The bundles were stretched from their zero load lengths to their reference lengths, producing a strain field mimicking the in vivo strain conditions of the ACL at full knee extension. A failure locus was created by performing multiple FE simulations of knee joint motion combinations until ACL failure.

Result: The relationship between knee joint orientation and ligament rupture was plotted providing a spectrum for the propensity of ACL injury based on knee joint orientations, known as a failure locus. The locus shows which combinations of internal/external femoral rotation and varus/valgus angle cause ACL failure. The results show the posterolateral bundle more susceptible to rupture than the anteromedial bundle in 17 of the 22 simulations. The results also show 45% less varus angle needed for ligament failure relative to valgus angle. The results highlight femoral external rotation as an important factor for ACL injury as it decreases the failure angle by an average of 23% compared to femoral internal rotation.

Conclusion: These results have various clinical applications. In sports where ACL injuries are prevalent, training programs can be adapted to address the avoidance of harmful knee orientations.

Transient Receptor Potential (TRP) ion channels are formed by either homomeric or heteromeric assembly of four TRP subunits that have six transmembrane domains (TM) and a P-loop located between TM5 and TM6 defining the channel pore. So far 30 different subunits classified in 6 families sharing as low as 20% homology, have been identified in mammals and about 20 in Xenopus. Although heterotetramers can be formed within a family, the assembly of subunits from members of different families was thought to be unlikely. We here propose that TRPV6 and TRPC1 subunits can co-assemble in the Xenopus oocyte. Western blots performed on naive oocytes lysates revealed that they express both TRPV6 and TRPC1 proteins. Furthermore, co-immunoprecipitation of tagged xTRPV6 and xTRPC1 proteins expressed in oocytes suggested a heteromeric assembly. In mammals, TRPV6 is mainly expressed in intestinal epithelia and in the placenta where it forms a homomeric channel with high calcium permeability whereas TRPC1 is a ubiquitous protein forming cationic channels with an unclear gating mechanism.

In oocytes, over expression of xTRPC1, as previously reported, did not induce any resting ionic current. Conversely, oocytes overexpressing xTRPV6 alone did show an inward rectifying cationic current that could be blocked by La3+ ions and Ruthenium red. As described for mammals, the xTRPV6 channel was largely permeable to calcium ions and displayed an anomalous mole fraction effect. A striking distinction with its mammalian counterpart is the high permeability of the xTRPV6 channel to Mg2+ over other divalents (Mg2+>Sr2+>Ba2+>Ca2+), the Mg2+/Ca2+ ratio being 2.08.

This high permeability to Mg2+ could be abolished by a point mutation in the P-loop (D525N) lowering the ratio to 1.18. Co-expression of xTRPC1 and xTRPV6 lead to a very strong reduction in the current amplitude and in cationic selectivity, the Mg2+/Ca2+ ratio also dropping to 1.18. Further investigations using heterologously expressed xTRPV6 and xTRPC1 in HEK293, allowing whole-cell recordings, will help us clarify the selectivity and the regulation of the channel. The potential interaction between TRPV6 and TRPC1 in mammals will also be examined using tissue extracts and selected cell lines.

The oscillation of cytoplasmic calcium concentration exists in a large number of non-excitable cells such as astrocytes in the brain, intestinal cells and oocytes (Fig.1). This rhythmic activity carries information into the cell, regulating various processes such as gene expression or transmitter release. In some cells, such as Xenopus oocytes, the oscillations can rely only on calcium being released in the cytoplasm from intracellular stores after stimulation of inositol triphosphate (IP3) receptors located on the endoplasmic reticulum (ER). To refill the intracellular stores in the ER, the cell has to import extracellular calcium. This is achieved through the STIM/Orai pathway where an ER calcium sensor (STIM) is activated by store depletion and in turn interacts and opens a calcium channel in the plasma membrane (Orai) leading to calcium influx, a process known as Store Operated Calcium Entry (SOCE). In the present work we evaluated the interactions between SOC and intracellular calcium oscillations in Xenopus oocytes. Intracellular calcium levels were monitored using the amplitude of the endogenous calcium-activated chloride currents. Following injection into the cells of a non-hydrolysable analog of IP3 (IP3-DF), long lasting calcium oscillations could be triggered as well as SOCE ( Fig. 1 ). The calcium oscillations were more efficient in promoting SOC than store depletion by ionomycin (2.34 ± 0.55 nA, n=13 vs 0.05 ± 0.02 nA, n=8), the mechanisms involved are currently under study. We also observed that activation of SOC could trigger a calcium wave in a dose-dependent manner following a “Calcium Induced Calcium Release” pattern. This suggests that SOC might not only be involved in replenishing the calcium stores but also in the regulation of the amplitude and timing of calcium oscillations. The well known down-regulation of SOC during oocyte maturation should therefore have important functional consequences on intracellular calcium oscillations.

Recently, a speculative model is proposed that tumor tissue is continuously regenerated by a small population of self-renewing cancer stem cells responsible for initiation, growth and propagation of tumor. Tumor cells reside in a “niche” and have constant interaction with the niche components including tumor vasculature, bone marrow mesenchymal stem cells (BM-MSCs) and the components of the extracellular matrix. It is believed that the growth advantage acquired by tumor cells as well as the ability of cancer stem cells (CSCs) to maintain tumor propagation is partly the outcome of this interaction.

Our study intends to better characterize this crosstalk by looking into the possibility of BM-MSC/endothelial cell-mediated activation of Notch pathway in breast cancer cells through production of certain cytokines aiming at proposing novel therapeutic approaches for targeting this disease.

The CSCs were enriched by culturing breast cancer cells (BCC) in 3D media to obtain multicellular spheres for further analysis. BM-MSC and Akt-activated endothelial cells were co-cultured with BCC and spheres to assess their capability in promoting cancer cell growth. RNA interference approach was applied to Notch3 to determine its role in BCC survival by the niche players. A human cytokine array was used to identify the cytokines that showed different expression patterns in the BCC co-cultures with the niche residents.

BM-MSC or Akt-activated Endothelium was able to increase BCC growth up to 4- and 5-fold, respectively, in contact and under serum-free condition. Blocking Notch3 in BCC or spheres by siRNA or inhibition of notch pathway by GSI reduced the proliferation rate of both entities to 2.5-fold, which might underline the role of notch in the interaction between tumor cells and the niche components. Also, several cytokines were identified that were differentially regulated in co-cultures system among which IL6, IL8, and GROa showed significant up-regulation.

Our preliminary data suggest the role of BM-MSC and endothelial cells in breast cancer growth and survival. Our results propose the idea of notch involvement in this interaction through stimulation by certain cytokines. Further investigation is required to elucidate the exact mechanism that is triggered by these cytokines in tumor propagation and maintenance.