Differentiation of human pluripotent stem cells into corneal epithelial like cells

PhD ThesisCornea is the clear outermost protective layer of the eye which enables transmission of light onto the retina. The corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in limbal stem cell deficiency (LSCD). Transplantations of ex vivo expanded autologous LSCs from patient’s healthy eye onto the affected eye have provided a successful treatment for unilateral LSCD. This however is not applicable to patient with total bilateral LSCD, whose both eyes are affected. This thesis investigated the potential of human induced-pluripotent stem cell (hiPSCs) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD, and tested the engraftment of the differentiated cells in LSCD mouse model. Combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid (RA) and epidermal growth factor (EGF) for the first nine days of differentiation followed by cell-replating on collagen-IV coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESCs) to corneal epithelial progenitors and mature corneal epithelial-like cells. Differences in the ability of hiPSCs lines to undergo differentiation to corneal epithelial-like cells were observed. These were dependent on the level of endogenous BMP signalling and could be restored via activation of this signalling pathway by a specific TGFβ inhibitor (SB431542). The hESC and hiPSCs-derived corneal epithelial cells were transplanted into a LSCD mouse model where they survived up to 14 days, but failed to provide long term engraftment and corneal surface regeneration. The findings showed a differential ability of hESCs and hiPSCs lines to generate corneal epithelial cells which is underlined by the endogenous BMP signalling pathway activity. However, the engraftment and functionality of the differentiated cells in the LSCD animal model has yet to be improved.Ministry of Education Malaysia and my employer, Universiti Kebangsaan Malaysia in a form of financial assistance and sponsorship. I would also like to acknowledge the Fund for Women Graduates (FfWG), UK organisation for awarding me with financial assistance during the final stage of my study

Protective effect of curcumin on experimentally induced arthritic rats

Curcuma longa (turmeric) rhizomes contains curcumin, an active compound which possesses anti-inflammatory effects. Collagen-induced arthritis (CIA) is an accepted experimental animal model of rheumatoid arthritis. The present study aimed to observe the histological changes in the joints of experimental arthritic rats treated with curcumin. Twenty four male Sprague-Dawley (approximately 7 weeks-old) rats were randomly divided into four groups. Three groups were immunized with 150 μg collagen. All rats with established CIA, with arthritis scores exceeding 1, were orally treated with betamethasone (0.5 mg/ml/kg body weight), curcumin (110 mg/ml/kg body weight) or olive oil (1.0 ml/kg body weight) daily, for two weeks. One remaining group was kept as normal control. Treatment with 110 mg/ml/kg curcumin showed significant mean differences in the average white blood cell (WBC) count (p<0.05), cell infiltration, bone and cartilage erosion scores (p<0.05) compared to the olive oil treated group. Pannus formation scores showed that curcumin supplementation successfully suppressed the pannus formation process that occurred in the articular cartilage of the CIA joints. The mean difference for histological scores for the curcumin group was insignificant compared to the betamethasone treated group. It is concluded that supplementation of curcumin has protective effect on the histopathological and degenerative changes in the joints of CIA rats which was at par with betamethasone

Oral administration of tocotrienol ameliorates lead-induced toxicity in the rat brain

The occurrence of severe lead (Pb) poisoning has risen in certain countries. There is increasing evidence that chronic lead exposure disturbs the prooxidant: antioxidant balance in the brain tissue and alters brain histology. The present study observed the antioxidant effect of tocotrienol-rich fraction (TRF) on brain tissues of the experimental rats following lead poisoning. Eighteen (n=18) male Sprague-Dawley rats, 6-weeks old, were randomly divided into control (CTRL) group and experimental groups; fed with 0.2% w/v lead acetate, as PB2 group; and fed with 0.2% w/v lead acetate and daily TRF supplementation (200 mg/kg body weight) as PB2T group. The experiment was conducted for 30 days. At the end of the study, the brain tissues were harvested and histopathological changes of the hippocampal region were observed. Biochemical findings such as brain lead, TRF and malondialdehyde (MDA) levels, and erythrocyte superoxide dismutase (SOD) activity were determined. It was observed that atypical apoptotic-like and disorganized neurons were present in the hippocampal region of the untreated PB2 group compared to PB2T group. Biochemical parameters showed a significant decrease (p 0.05) was obtained for MDA level, there was a significant increase (p < 0.05) in the erythrocyte SOD activity in PB2T compared to PB2 and CTRL. Supplementation with TRF improved histopathological changes in the brain tissues caused by lead exposure in drinking water by reducing lead accumulation in the brain of experimental rats

Curcumin as an anti-arthritic agent in collagen-induced arthritic sprague-dawley rats

Curcuma longa or turmeric has long been used in traditional medicine by the local population in Malaysia as an antiinflammatory agent. It has been proven to contain natural antiarthritic compound called curcumin. Joints abnormality and destruction have been implicated in the pathogenesis of rheumatoid arthritis (RA) due to inflammatory reactions. In this study, collagen-induced arthritis (CIA) model was utilized to study the effects of curcumin on joint inflammation in Sprague-Dawley rats. Body weight measurement, arthritis score assessment and radiology score assessment were carried out at specific intervals throughout this study. The results showed that the mean arthritis and radiology scores for animal groups designated as CIA CurcuminC and CIA CurcuminT were significantly lower compared with the negative control (CIA OV) group respectively. The mean arthritis scores for CIA CurcuminC group is significantly lower compared with CIA CurcuminT group but there is no significant difference in the mean radiology scores between the CIA CurcuminC and CIA CurcuminT groups. In conclusion, the oral supplementation of curcumin at the dose of 110 mg/mL/kg/day has a potential to delay and improve joint abnormality and injury in Sprague-Dawley rats with CIA

Curcumin minimises histopathological and immunological progression in the ankle joints of collagen-induced arthritis rats

Curcumin is a traditionally used spice with a potential to treat various inflammatory diseases including arthritis. This study was aimed at observing curcumin’s effects on the histopathological progression and interleukin-1β (IL-1β) levels in collagen�induced arthritis (CIA). Thirty male Sprague-Dawley rats (150+50 g) were divided into five random groups. A group was assigned as the normal control (CTRL), while the remaining were subcutaneously immunised with 150 µg of collagen emulsion on day 0. CTRL and CIA-Curcumin-d0 groups were supplemented daily with olive oil (1 ml/kg) and curcumin (110 mg/ml/kg) from day 0, respectively. The CIA-OV (negative control), CIA-Beta and CIA-Curcumin-d14 groups were given daily supplementation of olive oil (1 ml/kg), Betamethasone (0.5 mg/ml/kg), and curcumin (110 mg/ml/kg) from day 14, respectively. The daily oral supplementations continued until day 42. The study showed that CIA-Beta (**P<0.05) and CIA�Curcumin-d0 (**P=0.01) groups had significantly lower mean histological scores compared to CIA-OV, respectively. Serum IL-1β levels for CIA-Beta and CIA�Curcumin-d0 were not significantly raised on day 42 as to compared to day 0, and the mean increment of IL-1β levels from day 0 to day 42 were significantly lower (**P≤0.01) for all the CIA groups compared to CIA-OV. There was no significant difference in both mean histological scores and IL-1β levels of CIA-Curcumin-d0 compared to CIA-Beta. Early supplementation of curcumin could potentially minimise disease progression of CIA in rats

Curcumin Protects against Ovariectomy-Induced Bone Changes in Rat Model

Osteoporosis is a metabolic disease affecting both men and women especially in postmenopausal women. Curcumin possesses many medicinal properties. In this study, thirty two female Sprague-Dawley rats were used to determine the potential effect of curcumin in prevention of bone loss following ovariectomy. The animals were divided into Sham group, ovariectomised control, ovariectomised treated with curcumin 110 mg/kg and ovariectomised treated with Premarin 100 μg/kg. The treatments were given via daily oral gavages for 60 days. The structural parameters such as bone volume, trabecular number, trabecular thickness and trabecular separation were found to be deteriorated in ovariectomised rats compared to Sham group. Moreover, the reduced osteoblast count, the increased osteoclast count and increased eroded surface were found in ovariectomised groups. Treatment with curcumin was able to reverse all these ovariectomy-induced deteriorations. Curcumin treatment was as effective as Premarin in most parameters except the bone volume and eroded surface, which were better than Premarin. The high dose of curcumin treatment was not only able to reduce the osteoclast number but also increase the osteoblast count. Therefore, the potential effect of curcumin can be applied as an alternative to oestrogen for prevention of postmenopausal osteoporosis

Knowledge, information needs and behavior regarding HIV and sexually transmitted infections among migrants from sub-Saharan Africa living in Germany: Results of a participatory health research survey

Background: A total of 3,419 new HIV diagnoses were reported in Germany in 2016, with migrants from sub-Saharan Africa (misSA) accounting for 14.1%. To understand the driving factors behind the epidemiological situation, we conducted a quantitative cross-sectional survey on knowledge, attitudes, behavior, and practices regarding HIV and sexually transmitted infections (STIs) among misSA living in six German cities utilizing participatory health research. Methods: Participants were recruited by peer researchers. Levels of knowledge, information needs, and preferred methods of information dissemination were analyzed to inform future prevention planning. Additionally, we analyzed sexual behavior and other risk factors for contracting HIV and STIs. The results may facilitate the formulation of targeted prevention messages in the future. Results: We included 2,432 participants in the analysis. General knowledge about HIV was adequate, as 86.9% were aware of the presented information. Statements about HIV co-infections were prior knowledge for 53.4% of the participants and about German HIV policies and HIV testing for 54.7%. Knowledge about other STIs differed, ranging from 69.6% who have ever heard of gonorrhea to 23.8% who have ever heard of genital warts. Groups with particular knowledge gaps were i) younger misSA, ii) recent migrants, iii) misSA without regular access to the German health care system, iv) misSA of lower socioeconomic status, and v) misSA with Muslim religious affiliation. The majority of participants reported information needs (72.8%), and 71.3% wanted to obtain this information from health professionals. Male misSA were more likely to report five or more sexual partners compared to females. Less than half of participants reported always using condoms with non-steady sexual partners (46.8%). Reasons for not using condoms differed between males and females. A considerable proportion of females (16.3%) and males (6.8%) experienced sexualized violence. More than one fourth of women (26.9%) were affected by female genital mutilation/cutting. Discussion: Future prevention planning should focus on sub-groups with particular knowledge gaps, recognizing their preferred methods of information dissemination. Prevention messages for male misSA should focus on their own risk perception and for female misSA on empowerment, e.g. to negotiate condom use.Peer Reviewe

Impact of HIV knowledge and stigma on the uptake of HIV testing – Results from a community-based participatory research survey among migrants from sub-Saharan Africa in Germany

Background: In 2015, 3,674 new HIV diagnoses were notified in Germany; 16% of those newly diagnosed cases originated from sub-Saharan Africa (sSA). One quarter of the newly diagnosed cases among migrants from sSA (MisSA) are notified as having acquired the HIV infection in Germany. In order to reach MisSA with HIV testing opportunities, we aimed to identify which determinants influence the uptake of HIV testing among MisSA in Germany. Methods: To identify those determinants, we conducted a quantitative cross-sectional survey among MisSA in Germany. The survey was designed in a participatory process that included MisSA and other stakeholders in HIV-prevention. Peer researchers recruited participants to complete standardized questionnaires on HIV knowledge and testing. We conducted multivariable analyses (MVA) to identify determinants associated with ever having attended voluntary HIV testing; and another MVA to identify determinant associated with having had the last voluntary HIV test in Germany. Results: Peer researchers recruited 2,782 participants eligible for inclusion in the MVA. Of these participants, 59.9% (1,667/2,782) previously had an HIV test. For each general statement about HIV that participants knew prior to participation in the study, the odds of having been tested increased by 19% (OR 1.19; 95%-CI: 1.11–1.27). Participants reporting that HIV is a topic that is discussed in their community had 92% higher odds of having been tested for HIV (OR 1.92; 95%-CI: 1.60–2.31). Migrants living in Germany for less than a year had the lowest odds of having had their last HIV test in Germany (OR 0.17; 95%-CI: 0.11–0.27). Additionally, MisSA 18 to 25 years (OR 0.55; 95%-CI: 0.42–0.73) and participants with varied sexual partners and inconsistent condom use (OR 0.75; 95%-CI: 0.44–0.97) had significantly lower odds of having had their last HIV test in Germany. Discussion: Through participatory research, we were able to show that knowledge about HIV and discussing HIV in communities increased the odds of having attended HIV testing among MisSA. However, recent migrants and young sexually active people are among the least reached by testing offers in Germany. Community-based interventions may present opportunities to reach such migrants and improve knowledge and increase discussion about HIV

From peer to peer: Reaching migrants from sub-Saharan Africa with research on sexual health utilizing community-based participatory health research

Migrants from sub-Saharan Africa (misSA) in Germany are disproportionally affected by HIV. To develop targeted interventions, it is necessary to collect data on knowledge, attitudes, behaviour and practices (KABP) regarding HIV and sexual health. However, misSA are difficult to reach and to sample: a) it is unknown how many people with an African migration background are living in Germany, and b) HIV and sexual health topics are highly stigmatized in these communities. We utilized a community-based participatory health research approach to develop a study protocol and conducted a KABP survey on HIV and sexual health among misSA in six German cities between 2015 and 2016. A convenience sample of 2,879 participants was recruited by 99 trained peer researchers through outreach in their local communities. Due to steering of recruitment, the study population reflected the official registered misSA population well and was diverse in terms of sociodemographic characteristics. Peer researchers mainly recruited participants that were similar to themselves with regard to gender, age and regions of origin. Male and younger peer researchers more often recruited participants from vulnerable sub-groups like migrants with a probably undocumented legal status who could not have been reached by probability sampling based on population registers.Peer Reviewe