1.Antiplasmodial and chloroquine chemosensitizing and resistance reversal effects of coumarin derivatives against Plasmodium falciparum 3D7 and K1
Tropical Biomedicine 2016;33(1):14-26
Background Emergence of chloroquine (CQ) resistance among different strains
of Plasmodium falciparum is the worst incident that has ever faced the dedicated efforts to
eradicate malaria. The main cause of CQ resistance is over-activity of the pumping mechanism
that ousts CQ outside the DV. This urged the scientists to look for other alternatives or
adjuvants that augment its action. CQ The study aimed to test the potential of five coumarin
derivatives, namely; umbeliferon, esculetin, scopoletine, herniarin and 3-aminocoumarine to
inhibit plasmodium growth and reverse CQ resistance in Plasmodium falciparum K1 and
3D7. They are highly ubiquitous in nature and are famous by their diverse pharmacological
effects. SYBRE green-1 based drug sensitivity assay was used to screen the effect of CQ and
each coumarin on the parasite growth and isobologram technique was to assess the interaction
of the coumarins with CQ. Effect of each coumarin on both RBCs and Vero cells stability as
well as on RBCs fragility were screened to exclude any toxic impact on normal cells. On the
other hand, their effect on hemozoin formation was screened to investigate about their
molecular mechanism. For molecular characterization, Their antioxidant properties were
determined using the conventional in vitro tests and their characters were obtained from
Molinspiration Simulation Software. Results showed that all of them were safe to human
cells, have weak to moderate plasmodial growth inhibitory effect and only umbeliferon, 3-
aminocoumarin and esculetin has interacted effectively with CQ. These actions are neither
correlated with hemozoin formation inhibition nor to the antioxidant mechanisms. Further
studies recommended to investigate the mechanism of their action. Overall, all the tested
coumarins are not ideal to be used in the conventional malaria therapy and only umbeliferon,
3-aminocoumarin and esculetin can be suggested to potentiate CQ action.
2.Interleukin-27 exhibited anti-inflammatory activity during Plasmodium berghei infection in mice.
Fazalul Rahiman, S S ; Basir, R ; Talib, H ; Tie, T H ; Chuah, Y K ; Jabbarzare, M ; Chong, W C ; Mohd Yusoff, M A ; Nordin, N ; Yam, M F ; Abdullah, W O ; Abdul Majid, R
Tropical Biomedicine 2013;30(4):663-80
Interleukin-27 (IL-27) has a pleiotropic role either as a pro-inflammatory or anti-inflammatory cytokine in inflammatory related diseases. The role and involvement of IL-27 during malaria was investigated and the effects of modulating its release on the production of major inflammatory cytokines and the histopathological consequences in major affected organs during the infection were evaluated. Results showed that IL-27 concentration was significantly elevated throughout the infection but no positive correlation with the parasitaemia development observed. Augmentation of IL-27 significantly elevated the release of anti-inflammatory cytokine, IL-10 whereas antagonising and neutralising IL-27 produced the opposite. A significant elevation of pro-inflammatory cytokines (IFN-γ and IL-6) was also observed, both during augmentation and inhibition of IL-27. Thus, it is suggested that IL-27 exerts an anti-inflammatory activity in the Th1 type response by signalling the production of IL-10 during malaria. Histopathological examination showed sequestration of PRBC in the microvasculature of major organs in malarial mice. Other significant histopathological changes include hyperplasia and hypertrophy of the Kupffer cells in the liver, hyaline membrane formation in lung tissue, enlargement of the white and red pulp followed by the disappearance of germinal centre of the spleen, and tubular vacuolation of the kidney tissues. In conclusion, it is suggested that IL-27 may possibly acts as an anti-inflammatory cytokine during the infection. Modulation of its release produced a positive impact on inflammatory cytokine production during the infection, suggesting its potential in malaria immunotherapy, in which the host may benefit from its inhibition.
3.GSK3β: A plausible molecular target in the cytokinemodulating effect of exogenous insulin in a murine model of malarial infection
Aizuddin, N.N.F. ; Ganesan, N. ; Ng, W.C. ; Ali, A.H. ; Ibrahim, I. ; Basir, R. ; Embi, N. ; Hasidah, M.S.
Tropical Biomedicine 2020;37(No.4):1105-1116
Malaria is a life-threatening disease caused by the Plasmodium sp. parasite.
Infection results in heightened pro-inflammatory response which contributes to the
pathophysiology of the disease. To mitigate the overwhelming cytokine response, host-directed
therapy is a plausible approach. Glycogen synthase kinase-3β (GSK3β), a serine/threonine
kinase plays a pivotal role in the regulation of inflammatory response during pathogenic
infections. The present study was conducted to investigate the chemo-suppressive and
cytokine-modulating effects of insulin administration in malaria-infected mice and the
involvement of GSK3β. Intraperitoneal administrations of 0.3 and 0.5 U/kg body weight
insulin each for four consecutive days into Plasmodium berghei NK65 (PbN)-infected mice
resulted in chemo-suppression exceeding 60% and improved median survival time of infected
mice (20.5 days and 19 days respectively compared to 15.5 days for non-treated control).
Western analysis revealed that pGSK3β (Ser9) intensity in brain samples from insulin-treated
(0.3 and 0.5 U/kg body weight) infected mice each were 0.6 and 2.2 times respectively than
that in control. In liver samples, pGSK3β (Ser9) intensity from insulin-treated infected mice
were significantly higher (4.8 and 16.1 fold for 0.3 and 0.5 U/kg bw respectively) than that in
control. Insulin administration decreased both brain and liver pNF-κB p65 (Ser536) intensities
(to 0.8 and 0.6 times for 0.3 U/kg bw insulin; and to 0.2 and 0.1 times for 0.5 U/kg bw insulin
respectively compared to control). Insulin treatment (0.5 U/kg bw) also significantly decreased
the serum levels of pro-inflammatory cytokines (TNF-α (3.3 times) and IFN-γ (4.9 times))
whilst significantly increasing the levels of anti-inflammatory cytokines (IL-4 (4.9 fold) and
IL-10 (2.1 fold)) in PbN-infected mice. Results from this study demonstrated that the cytokinemodulating effects of insulin at least in part involve inhibition of GSK3β and consequent
inhibition of the activation of NF-κB p65 suggesting insulin as a potential adjunctive therapeutic
for malaria.
4.Anti-malarial and cytokine-modulating effects of andrographolide in a murine model of malarial infection
Hassan, W.R.M. ; Basir, R. ; Ali, A.H. ; Embi, N. ; Sidek, H.M
Tropical Biomedicine 2019;36(3):776-791
Malarial pathogenesis involves among others, uncontrolled or excessive cytokine
production arising from dysregulated immune responses mounted by the host to eliminate
the plasmodial parasite. The ubiquitous serine/threonine kinase, glycogen synthase kinase-
3β (GSK3β) is a crucial regulator of the balance between pro- and anti-inflammatory
cytokine productions in the inflammatory response to pathogenic infections.
Andrographolide, a bioactive compound in Andrographis paniculata, displays GSK3-
inhibitory effects. A previous study elsewhere has shown that this compound has antimalarial
activity but the molecular basis of its action is yet to be elucidated. Here we
aimed to study the anti-malarial activity of andrographolide in a murine model of malarial
infection to investigate whether its mechanism of action involves cytokine modulation
and inhibition of GSK3β. Andrographolide showed strong and selective anti-plasmodial
activity (IC50 = 13.70±0.71 μM; SI = 30.43) when tested against cultures of P. falciparum
3D7. Intraperitoneal administration of andrographolide (5 mg/kg body weight (bw)) into P.
berghei NK65-infected ICR mice resulted in chemo-suppression of 60.17±2.12%, and
significantly (P<0.05) improved median survival time of infected mice compared to nontreated
control. In addition, andrographolide treatment significantly (P<0.05) decreased
the level of serum pro-inflammatory cytokine, IFN-γ (1.4-fold) whilst the anti-inflammatory
cytokines, IL-10 and IL-4 were increased 2.3- and 2.6-fold respectively. Western blot analyses
revealed that andrographolide treatment of P. berghei NK65-infected mice resulted in an
increased level of phosphorylated GSK3β (Ser9) in liver of infected mice. Andrographolide
administration also decreased the levels of phosphorylated NF-κB p65 (Ser536) and
phosphorylated Akt (Ser473) in liver of malaria- infected animals. Taken together, our
findings demonstrate that the cytokine-modulating effect of andrographolide in
experimental malarial infection involves at least in part inhibition of NF-κB activation as a
consequence of GSK3β inhibition. Based on its cytokine-modulating effects, andrographolide
is thus a plausible candidate for adjunctive therapy in malaria subject to clinical evaluations.
5.IL-8 as a potential in-vitro severity biomarker for dengue disease
Soo, K.-M. ; Tham, C.L ; Khalid, B. ; Basir, R. ; Chee, H.-Y.
Tropical Biomedicine 2019;36(4):1027-1037
Dengue is a common infection, caused by dengue virus. There are four different
dengue serotypes, with different capacity to cause severe dengue infections. Besides,
secondary infections with heterologous serotypes, concurrent infections of multiple dengue
serotypes may alter the severity of dengue infection. This study aims to compare the severity
of single infection and concurrent infections of different combinations of dengue serotypes
in-vitro. Human mast cells (HMC)-1.1 were infected with single and concurrent infections of
multiple dengue serotypes. The infected HMC-1.1 supernatant was then added to human
umbilical cord vascular endothelial cells (HUVEC) and severity of dengue infections was
measured by the percentage of transendothelial electrical resistance (TEER). Levels of IL-
10, CXCL10 and sTRAIL in HMC-1.1 and IL-8, IL-10 and CXCL10 in HUVEC culture supernatants
were measured by the ELISA assays. The result showed that the percentage of TEER values
were significantly lower in single infections (p< 0.05), compared to concurrent infections on
day 2 and 3, indicating that single infection increase endothelial permeability greater than
concurrent infections. IL-8 showed moderate correlation with endothelial permeability (r >
0.4), indicating that IL-8 may be suitable as an in-vitro severity biomarker. In conclusion, this
in-vitro model presented few similarities with regards to the conditions in dengue patients,
suggesting that it could serve as a severity model to test for severity and levels of severity
biomarkers upon different dengue virus infections.
6.COMPARISON OF PATHOGENESIS OF P. BERGHEIINFECTION IN MOUSE AND RAT MODELS
Chin VK ; Chong WC ; Nordin N ; Lee TY ; Zakaria ZA ; Hassan H ; Basir R
Journal of University of Malaya Medical Centre 2019;22(2):4-12
Background: The cytokine cascade in the immunopathogenesis of malaria infection had been widely studied. However, their specific association with survival and severe infection remained obscure.Methods: Thestudy investigated the cytokine profiles and histopathological features of malaria in the severe infection and survival models by using male ICR mice and male Sprague Dawley rats respectively.Results: The severe model, the infected ICR mice, exhibited a high parasitemia with 100% mortality after peak parasitemia at day 5 post-infection. The survival model, the infected Sprague Dawley rats, showed mild parasitemia with full recovery by day 14 of infection. Both severe and survival models showed similar histopathological severity during peak parasitemia. The severe model produced highly elevated levels of pro-inflammatory cytokines, TNF-α and IL-1α, and low levels of the anti-inflammatory cytokine, IL-4; while the survival model showed low levels of TNF-α and IL-1α with high levels of IL-4.Conclusion: There were differences in the pathogenesis of the severe and survival models of malaria infection. These could be a basis for immunotherapy of malaria in the future
7.Medicinal plants with antimalarial activities mediated via glycogen synthase kinase-3 beta (GSK3β) inhibition
Hassan, W.R.M. ; Ali, A.H. ; Basir, R. ; Embi, N. ; Sidek, H.M.
Tropical Biomedicine 2022;39(No.3):384-393
Many of the therapeutic effects of plant extracts and bioactive compounds appear related to their
immunomodulatory effects and impact on the host immune system. The immune response is desirable
to mitigate established infections and, in the case of severe malaria, is a feasible approach to dealing
with the overwhelming cytokine response. Glycogen synthase kinase-3 (GSK3), a Ser/Thr kinase that
is a central regulator of the cytokine response, is a promising antimalarial drug target. In this review,
we discussed our ongoing research projects, which include assessing the antimalarial activities of
medicinal plants and their bioactive compounds, immunomodulatory activities mediated by GSK3, and
the potential inflammatory pathway involved in malarial infection.
8.Inhibition of Activin A suppressed tumor necrosis factor-α secretion and improved histopathological conditions in malarial mice
Chin, V.K. ; Tie, T.H. ; Abd Majid, R. ; Hassan, H. ; Nordin, N. ; Abas, R. ; Basir, R.
Tropical Biomedicine 2021;38(No.1):187-204
Malaria infection still remains as one of the most prominent parasitic diseases afflicting
mankind in tropical and subtropical regions. The severity of malaria infection has often
been associated to exuberant host immune inflammatory responses that could possibly
lead to severe immunopathological conditions and subsequent death of host tissues. Activin
A is a protein belonging to the transforming growth factor-beta (TGF-β) family that regulates
multiple physiological processes and pathological-associated diseases. The biological
roles of activin A have been associated with manipulation of inflammation-related processes
and modulation of host immune responses. This implies that activin A protein could play a
role in malaria pathogenesis since malaria infection has been closely linked to severe
immune responses leading to death, However, the actual in vivo role of activin A in malaria
infection remains elusive. Hence, this study was undertaken to investigate the involvement
of activin A in malaria infection as well as to assess the modulating effects of activin A on
the cytokine releases (TNF-α, IFN-γ and IL-10) and histopathological changes in major affected
organs (kidney, liver, lung, brain and spleen) in malarial mice infected with Plasmodium
berghei ANKA. Our results showed that the concentrations of plasma activin A were significantly
increased in malarial mice throughout the study periods. Also. the systemic activin A level
was positively correlated with malaria parasitemia. This indicates that activin A could play
a role in malaria pathogenesis and malaria parasitemia development. Plasma TNF-α,
IFN-γ and IL-10 cytokine levels were significantly increased in malarial mice at day-5 post
infection, suggesting that these cytokines attributed to severe malaria pathogenesis.
Histopathological features such as sequestration of parasitized red blood cells (pRBCs)
and hemozoin formation were amongst the most common pathological conditions observed
in tissues of major affected organs (kidney, liver, lung, brain and spleen) in malarial mice.
Neutralization of activin A production via recombinant mouse activin RIIA Fc chimera (rmActivin
RIIA Fc chimera) had significantly reduced the parasitemia levels in malarial mice. The
release of TNF-α cytokine was significantly reduced as well as the sequestration of
parasitized pRBCs and hemozoin formation in major affected organs in malarial mice were
also alleviated following inhibition of activin A production. Overall, this preliminary study
suggests that activin A could play an immune modulation role in malaria pathogenesis
through modulation of TNF-α release that benefits host from severe pathological destructions
provoked by intensified inflammatory responses. Further studies are warranted to elucidate
the precise mechanism of immune modulation mediated by activin A and its associated
immune-modulation mediators in regulating the inflammatory responses elicited during
the course of malaria infection.
9.Progression of malaria induced pathogenicity during chloroquine therapy
Zaid, O.I. ; Abd. Majid, R. ; Sidek, H.M. ; Noor, S.M. ; Abd Rachman-Isnadi, M.F. ; Bello, R.O. ; Chin, V.K. ; Basir, R.
Tropical Biomedicine 2020;37(No.1):29-49
Treatment Failure with chloroquine is one of the challenges that faced the dedicated efforts to eradicate malaria This study aims at investigating the impact of treatment failure with chloroquine on the progression of the disease-induced histo-pathogenic and immunogenic outcomes. To achieve this, Rane’s protocol with modifications was applied on a model of Plasmodium berghei ANKA infected ICR mice to determine the dose response curve of chloroquine and to screen the treatment impact on the disease progression. Chloroquine was given at 1, 5, 10, 15 and 20 mg/kg once the parasitemia reached to 20-30% (the experimental initiation point). During the subsequent days, the mice were monitored for changes in the clinical signs, hematology parameters and the progress of the parasitemia until the parasitemia reached to 60-70% (the experimental termination point) or up to 10 days after chloroquine administration in case of achieving a complete eradication of the parasite. At the end, the mice were exsanguinated and their blood and organs were collected for the biochemistry and the histology study. A complete eradication of the parasite was achieved at 20 mg/kg while recrudescence was observed at the lower doses. At 1 mg/kg, the parasite growth was comparable to that of the positive control. The histo-pathogenic and immunogenic changes were stronger in the groups that experienced recrudescence (at 5 and 10 mg/kg). All in all, the study highlights the possibility of having a worsened clinical condition when chloroquine is given at its sub-therapeutic doses during malaria treatment.