Effective Inhibition of TNF-α/mTOR Axis-Mediated Liver Inflammation and Fibrosis Induced by TAA Using a Combination of Metformin and Resveratrol in Association with the Inhibition of the Profibrotic Gene and Protein Expression

SUMMARY: The response of the immune system to harmful stimuli leads to inflammation, and the adverse effects of the toxic hepatitis chemical, thioacetamide (TAA) on the human body are well documented. This article investigated the degree of protection provided by the combined pleotropic drug, metformin (Met) and the plant polyphenolic and the antiinflammatory compound, resveratrol (Res) on liver tissue exposed to TAA possibly via the inhibition of the inflammatory cytokine, tumor necrosis factor-α (TNF-α) / mammalian target of rapamycin (mTOR) axis-mediated liver fibrosis, as well as amelioration of profibrotic gene and protein expression. Rats were either given TAA (200 mg/kg via intraperitoneal injection) for 8 weeks beginning at the third week (experimental group) or received during the first two weeks of the experiment combined doses of metformin (200 mg/kg) and resveratrol (20 mg/kg) and continued receiving these agents and TAA until experiment completion at week 10 (treated group). A considerable damage to hepatic tissue in the experimental rats was observed as revealed by tissue collagen deposition in the portal area of the liver and a substantial increase (p<0.0001) in hepatic levels of the inflammatory marker, tumor necrosis factor-α (TNF-α), as well as blood levels of hepatocellular injury biomarkers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). TAA also augmented hepatic tissue levels of the signalling molecule that promotes liver fibrosis (mTOR), and profibrogenic markers; alpha-smooth muscle actin (α-SMA) protein, tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA, and matrix metalloproteinase-9 (MMP-9) mRNA. All these parameters were protected (p≤0.0016) by Met+Res. In addition, a significant correlation was detected between liver fibrosis score and inflammation, liver injury enzymes, mTOR, and profibrogenesis markers. Thus, these findings suggest that Met+Res effectively protect the liver against damage induced by thioacetamide in association with the downregulation of the TNF-α/mTOR/fibrosis axis.

La respuesta del sistema inmunológico a estímulos dañinos conduce a la inflamación y los efectos adversos de la tioacetamida (TAA), una sustancia química tóxica para el hígado, están bien documentadas. Este artículo investigó el grado de protección proporcionado por el fármaco pleotrópico combinado metformina (Met), el polifenólico vegetal y el compuesto antiinflamatorio resveratrol (Res) en el tejido hepático expuesto a TAA, posiblemente a través de la inhibición de la citoquina inflamatoria, factor de necrosis tumoral α (TNF-α)/objetivo de la fibrosis hepática mediada por el eje de rapamicina (mTOR), así como mejora de la expresión de genes y proteínas profibróticas. Las ratas recibieron TAA (200 mg/kg mediante inyección intraperitoneal) durante 8 semanas a partir de la tercera semana (grupo experimental) o recibieron durante las dos primeras semanas del experimento dosis combinadas de metformina (200 mg/kg) y resveratrol (20 mg/kg) y continuaron recibiendo estos agentes y TAA hasta completar el experimento en la semana 10 (grupo tratado). Se observó un daño considerable al tejido hepático en las ratas experimentales, como lo revela el depósito de colágeno tisular en el área portal del hígado y un aumento sustancial (p<0,0001) en los niveles hepáticos del marcador inflamatorio, el factor de necrosis tumoral-α (TNF- α), así como los niveles sanguíneos de biomarcadores de lesión hepatocelular, alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). TAA también aumentó los niveles en el tejido hepático de la molécula de señalización que promueve la fibrosis hepática (mTOR) y marcadores profibrogénicos; proteína actina del músculo liso alfa (α- SMA), inhibidor tisular de las metaloproteinasas-1 (TIMP-1) mRNA y matriz metaloproteinasa-9 (MMP-9) mRNA. Todos estos parámetros fueron protegidos (p≤0.0016) por Met+Res. Además, se detectó una correlación significativa entre la puntuación de fibrosis hepática y la inflamación, las enzimas de lesión hepática, mTOR y los marcadores de profibrogénesis. Por lo tanto, estos hallazgos sugieren que Met+Res protege eficazmente el hígado contra el daño inducido por la tioacetamida en asociación con la regulación negativa del eje TNF-α/mTOR/fibrosis.

Raised plasma insulin level and homeostasis model assessment (HOMA) score in cerebral malaria: evidence for insulin resistance and marker of virulence.

OBJECTIVE: To study the glycaemic profile of patients with severe malaria (SM). METHODS: For this purpose, 110 SM patients were recruited. Pre-treatment random blood glucose and plasma insulin were measured in a subset of donors. An ex-vivo experiment was developed for estimation of glucose consumption by parasitized erythrocytes. RESULTS: Hyperglycaemia was frequent in SM but more commonly associated with cerebral malaria (CM), while hyperinsulinaemia was recognized in severe-malarial-hypotension (median, 25 %-75 %, 188.2, 93.8-336.8 pmol/L). The plasma insulin level was positively correlated with age (CC = 0.457, p < 0.001) and negatively with parasitaemia (CC = -0.368, p = 0.045). Importantly, fatal-CM was associated with hyperglycaemia (12.22, 6.5-14.6 mmol/L), hyperinsulinaemia (141.0, 54.0-186.8 pmol/L) and elevated homeostasis model assessment (HOMA) values. However, there was a trend of higher glucose consumption by parasites in CM compared with that in uncomplicated malaria (UM). CONCLUSION: Hyperglycaemia, hyperinsulinaemia and elevated HOMA are evidence for insulin resistance and possibly pancreatic B-cell dysfunction in fatal-CM.

The profile of IgG-antibody response against merozoite surface proteins 1 and 2 in severe Plasmodium falciparum malaria in Eastern Sudan.

In this study, antibodies (Ab) directed against three MSP antigens; MSP1(19), MSP2(A), and MSP2(B) were analyzed in blood samples obtained from 223 Sudanese patients who presented with either severe malaria (SM) or uncomplicated malaria (UM) and from 117 malaria-free donors (MF). The results showed that the prevalence of MSP Abs was associated with the clinical outcome of malaria infection, and the Ab prevalence was age-dependent (P<0.0005). More importantly, the prevalence of MSP Abs against the test antigens was lower in SM compared to UM (P=0.001 to 0.020), suggesting a protective role for these Abs against SM. Furthermore, the Ab responses between individual complications of SM were significantly different.

Allelic polymorphism of MSP2 gene in severe P. falciparum malaria in an area of low and seasonal transmission.

The severe malaria (SM) and uncomplicated malaria (UM) infections are expected to have different genetic makeup. In this study, blood samples were obtained from 325 donors with SM and UM and malaria-free donors (including asymptomatic submicroscopic malaria--ASUM), from Eastern Sudan. The SM group included patients with cerebral malaria (CM), severe malarial anemia (SMA), and other complications. The MSP2 locus was exploited for parasite genotyping. We found that the genetic diversity of the parasite population was marked (51 genotypes). The overall multiplicity of infection (MOI) was 1.5, and it was comparable between SM and UM. However, the MOI in ASUM (1.0) and fatal CM (1.14) was comparable and significantly lower than in UM (1.53), SMA (1.52), and nonfatal CM (1.7). The ratio of the IC1 to FC27 allele families was comparable between SM and UM, and the distribution of the allele sizes was correlated (correlation coefficient = 0.59 and 0.718; P < 0.001). It is interesting to note that the FC27 genotype was overrepresented in ASUM (68.2%) and was not recognized in fatal CM, while in mixed-clone infections, the clearance of IC1 after quinine treatment was faster than FC27 clearance. Finally, the composition of the multiclone infections (IC1 and FC27) was suggesting a stronger cross-immunity within rather than between MSP2 gene families.

Genetic fingerprints of parasites causing severe malaria in a setting of low transmission in Sudan.

In this study we intended to examine the extent of genetic diversity of Plasmodium falciparum parasites causing severe malaria (SM). For this purpose, 100 parasite isolates were obtained from patients with SM and uncomplicated malaria, from an area of low and unstable malaria transmission in Sudan. The diversity of infection (DOI) was estimated by relating the number of the different parasite genotypes that were detected to the total number of parasites that were genotyped (parasite population/subpopulation). We used different molecular markers individually (pfcrt-76, pfmr1-86, GLURP size and MSP2 family and size) and as a group to set a multilocus genetic profile for each parasite isolate. The DOI as estimated by MSP2 and GLURP was 0.553 and 0.435, respectively. However, combination of all four molecular markers (multilocus genetic profile) revealed a fingerprint pattern of genetic diversity with a DOI of 0.936, indicating that in SM infection, diversity is the rule and homogeny is the exception. Furthermore, our clinical data suggest that the virulence markers might also be more diverse than expected. In conclusion, the results are unexpected and overturn the assumption that parasites causing SM are a limited subpopulation of virulent parasites or of a clonal nature. However, it was more likely that there was a genetically unique parasite in each infection.

Drug resistance-virulence relationship in Plasmodium falciparum causing severe malaria in an area of seasonal and unstable transmission.

The pathogenesis of severe Plasmodium falciparum malaria is still obscure, but is believed to be multi-factorial, and among the important factors are intrinsic parasite-properties. Here we investigated the association between clinical manifestation of P. falciparum malaria (an indicator of virulence) and two parasite properties--drug resistance and gametocyte production. Among 996 P. falciparum infections detected in the out-patient clinic of Gedarif Hospital in eastern Sudan, there was no significant association between the incidence of severe versus mild disease and the presence of resistant alleles at the chloroquine-resistance transporter locus (pfcrt-T76) and the multi-drug-resistance locus (pfmdr1-Y86). However, among severe cases, there was a significantly lower prevalence of parasites carrying resistant alleles among patients that died versus survived. There was a trend towards a higher gametocyte rate among severe malaria patients compared with uncomplicated malaria cases. These results are discussed in relation to the fitness of drug resistant parasites.

Differential induction of immunoglobulin G to Plasmodium falciparum variant surface antigens during the transmission season in Daraweesh, Sudan.

BACKGROUND: The acquisition of immunoglobulin (Ig) G to variant surface antigens (VSAs) seems important for the development of protective immunity against malaria. Unlike VSAs expressed by parasite isolates associated with uncomplicated malaria, VSAs expressed by parasite isolates associated with severe malaria (VSA(SM)) are frequently recognized by IgG. METHODS: We analyzed levels of anti-VSA IgG in 57 individuals in Daraweesh, Sudan, before and after the transmission season. IgG responses to 79 Plasmodium falciparum isolates from children with defined malaria syndromes and exposed to high transmission in a different part of Africa were also analyzed. RESULTS: After the transmission season, individuals with malaria had an increase in IgG recognition to 25.8% (95% confidence interval [CI], 19.9%-31.7%) and a decrease in IgG recognition to 7.6% (95% CI, 4.4%-10.8%) of 79 parasite isolates, and individuals without malaria had an increase in IgG recognition to 8.1% (95% CI, 6.0%-10.2%) and a decrease in IgG recognition to 11.9% (95% CI, 7.0%-16.8%) of 79 parasite isolates. Most newly acquired IgG responses were against parasite isolates expressing VSAs(SM) that are frequently recognized by IgG. CONCLUSIONS: Anti-VSA IgG levels decrease in the absence of infection, and an episode of clinical malaria induces IgG against a range of VSAs, particularly VSAs(SM).

Cerebral malaria is frequently associated with latent parasitemia among the semi-immune population of eastern Sudan.

The accurate diagnosis of malaria starts with clinical suspicion, confirmed by reliable laboratory results. A hospital-based study, described here, was carried out in a malaria mesoendemic area in eastern Sudan, where the inhabitants are semi-immune to malaria, and the fever threshold of parasitemia is not above the detection level of microscopy. Thus, we hypothesized that patients with symptoms highly suggestive of cerebral malaria (CM), but aparasitemic by microscopy, may have submicroscopic parasitemia. Patients in our malaria clinic were screened by microscopy, and 120 individuals were selected for the study, including febrile patients with and without microscopically detectable parasitemia, and apparently healthy individuals. In the two former groups there were patients with severe anemia and deep coma. Polymerase chain reaction (PCR) for parasite detection and ELISA tests for measuring serum antibody levels were carried out on all blood samples. A majority of the febrile patients who were parasite negative by microscopy showed the presence of a Plasmodium falciparum infection by PCR. The occurrence of P. falciparum infection with parasitemia below the detection level of microscopy was recognized more often in patients with CM symptoms than in those with severe malarial anemia (SMA), and in older rather than younger patients. Patients clinically suspected (CS) of having CM ((CS)CM) mostly were infected with a single clone, and a large proportion of them acquired antibodies (Abs) against merozoite surface protein (MSP) antigens (Ags). The therapeutic response to quinine treatment was comparable between patients with (CS)CM and CM. In conclusion, uniquely in this setting, CM can be associated with sub-patent parasitemia; thus, a diagnostic tool more sensitive than microscopy is needed.