Authors:
1.Umair Farid
DHQ hospital Jhang
2.Ahsan Raza Khan
DHQ Hospital Jhang
3. Usman Latif
Mayo Hospital Lahore
4.Muhammad Waqar Haider
University of Agriculture Faisalabad
5.Mubashir Abbas
FQDMF College of medicine, Lyceum North Western University Dagupan City, Philippines.
6.Roha Tariq
Department of Biotechnology, Lahore College for Women University
Upcoming in PJMS with ID8550-1D
Abstract:
Liver diseases are common in inhabitants and migrants of tropical countries, where the liver can be exposed not only to toxins but also to many viral, bacterial, fungal, and parasitic infections. Schistosomiasis a common parasitic infection that affects at least 240 million people worldwide, mostly in Africa—is regarded as the most frequent cause of liver fibrosis worldwide. We present a case of a 19-year-old male refugee from Guinea with recurrent oesophageal variceal bleeding due to schistosomal liver fibrosis refractory to endoscopic therapy. This case was an indication for portosystemic surgery, which is a highly invasive non-reversible intervention. An alternative, less invasive, reversible radiological procedure, used in liver cirrhosis, is the placement of a transjugular intrahepatic portosystemic shunt. After thorough considerations of all therapeutic options we placed a TIPS in our patient. In more than 3 years of observation, he is clinically well apart from one episode of hepatic encephalopathy related to an acute episode of viral gastroenteritis. Bleeding from oesophageal varices has not recurred. In this Grand Round, we review the diagnostic approaches and treatment options for portal hypertension due to schistosomal liver fibrosis with involvemet of zinc and checked its histology.
Introduction
Liver diseases are common in inhabitants and migrants of tropical countries. The liver can be exposed not only to various toxins but also to the effects of malnutrition and many viral, bacterial, fungal, and parasitic infections.1 Moreover, specific genetic and autoimmune disorders further contribute to hepatic disease. Frequently, various harmful substances, genetic and autoimmune disorders, and other infections coincide to produce liver damage. The prevalence of hepatopathies varies according to the endemicity of these causal agents. The most important causes of chronic hepatopathies are preventable by hygienic and environmental measures, timely anti- schistosomal therapy, and hepatitis B virus (HBV) vaccination.Schistosomiasis is a parasitic disease caused by trematodic worms. Schistosoma haematobium causes urogenital schistosomiasis, and the other species cause intestinal schistosomiasis. The lifecycle of schistosomes involves freshwater snails as intermediate hosts, and human beings and other mammals. The infection is transmitted when worm larvae released from the snails penetrate the skin of the host exposed to a freshwater body such as an irrigation canal, a river, or a lake. Disease manifestations result from the inflammatory response of the host to the ova produced by the female schistosomes. These range from subclinical symptoms including anaemia and growth retardation to overt multiorganic systemic and organ pathological abnor- malities. About two-thirds of schistosomiasis cases are due to infection by S haematobium. S mansoni in Africa, South America, and the Arabian peninsula and S japonicum and S mekongi in east Asia cause hepatic fibrosis, which occurs in 5–10% of infected people. S mansoni causes most of these cases. An estimated 4•4 million people with S mansoni have bloody diarrhoea and bowel ulceration, and 8•5 million develop hepatomegaly or associated portal liver fibrosis, portal hypertension, and haematemesis with a conservative estimate of 130 000 deaths yearly. Severe disease manifestations can be prevented in most cases by early repeated anthelmintic single-dose chemotherapy with praziquantel, even when transmission persists.Schistosomiasis remains a public health problem in many parts of the world, especially in Africa where 93% of all people who need chemotherapy for schisto- somiasis to prevent its complications live. Complications include liver fibrosis, portal hypertension, urinary tract obstruction, hydronephrosis, bladder cancer, and reproductive tract sequelae. Data on treatment are collected to measure the progress being made towards achieving the targets set by World Health Assembly resolutions to reach more than 75% of all school-aged children who are at risk of morbidity from schistosomiasis. 52 of the 78 countries regarded as endemic for the disease have populations that need preventive chemotherapy. In 2012, the estimated number of people needing preventive chemotherapy worldwide was 230–249 million, of whom 114•3 million were school- aged children. These data suggest that early treatment, starting in early childhood, might prevent the occurrence of severe morbidity frequently seen in adolescence. In adolescents, bleeding from oeosophageal varices due to portal hypertension is not uncommon. Of all people worldwide treated for schistosomiasis in 2012, 85% were from sub-Saharan Africa, yet the number of people covered by chemotherapy is equivalent to only 14% of the population who need it. Although accurate data are not available, schistosomiasis should still be regarded as the most frequent cause of liver fibrosis worldwide
Case description.
A 19-year-old male refugee from Guinea on his journey from Africa to Europe had had recurrent haematemesis in Morocco and Spain where ligation for third-degree oesophageal varices was done. Upon his arrival in Germany he underwent a liver biopsy, which showed liver fibrosis but no cirrhosis. He was referred to our tropical medicine service because schistosomiasis was suspected. A hardened liver and massive splenomegaly were palpable. Haematology results showed decreased platelets , mild leucopenia without eosinophilia, and anaemia due to iron deficiency and to a sickle-cell trait as confirmed by haemoglobin electrophoresis. Thromboplastic was 51% , with international normalised ratio 1•5, and fibrinogen was 1•15 g/L , whereas activated partial thromboplastin time was delayed to 45 s. IgE was 2390 IU/mL, and creatine kinase was 266 U/L. γ-glutamyl transpeptidase was 95 U/L, and he had mild hypoalbuminaemia (55•4%, 55•8–66•1), mild hyper gamma globulinaemia, and normal transaminases and bilirubin, among other normal biochemistry results. Anti-HBc antibodies were positive but active HBV and hepatitis C virus (HCV) infections were excluded by further antigen and antibody tests and PCR. Past infections with Epstein- Barr virus, filariasis, and malaria were detected serologically; active infections were excluded by microscopy of stained thick and thin blood films, PCR, and microfiltration of blood and skin snips. Antischistosome antibodies were positive by ELISA and by indirect haemagglutination test, but schistosoma ova were not seen microscopically in several enriched stool samples, in microfiltration of 24 h urine, or in rectal snips. Antibody tests for HIV, human T-lymphotropic virus, syphilis, Entamoeba histolytica, and Trypanosoma brucei were negative.Left, typical echogenic areas (white) along the portal tree. Right, echogenic thickening of the gallbladder wall and neck ; the gallbladder was typically not tender under ultrasound-guided palpation. Arrows point to hyperechogenic areas indicating fibrotic areas in the liver and gallbladder wall and neck.Abdominal ultrasonography showed the typical pattern of schistosomal liver fibrosis, with echogenic areas accompanying portal branches.Splenic, gallbladder, and gastro-oesophageal varices were seen.Centripetal portal flow was delayed to 10 cm/s. The spleen was grossly enlarged and contained hyperechogenic nodules. The gallbladder wall was typically thickened with external wall protrusions; postprandial contractility was impaired but the gallbladder was not tender at ultrasound-guided palpation. Liver stiffness was increased by transient elastography to 11•6 kPa. Acoustic radiation force impulse elastography showed increased spleen stiffness, which in liver cirrhosis suggests a high risk of gastrointestinal bleeding; we also noted increased stiffness of the fibrotic hepatic areas but not of the remaining liver parenchyma. On ultrasound, the urinary tract was normal.MRI and CT confirmed ultrasound findings.On contrast-enhanced CT images in the venous contrast phase, paragastric collaterals and a spontaneous splenorenal shunt were seen. Portal fibrosis was confirmed on native T1-weighted MRI images; the spleen showed multiple Gamna-Gandy bodies on these images. Region-of-interest-based apparent diffusion coefficient (ADC) measurement of the spleen parenchyma yielded increased average values of 905 × 10−⁶. In liver cirrhosis, this spleen ADC value is a sign of portal hypertension. Although mean ADC value of liver parenchyma was moderately decreased, the strongly increased mean ADC of periportal areas suggested that a vasogenic oedema might have contributed to these changes. Endoscopy revealed portal gastropathy and persisting oesophageal varices at risk for rebleeding.24,25 Colonoscopy showed a macroscopically normal colon apart from grade 2 haemorrhoids. Histological examination of colon biopsy samples revealed fibrosis. Echocardiography did not show specific abnormalities suspicious for cardiopulmonary schistosomiasis or for endomyocardial fibrosis.Praziquantel was given at 40 mg/kg in three separate doses. PCR analysis of rectal biopsy samples and EDTA treated blood 6 weeks later confirmed the complete eradication of schistosomes. Iron was supplemented by mouth. After thorough consideration of therapy options for recurrences of refractory variceal bleeding, a transjugular portosystemic shunt was created. After transjugular catheterisation of the right hepatic vein and wire-guided introduction of an 18-gauge Chiba needle, the right portal vein was punctured under ultrasound guidance. Angiography showed peculiar node-like protuberances of the wall of the dilated portal vein and small portal vessels. After dilation of the intrahepatic tract to 6 mm, a self-expandable stent was inserted and subsequently distended to 8 mm to keep the tract patent. TIPS insertion resulted in a 48% decrease of the portal-venous pressure gradient from 23 mm Hg to 12 mm Hg. Contrast ultrasonography confirmed the perfusion of echogenic hepatic areas.The patient has now been regularly monitored for more than 3 years. Variceal bleeding has never recurred. Endoscopy confirmed the regression of oesophageal varices to grade I with no more signs of bleeding risk.Only one episode of hepatic encephalopathy occurred when the patient heavily vomited because of an acute viral gastroenteritis leading to hyperammonaemia. During the following weeks ammonia decreased to almost normal values. Flicker frequency always remained normal. TIPS was revised without complications 10 months later because the tube was displaced in the direction of the hepatic veins and inferior vena cava as seen by duplex ultrasonography.
Preparation of ZnO nano particles
Zinc Oxide nanoparticles have been synthesized by two simple routes using Aloe vera or Cassava starch. The XRD patterns and Raman spectra show that both synthesis routes lead to single-phase ZnO. XPS results indicate the presence of zinc atoms with oxidation state Zn2+. SEM images of the ZnO nanoparticles synthesized using Cassava starch show the presence of pseudo-spherical nanoparticles and nanosheets, while just pseudo-spherical nanoparticles were observed when Aloe vera was used. The UV-Vis spectra showed a slight difference in the absorption edge of the ZnO particles obtained using Aloe vera and Cassava starch. The ZnO nanoparticles were tested as adsorbents for the removal of copper in wastewater, it is shown that at low Cu2+ ion concentration the nanoparticles synthesized by both routes have the same removal efficiency, however, increasing the absorbate concentration the ZnO nanoparticles synthesized using Aloe vera have a higher removal efficiency. The synthesized ZnO nanoparticles can be used as effective and environmental-friendly metal trace absorbers in wastewater.
Experiment
Materials
All the chemicals used were of analytical grade. Zinc nitrate hexahydrate (Zn(NO3)2•6H2O, 98%) was purchased from Dynamic and copper nitrate trihydrate (Cu(NO3)2•3H2O, 99%) was purchased from Vetec (Sigma-Aldrich). All solutions were prepared with deionized water. Natural Cassava starch in the form of colloidal suspension was used as fuel. Aloe vera leaves were harvested in the São José region of Parana-Brazil. To obtain the extract of Aloe gel, about 200 g of Aloe vera leaves were washed with deionized water and the internal mucilaginous gel was extracted. Afterward, the mucilaginous gel was crushed using a pistil and a ceramic mortar to obtain the complete extract. Finally, the solution was washed, filtered and the resulting Aloe vera gel broth extract was stored under refrigeration (2°C).
Synthesis of Zinc Oxide
Two different routes, both easy to reproduce, were used for synthesizing Zinc oxide nanoparticles. In the route of green synthesis , Aloe vera gel broth extracts at the concentration were prepared with distilled water, the volume was made up to 100 ml. Subsequently, zinc nitrate was dissolved in the aloe extract solution under constant magnetic stirring and left at rest for 12 h. The suspension was calcined in a muffle furnace at temperatures for 1 h. In the route of gelatinization method: first starch was extracted from 100 g of natural Cassava starch in 300 ml of distilled water under mechanical stirring for 2 h. It was then sieved, and in the colloidal starch suspension was added 9.40 g of zinc nitrate. After 60 min of mechanical stirring, the suspension was calcined in a muffle furnace at a temperature of 750 °C for 1 h. The ZnO nanoparticles obtained were named Zn-AL and Zn-ST.
Characterization Techniques
X-ray powder diffraction profile shows XRD1 beamline, 12 keV energy, λ = 1.033 Å, 2θ of 0°-80°. Scanning electron microscopy images were recorded and operated 15 kV, the spatial resolution was 2.5 nm. The Raman spectra were recorded using a Micro-Raman system, λ = 532 nm, laser power 5 mW, time 10 s, resolution 4 cm−1. The optical diffuse reflectance was measured in the range of 300–800 nm. Zeta potential was recorded using ZETASIZER NANO ZS90 , model ZEN 1,010 at 25°C. The zeta potentials of the nano particles were determined from their electrophoretic mobilities according to Smoluchowski’s equation; the pH of these nano particles was adjusted between 3 and 11 using HCl or NaOH solutions. The chemical composition was evaluated by X-ray photoelectron spectroscopy (XPS). The XPS spectra were collected at a take-off angle of 45° with respect to the electron energy analyzer and the spot size was 200 μm. Pass energy (PE) of 20 eV was used for the high-energy resolution spectra (Zn 2p, O 1s, and C 1s). The spectra were analyzed using the CASA-XPS software.The metal ion solutions were analyzed using a Flame Atomic Absorption Spectrometer (FAAS). The FAAS was equipped with an air-acetylene burner. The hollow cathode lamp was set at 4 mA and the analytical wavelength was adjusted at 324.8 nm. The slit size was adjusted to 0.2 nm. The standard curve was drawn by using copper standard solutions. After the adsorption, the ZnO nanoparticles were characterized concerning their composition by energy dispersive X-ray spectrometer (EDX) from Shimadzu, model EDX-7000, containing a Rh tube, operating at 50 and 15 W. The crystalline phases were identified by powder X-ray diffraction (XRD) performed on a Bruker model D2 Phaser with Cu Kα radiation, with scan in 2θ from 10° to 90° and step rate of 0.2°/s. The zeta potential was recorded using ZETASIZER NANO ZS90 from MALVERN, model ZEN 1010. The electronic spectra of the powdered pigments samples were measured on the range of 400–900 nm with a UV-Vis Ocean Optics spectrophotometer model USB-2000.
Adsorption Measurements
To investigate the efficiency of the ZnO nanoparticles as adsorbents for the removal of copper metal ions from water, an adsorption test was performed. The parameters: contact time; initial pH and initial metal ion concentration were investigated. The adsorption experiments were carried out in conical flasks containing 25 mL of copper solution with an initial concentration ranging from 40 to 120 mg L−1. To this end, 250 mg of the ZnO particles were added, and the solutions were kept under continuous shaking for 240 min in a heating bath at 25°C. To study the adsorption kinetics and the pH parameters, 50 mg L−1 of a solution containing Cu (II) and the same amount of ZnO particles was prepared; its pH was adjusted using 0.1 HCl and 0.1 NaOH solutions. The resulting solutions were centrifuged at 1,200 rpm for 15 min. The ion concentration measurements were performed before the adsorption test without the presence of the adsorbent and after 4 h of adsorption in a flame atomic absorption spectrometer (FAAS).
Test Leaching of Nanoparticles
To check the stability of the nanoparticles a method adapted from was used. Thus, 50 mL of simulated sample was treated separately with 250 mg of ZnO synthesized. The initial pH of the experiment was 4 or 6 and the contents were allowed to remain in contact for 240 min while maintaining the temperature at 25°C. After centrifugation and filtration, the residue was washed with deionized water followed by oven drying at 60°C.
Results and Discussion
Characterization of the Zinc Oxides Nanoparticles
Figure 1A presents the X-ray diffractograms of the zinc oxides nanoparticles obtained after heat treatment at 750°C for 60 min. The crystalline phase is identified by the presence of the characteristic peaks of the Wurtzite ZnO phase , belonging to the compact hexagonal system with a space group P63mc to the crystallographic chart . Additional peaks were not detected, evidencing that the single-phase ZnO was successfully obtained regardless of the synthesis route used and the precursors were completely decomposed. The XRD patterns allowed to determine the average crystallite size of the ZnO nanoparticles, estimated by Scherrer’s equation , with the average size of 43.3 nm for Zn-AL and 44.9 nm for Zn-ST. According to these results, the crystalline size is affected by the polysaccharide.The E2low mode is attributed to the vibrations of zinc sublattice in ZnO and E2high mode is assigned to the oxygen vibration, the strong E2high mode indicates the high crystallinity of the oxide , the same vibrational mode has been identified for the zinc oxide nanoparticles obtained via the Starch-assisted synthetic route. The bands at 380 and 408 cm−1 correspond to the first-order optical modes A1(TO) and E1(TO), bands at 202 and 330 cm−1 are characteristic of second-order modes 2E2low and E2high—E2low, caused by multiphonon processes. The bands located at 573 and 584 cm-1 are assigned to A1(LO) and E1(LO) modes, these bands are associated to the presence of structural defects in the ZnO structure, being the E1(LO) mode strongly affected.For Zn-ST, uniform spherical particles are formed. The two samples show particle aggregation, related to the self-assembly effect . The Zn-AL particles tend to agglomerate in plaques, this was attributed to the Aloe vera gel acting as a sacrifice complexant in the formation of the ZnO nano particles during the combustion. The two synthesis routes have polysaccharides as fuel for the formation of ZnO nano particles; their formation mechanism can be described by the “egg-box” model. Therefore, their difference in morphology can be associated with the complex polymeric network of each polysaccharide. Aloe vera gel consists of a combination of organic chains, such as soluble polysaccharides, monosaccharides, proteins, amino acids, among others. The colloidal suspension of Cassava starch is more homogeneous and less complex because it consists basically of amylopectin and amylose leading to the formation of uniform particles, since the Zn (II) ions occupy the “egg-box” more efficiently, with more regular distance. The Aloe vera gel presents a greater variation in its natural components than Cassava starch, affecting directly the shape and reproducibility of ZnO nanoparticles.The chemical environment of the zinc and oxygen atoms were analyzed using X-ray photoelectron spectroscopy (XPS). The O 1s and Zn 2p XPS core-level spectra are shown in Figure 3. The binding energy of the XPS data was calibrated using the C 1s peak at 284.6 eV (Das et al., 2010). The O 1s spectrum was fitted with three components centered on 530.2 ± 0.1, 531.4 ± 0.6, and 532.3 ± 0.7 eV, for both samples . The low binding energy component located at 530.2 ± 0.1 eV is attributed to O2− ions participating in the Zn-O bond in the wurtzite structure of the hexagonal Zn2+ ions of ZnO. The component centered at 531.4 ± 0.6 is associated with photoelectrons emitted from O2− ions in oxygen-deficient regions in the matrix of ZnO . The high binding energy component located at 532.7 ± 0.7 is reported to be associated with oxygen species adsorbed on the surface of the ZnO, such a -CO3, H2O, or O2. The Zn 2p high-resolution XPS spectra show the 2p doublet with components centered at 1020.6 eV (Zn 2p3/2) and 1043.5 eV (Zn 2p1/2). For both samples, the binding energy difference between these core levels is 23.0 eV, reference value denoting the presence of zinc in Zn2+ oxidation state, the chemical state is confirmed by the Zn LMM Auger data.It can be observed in Figure 4A an increase in the reflectance at wavelengths larger than 380 nm, this can be attributed to the direct band-gap of ZnO due to the electron transitions from the valence band to the conduction band (O2p Zn3d), with a lower percentage of reflectance for Zn-AL (~65%). The band energy gaps (EGAP) of the samples were calculated using the Kubelka-Munk method, the EGAP were determined by linear extrapolation of the curve [F(R) x E]2 vs. energy (E) in (Figure 4B), with values: 3.24 eV (Zn-ST) and 3.18 eV (Zn-AL), similar values have been reported in for zinc oxides obtained with Starch. The variation in the optical gap of the ZnO nanoparticles can be associated with a variation in the average particle size and morphology. The synthesized ZnO nanoparticles exhibit a slight red shift in the absorption edge (Figure 4A), this increase in the response range toward the visible radiation region can be explored in the future as photocatalysts with visible light activity.
Review and discussion
Assessment of schistosomal liver fibrosis
Schistosomal hepatopathy described by Symmers in 1904 is a specific form of liver fibrosis that radiates from the walls of the portal branches, which macroscopically resemble clay-pipe stems perforating the liver parenchyma. Also known as pipe-stem, portal, or periportal fibrosis, Symmers’ fibrosis differs in almost all respects from non-cirrhotic portal hypertension, a disease reported from India, where schistosomiasis is not endemic.Most patients with severe schistosomal portal hypertension live in remote rural areas; therefore the best screening method in use is a portable ultrasound device connected to a portable power generator that can be used in rural areas where electricity might not be available.Of the ultrasound protocols that have been developed for assessment of the risk for variceal bleeding in schistosomiasis, the descriptive methods of the typical features of Symmers’ schistosomal liver fibrosis and the diameter of the portal stem have proved valuable, unlike approaches measuring peripheral or segmental portal branches.Liver fibrogenesis is mainly maintained by chronic activation of the wound healing response and oxidative stress. Fibrosis can be reversed, or stabilized in 57%–79% of patients, mainly by antiinflammatory compounds, nonetheless response is usually incomplete or inconsistently completed. Thus, searching for an appropriate therapeutic agent/s for liver fibrosis among the already marketing drugs is a great opportunity and challenge. This experimental work is an integral to our previous study, for evaluating the inhibitory effect of vinpocetine on NF-κB signalling pathway, as well as the possible influence of supplying NO through the NO donor, IS-5-MN. Furthermore, it quantifies hydroxyproline content in the liver as an index of fibrosis, and hepatic enzymes as biochemical markers of liver damage in S. mansoni -infected Swiss albino mice. The first part of our work archived that PZQ monotherapy or combined with vinpocetine or IS-5-MN significantly reduced the total worm count and the liver egg load. Also, combined vinpocetine and IS-5-MN dosing regimen significantly reduced the liver egg burden, without significant reduction in the adult count. While vinpocetine and IS-5-MN monotherapy did not significantly reduce adult worm burden or liver egg load. Regarding serum enzymes levels, vinpocetine regimen did not significantly reduce hepatic enzymes in S. mansoni-infected mice. It was assessed that the drug effects on CCl4-treated Sprague-Dawley rats and ischaemia-reperfusion injury in albino Wistar rats. The discrepancy in findings could be attributed to the differences in the hepatic injury and experimental models. This can also be explained by the role of TNF in limiting hepatocellular damage evoked by schistosome eggs as some amount of NO released as a result of over expression of TNF is believed to guard against excessive hepatic pathology. Besides, TNF is capable of inducing hepatocyte apoptosis via TNF receptor1 (TNFR1), hence it is logic to expect that TNF can play a role in restricting immunopathology induced by upregulation of pro-inflammatory cytokines. Since vinpocetine was documented to decrease TNF level, its administration is expected to increase hepatocellular damage. Besides, drugs that target NF-κB activity might cause elevated hepatocyte death. Similarly, vinpocetine and PZQ regimen caused non-significant increase in enzymes levels, which might be related to the combined injurious effects of NF-κB inhibition and PZQ hepatotoxicity. Compared with the untreated group, administration of IS-5-MN alone caused non-significant reduction in ALT and AST levels, which might be related to persistent oxidative stress status, caused by failure to eradicate or kill Schistosoma eggs, as the ROS generated during S. mansoni infection impair mitochondrial function, increase lipid peroxidation, and induce oxidative stress. Similarly,it was investigated that effect of 24-nor-ursodeoxycholic acid (NO donor) in S. mansoni-infected NMRI mice, and recorded unchanged ALT level. It was reported that addition of silymarin to IS-5-MN reduced ALT and AST levels in CCl4-inflicted liver injury in Sprague-Dawley rats. Likewise, the co-administration of vinpocetine and IS-5-MN in our study significantly reduced the enzymes levels. This may be attributed to the regression of inflammation and necrosis caused by addition of IS-5-MN to vinpocetine. Because of the ability of NO to increase TNF production, it can antagonize the deleterious inflammation detected when vinpocetine was given alone to mice. In addition, NO serves to prevent hepatic damage during S. mansoni infection, as it is implicated in modulating hepatic microcirculatory perfusion, improving hepatic and portal blood flow and sinusoidal oxygenation, decreasing hepatic sinusoidal blood flow perturbations after entry of transplanted hepatocytes into sinusoids, and suppressing the pro-inflammatory cytokines. Moreover, the significant reduction in ALT level caused by administration of IS-5-MN and PZQ, and in AST level in response to treatment with IS-5-MN, compared with PZQ-treated group could be attributed to improved oxidative status of the cells, as detected by reduced microvesicular steatosis, with its well-known association with impaired mitochondrial functions and oxidative stress. Also, the protective effects of NO donation cannot be ruled out. The variations seen in the hepatic enzymes among mice groups may be attributed to the different nature of the two transaminases, as AST is a cytosolic and mitochondrial isoenzyme found in the hepatocytes and many other cells, while ALT is a cytosolic enzyme and is more specific to the liver. In spite of the changes recorded in liver enzymes in all S. mansoni-infected groups, total serum bilirubin levels were within the normal range in both non-infected and infected groups. This can be explained by the fact that liver enzymes and bilirubin belong to two different categories of liver function tests i.e. cell injury or necrosis and cholestasis, respectively. Only micromolar levels of NO are required for protection against the toxicity of ROS. In this study, PZQ monotherapy did not increase NO level, it exerted anti-schistosomal effect, but did not significantly alter inflammation or necrosis. Also, administration of vinpocetine or IS-5-MN mono therapy or combined regimen did not increase NO level, because the drugs failed to significantly decrease hepatic egg load. In contrast, administration of IS-5-MN and PZQ induced a better interactive effect and significantly increased NO level that may be owing to the combined anti-schistosomal activity of PZQ and the hepato protective effect of NO donation caused by IS-5-MN. Infection of mice with S. mansoni increased hepatic contents of hydroxyl proline and collagen. Combined therapy using IS-5-MN and PZQ or vinpocetine and IS-5-MN significantly reduced liver fibrosis markers. These findings reflect a beneficial shift favouring ECM degradation, that might be due to strong additive interactions between the drugs used. Moreover, IS-5- MN acts by releasing NO, which inhibits the proliferation, motility, and contractility of HSCs, in addition to decreasing excessive ECM deposition. Our results are lower showed Immuno histochemical stain for nuclear factor-kappaB antibody of liver sections of different groups of mice euthanized 10 weeks after the beginning of the study. (A) Non-infected mice showing negative staining of hepatocytes. (B) Infected untreated mice showing marked nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma. (C) Infected mice treated with PZQ (6 WPI as 500 mg/kg/day for two successive days), showing moderate nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma. (D) Infected mice treated with vinpocetine (11.76 mg/kg, 4th–10th WPI, 5 days/week), showing weak nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma. (E) Infected mice treated with IS-5-MN, showing weak nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma. (F) Infected mice treated with IS-5-MN and PZQ, showing moderate nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma. (G) Infected mice treated with vinpocetine and IS-5-MN, showing weak nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma. (H) Infected mice treated with vinpocetine and PZQ, showing marked nuclear and cytoplasmic positive staining of hepatocytes and inflammatory cells surrounding granuloma.. It was reported that combined PZQ and silymarin significantly reduced hepatic hydroxyproline content by 46.81%, however; they identified that silymarin monotherapy caused significant reduction by only 19.06%. In infected mice, S. mansoni ova delivered to the liver provoke inflammatory reaction. Together with oxidative stress, inflammation elicits NF-κB activation, leading to increased expression of NF-κB positive cells in the granulomas. Activation of NF-κB results in development of the activated phenotype of HSCs, besides boosting survival of activated HSCs by protecting activated HSCs against TNF-induced apoptosis. In our study, the lowest expression of NF-κB was detected in response to vinpocetine, and its combination with IS-5-MN, which can partially account for reduction in granuloma size, as a consequence of NF-κB suppression and decreased IFN-γ activity. In addition, reduced expression of NF-κB was seen in mice groups administered IS-5-MN and its combination with PZQ. This might be related to the anti-inflammatory effect of IS-5-MN monotherapy, and the significant increase in NO level in mice given the combined regimen. Our findings are to some extent in agreement with it was assessed assessed the effect of the antiinflammatory drugs leflunomide and colchicine on CCl4-induced liver injury in Sprague-Dawley rats, and reported their role in reducing NFκB expression, hence improved liver pathology and significantly inhibited fibrogenesis. Similarly, it was revealed that adenoviral gene transfer in Sprague-Dawley rats resulted in overexpression of hepatic inhibitor κBα superrepressor (IκBα SR) and subsequent inactivation of NF-κB that markedly attenuated hepatic histopathological changes induced by ischaemia/reperfusion injury. It was assessed that effect of transfer of NF-κB decoy in CCl4-treated Balb/c mice, and documented that suppression of NF-κB activation improved liver healing, as well as reduced necrosis and inflammatory cells infiltrate. In addition evaluated the ability of l-theanine to inhibit NF-κB activation in experimental hepatic cirrhosis induced by CCl4 in Wistar rats, and recorded up-regulation of the anti-inflammatory cytokine IL-10 and the fibrolytic enzyme metalloproteinase-13, as well as normal appearance of the liver parenchyma. In spite of the promising results seen in mice treated with combined PZQ and IS-5-MN followed by its combination with vinpocetine, yet the death rate was high in these groups as well as in mice received either IS5-MN or vinpocetine monotherapy, or their combined regimen. This may be attributed to repeated drug/s administration (35–37 doses vs 2 doses only for PZQ montherapy) using oral gavage induces stress in mice and results in increased death rate, rather than any drug toxicity. Conclusively, the best anti-schistosomal and anti-fibrotic effects were recorded with PZQ and IS-5-MN combined therapeutic regimen. A careful interpretation of results from mouse studies might be helpful to clinical hepatology that can establish IS-5-MN as potential drug candidate for patients with chronic hepatic schistosomiasis.The ultrasound findings of echogenic areas accompanying portal branches and of gallbladder wall fibrosis are pathognomonic for schistosomiasis. Thus, a liver biopsy is usually not indicated. The finding of a portal vein enlarged to 15 mm is associated with a 20–35% risk of bleeding from oesophageal varices and suggests a roughly 33% future rebleeding risk even with antiparasitic treatment and endoscopic sclerotherapy. Well known to the clinician practising in endemic countries is that HBV, HCV, and hepatitis D virus co-infections as well as chronic alcohol use importantly increase the risk of variceal bleeding and affect ultrasound findings in schistosomiasis, although these features did not apply to our A wire-guided catheter is introduced via the jugular vein into the hepatic veins. Subsequently, by use of a needle, the portal vein is reached and the channel for the shunt is created by inflating a wire-guided angioplasty balloon within the liver along the tract created by the needle. Finally, a self-expandable mesh stent is placed and distended to keep the anastomosis patent. TIPS=transjugular intrahepatic portosystemic shunt. Arrows show dilated portal vein and tortuous collateral vessels.Left, portal flow (arrow) after transjugular intrahepatic portosystemic shunt (TIPS) implantation. Centre and right, contrast ultrasonography showing TIPS in situ (arrows) and perfusion of fibrotic areas.patient. Furthermore, because of the need for blood transfusions during variceal bleeding, patients with decompensated schistosomiasis are at an increased risk of acquiring viral hepatitis in areas where transfusions are not safe. Some portable ultrasound devices are equipped with Doppler ultrasound facilities. However, duplex ultrasound studies from centres in Brazil show that the interpretation of results is more difficult in schistosomiasis than in cirrhosis since in hepatosplenic schistosomiasis portal hypertension is multifactorial and is partly due to splenic hyperafflux. This difficulty results in a tendency to underestimate portal hypertension in schistosomiasis. Doppler ultrasonography is, however, useful to detect portosystemic collateral vessels. Although ultrasonographic protocols have been developed for field use, they can also be practical in well equipped hospitals because they enable an initial staging of liver fibrosis and international comparison of ultrasound findings. In such hospitals, endosonography, CT, and especially MRI can contribute further to the assessment of liver fibrosis and associated haemodynamic abnormalities.
Current treatment options
Treatment of variceal bleeding in patients with schistosomiasis includes endoscopic sclerotherapy, band- ligation, and devascularisation including various combinations of gastro-oesophageal devascularisation, portal systemic shunts, and splenectomy.Patients with advanced disease should always receive at least one full dose of antiparasitic praziquantel therapy to arrest the fibrotic process and to prevent cardiopulmonary schistosomiasis due to an occult persisting infection with a very low worm burden. For antibiotic therapy, knowledge of locally prevalent superinfecting bacteria and their sensitivity to antibiotics should be taken into account.Studies into the use of β antagonists have yielded controversial results.Other possible medical options, such as vasopressive substances used in liver cirrhosis, have not been systematically studied in patients with schistosomiasis.Presently, our young patient’s severe clinical situation is an indication for surgery. However, studies published on the outcome of surgery in schistosomiasis are not sufficiently systematic, differ in surgical methods, and do not report on indirect adverse events of splenectomy, such as superinfections and their prevention.In Brazil, techniques are being attempted to maintain the immunological functions of the spleen, such as partial splenectomy and reimplantation of spleen tissue in the omentum, but patients have never been followed up in malaria-endemic regions.Since our patient came from a malaria-endemic region, we did not remove the spleen despite confirmation of hypersplenism. The possibility of embolisation of the splenic artery has been offered to him, but for the time being he does not want to undergo another intervention because he is feeling well.TIPS is a reversible and a far less invasive alternative to surgery. A percutaneous, radiologically guided meso- caval shunt can be offered even to patients who have had portal vein thrombosis, a well known complication of surgery. Unfortunately, according to consensus conferences and existing guidelines on the indications for TIPS, schistosomiasis is neglected or only marginally mentioned and is misunderstood as merely a form of non-cirrhotic portal hypertension. Some investigators have argued that TIPS should not be used in schistosomiasis because hepatic synthetic function is normal in this disease and procedures that reduce portal pressures would lower hepatic perfusion and cause hepatic impairment. The risk of encephalopathy after shunt surgery would therefore be especially high in patients with schistosomiasis.We disagree with these assumptions because TIPS also reduces hepatic perfusion in the cirrhotic patient, whose liver is more susceptible and at a higher risk for further hepatic impairment than is a schistosomal fibrotic liver with a preserved parenchymal function.Furthermore, in schistosomiasis, portal hyperafflux related to hypersplenism contributes to portal hypertension to a higher extent than in cirrhosis, so the reduction of liver perfusion can be expected to be less. Hepatic encephalopathy should therefore also occur less frequently in patients with schistosomiasis, an observation confirmed in our case. Moreover, unlike in most surgical procedures, the spleen and its immunological function are preserved. Since liver function is preserved even in advanced schistosomal liver fibrosis, liver transplantation is, in our opinion, rarely indicated in schistosomiasis unless it is complicated by advanced liver cirrhosis or hepatocellular carcinoma of other aetiology such as chronic active viral hepatitis.
Global aspects
The technical facilities in specialised medical services in industrialised countries and emerging countries such as Brazil and China should be available to patients with severe schistosomal liver fibrosis, which can be also useful to assess the validity of field use of diagnostic methods in resource-poor countries. In this setting, TIPS is an alternative to surgery in patients with refractory schistosomal liver fibrosis and should be further assessed in centres experienced with this technique. The cost of one dose of schistosomiasis treatment in Africa is around $0•32. Even if re-exposure is not interrupted and reinfections continue to occur, repeated courses of single- dose therapy might suffice to prevent the development of severe liver fibrosis. By contrast, the annual cost of a 12-month follow-up of a patient treated with a TIPS in an industrialised country is around US$28 000, which is still cost effective compared with other therapeutic options. Several hundred cases of severe liver fibrosis could therefore be prevented with timely and cheap antiparasitic treatment.Although substantial progress has been achieved, the World Health Assembly’s treatment target of at least 75% of school-aged children at risk for both soil-transmitted helminth infections and schistosomiasis by 2010 has clearly not been met. Since progression of the disease is prevented by timely treatment and an affordable drug is available, there is no reason why severe morbidity due to this disease should not be consigned to history.
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