Development of liver microtissues with functional biliary ductular network.

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

MicroRNA-195 vector influence on the development of gradually induced hepatocellular carcinoma in murine model.

Background: Recent studies implicate the role of microRNAs in the pathogenesis of hepatocellular carcinoma (HCC). This study was designed to induce HCC, in an experimental model, with the prospect to study the molecular pathophysiologic changes accompanying the development of HCC and the effect of miRNA-195 vector on the process of hepatocarcinogenesis.Methodology: This study incorporated three groups of male albino mice; one control group and two other groups injected intraperitoneal with diethylnitrosamine (DEN) weekly for 12 weeks for the gradual induction of HCC. The third group was injected intra-hepatic with miR-195 vector 1 month after DEN injection. At the 8th and 12th weeks post-DEN treatment, the tumor-associated biomarkers alpha-fetoprotein (AFP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α) were assessed in the serum of all mice. Hepatic specimens were subjected to ultra-structural pathological examination as well as to caspase-3 and survivin genes expression analysis.Results: All the assessed serological and molecular parameters of HCC development, in the miRNA-195-treated group of mice, showed a significant increase, versus the DEN-treated group, whereas survivin was significantly down-regulated, in the miR-195-treated group (P < 0.001). Additionally, ultra-structural criteria of HCC were depicted, in the 12th week, in DEN-injected group, versus the 8th week, in the miRNA-195-treated group.Conclusions: Intra-hepatic injection of miRNA-195 vector induced apoptotic gene expression and suppressed anti-apoptotic gene but these favorable anti-cancer effects could not counteract the inflammatory, and subsequently, the oncogenic effect probably caused by vector administration. Therefore, further studies are required to investigate the effect of miRNA in combination with anti-inflammatory medications.

Formulation and Evaluation of Novel Hybridized Nanovesicles for Enhancing Buccal Delivery of Ciclopirox Olamine.

Ciclopirox olamine (CPO) is a topical wide-spectrum antimycotic agent that possesses antifungal, antibacterial and anti-inflammatory activities. Loading CPO into a hybridized vesicular system is expected to enhance its buccal permeation and hence, therapeutic activity, whereas the frequent administration and side effects are reduced. Vesicular systems with high penetration ability were prepared based on cholesterol, Lipoid S45 or Phospholipon 90H, with span 60 while incorporating a penetration enhancer (Labrafac or labrasol) followed by full assessment of their size, entrapment efficiency, and drug release profiles. The optimum formulation, composed of Lipoid S45 and Labrafac, possessed the smallest vesicle size (346.1 nm), highest entrapment efficiency (94.4%), and sustained CPO release pattern, and was characterized for its morphology and thermal properties. This powerful mixture of the penetration enhancers (Lipoid S45 and Labrafac) in the designed hybridized vesicles was thoroughly investigated for their characteristics after being incorporated in bioadhesive gel. Moreover, enhanced antifungal activity was demonstrated either upon testing the designed formulation on agar plates or in vivo upon treating infected rabbits with the proposed formulation. Results suggest that the presented bioadhesive gel incorporating the CPO-loaded vesicles can be a promising delivery system that can offer a prolonged localized antifungal treatment with enhanced therapeutic effect.

Metformin inhibits mTOR-HIF-1α axis and profibrogenic and inflammatory biomarkers in thioacetamide-induced hepatic tissue alterations.

The potential inhibitory effect of the antidiabetic and anti-inflammatory drug, metformin on thioacetamide (TAA)-induced hepatotoxicity associated with the inhibition of mammalian target of rapamycin (mTOR)-hypoxia-inducible factor-1α (HIF-1α) axis has not been investigated before. Therefore, we tested whether metformin can protect against liver injuries including fibrosis induced by TAA possibly via the downregulation of mTOR-HIF-1α axis and profibrogenic and inflammatory biomarkers. Rats either injected with TAA (200 mg/kg; twice a week for 8 weeks) before being killed after 10 weeks (model group) or were pretreated with metformin (200 mg/kg) daily for 2 weeks before TAA injections and continued receiving both agents until the end of the experiment, at Week 10 (protective group). Using light and electron microscopy examinations, we observed in the model group substantial damage to the hepatocytes and liver tissue such as collagen deposition, infiltration of inflammatory cells, and degenerative cellular changes with ballooned mitochondria that were substantially ameliorated by metformin. Metformin also significantly ( p < 0.05) inhibited TAA-induced HIF-1α, mTOR, the profibrogenic biomarker α-smooth muscle actin, tissue inhibitor of metalloproteinases-1, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), alanine aminotransferase (ALT) and aspartate aminotransferase in harvested liver homogenates and blood samples. In addition, a significant ( p < 0.01) positive correlation between hypoxia scoring (HIF-1α) and the serum levels of TNF-α ( r = 0.797), IL-6 ( r = 0.859), and ALT ( r = 0.760) was observed. We conclude that metformin protects against TAA-induced hepatic injuries in rats, which is associated with the inhibition of mTOR-HIF-1α axis and profibrogenic and inflammatory biomarkers; thus, may offer therapeutic potential in humans.

Immunohistochemical and electron microscopic morphometric image analysis of hepatocellular carcinoma in association of HCV infection.

Early detection of hepatocellular carcinoma (HCC) is crucial for successful therapy. The present work examined the value of ultrastructural morphometric image analysis of hepatocyte nuclei in patients with chronic hepatitis C virus (HCV) versus HCC cases with chronic HCV and the corresponding surgical tumor-free safe margins (TFMs), to highlight any early predictive signs of neoplastic cellular transformation. This work also performed an immunohistochemical assessment of cytokeratin 19 (CK19) and Ki-67-positive cells to visualize any associated proliferative activity in the examined groups. The results showed significant decrease in the hepatocyte nuclear surface areas in the HCC and TFMs versus those in the HCV cases. The hepatocyte nucleolar surface area was significantly increased in the HCC cases versus that in the HCV cases. This increase was associated with a significant increase in Ki-67-positive cells in the HCC cases compared to those in the other groups. Conversely, the mean number of CK 19-positive cells was significantly reduced in the HCC cases compared to the cell numbers in TFMs and HCV cases with severe hepatic fibrosis. Liver progenitor cells (LPCs) were discerned in the reactive ductules and canaliculo-ductular junctions that characterized TFMs. LPCs were sporadically distributed in the liver lobules and reactive bile ductules in the HCC samples. In conclusion, CK 19 represents an important marker for distinguishing between dysplastic and malignant liver nodules. Electron microscopic morphometric image analysis may be considered as adjunct factor for assessing hepatocyte malignant transformation. Wider scale studies are needed to authenticate these results.

Potential ultrastructure predicting factors for hepatocellular carcinoma in HCV infected patients.

Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition. In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells. For reliability, the results can be subjected to cohort longitudinal study.

Spotlight on the three main hepatic fibrogenic cells in HCV-infected patients: Multiple immunofluorescence and ultrastructure study.

The present work deals with the simultaneous ultrastructure and triple immunofluorescence study of the three main hepatic fibrogenic cells, hepatic stellate cell, myofibroblast (MF), and fibroblast, in a group of hepatitis C virus (HCV) RNA positive patients, as their exact interrelation behavior in vivo with the progress of hepatic fibrosis is still inadequate. In this study, for the first time, cells having the morphological characteristic of MF and not bone marrow fibrocytes were revealed in liver portal vessels. This necessitates the reevaluation of the available knowledge concerning bone marrow fibrocyte. Also, the distribution, cellular interrelations, and the fate of MF were highlighted.

Value of reelin for assessing hepatic fibrogenesis in a group of Egyptian HCV infected patients.

BACKGROUND: Development of non-invasive markers that can predict the stages of hepatic fibrosis without resorting to repeated liver biopsies is still an important goal to evaluate the effectiveness of antifibrotic treatment. The present work investigates the value of the assessment of peripheral circulating reelin, in which the liver represents its prime source, as a marker for monitoring hepatic fibrogenesis. METHODS: Seventy-four cases with chronic hepatitis positive for serum HCV RNA and 15 healthy volunteers were enrolled in this study. Assessment of reelin in the harvested serum and in 64 corresponding liver biopsies using immunofluorescence technique was done. The results were evaluated in relation to the stages and quantitative morphometric analysis of hepatic fibrosis as well as the serum levels of the validated biomarker hyaluronic acid. RESULTS: Significant correlation was detected between the levels of serum reelin and the semiquantitative assessment of reelin immunoreactivity in liver tissue, the stages of hepatic fibrosis, the morphometrically determined collagen and serum hyaluronic acid with a correlation coefficient of 0.675, 0.623, 0.479, 0.772, respectively with p<0.001. The sensitivity and the specificity of reelin for the determination of advanced (F2+F3) and significant fibrosis (F2-F4) were nearly comparable to the result of hyaluronic acid. In addition the area under curve (AUC) were 0.859, 0.871 for the reelin versus 0.878, and 0.891 for the hyaluronic acid. CONCLUSIONS: In conclusion serum reelin may be considered an additional useful parameter for monitoring the progression of hepatic fibrosis in HCV-infected patients specially in those with active rheumatological conditions which result in an increase in serum hyaluronic acid.

Innovated pirfenidone loaded lecithin nanocapsules for targeting liver fibrosis: Formulation, characterization and in vivo study.

Increasing interest in developing antifibrotic therapies became a paramount priority due to the globally raised incidence of deaths secondary to hepatic cirrhosis. This work deals with the development of innovative antifibrotic pirfenidone -loaded lecithin core nanocapsules. This with the intention to target the liver and to increase the drug bioavailability, reducing drug liver toxicity, and studying the associated hepatic microenvironment changes. PFD-loaded lecithin nanocapsules (PFD-LENCs) were prepared using the natural lipoid S45 for its dual benefits of being both a lipid and an amphiphilic surfactant. The selected formulation exhibited in vitro sustained drug release up to 24 h compared to free PFD, which is consistent with the studied pharmacokinetic profile. The studied cytotoxicity of PFD as well as PFD-LENCs exhibited negligible cytotoxicity in normal oral epithelial cells. For exploring the capability of the PFD-LENCs in reaching the liver; in vivo tracing using CLSM, in vivo biodistribution to the vital organs were conducted and electron microscopic examination for depicting nanoparticles in liver tissue was performed. Results revealed the capability of the prepared fluorescent LENC2 in reaching the liver, PFD-LENCs detection in the Disse space of the liver and the significant accumulation of PFD-LENCs in liver tissue compared to the other tested organs. The assessment of the necro-inflammatory, antioxidant and the anti-fibrotic effect of PFD-LENCs (50 & 100 mg/kg) exhibited a significant decrease of liver enzymes, TNF-α, TGF-ß, Col-1, α-SMA, and TIMP-1, and a significant increase of catalase enzyme and MMP2 compared to free PFD. EM studies, revealed often detection of dendritic cells in PFD-LENCs (100 mg/kg) treated mice and abnormal collagen structure which can represent an adjunct contribution to the antifibrotic mechanism of PFD-LENCs. In conclusion, the development of this innovative PFD loaded lecithin nanocapsules achieved a targeting ability to the liver, controlled drug release, thereby increase the PFD therapeutic value in downregulating hepatic fibrosis in adjunct with the reduction of liver toxicity.

Promising bioadhesive ofloxacin-loaded polymeric nanoparticles for the treatment of ocular inflammation: formulation and in vivo evaluation.

Our work tackles the combined advantages of both nanotechnology and the bioadhesive gel properties which were utilized to design an ocular drug delivery system that is capable to treat ocular inflammation. Nanoparticles encapsulating an antibiotic drug, ofloxacin, were fabricated using emulsion solvent evaporation technique adopting 23 full factorial design to evaluate the effect of formulation parameters: that is to say, the molecular weight of the polymer (polycaprolactone), amount of Kolliphor P188, and presence of the charge inducer (chitosan hydrochloride) on the measured responses: drug entrapment efficiency (EE%), particle size (PS), polydispersity index (PDI) and zeta potential (ZP). The results show that the optimized LPCL-NP2 formulation (composed of low molecular weight polycaprolactone, 500 mg of Kolliphor P188, 0.25% chitosan hydrochloride, and 50 mg ofloxacin)  displayed a sphere shape with EE%, PS, PDI, and ZP values of 89.73 ± 0.04%, 195.4 ± 13.17 nm, 0.323 ± 0.01, and 55.4 ± 0.66 mV, respectively. DSC study confirmed the amorphous nature of the drug. The optimized nanoparticle formulation was then further incorporated into the following two ocular formulations: gel (LPCL-NP2-G4) and in situ forming gel (LPCL-NP2-ISG4). The penetration of optimized ocular formulations was assessed by confocal laser scanning microscopy. The antimicrobial study was conducted  for the following three ocular formulations: LPCL-NP2 presented as eye drops, LPCL-NP2-G4, and LPCL-NP2-ISG4 as well as the market product using rabbits which were infected in their eyes with Escherichia coli. Results revealed that rabbits treated with LPCL-NP2-ISG4 demonstrated a remarkable antibacterial efficacy and evident low bacterial growth which was additionally assured by the histopathological examination of eye biopsies compared with the other investigated groups. Thus, a novel ofloxacin-loaded nanoparticle formulation based on polycaprolactone is presented in the form of mucoadhesive non-irritating in situ forming ocular gel possessing a superior antibacterial activity. Graphical abstract.

Hepatic stellate cells and fibrogenesis in hepatitis C virus infection: an ultrastructural insight.

An ultrastructural quantitative assessment of hepatic stellate cells (HSCs) was made in relation to hepatic fibrosis, apoptotic cellular changes, intracellular fat deposition, circulating inflammatory cells in the sinusoids, and the necroinflammatory activity in liver specimens of 33 patients proven to be positive for hepatitis C virus (HCV)-RNA by polymerase chain reaction with the intention that electron microscopy may throw more light on the role of HSCs in the complicated process of fibrogenesis. A detailed review concerning these parameters and observed evidence suggesting the potential properties of HSCs to recycle cellular debris into collagen fibers are reported.

HCV has transforming potential to retrovirus: an ultrastructure hypothesis.

The present work highlights intracellular viral morphogenesis and virus-host cell interactions in patients proved to be infected with HCV. The material of this study consisted of 28 liver biopsies taken from patients positive for serum HCV-RNA by polymerase chain reaction. Liver biopsies were processed for light and electron microscopic examination. Ultrastructural findings of this work supported a new hypothesis for the turnover of HCV to retrovirus and described the presumed involved mechanism. This novel perception offers important insights that can explain the vague mechanisms of HCV behavior in the infected hepatocytes.

Carbamoyl phosphate synthetase 1 (CPS1) as a prognostic marker in chronic hepatitis C infection.

This study aims to assess the value of carbamoyl phosphate synthetase 1 (CPS1), as a non-invasive serum marker, for the evolution of chronic HCV infection and hepatic fibrosis. Seventy-two patients with HCV positive serum RNA and 15 health volunteers were enrolled in this study. Out of 72 patients, 10 patients had decompensated liver with ascites. Quantitative analysis of CPS1 was performed in the harvested sera and corresponding liver biopsies using ELISA and immunohistochemistry techniques respectively. Also, mitochondrial count using electron microscopy, urea analysis and conventional liver tests were done. Patients were grouped into (F1 + F2) and (F3 + F4) representing stages of moderate and severe fibrosis respectively. Tissue and serum CPS1 (s.CPS1) correlated significantly in moderate and severe fibrosis. Patients with severe fibrosis showed significantly higher levels of s.CPS1 (p-value ≤ 0.05) and significantly lower mitochondrial counts (p-value = 0.0065) than those with moderate fibrosis. S.urea positively correlated with s.CPS1 only in the decompensated group, at which s.urea reached maximal levels. In conclusion, s.CPS1 is a potential non-invasive marker for the assessment of severity and progression of HCV in relation to mitochondrial dysfunction. Also, increased s.urea with the progression of the disease is mainly due to a concurrent renal malfunction, which needs further investigation.

A potential antibacterial wound dressing of cefadroxil chitosan nanoparticles in situ gel: Fabrication, in vitro optimization and in vivo evaluation.

Wound healing following skin injury is a natural phenomenon that usually lacks quality, rapidity, and aesthetics. Thus, the purpose of this study was to fabricate a new easily applied in situ gel of cefadroxil (CDX) loaded chitosan nanoparticles (CDX-CSNPs) that could promote wound healing, capable of inhibiting the possible accompanying bacterial infection. The nanoparticles were prepared by double emulsion technique and the influence of formulation parameters on drug entrapment efficiency (EE%), particle size (PS), polydispersity index (PDI) and zeta potential (ZP) were investigated using a full factorial design. The results show that the optimized CDX-CSNP1 composed of low molecular weight chitosan (0.2%w/v) was spherical with EE%, PS, PDI and ZP of 84.25 ±â€¯0.02, 408.30 ±â€¯53.17 nm, 0.458 ±â€¯0.048 and 22.80 ±â€¯0.57 mV, respectively. DSC and XRD studies confirmed the amorphous nature of the drug. After ensuring the safety and non toxicity of CDX-CSNP1 in situ gel through cytotoxic study, the antibacterial activity was evaluated using a rat skin infection model against Staphylococcus aureus. Compared to the rats treated with free CDX, the CDX-CSNP1 treated group revealed a remarkable accelerated wound healing process and bacterial clearance which was further confirmed by the histopathological examination of skin biopsies.

Value of the innovated technique agarose cell block in improving the sensitivity of urine cytology in cases of bladder carcinoma.

Proper handling and processing of urine sample can greatly improve diagnostic sensitivity. This work investigates the value of agarose cell block technique in processing urine samples simultaneously for light and electron microscopic examination, with the prospect to enhance the quality of diagnosis. The material of this study consisted of 45 voided urine samples, processed for the performance of Papanicolaou-stained urine smears, agarose cell blocks paraffin sections stained with hematoxylin & eosin, and electron microscopy-contrasted ultrathin sections. The studied technique increases the sensitivity of urine cytology and opens a new prospect for cytomorphological study.

Agarose cell block: innovated technique for the processing of urine cytology for electron microscopy examination.

Easy manipulation and preservation of cells in suspension through the different steps of sample processing for electron microscopy examination is essential for proper diagnosis. The author used agarose gel as an embedding media for processing cells in suspension for electron microscopic examination. The AgarCyto cell block procedure of Kerstens et al. (J Histochem Cytochem. 2000; 48: 709--718) was used to begin electron microscopic processing of exfoliated urothelial cells in voided urine or cells in suspension. Processing of agarose cell block simultaneously for light and electron microscopic examination represents a great advantage offered by this innovated technique.

Effect of Ferula assafoetida on experimental murine Schistosoma mansoni infection.

Ferula assafoetida is a hard, resinous, oily herbaceous gum belonging to plant family Umbelliferae. It is used as a traditional medicine in many parts of the world and its wide use in medicine was listed by many authors. In the current study, the effect of F. assafoetida on Schistosoma mansoni in experimentally infected mice is investigated. F. assafoetida was given orally via intragastric tube in an oil-form and a powder-form in different concentrations. Four test groups of 30 mice each were studied. Gs I & II mice were given F. assafoetida in an oil-form in different concentrations at 4 and 6 weeks post infection (PI) respectively. Mice in Gs III & IV were given re-constituted F. assafoetida powder in different concentrations (conc.) at 4 & 6 weeks PI. respectively like the previous groups. Oil-form F. assafoetida was given at conc. of 50, 25 & 15 mg/ml. Powder-form was given at conc. of 32, 16 & 8 mg/ml. A highly significant statistical difference between the test Gs (I, II, III & IV) was recorded in comparison to the infection control G with p value < 0.0001 and also between powder and oil forms of F. assafoetida (p < 0.0001) as regards the mean worm burden and tissue egg count. The highest reduction in worm burden and egg counts was found with powder form of F. assafoetida (Gs III & IV) when compared to oil form (Gs I & II), as confirmed histopathologically and by ultrastructural profile alteration.