Cholangiocyte Organoids in Liver Transplantation; a Comprehensive Review.

Liver transplantation is the only curative option for many liver diseases that end up in liver failure, and cholangiopathy remains a challenging complication post-liver transplant, associated with significant morbidity and potential graft loss. The low availability of organs and high demand for transplantation motivate scientists to find novel interventions. Organoids, as three-dimensional cell cultures derived from adult cells or induced pluripotent cells, may help to address this problem. Different types of organoids have been described, from which cholangiocyte organoids offer a high level of versatility and plasticity for a deeper study of liver disease mechanisms. Cholangiocytes can be obtained from different segments of the biliary tree and have shown a remarkable capacity to adapt to new environments, presenting an effective system for studying cholangiopathies. Studies using cholangiocyte organoids show promising results for disease modeling, where organoids offer fundamental features to recapitulate the complexities of tissues in vitro and uncover fundamental pathological pathways to potentially reveal therapeutic strategies for personalized medicine. Organoids could hold the potential for regeneration of injured livers, representing tools of clinical impact in regenerative medicine when tissue damage is already present.

Extrahepatic Bile Duct Organoids as a Model to Study Ischemia/Reperfusion Injury During Liver Transplantation.

Biliary complications are still a major cause for morbidity and mortality after liver transplantation (LT). Ischemia/reperfusion injury (IRI) leads to disruption of the biliary epithelium. We introduce a novel model to study the effect of IRI on human cholangiocytes using extrahepatic cholangiocyte organoids (ECOs). Extrahepatic bile duct tissue was collected during LT at static cold storage and after reperfusion (n = 15); gallbladder tissue was used for controls (n = 5). ECOs (n = 9) were cultured from extrahepatic biliary tissue, with IRI induced in an atmosphere of 95% air (nitrogen), 1% O2 and 5% CO2for 48 h, followed by 24 h of reoxygenation. Qualitative and quantitative histology and qRT-PCR were performed to discern phenotype, markers of hypoxia, programmed cell death and proliferation. ECOs self-organized into circular structures resembling biliary architecture containing cholangiocytes that expressed EpCAM, CK19, LGR5 and SOX-9. After hypoxia, ECOs showed increased expression of VEGF A (p < 0.0001), SLC2A1 (p < 0.0001) and ACSL4 (p < 0.0001) to indicate response to hypoxic damage and subsequent programmed cell death. Increase in cyclin D1 (p < 0.0001) after reoxygenation indicated proliferative activity in ECOs. Therefore, ECO structure and response to IRI are comparable to that found in-vivo, providing a suitable model to study IRI of the bile duct in-vitro.

Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.

Nociceptive and inflammatory effects of subcutaneous TNFalpha.

Tumor necrosis factor alpha (TNF) is a potent pro-inflammatory cytokine that produces pain and hyperalgesia following injection. Its algesic effects are due to sensitizing actions on nociceptive primary afferents and to the upregulation of other pro-inflammatory and algesic proteins. In anesthetized rats, we investigated the effect of subcutaneously injected TNF on background activity and mechanical sensitivity of C nociceptors of the sural nerve, as well as its effects on cutaneous plasma extravasation. TNF sensitized C nociceptors dose-dependently; the optimal dose (5 ng) lowered threshold in 66.7% of the tested fibers. This sensitization occurred within 30 min and could last for 2 or more hours. Injected TNF had no effect on Abeta mechanoreceptive fibers. In addition, TNF evoked ongoing activity in 14% of C nociceptors and caused significant and dose-related increases in vascular permeability in glabrous skin. Our data suggest that TNF released during disease or after tissue injury participates in the generation of hyperalgesia and inflammation.

Hypertonic saline resuscitation: a tool to modulate immune function in trauma patients?

Hypertonic saline (HS) resuscitation has recently gained attention from trauma physicians because it may benefit the immune system of trauma patients. We have found that HS augments in vitro and in vivo immune function of healthy T-cells. In addition, HS restored the function of suppressed T-cells in vitro and in vivo and reduced immunosuppression after hemorrhage, protecting mice from subsequent sepsis. These effects of HS are based on its direct influence on cellular signaling events through specific signaling pathway(s) that include protein tyrosine kinase and mitogen-activated protein kinase p38 activation. HS provides a costimulatory signal that enhances the proliferation of activated T-cells. HS may be able to substitute signals lost through blockage as a result of trauma induced suppressive factors, thereby restoring the function of suppressed T-cells. Although further work is needed to determine the optimal conditions and possible risks of HS resuscitation, the data presented in this short review of our recent work shed a favorable light on HS as a simple but effective tool to modulate cellular immune function after trauma.

CNTF and GDNF, but not NT-4, support corticospinal motor neuron growth via direct mechanisms.

Axotomy and neurodegenerative diseases cause corticospinal motor neuron (CSMN) degeneration. We previously showed that CNTF, NT-4 and GDNF can support CSMN survival in enriched preparations. Here we developed a fluorescence-activated cell sorting method to highly purify CSMN (97+/-4.6%). We tested the neurotrophic activities of CNTF, NT-4 and GDNF on enriched and purified CSMN preparations. Similar to their effects on enriched CSMN preparations, CNTF and GDNF sustained the survival of purified CSMN for at least 5 days with ED50 values of 1.28+/-0.46 nM and 0.59+/-0.39 nM, respectively. In contrast, NT-4 supported survival of enriched but not of purified CSMN, indicating that CNTF and GDNF sustain motor neuron survival by direct action of CSMN, while NT-4 requires accessory cells present in enriched CSMN preparations.

Neurotrophin-4 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) promote the survival of corticospinal motor neurons of neonatal rats in vitro.

We have used enriched dissociated, low density cultures of neonatal rat corticospinal motor neurons to evaluate the survival-promoting effect of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) and the ciliary neurotrophic factor (CNTF). Our current findings demonstrated that CNTF promoted the survival of corticospinal motor neurons, in the same fashion and at an equivalent potency, as was previously described using a different assay system. Among the other factors tested, we also found that NT-4 and GDNF increased the number of surviving neurons in a dose-dependent manner, whereas NGF, BDNF and NT-3 showed no survival promoting effect on corticospinal motor neurons.

C-nociceptor sensitization by isoprostanes is cyclooxygenase dependent.

Isoprostane E(2) (8-iso-PGE) and F(2alpha) (8-iso-PGF) sensitize nociceptors and capsaicin-sensitive DRG neurons. In this study we investigated the cyclooxygenase-dependence of isoprostane-induced C-nociceptor sensitization. Systemic pretreatment of rats with ketorolac (1 and 10 mg/kg) abolished 8-iso-PGF sensitization and reduced the effects of 8-iso-PGE. Ibuprofen (30 mg/kg) blocked all sensitizing effects. These data suggest that some algesic properties of isoprostanes are mediated via prostanoid synthesis.

Antibody directed against GD(2) produces mechanical allodynia, but not thermal hyperalgesia when administered systemically or intrathecally despite its dependence on capsaicin sensitive afferents.

Anti-GD(2) antibodies have been shown to be effective for immunotherapy of neuroblastoma and other GD(2) enriched malignancies. Infusion of anti-GD(2) antibodies frequently causes spontaneous pain and allodynia for the duration of the immunotherapy and occasionally longer lasting neuropathic pain. Bolus intravenous injection of anti-GD(2) in rats initiates mechanical allodynia as measured by withdrawal threshold of the hindpaws. In this study, thermal thresholds were measured prior to and for up to 6 h following systemic anti-GD(2) administration in adult rats. In addition, both thermal and mechanical thresholds were tested following intrathecal administration of anti-GD(2) and IgG(2a). Murine anti-GD(2) elicited mechanical allodynia when administered into either the vasculature or the intrathecal space. Effective systemic doses were 1--3 mg/kg as previously shown. Intrathecally, optimal doses ranged from 0.01 to 0.1 ng; a higher dose was ineffective. Thermal hyperalgesia was not observed via either route of administration. Intrathecal pretreatment 48--72 h prior to the experiment with capsaicin at doses sufficient to cause a 50% depletion of dorsal horn CGRP, caused a total blockade of the mechanical allodynia indicating an involvement of peptidergic fine afferent fibers. It is likely that the antibody reacts with an antigen on peripheral nerve and/or myelin to initiate its effect. The lack of observed thermal hyperalgesia is surprising especially in light of the capsaicin-associated blockade, however, it is consistent with several other immune system related models of pain.

Isoprostanes induce plasma extravasation in rat skin.

Isoprostane E2 (8-iso PGE) and isoprostane F2 alpha (8-iso PGF) contribute to numerous vascular, proinflammatory, and nociceptive functions. The underlying mechanisms for many of their actions are still under investigation. We examined the ability of isoprostanes to promote cutaneous inflammation using the Evan's blue dye method. Our data show that 4 micrograms subcutaneously (s.c.) injected 8-iso PGE or 8-iso PGF induced plasma extravasation in glabrous rat skin. Dye extravasation was also elicited in hairy skin after injections of 8-iso PGE, but not after 8-iso PGF. Isoprostane-evoked dye extravasation can be reduced by pretreatment with both the S+ and R- isomers of the cyclooxygenase (COX)-inhibitor ibuprofen (30 mg/kg intraperitoneally), indicating perhaps a nonspecific inhibition; pretreatment with ketorolac (1 and 10 mg/kg i.v.) was without effect. Unlike isoprostane-induced cutaneous nociceptor sensitization, which is blocked in a stereospecific and dose-dependent manner by COX-inhibitors, the effect of these drugs on isoprostane-induced cutaneous plasma extravasation is less consistent. We conclude that at least a large component of the isoprostane effect on cutaneous plasma extravasation is COX-independent.

Retinal morphology of cyprinid fishes: a quantitative histological study of ontogenetic changes and interspecific variation.

Morphological patterns of the retina, cone size and density, rod density, rod-cone ratio, ganglion cell density, convergence of receptor cells, resolving power (RP) and regionalization were examined throughout life history in roach and in adults of asp, bream, common carp, roach and sabre carp. The retina of hatchlings is packed with small cones. During larval and juvenile growth the retina stretches, cones increase in diameter and rods are present in increasing numbers. Photopic and scotopic sensitivity as well as resolving power increase. Comparison of adults shows distinct interspecific differences in retinal parameters, which can be related to life style.

Stability behaviour of molsidomine-containing pellet formulations.

Molsidomine in mixtures with different inactive ingredients has been subjected to a stability test. The fingerprint chromatogram obtained by HPLC with diode-array detection of mixtures of molsidomine with povidone 25 revealed decomposition products; the detection wavelength of 210 nm resulted in easy detection of the degradation products. Molsidomine-containing pellets were manufactured according to a compact procedure and by applying the active ingredient to placebo pellets. Compared with the nonpareil pellet formulations, compact pellets have a considerably higher water content and undergo decomposition of the active ingredient after storage for 50 months under different conditions. It is assumed that the decomposition of molsidomine is accelerated by the peroxide found in povidone.

Oral supplements in HIV-infected patients with chronic wasting. A prospective trial.

AIM: Supplementation with polymeric formula diet was evaluated as second step of a nutritional intervention program for malnourished, but otherwise clinically stable HIV-infected patients. PATIENTS AND METHODS: 34 patients with weight loss, without opportunistic infections, diarrhea or fever, not responding to nutritional counselling as first intervention, were included. They were given 1000 kcal per day as polymeric formula. If further weight loss occurred, dose was increased to 1500 kcal per day. Nutritional status was determined by body weight and bioelectrical impedance. RESULTS: After four weeks, 7/34 patients already stopped intake. Only 10/34 patients completed three months of treatment. The average intake during three months was 496 +/- 363 kcal/d. Reasons for drop-out were intervening clinical deterioration in eight, early improvement in six, but intolerance or loss to follow-up in ten patients. On average, body weight did not change. Intake of formula diet and weight changes were not correlated. CONCLUSION: Oral supplements are a feasible, cost-effective and safe treatment of malnutrition, but they are accepted only for short time and in limited amount. To estimate efficacy of this treatment, clinical reasons for malnutrition must be considered. More effective nutritional treatments are needed for chronically malnourished HIV infected patients.

Aplasia cutis congenita of the scalp: how much therapy is necessary in large defects?

AIM: To show that local antibiotic management and a regular inspection of aplasia cutis congenita of the skull can give an excellent result. METHOD: This case reports a girl born with aplasia cutis congenita of the skull presenting with a large aplasia of the epidermis, dermis, subcutaneous tissue and galea, including a bone defect without any additional risk factor, e.g. early eschar formation, cerebrospinal fluid leakage or uncommon dural blood vessels. RESULTS: A primarily conservative treatment with local wet and antibiotic dressings together with a systemic antibiotic treatment for the first 2 wk led to an excellent result and thus prevented untimely operative and peri-operative procedures. CONCLUSIONS: Here we have shown that conservative treatment might be an option, even if the wound diameter is greater than 1 cm(2), to prevent infants from any untimely operative procedure with an elevated operative risk if any additional risk factors are excluded.