Dynamic distribution of ERK,p38 and JNK during the development of pancreatic ductal adenocarcinoma

We recently discovered that oncogenic c-kit is highly expressed concomitantly with the development of pancreatic ductal adenocarcinoma (PDAC). Since oncogenic c-kit may activate major pathways of protein tyrosine phosphorylation, we decided to investigate this issue in the major protein phosphorylation cascades. In normal pancreas labeling with antiphosphorylated ERK1/2 (pERK1/2) antibody was mainly confined to islets of Langerhans in close overlapping with insulin containing cells. Phosphorylated p38 (pp38) showed a similar pattern of distribution, while only weak labeling was evident for pJNK and no labeling of pMEK was observed. As expected, general ERK1/2 (gERK1/2), general p38 (gp38), general JNK (gJNK) as well as general MEK (gMEK) were all evident in islets of Langerhans and in the exocrine tissue. In early development of PDAC, pERK1/2 and pp38 retained their localization in islets of Langerhans. Intensive staining of pERK1/2 was also evident in the cancerous ducts, while the labeling with antibodies to pp38 was more moderate. While pJNK staining in islets of Langerhans was weak, with no labeling in the cancerous ducts, antibodies to gJNK revealed intensive staining suggesting the weak staining of pJNK is not due to the lack of the enzyme. In a more advanced stage of PDAC the carcinomas were clearly stained with pERK1/2 and pp38, while moderate staining with pJNK was also evident. In liver metastases, the cancer cells were heavily labeled with all three phospho-MAPKs. It should be noted that the localization of all three kinases was mainly in the cell nuclei. In the more advanced stage of PDAC, heavy labeling was evident using antibodies to gERK1/2, gp38, gJNK and gMEK. However, no labeling to pMEK was evident in parallel sections. Our data suggest that both in normal and cancerous pancreas, most of the MAPK activities are located in islets of Langerhans and cancerous ducts. It is suggested that using inhibitors to protein kinases may attenuate the progression of the disease.     

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30 μM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H₂O₂, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H₂O₂ with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.     

Treating inflammation by blocking interleukin-1 in humans

IL-1 is a master cytokine of local and systemic inflammation. With the availability of specific IL-1 targeting therapies, a broadening list of diseases has revealed the pathologic role of IL-1-mediated inflammation. Although IL-1, either IL-1α or IL-1β, was administered to patients in order to improve bone marrow function or increase host immune responses to cancer, these patients experienced unacceptable toxicity with fever, anorexia, myalgias, arthralgias, fatigue, gastrointestinal upset and sleep disturbances; frank hypotension occurred. Thus it was not unexpected that specific pharmacological blockade of IL-1 activity in inflammatory diseases would be beneficial. Monotherapy blocking IL-1 activity in a broad spectrum of inflammatory syndromes results in a rapid and sustained reduction in disease severity. In common conditions such as heart failure and gout arthritis, IL-1 blockade can be effective therapy. Three IL-1blockers have been approved: the IL-1 receptor antagonist, anakinra, blocks the IL-1 receptor and therefore reduces the activity of IL-1α and IL-1β. A soluble decoy receptor, rilonacept, and a neutralizing monoclonal anti-interleukin-1β antibody, canakinumab, are also approved. A monoclonal antibody directed against the IL-1 receptor and a neutralizing anti-IL-1α are in clinical trials. By specifically blocking IL-1, we have learned a great deal about the role of this cytokine in inflammation but equally important, reducing IL-1 activity has lifted the burden of disease for many patients.     

Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant Mycobacterium tuberculosis strains isolated in Australia.

Control of tuberculosis, the single largest killer among the infectious diseases, has been threatened by the emergence of multidrug-resistant Mycobacterium tuberculosis (MDRTB) infection due to the limited treatment options. Rifampicin (RIF) resistance is considered as a marker for MDRTB. The aim of this study was the detection of rpoB gene mutations and rifabutin resistance in MDRTB strains recently isolated in Australia by a line probe assay (INNO-LiPA Rif. TB, Innogenetics). Rifabutin and RIF susceptibility of 20 MDRTB and 16 RIF-sensitive M. tuberculosis complex clinical isolates were studied. The overall concordance of the line probe assay (LiPA) with phenotypic RIF susceptibility test was 96%. Seven distinct nucleotide substitutions were identified in 21 of 22 RIF-resistant isolates of diverse geographical origins, but in none of the RIF-sensitive strains. The majority (71%) of mutations occurred in the 526-533 codons and were associated with resistance to rifabutin and RIF. Of the RIF-resistant MDRTB strains, 18% appeared to be rifabutin-sensitive and produced delta S2 and delta S3 INNO-LiPA patterns. We conclude that amino acid substitutions at Asp516 and Ser522 in the rpoB gene in RIF-resistant M. tuberculosis predict rifabutin susceptibility for MDRTB. Use of the LiPA for RIF and rifabutin resistance may facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.     

Broadsheet. Number 50: Diagnosis of human cytomegalovirus infection and disease.

Human cytomegalovirus (CMV) remains an important cause of illness in immunocompromised individuals and the most common viral cause of congenital malformation. The tests available for diagnosis of CMV include serology, antigen detection, virus culture, tissue histopathology and nucleic acid detection. The diagnosis of CMV remains difficult because of the issues of virus latency, virus infection versus clinical disease and virus reactivation. The tests available and the use of these tests are undergoing significant changes. This Broadsheet presents a review of these tests, particularly in the diagnosis of congenital infection and infection in pregnant women and immunocompromised individuals.     

Dendritic cells in host response to biologic scaffolds

Tissue regeneration and repair require a highly complex and orchestrated series of events that require inflammation, but can be compromised when inflammation is excessive or becomes chronic. Macrophages are one of the first cells to contact and respond to implanted materials, and mediate the inflammatory response. The series of events following macrophage association with biomaterials has been well-studied. Dendritic cells (DCs) also directly interact with biomaterials, are critical for specific immune responses, and can be activated in response to interactions with biomaterials. Yet, much less is known about the responses by DCs. This review discusses what we know about DC response to biomaterials, the underlying mechanisms involved, and how DCs can be influenced by the macrophage response to biomaterials. Lastly, I will discuss how biomaterials can be manipulated to enhance or suppress DC function to promote a specific desirable immune response – a major goal for implantable biologically active therapeutics.     

Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na⁺–K⁺-ATPase α/β, Na⁺–K⁺–2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na⁺–K⁺-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na⁺–K⁺-ATPase β in the secretory coils was the same as Na⁺–K⁺-ATPase α, but Na⁺–K⁺-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.     

Epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital

BackgroundThe prevalence of respiratory viruses in adults is largely underexplored, as most studies focus on children. Additionally, in severely ill or immunocompromised adults, where respiratory infections are mostly attributed to bacteria and fungi; respiratory viruses can lead to severe complications.ObjectivesTo evaluate the epidemiology of respiratory viruses in bronchoalveolar lavage fluid (BAL) specimens from patients with lower respiratory tract disease. The study population consisted of different groups including immunocompetent patients (control patients), solid organ transplant recipients, patients with haematological malignancies and other immunocompromised adults.Study designA total of 134 BAL fluid specimens collected during 2009–2011 were retrospectively assessed with the new commercial multiplex real-time PCR FTD Respiratory 21 Plus^®, targeting 18 different viruses and 2 atypical bacterial pathogens.ResultsViral or atypical bacterial pathogens were detected in 29.1% of BAL fluid specimens. Coronaviruses were most prevalent (13.4%), followed by rhinoviruses (5.2%), RSV (4.5%) and bocaviruses (3.7%). Comparing the total number of viruses detected, a statistically significant difference was observed between the control group and patients with haematological malignancies (27.5% vs. 57.1%, p < 0.05).ConclusionIn conclusion, our study highlights the high prevalence of respiratory viruses in BAL fluid specimens from adult patients with lower respiratory tract disease. The methods to be used should be sensitive and cover a wide range of potential pathogens. The specific patient population can also influence the detection rates of respiratory viruses.     

MASP-1 of the complement system promotes clotting via prothrombin activation

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.     

Distribution of methionine-enkephalin in the minipig brainstem

We have studied the distribution of immunoreactive cell bodies and axons are containing methionine-enkephalin in the minipig brainstem. Immunoreactive axons were widely distributed, whereas the distribution of perikarya was less widespread. A high or moderate density of axons containing methionine-enkephalin were found from rostral to caudal levels in the substantia nigra, nucleus interpeduncularis, nucleus reticularis tegmenti pontis, nucleus dorsalis raphae, nucleus centralis raphae, nuclei dorsalis and ventralis tegmenti of Gudden, locus ceruleus, nucleus sensorius principalis nervi trigemini, nucleus cuneatus externalis, nucleus tractus solitarius, nuclei vestibularis inferior and medialis, nucleus ambiguus, nucleus olivaris inferior and in the nucleus tractus spinalis nervi trigemini. Immunoreactive perikarya were observed in the nuclei centralis and dorsalis raphae, nucleus motorius nervi trigemini, nucleus centralis superior, nucleus nervi facialis, nuclei parabrachialis medialis and lateralis, nucleus ventralis raphae, nucleus reticularis lateralis and in the formatio reticularis. We have also described the presence of perikarya containing methionine-enkephalin in the nuclei nervi abducens, ruber, nervi oculomotorius and nervi trochlearis. These results suggest that in the minipig the pentapeptide may be involved in many physiological functions (for example, proprioceptive and nociceptive information; motor, respiratory and cardiovascular mechanisms).     

The role of aquaporin-1 in idiopathic and drug-induced intracranial hypertension

Idiopathic intracranial hypertension is a common disorder affecting mainly healthy, young, overweight women. The pathogenesis of this condition is unknown, but it has been shown to follow treatment with several compounds including corticosteroids and vitamin A derivatives. This paper will offer a novel hypothesis and insight on the pathogenesis of drug induced intracranial hypertension following a review and analysis of the literature. Both corticosteroids and vitamin A derivatives have been shown to upregulate the expression of aquaporin 1, a water channel protein. Aquaporin 1 is widely distributed in the human brain and is associated with water secretion into the subarachnoid space. Aquaporin 1 was also shown to participate in the regulation of weight. Agents used for treating idiopathic intracranial hypertension reduce aquaporin 1 expression. Based on these observations, we propose that aquaporin 1 has a pathogenetic role in drug induced idiopathic intracranial hypertension. Over expression of this gene causes increased intracranial pressure, and downregulation reduces pressure and alleviates the symptomatology and complications of idiopathic intracranial hypertension.     

Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium

We investigated the relationship between mouse taste bud development and innervation of the soft palate. We employed scanning electron microscopy and immunohistochemistry using antibodies against protein gene product 9.5 and peripherin to detect sensory nerves, and cytokeratin 8 and α-gustducin to stain palatal taste buds. At E14, nerve fibers were observed along the medial border of the palatal shelves that tracked toward the epithelium. At E15.5, primordial stages of taste buds in the basal lamina of the soft palate first appeared. At E16, the taste buds became large spherical masses of columnar cells scattered in the soft palate basal lamina. At E17, the morphology and also the location of taste buds changed. At E18–19, some taste buds acquired a more elongated shape with a short neck, extending a variable distance from the soft palate basal lamina toward the surface epithelium. At E18, mature taste buds with taste pores and perigemmal nerve fibers were observed on the surface epithelium of the soft palate. The expression of α-gustducin was demonstrated at postnatal day 1 and the number of pored taste buds increased with age and they became pear-shaped at 8 weeks. The percent of pored fungiform-like papillae at birth was 58.3% of the whole palate; this increased to 83.8% at postnatal day 8 and reached a maximum of 95.7% at 12 weeks. The innervation of the soft palate was classified into three types of plexuses in relation to taste buds: basal nerve plexus, intragemmal and perigemmal nerve fibers. This study reveals that the nerve fibers preceded the development of taste buds in the palate of mice, and therefore the nerve fibers have roles in the initial induction of taste buds in the soft palate.     

Orexinergic innervation of urocortin1 and cocaine and amphetamine regulated transcript neurons in the midbrain centrally projecting Edinger–Westphal nucleus

Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger–Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2 h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either.     

Histological studies of neuroprotective effects of Curcuma longa Linn. on neuronal loss induced by dexamethasone treatment in the rat hippocampus

Long term exposure to dexamethasone (Dx) is associated with brain damage especially in the hippocampus via the oxidative stress pathway. Previously, an ethanolic extract from Curcuma longa Linn. (CL) containing the curcumin constituent has been reported to produce antioxidant effects. However, its neuroprotective property on brain histology has remained unexplored. This study has examined the effects of a CL extract on the densities of cresyl violet positive neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes in the hippocampus of Dx treated male rats. It showed that 21days of Dx treatment (0.5 mg/kg, i.p. once daily) significantly reduced the densities of cresyl violet positive neurons in the sub-areas CA1, CA3 and the dentate gyrus, but not in the CA2 area. However, CL pretreatment (100 mg/kg, p.o.) was found to significantly restore neuronal densities in the CA1 and dentate gyrus. In addition, Dx treatment also significantly decreased the densities of the GFAP-ir astrocytes in the sub-areas CA1, CA3 and the dentate gyrus. However, CL pretreatment (100 mg/kg, p.o.) failed to protect the loss of astrocytes in these sub-areas. These findings confirm the neuroprotective effects of the CL extract and indicate that the cause of astrocyte loss might be partially reduced by a non-oxidative mechanism. Moreover, the detection of neuronal and glial densities was suitable method to study brain damage and the effects of treatment.     

The centrally projecting Edinger–Westphal nucleus—I: Efferents in the rat brain

The oculomotor accessory nucleus, often referred to as the Edinger–Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW—EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW—EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW–LHA and EW–CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.