Characterization of biliary epithelial cells isolated from needle biopsies of human liver in the presence of hepatocyte growth factor.

Current methods for the isolation of intrahepatic biliary epithelial cells from human liver rely upon relatively large segments of tissue, thereby limiting studies to cells isolated from patients with end-stage disease. To investigate a greater range of diseases and those at an earlier stage, we have developed a method to isolate biliary epithelial cells from biopsy-sized fragments of human liver. Tissue explants are cultured for > 4 weeks, and, in approximately 50% of samples incubated with medium containing hepatocyte growth factor, biliary epithelial cells begin to migrate from the fragments and proliferate. With time they form confluent pavements of cells that express cytokeratin 19 and gamma-glutamyl transpeptidase and are negative for markers of non-biliary cell phenotype. After subculturing, cells can be expanded, yielding substantial numbers for subsequent study in vitro. Cells can be isolated with a similar degree of success from adult normal liver, from a variety of liver diseases, and from post-transplant liver biopsies. Overall, pediatric tissue yielded cells less frequently than adult tissue. This novel technique is likely to have a major impact on the study of biliary pathophysiology, as small fragments of tissue removed from biopsies taken for diagnostic purposes can be used.     

Regulation of the human T-cell response toSchistosoma japonicum egg antigen by concomitant cellular and humoral mechanisms in vitro

Serum-mediated regulation of T-cell responses specific for soluble egg antigen (SEA) ofSchistosoma japonicum was tested in human hosts. When we added autologous serum to SEA-specific human T-cell lines (CD3+, 4+, 8–), we observed suppression of T-cell proliferation, and this suppressive activity was detected in the immunoglobulin-G2 (IgG₂) subclass. Suppression was dose-dependent and antigen-specific. T-cell proliferation induced by only one SEA fraction of >18 kDa was modulated in the presence of 100 g/ml autologous as well as allogeneic infected IgG₂. This SEA fractiondriven proliferation was also regulated by suppressor T-cells through distinct suppressive mechanisms. Our results suggest that T-cell responses to a particular component(s) of SEA are strictly regulated through both cellular and humoral mechanisms in human chronicS. japonicum infection.     

Astroglial responses against Abeta initially occur in cerebral primary cortical cultures: species differences between rat and cynomolgus monkey.

In the present study, we investigated how amyloid beta (Abeta) peptides initially affect neuronal cells in primary cerebral cortical cultures from rat and cynomolgus monkey. In these cultures, complicated interactions between glial and neuronal cells occur; moreover, synaptic interactions similar to those observed in vivo also occur between neuronal cells in these cultures. In this study, we applied low concentrations of Abeta to these well-characterized primary cultures to investigate how Abeta initially affects neurons or astroglial cells. In both rat and monkey cortical cultures, treatment with low concentrations of Abeta failed to drastically change or damage of neurons. Abeta treatment, however, significantly activated astrocytes, resulting in increased apolipoprotein E (ApoE) production. Rat astrocytes were more sensitive to Abeta than monkey astrocytes, and responded to Abeta via a different mechanism. In monkey astrocyte cultures, only direct treatment with Abeta increased ApoE production. In rat astrocyte cultures, however, treatment with conditioned media from cortical cultures grown with Abeta increased ApoE production, indicating that some sort of neuron-derived soluble factor(s) was also involved in activating rat astrocytes. These species differences suggest that monkey cortical cultures would be more useful as an in vitro model system to understand the details of how Abeta accumulates in the human brain, since monkeys are phylogenetically more similar to humans.     

The role of saliva of Anopheles stephensi in inflammatory response: identification of a high molecular weight neutrophil chemotactic factor

Mosquito bites can elicit dermal hypersensitivity reactions, but little is known about the chemotactic factors for host leukocytes in mosquito saliva. In this study, we determined that saliva from a malarial vector mosquito, Anopheles stephensi, possesses intense neutrophil chemotactic activity. In contrast, the midgut extract had only marginal neutrophil chemotactic activity. Eosinophil chemotactic activity was detected in the midgut but not in the saliva. According to the results of size-exclusion HPLC on a G3000SW column and Western blot analysis, the apparent molecular weight (MW) of the main neutrophil chemotactic factor (NCF) was estimated to be 200 kDa. NCF could bind with IgG from the pooled serum of Solomon islanders, whereas not with that of healthy Japanese. NCF activity was increased upon heating to 56 degrees C for 30 min or protease digestion, whereas it was affected by periodate treatment. Protease-digested NCF and naive NCF bound to lentil lectin-Sepharose, and both were eluted with a competitive sugar, methyl-alpha-D-glucoside. These results indicate that A. stephensi saliva-derived NCF is a high MW glycoprotein, and its protein moiety is important for neutrophil chemotactic activity. This NCF is thought to contribute to the inflammatory reactions through the accumulation of neutrophils at the site of the mosquito bite.     

Expression and localization of chromogranin A gene and protein in human submandibular gland

Human saliva chromogranin A (CgA) is clinically promising as a psychological stress marker. However, expression of CgA is poorly understood in humans, although salivary gland localization of CgA in other mammals, such as rodents and horses, has been demonstrated. In the present study, we investigated the expression and localization of CgA in the human submandibular gland (HSG) using various methods. CgA was consistently localized in serous and ductal cells in HSG, as detected by immunohistochemistry and in situhybridization. Reactivity was stronger in serous cells than in ductal cells. In addition, strong immunoreactivity for CgA was observed in the saliva matrix of ductal cavities. Western blotting gave one significant immunoreactive band of 68 kDa in the adrenal gland, HSG and saliva. Finally, CgA was detected in secretory granules of serous and ductal cells by immunoelectron microscopy. In conclusion, CgA in humans is produced by HSG and secreted into saliva.     

Excitability recovery curves of blink reflexes in patients with athetotic cerebral palsy

We evaluated the brainstem function or its excitability by the blink reflex evoked with the electrical stimulation to the supraorbital nerve in 10 patients with athetotic cerebral palsy compared with 10 normal subjects and 7 spastic type patients. There were no differences in stimulus intensity, latency of R1 and R2 components, and duration and area of EMG activity of the R2 component of the blink reflex elicited by single stimulation among the two patients' groups and normal subjects. R1 recovery cycle to paired stimuli in the athetotic group showed a facilitation of the test responses by the conditioning stimuli at 100 and 200 ms intervals, but were not significantly different from those in the normals. On the other hand, the R2 recovery curve in the athetotic group showed a significant hyperexcitability at all intervals from 100 to 600 ms compared to the normals. Our results from the R2 hyperexcitable recovery to paired stimuli are indicative of increased brainstem interneuronal excitability in athetotic patients and similar to the results reported in the disorders of the basal ganglia, i.e. Parkinson's disease, dystonia and blepharospasm. We suggest that this hyperexcitability might be caused by abnormal input possibly from the basal ganglia upon these brainstem interneurons.     

Treatment with cathepsin L inhibitor potentiates Th2-type immune response in Leishmania major-infected BALB/c mice

Prior to the activation of CD4 (+) T cells, exogenous proteins must be digested by endo/lysosomal enzymes in antigen-presenting cells (APC) to produce antigenic peptides that are able to be presented on class II molecules of the MHC. Studies described here inspect the functional significance of cathepsin L inhibition for antigen processing and T (h) 1/T (h) 2 differentiation in experimental leishmaniasis. We first demonstrated using in vitro systems that cathepsin L is one of the candidate endo/lysosomal enzymes in processing of soluble Leishmania antigen (SLA) and that its specific inhibitor, CLIK148, modulated the processing of SLA. BALB/c mice are known to be susceptible to infection with Leishmania major. Interestingly, treatment of BALB/c mice with CLIK148 exacerbated the infection by enhancing the development of SLA-specific T (h) 2-type response such as production of IL-4 and generation of T (h) 2-dependent specific IgE/IgG1 antibodies. Moreover, addition of CLIK148 in incubation of a SLA-specific CD4 (+) T cell line with APC up-regulated the production of IL-4. However, CLIK148 did not exert any direct influence on the function of T cells themselves. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APC, resulting in the potentiation of T (h) 2-type immune responses and thus leading to exacerbation of the infection. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.     

Voltammetry in unanesthetized rat: Increases of striatal dopamine turnover after unilateral haloperidol injection into the substantia nigra

In freely moving rats, effects of unilateral haloperidol injection into the substantia nigra were monitored with in vivo voltammetry in the bilateral striata. The electrochemical responses at 120 mV versus Ag-AgCl, reflecting mainly a level of 3,4-dihydroxyphenylacetic acid (DOPAC), increased both in the striata within 1.5 h after 5 μg of haloperidol treatment. In the experiments of high-performance liquid chromatography with electrochemical detection, the ratio of DOPAC to dopamine in the striata significantly increased at 2.75 h after drug treatment. These data support the idea that unilateral injection of haloperidol into the substantia nigra in freely moving rats increases dopamine turnover in the bilateral striata.     

Lymphocyte subpopulations and T-cell subsets in human oral cancer tissues: immunohistologic analysis by monoclonal antibodies

A series of T-cell-specific monoclonal antibodies (Leu-1, Leu-2a, and Leu-3a) and B-cell-specific monoclonal antibody (HLB-1) were used to detect the localization and intensity of infiltration of lymphocyte subpopulations and T-cell subsets in frozen sections of 17 patients with the oral cancer. The vast majority of the lymphocyte infiltrates in the oral cancer tissues were reactive with Leu-1. In contrast, B cells were detectable with HLB-1 in only 2 of 17 cases. Leu-2a-positive cells were dominant in four cases, whereas Leu-3a positive cells were dominant in only three cases. In seven cases, both cells infiltrated to the same degree. Leu-2a positive cells tended to be dominant in the cases with earlier clinical stages.