Amelioration of hepatic fibrosis via Padma Hepaten is associated with altered natural killer T lymphocytes.

Hepatic fibrosis is the end-stage consequence of chronic liver disease, affecting many people worldwide. Unlike the anti-fibrotic effect of natural killer (NK) cells, CD8 and NK T subsets are considered as profibrogenic subsets. Padma Hepaten is a multi-compound herbal preparation derived from Tibetan medicine and has proven efficacy in some clinical trials and tests at the cellular level. In this study, we evaluate the immune efficacy of Padma Hepaten administered intraperitoneally (i.p.) and/or orally in a mice model of hepatic fibrosis. Hepatic fibrosis was induced by 6 weeks of biweekly i.p. carbon tetrachloride (CCl4) injections in male C57Bl6 mice. There were four groups, including naive mice, non-treated fibrotic mice and fibrotic mice treated by Padma Hepaten at weeks 5-6 of fibrosis induction either orally or by i.p. injections. Padma Hepaten was prepared at 10 mg/ml in saline and 250 microl (2.5 mg) were administered four times per week. After week 6, animals were killed. To isolate a Padma Hepaten-associated effect on lymphocytes, splenocytes were harvested from either naive or Padma Hepaten-treated non-fibrotic donors. Isolated splenocytes were therefore reconstituted into two groups of irradiated recipients. Recipients were then administered the same CCl4 regimen. Hepatic fibrosis was determined by sirius red staining of liver sections and by assessment of alpha smooth muscle actin expression compared with beta-actin (both by mRNA as well as the protein liver extract western blotting). Hepatic fibrosis and alanine aminotransferase serum levels were decreased significantly in both Padma Hepaten-treated groups compared with the non-treated fibrotic group. Padma Hepaten treatment was associated with attenuation of lymphocyte subsets in both treated groups. Using a chemiluminescence technique to assess total anti-oxidant capacities (TAC), it was found that both the plasmas and livers of mice treated by CCl4 had significantly higher TAC compared with controls. However, the levels of TAC in animals treated either by CCl4 alone or CCl4 with Padma Hepaten were similar. Adoptive transfer of Padma Hepaten-treated lymphocytes was associated with fibrosis amelioration compared with recipients with naive lymphocytes. CCl4 generates higher levels of anti-oxidant capacities, probably as a response to oxidative stress. Padma Hepaten administration attenuated hepatic fibrogenesis significantly, accompanied by attenuation of lymphocyte but not anti-oxidant capacities.

Immunomodulatory effects of plasminogen activators on hepatic fibrogenesis.

Tissue-type plasminogen activators (tPA) and urokinase-type plasminogen activators (uPA) are involved in liver repair. We examined the potential immunomodulatory actions of uPA, tPA and uPA-receptor (uPAR) in carbon-tetrachloride-induced hepatic fibrosis in wild-type (WT), tPA-/-, uPA-/- and uPAR-/- mice. Carbon-tetrachloride treatment increased fibrosis in four groups but significantly less in three knock-out models. Serum cytokines and intrahepatic T cells elevated significantly following fibrosis process in WT animals but not in the knock-out groups. In culture, uPA increased lymphocyte proliferation significantly in WT and uPA-/- but not uPAR-/- animals. Following uPA exposure in vivo, there was CD8 predominance. To isolate uPA's effect on lymphocytes, WT mice were irradiated sublethally and then reconstituted with WT or uPA-/- lymphocytes. In these animals fibrosis was decreased and T cells were reduced in the uPA-/- recipients. Based on these data we postulate that plasminogen activators affect fibrosis in part by liver-specific activation of CD8 subsets that govern the fibrogenic activity of hepatic stellate cells.

Clinical characteristics of eyes demonstrating atypical patterns in scanning laser polarimetry.

PURPOSE: To characterize which clinical features are associated with the occurrence of atypical birefringence patterns (ABP) occasionally seen with scanning laser polarimetry (SLP). METHODS: Sixty-one subjects, including glaucoma patients, glaucoma suspects, and normal subjects, underwent a full clinical examination, standard visual field (VF) test, and a GDx-VCC SLP examination. One eye was selected from each patient. The magnitude of ABP was determined in two independent ways: using a support vector machine analysis (typical scan score (TSS)) and by a masked experienced observer. We assessed whether the magnitude of ABP was correlated with age, gender, the refractive state of the eye, corneal polarization axis and magnitude, GDx global parameters (TSNIT and NFI), and the VF status, as evident from glaucoma hemifield test (GHT), mean deviation (MD), and the pattern standard deviation (PSD). RESULTS: Of the 61 study eyes, 27 (44%) showed an ABP, based on a TSS cutoff (<82.5). A very high correlation was found between the TSS score and the masked experienced observer score (r(2)=0.80; P<0.001). The following clinical parameters were found, on bivariate analysis, to be significantly correlated with the presence of an ABP: age (r(2)=0.086; P=0.02); corneal polarization magnitude (r(2)=0.069; P=0.04); TSNIT (r(2)=0.16; P<0.001). CONCLUSION: The presence and magnitude of ABP did not seem to be closely correlated with most clinical parameters. A low, but statistically significant, correlation was found for age and corneal polarization magnitude (r(2)=0.086 and 0.069, respectively). A low-medium correlation was found for TSNIT (r(2)=0.16); however, we speculate that this might represent a confounding effect, rather than an underlying association. We conclude that none of the clinical parameters investigated in this study appears to be strongly correlated with the presence of an ABP on SLP scans performed using the commercially available GDx-VCC.

Test-retest variability in visual field testing using frequency doubling technology.

PURPOSE: To quantify the magnitude of test-retest variability (TRV) for normal subjects in serial visual fields (VF) using the frequency doubling technology (FDT) instrument. METHODS: Twenty-one healthy adults, aged 23 to 60 years, underwent four serial FDT VF tests, using the full-threshold C-20 program of the Zeiss-Humphrey FDT analyzer, on one randomly chosen eye. The VF tests were spaced 2 to 4 days apart. All subjects performed two preliminary FDT tests in order to minimize any learning effect. Test-retest variability was calculated as the standard deviation of each location's sensitivity value across the four VF tests. RESULTS: Mean TRV (+/-SD) for the entire field was 2.44+/-1.32 dB. Mean TRV (+/-SD) for the superior, inferior, nasal, and temporal hemifields were 2.48+/-1.3, 2.40+/-1.4, 2.40+/-1.3, and 2.48+/-1.3 dB, respectively. Mean TRV (+/-SD) for the foveal location, the 4 central, and the 12 peripheral locations were 2.49+/-1.4, 2.16+/-1.2, and 2.54+/-1.4 dB, respectively. CONCLUSIONS: TRV was found to be rather uniform across the visual field of the commercially available FDT device, with only a mild, clinically insignificant, effect of both eccentricity and age on TRV. Variability in the FDT VF, for normal subjects, was found to be more uniform than that of both standard and short wavelength automated perimetry. In addition, a strong inverse correlation was found, in normal subjects, between the mean sensitivity and TRV.

Lymphocyte-hepatic stellate cell proximity suggests a direct interaction.

Recent functional research studies suggest an anti-fibrotic role for natural killer (NK) cells coupled with a profibrotic role for CD8 cells. However, the morphological cellular interplay between the different cell types is less clear. To investigate lymphocyte/hepatic stellate cell (HSC) interactions, hepatic fibrosis was induced by administering carbon tetrachloride (CCl4) intraperitoneally (i.p.) for 4 weeks in C57Bl/6 mice. Animals were killed at 0, 1, 2, 3 and 4 weeks. Liver sections were stained for Sirius red. Confocal microscopy was used to evaluate alpha smooth-muscle actin (alphaSMA) and lymphocyte subsets in liver sections. At weeks 0 and 4, liver protein extracts were assessed for alphaSMA by Western blotting and isolated liver lymphocytes as well as HSC were analysed by fluorescence activated cell sorter (FACS). Similar to the results obtained from classical Sirius red staining and alphaSMA blotting, analysis of liver sections by confocal microscopy revealed a marked and continuous accumulation of alphaSMA staining along sequential experimental check-points after administering CCl4. Although the number of all liver lymphocyte subsets increased following fibrosis induction, FACS analysis revealed an increase in the distribution of liver CD8 subsets and a decrease of CD4 T cells. Confocal microscopy showed a significant early appearance of CD8 and NK cells, and to a lesser extent CD4 T cells, appearing only from week 2. Lymphocytes were seen in proximity only to HSC, mainly in the periportal area and along fibrotic septa, suggesting a direct interaction. Notably, lymphocyte subsets were undetectable in naive liver sections. Freshly isolated HCS show high expression of major histocompatibility complex (MHC) class II and CD11c. In the animal model of hepatic fibrosis, lymphocytes infiltrate into the liver parenchyma and it is thought that they attach directly to activated HSC. Because HSCs express CD11c/class II molecules, interactions involving them might reflect that HSCs have an antigen-presenting capacity.