Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.)

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.     

Pathogenesis of IgA nephropathy: In vitro activation of human mesangial cells by IgA immune complex leads to cytokine secretion

IgA immune complex (IC) plays a crucial role in the pathogenesis of IgA nephropathy (IgAN). As IgA-IC is not itself cytotoxic, other mediators may be involved in the pathogenesis. In order to elucidate the mechanisms by which IgA-IC mediates renal injury in IgAN, the ability of IgA-IC to ‘activate’ cultured human mesangial cells (HMC) was studied. HMC were incubated with nephritogenic IgA-IC, containing a MOPC-315 plasmacytoma-derived IgA anti-dinitrophenyl (DNP) and DNP-conjugated bovine serum albumin. The cells showed morphological changes, an accelerated rate of proliferation, and increased production of interleukin-1 (IL-1), interleukin-6 (IL-6), platelet activating factor (PAF) and generation of superoxide anion. The enhancement of IL-1 and IL-6 mRNA expression in HMC incubated with IgA-IC was identified by dot blot analysis. Northern blot hybridization also demonstrated an augmented IL-6 mRNA expression in HMC treated with IgA-IC. These results suggest that nephritogenic IgA-IC may amplify the proliferation of HMC and the production of immune/chemical mediators and superoxide anion thereby resulting in the renal lesions of IgAN.     

Chronic oral administration of dexamethasone to rats increases cytotoxicity, but not interleukin-1 elaboration, by alveolar macrophages.

Chronic administration of corticosteroids has been used to induce pulmonary infection in animals. but the specific mechanisms by which corticosteroids modulate pulmonary host defence have not been clarified. Specifically, little is known about how corticosteroids influence the function of lung cells, such as alveolar macrophages. Cytotoxicity and interleukin-1 elaboration are two important mechanisms of macrophage defence against pathogens. To determine whether chronic administration of dexamethasone alters cytotoxicity and interleukin-1 elaboration by alveolar macrophages, we studied alveolar macrophages from rats treated with oral doses of dexamethasone for 2 weeks. We found that unstimulated alveolar macrophages from dexamethasone-treated rats exhibited increased cytotoxicity compared with alveolar macrophages from control rats. Moreover, alveolar macrophages from both groups of rats showed enhanced cytotoxicity after in vitro interferon-gamma and lipopolysaccharide treatment, in a dose-dependent manner. Although no spontaneous interleukin-1 elaboration was detected from alveolar macrophages from either group of rats, lipopolysaccharide increased interleukin-1 elaboration by alveolar macrophages from both groups of rats equivalently. These results indicate that chronic oral administration of dexamethasone to rats increases cytotoxicity, and does not alter the capacity of alveolar macrophages to elaborate interleukin-1. Therefore, chronic corticosteroid administration appears to produce selective alterations in these defence mechanisms of alveolar macrophages.     

In vitro effects of interleukins on human mesangial cells: Implications for glomerulonephritis

Intrinsic glomerular cells, especially mesangial cells, are considered to be actively involved in the pathogenesis of glomerulonephritis (GN), but the precise mechanism(s) remains elusive. We have previously demonstrated that nephritogenic IgA immune complex can stimulate human mesangial cells (HMCs) to increase their production of interleukin-1 (IL-1) and interleukin-6 (IL-6). In order to evaluate the roles of cytokines such as IL-1 and/or IL-6 and mesangial cells as mediators of renal injury in GN, we have now examined the changes of HMCs and their secreted products in vitro, after stimulation with various concentrations of IL-1 and IL-6. Cytokine-activated HMCs showed the following changes: (1) increased cell size, with intracytoplasmic vaculoes, dilated endoplasmic reticulum, increased free ribosomes and polysomes, and mitochondrial swelling; (2) increased cell proliferation, reflected in thymidine incorporation and an increased proportion of S and G2/M phase cells by cell cycle analysis; (3) enhancement of IL-6 mRNA expression in HMCs with stimulation of IL-6 alone or IL-1 plus IL-6; and (4) release of large amounts of platelet activating factor (PAF), thromboxane B2 (TxB2), and superoxide anion. Taken together, these results strongly suggest that mesangial cell proliferation and increased production of immune/chemical mediators and superoxide anion can be directly induced by IL-1 plus IL-6. These changes may lead to ongoing renal injury.     

脱细胞基质材料体外激活巨噬细胞的作用

背景：因巨噬细胞在机体炎症反应中有着举足轻重的作用,所以检测生物材料对巨噬细胞行为的影响,可评估材料的免疫原性。 目的：分析脱细胞基质材料对巨噬细胞的激活作用。 方法：获取BALB/c小鼠腹腔巨噬细胞,分5组培养：对照组为单纯细胞培养组;实验1组为脱细胞基质膜材料与巨噬细胞直接接触培养,实验2组为新鲜心包膜材料与巨噬细胞直接接触培养,实验3组为脱细胞基质膜材料与巨噬细胞间接接触培养,实验4组为新鲜心包膜材料与巨噬细胞间接接触培养。培养48 h后,MTT法检测各组A值,判定毒性分级;培养24 h后,检测细胞培养上清中一氧化氮及细胞因子的分泌。 结果与结论：实验1-4组毒性分别为2级、4级、0级及2级。实验1组、实验2组一氧化氮水平高于对照组(P < 0.05),并且实验2组高于实验1组(P < 0.05)。5组间白细胞介素2、白细胞介素4、干扰素γ、白细胞介素17A和白细胞介素10水平比较差异无显著性意义;实验2组白细胞介素6水平高于对照组(P < 0.05);实验1组、实验2组、实验4组肿瘤坏死因子水平高于对照组(P < 0.05),并且实验2组高于实验1组(P < 0.05)、实验1组高于实验4组(P < 0.05)。说明脱细胞基质材料可在直接接触时激活巨噬细胞。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Characterization of cytokine production by human term placenta macrophages in vitro

Problem Macrophages are apparently the only immune cells within placenta villi, yet functions of these cells remain obscure. It has been postulated that placental macrophages accomplish regulatory roles at the fetal–maternal interface by means of wide variety of secreted cytokines. We attempt to analyze the patterns of cytokine production in an isolated population of placental macrophages. Method of study Macrophages were obtained from term placentas in the absence of spontaneous labor. The basal and lipopolysaccharide (LPS)‐stimulated levels of intracellular cytokines were detected by flow cytometry. The basal cytokine secretion was determined by BD^?Cytometry Bead Array (BD Biosciences, San Diego, CA, USA). Results Intracellular IL‐1α, IL‐1β, IL‐6, and TNFα were detected in 31, 27, 4, and 3% CD68+ cells, respectively. Stimulation with LPS increased the proportions of cytokine‐producing CD68+ cells to 48, 50, 28, and 49%, respectively. Under basal conditions, levels of released TNFα and IL‐6, respectively, were 20‐ and 25‐fold higher when compared with IL‐1β while IL‐10 was secreted in small but detectable amounts. When a secretory activity was estimated for cytokine‐producing cells, the secretion rate for TNFα and IL‐6 overwhelmingly surpassed that for IL‐1β (TNFα:IL‐6:IL‐1β ratio was 192:145:1). Conclusion These results suggest functional heterogeneity of the placental macrophage population and contribute to the elucidation of regulatory roles of these cells in gestation.     

The immunomodulatory glycan LNFPIII initiates alternative activation of murine macrophages in vivo

The early pathogen–macrophage interactions that help drive macrophage maturation towards classically or alternatively activated are largely unknown. To examine this question we utilized the immunomodulatory glycan Lacto-N-fucopentaose III (LNFPIII), which contains the Lewis X (LeX) trisaccharide, to activate murine peritoneal macrophages in vivo. Because LNFPIII is known to induce anti-inflammatory responses, we asked if LNFPIII stimulation of macrophages in vivo initiates alternative activation events such as upregulation of Arginase 1, Ym1, FIZZ-1, MGL-1 or macrophage mannose receptor (MMR). Examination of peritoneal exudate cells from mice 20 hr post-LNFPIII injection demonstrated increased Arginase 1 activity, at the mRNA and protein levels, coincident with undetectable inducible nitric oxide synthase expression or nitric oxide production. In addition to Arginase 1, Ym1 expression was also significantly upregulated at 20 and 48 hr after LNFPIII exposure in vivo. However, the expression of FIZZ-1, MGL-1, and MMR was not changed in these macrophages. In an attempt to determine activation requirements for functional activity, we adoptively transferred antigen-pulsed, in vivo LNFPIII activated macrophages into naïve recipients and found that they were capable of triggering recipient T cells to secrete elevated levels of interleukin (IL)-10 and IL-13 compared to mice receiving control macrophages. Together, these data demonstrate that upregulation of expression of Arginase 1 and Ym1 occur very early in activation of macrophages, and can be independent of other alternatively activated (AA) macrophage markers. Importantly, these early events appear to be IL-4/IL-13-independent in our model. In the future we hope to determine if upregulation of these initial AA maturational events is sufficient for these macrophages to exert immunoregulatory activity in vivo.     

Insulin release from islets of Langerhans entrapped in a poly(N-isopropylacrylamide-co-acrylic acid) polymer gel

Abstraet-A copolymer of N-isopropylacrylamide (98 mol% in feed) and acrylic acid, poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), was prepared by free radical polymerization for development of a thermally reversible polymer to entrap islets of Langerhans for a refillable biohybrid artificial pancreas. A 5 wt% solution of the polymer in Hanks' balanced salt solution forms a gel at 37°C that exhibits no syneresis. Diffusion of fluorescein isothiocyanate (FITC) dextrans having molecular weights of 4400 and 70000 were used to evaluate mass transport in the gel at 37°C. Insulin secretion from islets in the polymer gel was also investigated in both static and dynamic systems. The polymer gel exhibited excellent diffusion of FITC dextran 4400 and FITC dextran 70 000 with diffusion ratios, D/D₀ (ratio of diffusion in the gel to diffusion in water), of 0.20 ± 0.04 and 0.35±0.17, respectively. Human islets entrapped in the polymer gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free human islets. Rat islets showed prolonged insulin secretion in response to high (16.5 mM) glucose concentrations compared to free rat islets. Rat islets in the polymer gel maintained insulin secretion in response to the higher glucose concentration for over 26 days. Rat islets entrapped by the polymer also released higher quantities of insulin more rapidly in response to changes in concentrations of glucose and other stimulants than rat islets entrapped in an alginate control. These results suggest that this material would provide adequate diffusion for rapid insulin release in an application as a synthetic extracellular matrix for a biohybrid artificial pancreas.     

In vitro synthesis of human IgE is suppressed by human IgG

Recently it has been shown that intravenous immunoglobulin (IVIG) preparations suppress thein vitro synthesis of IgG, IgA, and IgM. In this paper we demonstrate that IVIG and IgG purified from a single donor's serum also suppress thein vitro synthesis of IgE. We had noticed this effect when we added human serum (HS) toin vitro cultures for IgE synthesis. The interleukin-4 (IL-4)-induced IgE synthesis from human peripheral blood mononuclear cells (PBMC) in the presence of fetal calf serum (FCS) was suppressed by HS in a dose-dependent fashion. The following results indicate that this suppression is mediated by IgG: (1) IVIG preparations, which consist mainly of IgG, suppressed the IgE synthesis from IL-4-stimulated PBMC in a dose-dependent way; (2) when HS was fractionated by protein G sepharose or anti-IgG sepharose, the eluate fractions (containing IgG), but not the effluent fractions (void of IgG) suppressed IgE synthesis, whereas the opposite was found when HS was fractionated by FCS-coupled sepharose. We conclude from these data that human IgG preparations suppress thein vitro synthesis not only of the IgG, IgA, and IgM isotypes, but also of the IgE isotype.     

Dose- and time-dependent activation of rat alveolar macrophages by glucocorticoids

Effects of glucocorticoids on immune functions are generally thought to be suppressive and anti-inflammatory. However, most reports dealing with this issue describe effects of long-term treatment with high doses of glucocorticoids on immune functions. In the present study we have investigated both dose and timing effects of exposure of alveolar macrophages with dexamethasone on lipopolysaccharide (LPS)-induced IL-1β and nitric oxide secretion. For this purpose, alveolar macrophages were preincubated with various doses of dexamethasone during varying intervals, followed by stimulation of the cells with endotoxin, either in the absence or presence of dexamethasone. Subsequently, the effects of this treatment on IL-1β and nitric oxide secretion were measured. It was shown that both short-term incubation of alveolar macrophages with high doses of dexamethasone and long-term incubation with low doses of dexamethasone lead to enhanced nitric oxide and enhanced IL-1β secretion upon subsequent stimulation of the cells with LPS. In contrast, long-term incubation of alveolar macrophages with high-dose dexamethasone leads to decreased IL-1β and nitric oxide secretion upon subsequent stimulation. Thus, it is concluded that the effects of dexamethasone on rat alveolar macrophages are both time- and dose-dependent. It is therefore argued that effects of glucocorticoids on immune functions are not a priori suppressive, but that both dose and timing effects should be taken into account.     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

In vitro effect of cetirizine on PGE2 release by rat peritoneal macrophages and human monocytes

Cetirizine was first described as a specific anti-H₁ molecule displaying potent antiallergic activity. It was later found that its pharmacological properties extended to cellular actions as on eosinophil recruitment at inflammatory sites in allergic patients. Monocytes and macrophages participate in allergic mechanisms, particularly through high affinity H₁ and H₂ membrane receptors and generation of pro- and anti-inflammatory agents; among them histamine-induced factors, IL-1 and prostanoids are of importance. The aim of this work was to investigate the effect exerted by various concentrations of cetirizine (0.1–10 g/ml) appliedin vitro to human monocytes and peritoneal rat macrophages cultured for 24 h. Peritoneal macrophages were collected either from normal or experimentally inflamed rats. Human monocytes, isolated from peripheral blood, were studied either in a resting state or after stimulation by LPS fromEscherichia coli (1 and 10 g/ml). Cetirizine (10 g/ml) significantly enhanced IL-1 release by human monocytes stimulated by a weak LPS concentration (1 g/ml) but could not modify the maximal increase of IL-1 release induced by 10 g/ml of LPS. It did not exert any effect on resting cells. Cetirizine (0.1–10 g/ml) enhanced PGE₂ release by resting human monocytes. Concentrations of 1 and 10 g/ml enhanced PGE₂ release by LPS-stimulated monocytes, and by healthy and inflamed rat macrophages. This effect was concentration-dependent. Our findings point to an anti-inflammatory action of cetirizine via PGE₂ release and histamine H₂ interactions. Cetirizine did not directly modify IL-1 generation by resting monocytes but the IL-1 production observed after LPS stimulation could promote the mechanisms by which PGE₂ is released.     

Large variability of the intracellular ATP content of human islets isolated from different donors

Observations in experimental heart, liver, kidney and pancreas transplantation indicated that graft function and survival correlates significantly with ATP content of transplanted tissue. The ATP content of cells can be reduced by several factors i.e. the nutritional donor status, storage technique, warm ischemia and cold ischemia time. This study investigates the intracellular ATP content of isolated human islets for the first time. Quantified samples of freshly isolated (digestion-filtration, continuous ficoll gradient purification) and cultured (22°C, CMRL+10% FCS) islet equivalents (IEQ) of consecutively processed human pancreata from multiorgan donors (UW vascular flush) were shock frozen in liquid nitrogen and stored at –196°C until rapid thawing, sonification and subsequent luminometric determination of ATP (Luciferin-Luciferase-reaction) and assessment of islet protein (IP). The ATP content was analysed for freshly isolated and subsequently 5±1 days cultured islets (n=10). The ATP content of freshly isolated human islets was 130.4±53.4 pg/μg IP (mean ± SEM) corresponding to 20.7±6.3 pg/IEQ. After culture ATP content increased to 265.5±113.3 pg/μg IP (204.2±41.5%) corresponding to 43.7±15.3 pg/IEQ (216.1±34.9%; p<0.05). The coefficient of variation was 129.5%, 96.5% (fresh) and 135.0%, 111.0% (cultured) for ATP/μg IP and ATP/IEQ, respectively. The present data show that: (1) the ATP content of freshly isolated human islets varies enormously; (2) intraislet ATP levels increase significantly during 22°C culture suggesting that the capacity to produce ATP is maintained despite hypothermic environment. More data are necessary to clarify the relevance of intraislet ATP content for graft function and survival after islet transplantation.     

Study of the infection of human blood derived monocyte/macrophages with hepatitis C virus in vitro

Hepatitis C virus (HCV) is essentially hepatotropic, but clinical observations based on quasispecies composition in different compartments or on viral RNA detection in cells suggest that the virus is able to infect and persist in cells other than liver cells. It was shown previously that peripheral blood mononuclear cells (PBMCs) are permissive to HCV replication in vitro but at a very low rate. Since different viruses associated with chronic infections are known to persist in monocyte/macrophages, it is important to determine whether these mononuclear blood cells are susceptible preferentially to HCV. In order to study HCV interaction with monocytes/macrophages, these cells were isolated from the blood of healthy donors and incubated with HCV genotype 1b positive sera. The detection by RT-PCR of the positive- and negative-strand RNA in the cells at different times and the increase in the amount of intracellular viral RNA measured by the branched DNA assay suggest that monocyte/macrophages can support HCV RNA replication. The rate, however, is very low. The analysis of hypervariable region 1 (HVR-1) nucleotide sequences indicated that some minor variant present in the inoculum might display a specific tropism for the monocytes/macrophages. These results provide evidence that human monocytes/macrophages might represent a reservoir for HCV. This cell tropism may be a crucial factor in the pathogenesis of hepatitis C.