Serum anti-cholesterol antibodies in chronic hepatitis-C patients during IFN-alpha-2b treatment

Previously we detected more than 3 times higher anti-cholesterol antibody (ACHA) levels in HIV positive patients compared to healthy individuals, however, this level significantly decreased during highly active anti-retroviral therapy (HAART). In our present study we examined whether these findings could also be detected in patients with chronic hepatitis C (CHC). We calculated the correlation between the ACHA levels and the C5b-9 complement activation product. 39 patients with CHC were treated with IFN-alpha-2b (Schering-Plough) 5 MU daily for 6 weeks, followed by 5 MU TIW. Serum levels of ACHA and complement activation products were measured with ELISA. Serum HCV RNA was measured by a highly sensitive branched DNA technique before and 3, 6 and 12 months after the beginning of IFN-alpha-2b therapy. 52 healthy persons served as controls. At the onset of treatment ACHA level was significantly (p = 0.0062) higher in patients (40 (24-69) AU/ml) (median (interquartile range)) than in control sera (26 (20-35) AU/ml). In the 26 responder patients ACHA levels decreased to the normal level during the therapy, but no change was observed in the 13 non-responders. In patients with a sustained response ACHA levels remained low till the end of the 12 months IFN treatment. ACHA levels were significantly (p = 0.0422) higher in the patients with low (< 4.0 mmol/l) than in those with normal (> or = 4.0 mmol/l) cholesterol concentrations. The ACHA level before the therapy strongly correlated (r = 0.5499, p = 0.0014) with C5b-9 serum levels. ACHA levels are elevated in CHC, but this elevation is not as high as in HIV. Decrease of viral load by IFN-alpha-2b treatment in the responders results in normalization of ACHA concentration. High ACHA levels in patients with low serum cholesterol concentration suggest that high ACHA levels may contribute to the decrease in cholesterol levels. The correlation between the ACHA and C5b-9 levels indicate, that the ACHA may play a role in the complement activation in CHC.     

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.     

Functional characteristics of coronary vasomotor function following intramyocardial gene therapy with naked DNA encoding for vascular endothelial growth factor165.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. VEGF gene therapy improves perfusion of ischemic myocardium in experimental models and possibly in patients with end-stage coronary artery disease. In addition to its proliferative and migratory effect on endothelial cells, it also activates and up-regulates endothelial nitric oxide synthase (eNOS). Therefore, the authors investigated coronary endothelium-dependent vasodilatation in patients before and after VEGF gene therapy. The effect of intracoronary acetylcholine infusion on coronary diameter was assessed at baseline and after 3 months follow-up in patients with end-stage coronary artery disease treated with VEGF gene and in controls scheduled for elective percutaneous transluminal coronary angioplasty (PTCA) (acetylcholine test at diagnostic angiography and before a subsequently scheduled PTCA). Five out of six VEGF patients experienced a reduction in anginal complaints. Angiographic evidence for improved collateral filling was evident in two out of six patients. The vasoconstrictive response to acetylcholine was partly converted into dilatation. In contrast, the acetylcholine response in control patients remained vasoconstrictive. In conclusion, VEGF gene therapy has an important beneficial effect on the functional characteristics of the myocardial vascular network. Therefore, this therapy can potentially play an important role in all stages of the atherosclerotic process.     

Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis.     

The effects of direct-acting antiviral agents on the frequency of myeloid-derived suppressor cells and natural killer cells in patients with chronic hepatitis C

Currently, hepatitis C antiviral therapy is entering a new era with the use of direct-acting antiviral (DAA) agents. However, the precise immunological influences of DAA therapy in patients with chronic hepatitis C (CHC) are insufficiently understood. This study aimed to investigate the effects of DAA therapy on the frequency of myeloid-derived suppressor cells (MDSCs), T lymphocytes, and natural killer (NK) cells in patients with CHC. Thirty-two treatment-naive CHC patients were treated with DAA therapy, and the frequency of immune cells was analyzed by flow cytometry at various time points during and after therapy. Sixteen healthy donors were recruited for comparison. DAA therapy decreased the frequency of MDSCs and monocytic MDSCs in patients with CHC to a normal level. DAA therapy also increased the CD8⁺ T and NK cell levels in patients with CHC. In addition, activation (NKp30 and NKp46) and inhibitory (NKG2A) receptors on NK cells were downregulated to yield an NK cell phenotype resembling that observed in the healthy controls. This study provides insight into the normalization of immune cell levels under DAA therapy and indicates that restoration of the immune system in patients with CHC strongly supports long-term curative hepatitis C virus eradication.     

Evaluation of cytokine mRNA in induced sputum from patients with allergic rhinitis: relationship to airway hyperresponsiveness

Background:  Although airway hyperresponsiveness (AHR) is a characteristic feature of asthma, it is also frequently present in allergic rhinitis (AR). However, the pathogenesis of AHR is unclear and the roles of cytokines in the airway have not been well established in AR. We sought to compare cytokine mRNA levels in the sputum of AR patients with or without AHR and those of asthma patients, and to evaluate whether differences in cytokine levels are associated with the development of an abnormal airway response and the absence of respiratory symptoms in AR patients with AHR.
Methods:  Airway cells were obtained by sputum induction from 18 AR patients with AHR, 58 AR patients without AHR, and 27 asthma patients. Airway cell cytokine levels, interleukin (IL) -4, IL-5, IL-13, vascular endothelial growth factor (VEGF), and interferon-γ (IFN-γ), were studied at the mRNA level by RT-PCR.
Results:  Vascular endothelial growth factor and IL-5 mRNA levels were significantly higher in AR patients with AHR than in AR patients without AHR, but these were lower than those of asthmatic patients. Eosinophils were significantly higher in AR patients with AHR and in asthmatic patients than in AR patients without AHR. Interleukin-4, IL-13, and IFN-γ levels were not elevated in AR patients with or without AHR vs asthma patients.
Conclusions:  These findings suggest that VEGF and IL-5 can be important determinants of the development of AHR in AR patients and that lower levels of other cytokines may be associated with the absence of asthmatic symptoms in AR patients with AHR.     

Impact of epidermal growth factor receptor and transforming growth factor–α on hepatitis C virus‐induced hepatocarcinogenesis

Epidermal growth factor receptor system plays a central hepato‐protective and pro‐regenerative role in liver. Transforming growth factor‐α (TGF‐α) is an important autocrine growth regulator of hepatocytes that plays a role in development of hepatocellular carcinoma (HCC) among patients with chronic hepatitis C (CHC). This study was done on 40 core liver biopsies from patients with CHC, 20 liver specimens from HCC cases on top of CHC as well as five normal controls. All were immunohistochemically stained with epidermal growth factor receptor (EGFR) and TGF‐α antibodies. Some selected HCC cases were submitted for FISH technique to detect EGFR gene alteration. By immunohistochemistry EGFR and TGF‐α were overexpressed in HCC and cirrhotic cases compared to CHC cases without cirrhosis. Also, their expression was stronger in CHC cases with higher grades of activity and stages of fibrosis compared to lower ones. FISH positive results for EGFR were detected in 33.3% of the examined HCC cases. EGFR and TGF‐α can be used as predictive markers for activity, fibrosis, and carcinogenesis in CHC patients. Overexpression of EGFR in HCC patients can be promising in selecting those who can get benefit from anti‐EGFR target therapy.     

Changes in circulating levels of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 after carotid endarterectomy

It has been shown that vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) are upregulated in severe carotid stenosis. However, it is unknown whether carotid endarterectomy (CEA) affects serum level of these molecules. We investigated changes in concentration of VEGF and VEGFR-2 in patients undergoing carotid endarterectomy. Forty-three patients with extracranial carotid stenosis (>70%), were studied. Patients with severe vertebrobasilar stenosis, recent (<1 month) vascular event (stroke, coronary infarction, arterial thromboembolism), critical ischemia of lower extremity, recent infection, autoimmune disease or malignancy were excluded from the study. Blood samples were taken before CEA and on the second post-operative day. Thirty healthy blood donors served as a control group. We used enzyme linked immuno-absorbent assay as a method for the determination of VEGF and VEGFR-2. Pre-operative levels of VEGF (371+/-42 pg/ml) and VEGFR-2 (8424+/-356 pg/ml) were significantly elevated. There was significant decrease in both VEGF (152 pg/ml) and VEGFR-2 (1297 pg/ml) after CEA, without however reaching normal values. In asymptomatic patients and in patients with a contralateral carotid stenosis of >50%, however, the observed reduction of VEGF did not reach statistical significance. On the other hand, in the same subgroups, a major decrease of VEGFR-2 values was observed. VEGF and VEGFR-2 showed a very significant increase in serum of patients with severe carotid stenosis. These pre-operative levels decreased significantly after endarterectomy, and the changes emphasize the importance of these molecules in carotid disease progression.     

Clinical Profiles of Chronic Hepatitis C in a Major County Medical Center Outpatient Setting in United States

The estimated prevalence of hepatitis C virus (HCV) infection in the US is 1.8 %. Data are limited on the clinical profile of the disease at first presentation and dynamic follow-up of ALT level, especially in publicly-funded patients. This information is critical for optimal management of these patients. The present study is aimed to assess the clinical profiles of chronic hepatitis C (CHC) at first presentation and clinical implication of dynamic follow-up of ALT level in a county medical center setting. A total of 294 patients were selected from the population consecutively evaluated in the Hepatitis Clinic at Los Angeles County-USC Medical Center between Jan. 1990 and Dec. 1998. Ethnicity of the patients was Hispanics-49.0%, Caucasian-28.6%, African American-13.6%, and Asian-8.8%. Risk factors were identifiable in 84.0% of patients, and injection drug use (IDU) represented the leading risk factor for HCV acquisition (47.4%). History of alcoholism was present in 39.1%. The initial clinical diagnoses were chronic hepatitis 76.9%; compensated cirrhosis 20.4%; and decompensated cirrhosis 2.7%. Elevation of ALT, alpha fetoprotein (AFP), ferritin, and anti-nuclear antibody (ANA) titer were seen in 219/294 (74.5%), 60/194 (30.9%), 20/83 (24.1%), and 35/97 (36.1%) patients, respectively. Anti-HBc (total) test was positive in 65/129 (50.5%) patients. The presence of cirrhosis was significantly associated with age greater than 55 years at entry, female gender, non-African American ethnicity, history of transfusion, lower level of albumin and elevated level of AFP. Longitudinal observation of ALT changes in 178 patients who had neither evidence of cirrhosis at entry nor received interferon treatment showed persistently normal, intermittently or persistently elevated ALT level in 15.2%, 38.3%, and 46.6% patients, respectively. The frequency of developing clinical evidence of cirrhosis during follow-up was significantly higher in patients with persistently (16.0%) or intermittently (7.0%) elevated ALT than that in patients with persistently normal ALT (4.0%). In conclusion, the present study analyzed the clinical profiles of CHC, assessed risk factors for developing cirrhosis, and demonstrated the clinical value of dynamic follow-up of ALT level in a cohort of publicly-funded patients. These data have major implications in designing optimal strategies for disease management, antiviral therapy, and screening for hepatocellular carcinoma in patients with CHC.     

Vascular endothelial growth factor, capillarization, and function of the rat plantaris muscle at the onset of hypertrophy

Capillary proliferation occurs during compensatory hypertrophy. We investigated whether the expression of vascular endothelial growth factor (VEGF) is elevated at the onset of hypertrophy when capillary proliferation is minimal, and whether muscle damage as assessed by muscle force deficits, may occur at the onset of hypertrophy. To investigate this, we induced in 9-month-old rats, under isoflurane anesthesia, hypertrophy of the left plantaris muscle by denervation of the gastrocnemius and soleus muscles. Capillarization was investigated in both the deep (oxidative) and the superficial (glycolytic) regions of the plantaris muscle. After 2 weeks, muscle mass had increased by 16% (p< 0.01), which was not accompanied by increases in fiber size. The maximal tetanic force (P(0)) and specific tension (P(0).g(-1) or P(0).cm(2)) and twitch characteristics were unaltered, and fatigue resistance of the overloaded muscle was improved (p< 0.05). However, the myosin heavy chain composition was unaltered. Capillary proliferation was not yet evident, but VEGF mRNA and protein levels were elevated 1.5- and 8-fold, respectively (p< 0.05). We concluded that the normal specific tension and the elevated VEGF expression after 2 weeks of overload indicate (1) an absence of or minimal muscle damage at this early time point, and (2) that elevated VEGF expression precedes and is involved in capillary proliferation that occurs during the later stages of compensatory hypertrophy.     

Selected angiogenic cytokines in systemic lupus erythematosus patients

The objective of this study was to determine the serum concentration of angiogenic factors (vascular endothelial growth factor, VEGF; transforming growth factor beta, TGF-β1; hepatic growth factor, HGF; basic fibroblast growth factor, bFGF; tumor necrosis factor alpha, TNF-α; soluble vascular endothelial growth factor receptor 1, sVEGF-R1; soluble vascular endothelial growth factor receptor 2, sVEGF-R2), the relationships among them and to assess the relation of their levels with the applied therapy in 48 females with systemic lupus erythematosus (SLE; 37 long-term treated +11 newly diagnosed). The control group consisted of 24 healthy women. A statistically significant increase of sVEGF-R2 and significant decrease of sVEGF-R1 were observed in the subgroup of newly diagnosed SLE patients as compared to the control subjects. No significant differences were found between serum angiogenic factors in the long-term treated subgroup and the control, the long-term treated subgroup and the newly diagnosed SLE patients after a 3-month treatment, and the subgroup of newly diagnosed SLE patients before therapy and after a 3-month treatment. The significant decrease in the serum of sVEGF-R2 was revealed in the subgroup treated for a long-time as compared to the subgroup of newly diagnosed untreated SLE patients. The analysis of relationships between serum concentration of sVEGF-R1 and other cytokines levels revealed positive correlation with concentration of VEGF and TNF-α in the total group of patients. In the newly diagnosed untreated subgroup, a strong positive correlation between concentration of sVEGF-R1 and bFGF was observed. Furthermore, a moderate positive correlation between concentration of sVEGF-R1 and the level of VEGF was revealed in the long-term treated patients. The association between sVEGF-R2 and HGF was also noted in this subgroup. The obtained data suggest the necessity of further investigations to determine the importance of angiogenic factors in pathogenesis and therapy of SLE.     

Asymptomatic systemic sarcoidosis arising 5 years after IFN-alpha treatment for chronic hepatitis C: a new challenge for clinicians.

Interferon (IFN)-induced sarcoidosis is well documented. Herein, we report the case of a patient with chronic hepatitis C (CHC) who developed IFN-alpha-induced sarcoidosis. The clinical features of this case make it unique among all cases so far described. The patient was, in fact, asymptomatic for sarcoidosis, and the disease, characterized by liver and lung granulomatosis, was discovered by chance during the CHC follow-up. The diagnosis was made 5 years after IFN-alpha discontinuation. A pathogenetic role for IFN-alpha in our patient is supported by a liver biopsy performed before the therapy with IFN-alpha was started, showing no evidence of granulomatous localizations. This case suggests that the incidence of sarcoidosis during IFN-alpha treatment is underestimated. A search for clinical and laboratory findings typical of the disease, as well as a liver biopsy, should always be included in the follow-up of CHC patients undergoing therapy with IFN-alpha.     

Effect of tamoxifen on serum IL-18, vascular endothelial growth factor and nitric oxide activities in breast carcinoma patients

Vascular endothelial growth factor (VEGF) is a multi-functional cytokine that has been suggested to be a major angiogenic factor in breast cancer. Nitric oxide (NO) is a potent biological molecule that participates in the multi-step process of carcinogenesis. Interleukin (IL)-18 has been shown to have potent anti-tumour effects. In this study, we investigated the effect of tamoxifen therapy on serum VEGF, NO and IL-18 activity in breast cancer patients. Serum levels of VEGF, nitrate + nitrite and IL-18 were measured in 34 postmenopausal breast cancer patients before and 3 months after the tamoxifen therapy. Both serum VEGF and IL-18 levels decreased after tamoxifen therapy (P = 0.051, P < 0.05, respectively). Serum VEGF levels increased in patients with endometrial thickness, while patients without endometrial thickness had a significant reduction in serum VEGF levels after therapy (P < 0.05). Serum nitrate + nitrite levels increased after the therapy, but this was not statistically significant (P > 0.05). A decrease in serum VEGF levels with tamoxifen therapy may be a reflection of reduced angiogenic activity in patients without endometrial thickness. The negative effect of tamoxifen therapy on IL-18, which is known to have a potent antitumour activity, may be related to the decreased tumour growth by induction of NO and reduction of VEGF activity as a feedback mechanism.     

Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1alpha

The contribution of chemokines toward angiogenesis is currently a focus of intensive investigation. Certain members of the CXC chemokine family can induce bovine capillary endothelial cell migration in vitro and corneal angiogenesis in vivo, and apparently act via binding to their receptors CXCR1 and CXCR2. We used an RNAse protection assay that permitted the simultaneous detection of mRNA for various CXC chemokine receptors in resting human umbilical vein endothelial cells (HUVECs) and detected low levels of only CXCR4 mRNA. Stimulation of HUVECs with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) up-regulated levels of only CXCR4 mRNA. CXCR4 specifically binds the chemokine stromal-derived factor-1alpha (SDF-1alpha). Competitive binding studies using 125I-labeled SDF-1alpha with Scatchard analysis indicated that VEGF or bFGF induced an average number of approximately 16,600 CXCR4 molecules per endothelial cell, with a Kd = 1.23 x 10(-9) mol/L. These receptors were functional as HUVECs and human aorta endothelial cells (HAECs) migrated toward SDF-1alpha. Although SDF-1alpha-induced chemotaxis was inhibited by the addition of a neutralizing monoclonal CXCR4 antibody, endothelial chemotaxis toward VEGF was not altered; therefore, the angiogenic effect of VEGF is independent of SDF-1alpha. Furthermore, subcutaneous SDF-1alpha injections into mice induced formation of local small blood vessels that was accompanied by leukocytic infiltrates. To test whether these effects were dependent on circulating leukocytes, we successfully obtained SDF-1alpha-induced neovascularization from cross sections of leukocyte-free rat aorta. Taken together, our data indicate that SDF-1alpha acts as a potent chemoattractant for endothelial cells of different origins bearing CXCR4 and is a participant in angiogenesis that is regulated at the receptor level by VEGF and bFGF.     

MxA and PKR expression in chronic hepatitis C.

The effectiveness of therapy for chronic hepatitis C (CHC) patients has greatly improved in the last few years, and the gold standard is currently held to be pegylated interferon (IFN) in combination with ribavirin. Overall, however, the percentage of patients achieving a sustained virologic response (SVR) is only around 50%,and it is not possible to predict those patients who will benefit from therapy. The molecular mechanisms underlying lack of therapeutic response remain unknown. In this study, we investigated the tissue expression of MxA and RNA-dependent protein kinase (PKR), two antiviral proteins modulated by IFN, in biopsy samples from hepatitis C patients before the beginning of therapy. Our results show that expression of MxA, but not of PKR, is significantly lower in responders compared with nonresponders. No differences were observed regarding the hepatitis C virus (HCV) genotype and the viral load. These results suggest that expression of the MxA protein could play a role among the mechanisms underlying responsiveness to therapy.     

Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval

The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates.