Donatella Marchesi

URAEMIC BLEEDING: ROLE OF ANAEMIA AND BENEFICIAL EFFECT OF RED CELL TRANSFUSIONS

Abstract Bleeding time, altered in most uraemic patients, may be influenced by packed cell volume (PCV). Uraemic patients are often anaemic, so the influence of PCV on bleeding time was assessed in several groups of patients with chronic uraemia. The findings were that bleeding times in uraemic patients are profoundly influenced by anaemia, and that the platelet-mediated haemorrhagic tendency in uraemia may be successfully managed by raising PCV values to above 30%.

HÆMOLYTIC-URÆMIC SYNDROME: DEFICIENCY OF PLASMA FACTOR(S) REGULATING PROSTACYCLIN ACTIVITY?

It is suggested that patients with the haemolytic-uraemic syndrome and related disorders (such as thrombotic thrombocytopenic purpura) lack a plasma factor which stimulates prostacyclin (P.G.I2) activity. Normal plasma would supply the missing factor and is a rational treatment for some life-threatening symptoms (thrombocytopenia, haemolytic anaemia, hypertension) of this syndrome.

Reduced umbilical and placental vascular prostacyclin in severe pre-eclampsia

Prostacyclin production was significantly depressed in foetal and placental vascular tissues from five patients with severe pre-eclampsia in comparison to vascular tissues from women with uncomplicated pregnancy. Such an abnormality may be responsible for a reduced blood flow and defective fetal nutrition thus playing a major role in the pathogenesis of this syndrome.

Prostacyclin and human foetal circulation.

Tissues from human umbilical cord arteries and placental veins generated much greater prostacyclin activity than vessels from normal adults. High prostacyclin generation could contribute to maintaining the low peripheral vascular resistance typical of foetal circulation in which blood pressure is low despite very high cardiac output.

Altered platelet and vascular prostaglandin-generation in patients with renal failure and prolonged bleeding times.

Abstract Venous tissues from 15 patients with renal failure (five acute and 10 chronic) generated significantly higher PGI2-like (platelet aggregation inhibitory) activity than venous tissues from 10 normal subjects. The longer the bleeding times, the higher the values for PGI2-like activity found in these patients. Both bleeding times and PGI2-like activity values returned to normal in three acute uraemic patients on restoration of their renal function. Two additional patients with acute renal failure and greatly prolonged bleeding times were under aspirin treatment at the moment of this study: venous specimens from neither of them generated measurable amounts of PGI2-like material. Malondialdehyde formation in platelets from 12 patients with chronic renal failure and prolonged bleeding times was significantly lower than in platelets from 11 controls. The defect was evident with each of the three stimuli used, i.e., collagen, arachidonic acid and thrombin. It is concluded that prostaglandin metabolism in platelets and in the vessel wall from uraemic patients is impaired in different ways, both contributing to the impaired primary haemostasis in these patients.

Plasmatic regulation of vascular prostacyclin in pregnancy.

Activity of prostacyclin-stimulating factor was measured in six normal, non-pregnant women, six women in early normal pregnancy, six in late normal pregnancy, and six in late pregnancy complicated by severe pre-eclampsia. The activity was lower in the women in late pregnancy than in those in early pregnancy and the controls but was about normal in those with severe pre-eclampsia. These results may be relevant to the physiology of pregnancy and the pathogenesis of pre-eclampsia.

Familial deficiency of a plasma factor stimulating vascular prostacyclin activity.

Abstract We previously reported successful treatment with plasma in patients with thrombotic microangiopathy and defective vascular PGI2 activity and suggested that a defect in a plasma factor stimulating PGI2 synthesis might be implicated in the pathogenesis. We report here that in one of these patients the plasma defect was still detectable one year after clinical remission (without recurrence). Two of this patients four children had a similar, though less severe, plasma defect. The proposita is a 54-year-old woman with a clinical and laboratory picture of haemolytic uraemic syndrome. Unlike normal plasma, the patients plasma had a low capacity to stimulate PGI2 production by rat aortic rings (previously washed until their endogenous PGI2 activity was exhausted). After plasma treatment the patients plasma behaved normally in this respect, but again appeared deficient at out-patient follow-up. PGI2 stimulating activity was normal in two daughters but consistently low (20–50% of control) in both patients sons. None of them had any history or clinical signs of microangiopathic disorders. Detection of this plasma defect in apparently healthy subjects and in patients who have recovered from thrombotic microangiopathy episodes could have clinical implications.

GB virus C Infection in patients with type II mixed cryoglobulinemia

Type II mixed cryoglobulinemia is characterized by purpura, arthralgias, weakness, glomerulonephritis, peripheral neuropathy, and other manifestations of systemic vasculitis. Its pathogenesis probably depends on cryoprecipitable circulating immunocomplexes consisting of polyclonal IgG and monoclonal IgM, the latter having rheumatoid factor activity [1-3]. Although mixed cryoglobulinemia is commonly associated with hepatitis C virus (HCV) [4-8], a variable proportion of cryoglobulinemic patients does not have markers of infection with any known viral agent. We investigated the possible role of GB virus C (GBV-C), a newly discovered RNA virus that shares some sequence homology with HCV [9-11], in type II mixed cryoglobulinemia. Methods We studied 58 patients with type II mixed cryoglobulinemia that was not associated with hematologic malignant conditions, autoimmune disorders, or infectious diseases other than chronic viral hepatitis. One hundred forty-five volunteer blood donors served as healthy controls. We also studied a second control group that included 20 noncryoglobulinemic blood donors who were age-matched with cryoglobulinemic patients and were positive for antibodies to HCV. All patients were tested for liver and kidney function according to standard methods. The serum concentration of immunoglobulins, rheumatoid factor activity, and C3 and C4 components were measured by using routine nephelometric assays. Cryoglobulins were measured as the protein concentration of isolated cryoprecipitate and were characterized by using standard immunoelectrophoresis and immunofixation procedures. Antibodies to HCV were measured with a second-generation enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, Illinois). To search for enrichment of antibodies to HCV in cryoglobulins, the isolated and washed cryoprecipitate was resuspended in a volume of phosphate-buffered saline equal to the volume of the original serum. The activity of the antibodies to HCV per mg of IgG was subsequently measured, as reported elsewhere [12], in serial fivefold dilutions of both cryoprecipitate and supernatant. Serum specimens were tested for HCV RNA with a reverse-transcription polymerase chain reaction (PCR) assay, as described elsewhere [5]. The HCV viral load was measured in both cryoprecipitate and supernatant by using a quantitative reverse-transcription PCR assay (Amplicor HCV Monitor, Roche Diagnostic Systems, Inc., Branchburg, New Jersey), according to the manufacturers instructions. The GBV-C genome was detected by using a reverse-transcription PCR method developed in our laboratory [13]. Total RNA was extracted from 100 L of serum and was reverse transcribed by using random esanucleotides as primers. Complementary DNA was amplified through 45 cycles by using a pair of primers spanning a 161-base pair sequence in the conserved GBV-C helicase region. An aliquot of the PCR product was hybridized to an internal probe with a DNA enzyme immunoassay [14]. The optical densities of this immunoassay were used to measure GBV-C RNA levels in serial tenfold dilutions of cryoprecipitate-supernatant pairs. Twenty-seven cryoglobulinemic patients received recombinant interferon-alpha2b, and 8 received human lymphoblastoid interferon-, 4.5 to 6 million U three times weekly for 6 months and then 3 million U three times weekly for 6 to 12 months. For our purposes, virologic response was defined as the disappearance from serum of GBV-C RNA, HCV RNA, or both as a result of interferon- therapy. A clear alleviation of the signs and symptoms of cryoglobulinemia, along with a reduction of 50% or more in serum cryoglobulin levels, was considered a clinical response. Statistical analyses were done by using the two-sample Wilcoxon test, the chi-square test, or the Wilcoxon matched-pairs signed-rank test, as appropriate. All P values are two tailed, and statistical significance was set at the 0.05 level. Results We detected GBV-C RNA in 23 of the 58 patients with type II mixed cryoglobulinemia (40%); 20 of the 23 were also infected with HCV. We found HCV RNA alone in 28 patients. We detected GBV-C RNA in 1 of 145 unselected blood donors; thus, the prevalence of this virus was much lower among unselected blood donors than among cryoglobulinemic patients (0.7% compared with 40%; P < 0.001). Similarly, only a small proportion of blood donors was positive for antibodies to HCV compared with cryoglobulinemic patients (2.7% compared with 81%; P < 0.001). In contrast, the prevalence of GBV-C infection was similar in cryoglobulinemic patients positive for antibodies to HCV and age-matched blood donors positive for these antibodies (43% compared with 35%; difference, 8% [95% CI, 18% to 33%]; P > 0.2). As indicated above, 20 of 58 cryoglobulinemic patients (34%) were co-infected with GBV-C and HCV. No obvious difference was found when the demographic, clinical, and laboratory features of these patients were compared with those of patients infected with HCV alone. However, the patients with GBV-C and HCV co-infection tended to have slightly higher levels of cryoglobulins (P = 0.18) and rheumatoid factor activity (P > 0.2) and lower concentrations of C4 (P = 0.05). We detected GBV-C RNA alone in the serum specimens of three cryoglobulinemic patients who seemed to be clinically similar in all respects to patients infected with HCV and patients without demonstrable HCV infection. We measured the concentration of viral markers in samples of cryoprecipitate and supernatant from 10 patients. Results in patients who were co-infected with HCV and GBV-C are shown in the (Table 1). The concentration of antibodies to HCV, as expressed by the antibody activity per mg of IgG, was clearly higher in the cryoprecipitate than in the supernatant (P = 0.022). Similarly, the HCV RNA level was consistently greater in the cryoprecipitates than in the supernatants obtained from the same patients (P = 0.036). In contrast, GBV-C RNA levels seemed to be lower in cryoprecipitate than in supernatant in 4 of the 5 patients studied in this group (P > 0.2). Finally, GBV-C RNA was undetectable in all three cryoprecipitate specimens obtained from patients infected with GBV-C alone. Table 1. Quantitative Evaluation of Antibodies to Hepatitis C Virus, Hepatitis C Virus RNA, and GB Virus C Supernatant from Cryoglobulinemic Patients Co-Infected with Hepatitis C Virus and GB Virus C* Twenty-eight of 35 patients receiving interferon- responded by clearing circulating GBV-C, HCV, or both. The clinical condition did not improve in either of the two patients in whom GBV-C RNA but not HCV RNA disappeared from serum. In contrast, a clinical response was evident in 5 of 6 patients who cleared HCV RNA alone and 6 of 7 patients who cleared both HCV RNA and GBV-C RNA. Of the 18 patients with isolated HCV infection, 13 had both a virologic and a clinical response. Discussion Infection with GBV-C or its homologue, hepatitis G virus, has been demonstrated in many patients with such conditions as acute and chronic hepatitis, intravenous drug use, hemophilia, aplastic anemia, and maintenance hemodialysis [9-11, 15-18]. However, the pathogenic role of these viruses has not been established. We show that GBV-C infection, usually associated with HCV, is frequently found in patients with type II mixed cryoglobulinemia. It is interesting that the prevalence of GBV-C infection is equally high in healthy blood donors and cryoglobulinemic patients who are positive for antibodies to HCV. This suggests a common route of transmission for both viruses and may be taken as evidence against a causal role for GBV-C in type II mixed cryoglobulinemia. Moreover, because risk factors for parenteral exposure were uncommon in our patients with cryoglobulinemia, it is likely that most of these patients had community-acquired infection with cotransmitted GBV-C and HCV. No clinical or biochemical characteristic clearly distinguished the cryoglobulinemic patients with GBV-C infection from those who were not infected. Nonetheless, the tendency of cryoglobulinemic patients with GBV-C and HCV co-infection to have more evident immunologic abnormalities than patients infected by HCV alone suggests a pathogenic role for GBV-C. This possibility is also supported by the fact that GBV-C was the only agent identified in three cryoglobulinemic patients. Because mixed cryoglobulinemia is commonly considered to be a vasculitic process mediated by cryoprecipitable circulating immunocomplexes, several authors have sought markers of HCV infection in cryoprecipitate; some have found increased concentrations of antibodies to HCV, HCV RNA, or both [5-8]. In our patients with GBV-C and HCV co-infection, we found that concentrations of both antibodies to HCV and HCV RNA were consistently higher in cryoprecipitates than in supernatants, whereas levels of GBV-C RNA were lower in most cryoprecipitates obtained from the same patients. Moreover, GBV-C RNA was not detected in the cryoprecipitates of any of the three patients with isolated GBV-C infection. Recent therapeutic trials [12, 19] indicate that the effectiveness of interferon- in HCV-associated mixed cryoglobulinemia is related to the drugs antiviral activity. We were able to study the effects of interferon- in patients co-infected with GBV-C and HCV. In these patients, we found that response with respect to GBV-C infection, expressed by the disappearance of viral genome from serum, was as frequent as response with respect to HCV infection. However, our most important finding was that clinical response was related to the virologic response with respect to HCV but not GBV-C infection. Thus, our results suggest that GBV-C does not have a primary role in the pathogenesis of type II mixed cryoglobulinemia in patients co-infected with GBV-C and HCV. Although it seems unlikely, the situation could be different in cryoglobulinemic patients with isolated GBV-C infection. These patients formed a small

Platelet and Vascular Prostaglandins in Uremia and Thrombotic Microangiopathy

A balance between vascular prostacyclin and platelet thromboxane has been proposed as a requisite for normal hemostatis. A derangment in such a balance would result in hemorrhage or thrombosis. Several factors regulate the interplay of prostacyclin and thromboxane. Albumin and haptoglobin inhibit prostaglandin (PG) synthesis, possibly acting on cyclooxygenase.1 On the other hand, bradykinin and angiotensin II stimulate arachidonic acid (AA) release from membrane phospholipids after binding to the cell surface.2,3 Thrombin is a potent stimulator of PGI2 synthesis by endothelial cells in culture,2,3 whereas β-thromboglobulin, a protein derived from platelet release reaction, reduces PGI2 generation4 after binding to a high-affinity receptor on cultured bovine endothelial cells.5