Polyamine biosynthesis and control of the development of functional pollen in kiwifruit.

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Major surgery for a gastric cancer in a haemophilic with high inhibitor titre successfully performed by the use of recombinant FVIIa.

A total gastrectomy with omentectomy and resection of the distal oesophagus in a 69-year-old haemophilia A patient with high inhibitor of 128 Bethesda units is described. Surgery was successfully performed after infusion of 112 microg kg-1 bw of recombinant FVIIa. Ninety-two microg kg-1 were given thereafter at time intervals of 2 h until 12 h, then every 3 h until 24 h, and every 4 h until 48 h after surgery. Doses were gradually reduced in the following days and finally discontinued on day 28 after surgery. The complete treatment schedule required the administration of a total of 708 mg of recombinant FVIIa. Using this approach, we observed normal haemostasis, and there were no signs of excessive postoperative bleeding or wound haematoma. No clinical side-effects or evidence of systemic activation of coagulation occurred during the treatment. As judged from the clinical course of this major surgery, recombinant FVIIa appears to be highly efficacious and safe and should be used as first line treatment in high titre inhibitor patients with cross-reactivity to porcine factor VIII, undergoing surgery.

Successful immune tolerance treatment with monoclonal or recombinant factor VIII concentrates in high responding inhibitor patients.

Very high purity factor VIII (FVIII) concentrates (plasma-derived or produced by recombinant-DNA technology) were used to achieve immune tolerance in five patients with high-responding inhibitors to FVIII. The mean time required for inhibitor disappearance was 5 months. Four out of five patients showed normalisation of the half-life of infused FVIII after 8-18 months of treatment with 100 IU FVIII/kg body weight administered once daily. Highly purified FVIII products thus appear to be suitable for achieving immune tolerance without negative effects on endogenous von Willebrand factor levels and activity.

The impact of a very high-purity factor VIII concentrate on the immune system of HIV-infected haemophiliacs: a randomized, two-year comparison with a high-purity concentrate.

Randomized and cohort studies have provided evidence confirming the hypothesis, based on in-vitro observations, that the use of very high-purity factor VIII (FVIII) concentrates, either immuoaffinity chromatography purified or produced by recombinant DNA technology, may slow immunological deterioration in human immunodeficiency virus (HIV)-infected haemophiliacs, while high-purity concentrates, produced by ion-exchange chromatography, did not produce a benefit. Even though these data clearly indicate that very high-purity concentrates should be preferred for the replacement therapy of HIV-positive haemophiliacs, there are little data, based on direct comparison, supporting the use of very high-purity concentrates rather than high-purity preparations, which are less expensive. In an attempt to address this issue, we prospectively compared CD4 cell counts and changes of clinical status in 18 HIV-positive haemophiliacs, randomly assigned either to receive the treatment with a very high-purity FVIII concentrate, purified by immunoaffinity chromatography, or a high-purity product, produced by ion-exchange chromatography. All patients had CD4 lymphocyte counts below 300 µL(-1) , were negative for the hepatitis B surface antigen and the HIV p24 antigen, and were receiving antiretroviral treatment with Zidovudine for at least 6 months. There were no significant changes of CD4 cell counts over the 96-week follow-up period or between the two groups. No signficant differences between the two groups were detected in the occurrence of AIDS-defining diagnoses (one in each group). On the whole, no striking benefit is conferred to the immune status of asymptomatic HIV-positive haemophiliacs by using either of these high-purity and very high-purity FVIII concentrates for 96 weeks. Larger prospective randomized trials are needed to establish definitely whether it is necessary to resort to very high-purity concentrates or it is sufficient to use high-purity concentrates to slow the fall of CD4 cell counts that occurs in HIV-positive haemophiliacs. Randomized trials, based on clinical end-points, are also needed to demonstrate whether slowing the fall in CD4 cells results in clinical benefits, delaying the occurrence of AIDS.

Epidemiology of hemophilia and of HIV infection in Italy. GICC. Gruppo Italiano Coagulopatie Congenite.

To evaluate the incidence and prevalence of hemophilia in Italy and the impact of HIV infection on the Italian hemophiliac population, data from a computerized national registry of patients from 95% of the hemophilia care centers in Italy were analyzed. A total of 4643 patients were included in the registry. The prevalence of hemophilia A was 8.2 per 100,000 males, with no significant regional differences; for hemophilia B the corresponding figure was 1.5 per 100,000. Temporal trends in hemophilia incidence suggest that the diagnosis of mild and moderate hemophilia has improved. The overall HIV prevalence was 26% and was significantly (p < 0.001) higher in patients with hemophilia B (47.1%) compared to those with hemophilia A (26.8%) or other diseases (16.5%). The highest rate of HIV seropositivity was among patients 20-29 years of age. The annual amount of clotting factor concentrates received was significantly (p < 0.001) higher in HIV seropositive patients than in those who were seronegative. Antibody testing was never performed on 10.1% of severely affected patients. The number of patients in the Italian registry was similar to the number that would have been expected based on prevalence estimates from other countries. In comparison with other countries, the prevalence of HIV infection recorded in Italy was lower in persons with hemophilia A, but higher in those with hemophilia B. Our study demonstrates the usefulness of a registry in delineating the epidemiology of hemophilia and in studying risk factors for HIV infection. It also underlines the need for continuing surveillance of this population.

Patterns of immunogenicity of an inactivated hepatitis A vaccine in anti-HIV positive and negative hemophilic patients.

Hepatitis A vaccination has been recommended to patients with hemophilia since they are exposed to potentially infectious clotting factor concentrates. Aim of this study was to assess the immunogenicity of vaccination in hemophiliacs, infected or not with the human immunodeficiency virus (HIV). A formalin-inactivated hepatitis A vaccine was injected subcutaneously to 113 susceptible adults and children and repeated after 1 and 6 months. 47 vaccinees were anti-HIV positive (28 asymptomatic, 15 with CD4 cell counts of less than 200/microliter and 4 with symptomatic disease). The first dose of vaccine induced seroconversion, with antibody titers of at least 20 mIU/ml, in 89% of the 66 anti-HIV negative patients, 100% of them responding after the second injection. In anti-HIV positive hemophiliacs seroconversion rates and antibody titers were significantly lower than in non-infected patients. After 12 months, only 76% of anti-HIV positive vaccinees and 40% of those with signs of HIV disease progression maintained the antibody, whereas all anti-HIV negative patients had titers of 20 mIU/ml or more. Our results indicate that there is an association between defective response to hepatitis A vaccine and stage of progression of HIV disease.

Incidence of factor VIII inhibitor development in hemophilia A patients treated with less pure plasma derived concentrates.

Very-high-purity Factor VIII concentrates produced by monoclonal or recombinant technology have been postulated to be more antigenic resulting in an increased risk of inhibitor development in hemophilia A patients. However, previous reports, mainly based on prevalence figures, may have underestimated the "true" risk of this complication in patients treated with less pure Factor VIII concentrates. The present study, started in 1975, has been designed to calculate the risk of inhibitor development in patients with severe or moderate hemophilia A, followed since their first exposure to intermediate or high-purity Factor VIII concentrates, produced by conventional technologies. Sixty-four hemophiliacs fulfilled the enrollment criteria. Inhibitors developed in 20.3% (13/64) of all patients and in 23% (11/48) of those with severe Factor VIII deficiency. Eleven patients manifested a strong anamnestic response after exposure to Factor VIII (high responders) and 2 had low inhibitor concentrations despite repeated Factor VIII infusions (low responders). The incidence of inhibitor development was 24.6 per 1000 patient-years of observation. The cumulative risk of inhibitor formation was 19.9% at age of 6 years, and 20.3% at 5 years after the first exposure. The risk was 19.3% at 70 days of exposure to Factor VIII concentrates, and 17.2% after a total of 50,000 units of Factor VIII given. Further studies are needed to confirm the above risk of acquiring an inhibitor, which indicates an under-estimation by previous studies. In addition, more data is needed to demonstrate whether very-high-purity Factor VIII concentrates may be more antigenic than conventional preparations.

Immune status of asymptomatic HIV-infected hemophiliacs: randomized, prospective, two-year comparison of treatment with a high-purity or an intermediate-purity factor VIII concentrate.

It has been postulated that high-purity factor VIII (FVIII) concentrates, since they contain less alloantigenic proteins than intermediate-purity concentrates, might cause lesser deterioration of the immune systems of hemophilic patients infected with the human immunodeficiency virus (HIV). To evaluate this hypothesis, we have prospectively compared T-lymphocytes subsets and delayed hypersensitivity reactions to skin tests in 17 asymptomatic HIV-positive hemophiliacs randomly assigned to continue treatment with an intermediate-purity concentrate with those of 16 hemophiliacs changed to a high-purity concentrate. For both groups, during the 24-month follow-up period CD4 cell counts showed similar rates of fall from baseline values. There was also no difference in the number of patients anergic to skin tests. Three patients treated with the intermediate purity concentrate and one treated with the high-purity concentrate developed symptoms of HIV infection. On the whole, no striking benefit is conferred to the immune status of asymptomatic HIV-positive hemophiliacs by using this high-purity concentrate for 2 years.

Low risk of transmission of the human immunodeficiency virus by a solvent-detergent-treated commercial factor VIII concentrate.

A study evaluating the risk of a commercial factor VIII (FVIII) concentrate's transmitting the human immunodeficiency virus (HIV) was carried out on hemophiliacs, by using multiple serological markers and the polymerase chain reaction (PCR). Twenty-nine hemophiliacs, negative for HIV antibodies, were treated for 18 months with a concentrate that had been inactivated by solvent-detergent. HIV-1 antibodies and antigen were assayed during the follow-up period. At the end of the study, all patients were also tested by the HIV 1 + 2 combined antibody assay; Western blot (WB) antibody analysis; and in eight cases, by an HIV-1 PCR technique. Patients received a yearly median FVIII dose of 35,330 IU (range 3,300-306,000); the median number of lots given to each patient was 6 (1-45). During the follow-up period and at the end of the study, HIV-1 antibodies and antigen were not detected in any of the subjects. The HIV 1 + 2 combined assay and WB analysis carried out only at the end of the study were negative. HIV-1 PCR was negative in all the tested patients. This study has shown that this solvent-detergent-treated FVIII concentrate did not transmit HIV.

The impact of clotting factor concentrates on the immune system in individuals with hemophilia.

All reported studies to date, whether comparative or not, have shown a tendency toward a slower decrease in CD4+ numbers in patients receiving very high-purity agents with the rapidity in fall-off being affected by level of CD4+ numbers (low CD4+ numbers decreasing more rapidly), age (older persons showing more rapid CD4+ cell fall-off), and anti-HIV therapy. Clearly, these observations need to be extended in numbers, and the high-purity agents should also be similarly compared with the very high-purity therapies.

The impact of a very high purity factor VIII concentrate on the immune system of human immunodeficiency virus-infected hemophiliacs: a randomized, prospective, two-year comparison with an intermediate purity concentrate.

Pathophysiologic considerations as well as non-comparative clinical results suggest that very high purity concentrates may slow immunologic deterioration in human immunodeficiency virus (HIV)-infected hemophiliacs. In an attempt to evaluate this hypothesis, we prospectively compared CD4 cell counts, skin testing responses, and changes of the clinical status in 20 asymptomatic HIV-positive hemophiliacs, randomly assigned to continue the treatment with an intermediate purity concentrate or to receive a very high purity product, purified by immunoaffinity chromatography with monoclonal antibodies. In the group switched to the very high purity concentrate there was no significant change of the CD4 cell counts over the 96-week follow-up period, whereas in the group continued on the intermediate purity concentrate, a highly significant decline was detected (P less than .013). Furthermore, in the very high purity group, four of six anergic patients at entry acquired reactivity to skin testing. The results of this study clearly support the use of very high purity concentrates for the replacement therapy of HIV-infected hemophiliacs.

Antibody to hepatitis C virus after a vapour-heated factor VIII concentrate. The Study Group of the Fondazione dell'Emofilia.

To evaluate whether or not clotting factor concentrates exposed to virucidal procedures transmitted hepatitis C, sera obtained in 1984-1986 from 27 previously untreated hemophiliacs infused with a vapour-heated factor VIII concentrate were tested retrospectively for the antibody to the hepatitis C virus (anti-HCV). A 2-year-old hemophiliac, negative for anti-HCV before administration of concentrate, seroconverted at week 12 and remained anti-HCV positive thereafter. Both his parents were anti-HCV negative and he had no other household contact. The patient had also become HBsAg positive at week 8 and had at the same time a marked elevation of alanine aminotransferase. His double infection with the hepatitis B and C viruses indicates that hot vapour was not completely effective in inactivating these viruses.

Recurrent mutations and three novel rearrangements in the factor VIII gene of hemophilia A patients of Italian descent.

Hemophilia A (HA), a common inherited bleeding disorder in humans, is due to the deficiency or absence of the factor VIII (FVIII) activity. The cloning of the FVIII gene has made molecular probes available for the characterization of the basic defect in this disease. In this study we describe six different mutations in the FVIII gene detected by DNA analysis of 100 HA patients of Italian descent. In two of them, with a severe clinical picture, we identified two novel deletions, one in the middle of the FVIII gene from exons 7 to 22 and the other encompassing the entire factor VIII gene. Both of these patients produced antibodies to factor VIII. In a patient with mild HA we detected a duplication of exon 13, which is a rearrangement not yet described within the FVIII gene. A possible explanation for the mild phenotype in this patient is that the molecular defect results in the production of an unstable FVIII protein with residual 10% FVIII activity. Screening by Taq I restriction endonuclease detected three mutations that were further characterized by direct sequencing on amplified DNA: a C-T substitution at codon 1960, in exon 18, converting the codon for arginine to a non-sense codon; and a G-A substitution at codon 2228 and 2326, in exons 24 and 26 respectively, resulting in the substitution of glutamine for arginine. All three of these mutations have been previously described. The non-sense mutation and the codon 2228 G-A mutation was found in patients with severe HA, while the codon 2326 G-A mutation was associated with a quite severe condition. These results confirm that the molecular bases of HA are very heterogeneous and provide further evidence that recurrent mutations are not uncommon in this system.

Long-term immunogenicity of a plasma-derived hepatitis B vaccine in HIV seropositive and HIV seronegative hemophiliacs.

Short-term studies indicate that hepatitis B vaccines are safe and satisfactorily immunogenic in hemophiliacs. The duration of immunity in these immunocompromised patients, however, is not known. To determine this, we studied 78 hemophiliacs prospectively 2, 3, and 4 years after the initial vaccination with a plasma-derived vaccine given as three monthly injections followed by a fourth booster injection at month 14. The duration of immunity clearly depended on whether the patients were infected with the human immunodeficiency virus (HIV). In HIV seronegative hemophiliacs (n = 67), there was a progressive decline in titers of antibody to the hepatitis B surface antigen (anti-HBs), but antibody was still detectable 4 years later in all of them. From the curves of decline of antibody titers, it appears that there is no need to revaccinate patients for at least 5 to 6 years. The HIV seropositive hemophiliacs (n = 11) not only started from much lower anti-HBs titers, but 5 of 11 lost anti-HBs. None of the 45 patients treated with concentrates during the postvaccination period developed serologic signs of hepatitis B, even though 6 of them had come into contact with live or inactivated hepatitis B virus as shown by the occurrence of spontaneous anamnestic antibody responses. This vaccine and schedule of vaccination afford a prolonged duration of immunity in HIV seronegative hemophiliacs, but HIV seropositive hemophiliacs have a risk of losing immunity early.

Kaposi's sarcoma as clinical manifestation of the acquired immunodeficiency syndrome in a hemophilic patient.

The authors report the natural history of HIV infection in a patient with severe hemophilia A who became HIV-seropositive in 1983 and, four years later, developed full-blown AIDS associated with a disseminated Kaposi's sarcoma. Neutralizing antibody titers against HIV were shown to be inversely disease-associated, while the progression of clinical symptoms was directly related to the decline of T4 cells and the increase of urinary neopterin levels. It is suggested that the presence of an HLA DR 5 phenotype and repeated CMV infection could have been crucial for the development of KS.

Chronic lymphocytic leukemia (CLL) in younger adults: a retrospective study of 133 cases.

Clinical and biological data have been evaluated, using both univariate and Cox's multivariate statistical analyses, in a series of 133 Chronic Lymphocytic Leukemia (CLL) patients with a mean age of 46.6 years (range 31-50). In univariate analyses, anemia (Hb less than 13 g/dl), peripheral blood (PB) lymphocytosis (greater than 40 x 10(9)/l) and bone marrow (BM) lymphocytosis (greater than 80 per cent) were shown to be of significant prognostic value. Multivariate analysis, through a forward stepwise procedure, showed that the most important and independent variable is the BM lymphocytosis. These results are different from those obtained in previous studies and particularly in a recent identical study performed by the same Cooperative Group on 1777 patients with a mean age of 64.2 years (Mandelli et al., 1987). No significance can be demonstrated in stratifying this series of younger patients according to different staging methods (Rai et al., 1975; Binet et al., 1981b; Mandelli et al., 1987). Therefore this population of CLL patients, with less than 50 years of age, has risk factors quite different from classical CLL. The results of the present study show that the diagnostic approach to B-CLL in younger adults must be more complete: using the common diagnostic criteria, established staging systems appear to be inadequate in this series of younger patients.

Immunogenicity of a recombinant hepatitis B vaccine in hemophiliacs.

Yeast-recombinant vaccines against hepatitis B virus (HBV) are now available, but there is no information about whether or not they are immunogenic in patients with hemophilia and other congenital bleeding disorders. Twenty micrograms of a recombinant vaccine expressing the adw serotype of the hepatitis B surface antigen (HBsAg) were given to 41 patients negative for HBV markers and again after 1 and 6 months. Ten percent of the vaccinees had anti-hepatitis B surface antibody (anti-HBs) responses, with titers of 10 mIU/ml or more, 1 month after the first dose of vaccine. The percentage of anti-HBs-positive patients increased to 54% after the second dose and to 98% after the third dose, with only one non-responder. Hence, the recombinant vaccine was immunogenic, with percentages of seroconversion and anti-HBs titers similar to those achieved with plasma-derived vaccines.