Methods for platelet preparation in Italy in 1996.
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Blood Platelets↗
Biomedical subjects
R Bruni
Publications and source records attributed to R Bruni.
Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery.
The herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a promiscuous transactivator, and by necessity, its functions must be mediated through cellular gene products. In an attempt to identify cellular factors interacting with ICP0, we used the carboxyl-terminal domain of ICP0 as "bait" in the yeast (Saccharomyces cerevisiae) two-hybrid system. Our results were as follows: (i) All 43 cDNAs in positive yeast colonies were found to encode the same translation factor, elongation factor delta-1 (EF-1delta). (ii) Purified chimeric protein consisting of glutathione S-transferase (GST) fused to EF-1delta specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) Fractionation of infected HEp-2 cells and immunofluorescence studies revealed that ICP0 was localized both in the nucleus and in the cytoplasm. In primary human foreskin fibroblasts, ICP0 was localized predominantly in the cytoplasm throughout HSV-1 infection even early in infection. (iv) Addition of the chimeric protein GST-carboxyl-terminal domain of ICP0 to the rabbit reticulocyte lysate in vitro translation system resulted in a dose-dependent decrease in protein synthesis. In contrast, GST alone or GST fused to the amino-terminal domain of ICP0 had no effect on the in vitro translation system. (v) The predominant forms of EF-1delta on electrophoresis in denaturing gels have apparent Mrs of 38,000 and 40,000. The higher-Mr form is a minor species in mock-infected cells, whereas in human fibroblasts and Vero cells infected with HSV-1, this isoform becomes dominant. These results indicate that ICP0 is present and may have a significant role in the cytoplasm of infected cells, possibly by altering the efficiency of translation of viral mRNAs.
Animals↗
Spiking Survanta with synthetic surfactant peptides improves oxygenation in surfactant-deficient rats.
The hypothesis that the in vivo function of Survanta (Beractant) can be improved by supplementation with synthetic surfactant peptides B and C was tested in a surfactant-deficient rat model. Full length surfactant protein-B (SP-B1-78) (B) and palmitoylated surfactant protein-C (SP-C1-35) (C), and synthetic KL4 peptide were added to Survanta after extraction, creating extracted Survanta (ES) with 1% B, 2% B, and 2% B plus 1% C, or mixed with Survanta without extraction, creating modified Survanta (S) with 2% B, 2% B plus 1% C, and 2% KL4. Adult rats were ventilated with 100% oxygen, tidal volumes (VT) of 7.5 ml/kg and a rate of 60/min, and were lavaged until the PaO2 dropped below 80 mm Hg, when 100 mg/kg of surfactant was instilled. After 15 to 60 min of ventilation, pressure-volume (P-V) curves were generated in situ. Instillation of ES or S with 2% B plus 1% C led to the greatest increase in oxygenation, closely followed by ES and S with 2% B, and more distantly by S plus 2% KL4. TLC was comparable among the ES and S groups, but greater than that of air-placebo controls. These data suggest that spiking of Survanta with synthetic SP-B and SP-C increased oxygenation more effectively than B or KL4 alone in this surfactant-deficient rat model.
Amino Acid Sequence↗
Erythrothrombocytapheresis and plasmathrombocytapheresis with storage in T-sol of platelets collected by the new Amicus cell separator.
The Amicus cell separator is the latest apparatus introduced into the international market for high-yield, low WBC contamination and short-procedure time thrombocytapheresis. In its original configuration the apparatus collects platelets for subsequent resuspension in plasma and no collection of PRBC can be carried out along with thrombocytapheresis. In this paper we present the results of plasma-thrombocytapheresis and erythro-thrombocytapheresis after the adaptation of the Amicus to the collection and storage of platelets in a non-plasma medium and the concurrent collection of PRBC. From our study it is concluded that PRBC collection doesn't modify the quality of the platelet product obtained from random donors (platelets pre-count 263x10e3/microliter) in terms of yield which is 4.6x10e11 platelets, WBC contamination (1.3x10e5) or procedure time (63 +/- 26 min.). The quality of the platelet products is satisfactory too, as measured by aggregation induced by collagen and ristocetin or by the substantial stability of membrane glycoproteins (CD62-63-51-36-42B). The concentrate had an average content of 58.8 g of hemoglobin per bag, a final volume of 376 +/- 13 ml, (after the addition of 100 ml of SAG-M) and a normal mechanic and osmotic fragility.
Blood Component Removal↗
[Ambulatory subcutaneous internal sphincterectomy].
The internal anal sphincterotomy operation represents the best therapy for the treatment of those anal lesions, above all anal fissures, which cause hypertrophy and spasms of the internal anal sphincter. The Authors report their experience of 253 operations of internal subcutaneous sphincterotomy carried out between 1989 and 1995 under local anaesthesia in the outpatient clinic. The excellent results achieved prove that the procedure can easily be carried out under local anaesthesia in an outpatient clinic and, therefore, it is preferable to the surgical technique of isolation of the internal sphincter, not only for its practicality but also for the improved results.
Adolescent↗
Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.
The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.
Carcinoma, Hepatocellular↗
Inactivation of surfactant in rat lungs.
Although surfactant replacement therapy has dramatically improved the outcome of premature infants with respiratory distress syndrome, approximately 30% of treated infants show a transient or no response. Nonresponse to surfactant replacement therapy may be due to extreme lung immaturity and possibly surfactant inactivation. Surfactant inactivation involves aspecific biophysical events, such as interference with the formation or activity of an alveolar monolayer, and specific interactions with serum proteins, including antibodies, leaking into the alveolar space. As formulations containing surfactant proteins appear to better tolerate serum inactivation, we used an excised rat lung model to compare the susceptibility to serum inactivation of a mixture of synthetic phospholipids selected from surfactant lipid constituents, Exosurf (a protein-free synthetic surfactant), Survanta [containing surfactant proteins B and C (SP-B and -C)], and a porcine surfactant (containing SP-A, -B, and -C). For each of these preparations, we used pressure/volume determinations as an in situ measure of surfactant activity and retested the same preparations after mixing with human serum, a nonspecific surfactant inactivator. Human serum inactivated porcine surfactant to a lesser extent than Survanta, Exosurf, or synthetic phospholipids. Temperature exerted a significant effect on deflation stability, as shown by a greater lung compliance in untreated, normal lungs and a larger improvement in compliance after treating lavaged lungs with synthetic phospholipids at 37 degrees C than at 22 degrees C. We conclude that surfactant containing SP-A, -B, and -C is only moderately susceptible to inactivation with whole serum and may therefore exert a greater clinical response than protein-free surfactants or those containing only SP-B and -C.
Animals↗
A synthetic segment of surfactant protein A: structure, in vitro surface activity, and in vivo efficacy.
Surfactant protein A (SP-A) is a 248-residue, water-soluble, lipid-associating protein found in lung surfactant. Analysis of the amino acid sequence using the Eisenberg hydrophobic moment algorithm predicts that the SP-A segment spanning residues 114-144 has high hydrophobic moments, typical of lipid-associating amphipathic domains. The secondary structure, in vitro surface activity and in vivo lung activity of this SP-A sequence were studied with a 31-residue synthetic peptide analog (A114-144). Analysis of the secondary structure using circular dichroism and Fourier transform infrared spectroscopy indicated association with lipid dispersions and a dominant helical content. Surface activity measurements of A114-144 with surfactant lipid dispersions and the hydrophobic surfactant proteins B and C (SP-B/C) showed that A114-144 enhances surface activity under conditions of dynamic compression and respreading on a Langmuir/Wilhelmy surface balance. Synthetic surfactant dispersions containing A114-144 improved lung compliance in spontaneously breathing, 28-d premature rabbits to a greater degree than surfactant dispersions with synthetic SP-B/C and synthetic surfactant lipids alone. These observations indicate that inclusion of A114-144 may improve synthetic preparations currently used for surfactant replacement therapy.
Amino Acid Sequence↗
Twelve years experience with high-dose therapy and autologous stem cell transplantation for high-risk Hodgkin's disease patients in first remission after MOPP/ABVD chemotherapy.
High-dose therapy followed by autografting can cure patients with aggressive Hodgkin's disease (HD) refractory or with early relapse to first-line combination chemotherapy. On the other hand, the eradication of the disease is rarely achieved in heavily pretreated patients. It has been suggested that patients with HD with very high risk characteristics at diagnosis, often relapse despite appropriate therapy with 7-8 drugs combination. Thus it seems to us that such patients are potential candidates for early autografting during first remission. Twelve years ago, we initiated a pilot study to investigate whether patients with very high risk characteristics, would benefit from early autografting. The application of early autografting was compared with our historical group of patients in complete remission after receiving MOPP/ABVD, who had the same negative prognostic characteristics, refused autografting and who did not receive other treatment after achieving complete remission. Among the 22 consecutive patients entered into the pilot study and autografted, 18 are alive and 17 (77%) remain alive in unmaintained remission at a median of 86 months. One patient (4%) died of interstitial pneumonitis in the transplantation group. Only 8/24 (33%) patients, who did not receive an autograft, are currently alive and disease free at a median of 89 months. In conclusion, the early application of autografting appears to improve the outcome in patients with very high risk HD who achieved remission with MOPP/ABVD.
Adolescent↗
Plateletpheresis: what's new?
1995 has brought several novelties into the field of thrombocytapheresis. The most important is the presentation by Baxter of the Amicus CFC apparatus that, in a two-vein collection time of 51-53 min, collects 3.93-4.13 x 10(11) platelets contaminated by 0.2 x 10(6) white blood cells (WBC) with an efficiency of 72-74%. Similar results are obtained in 55 min, in single-needle procedures, with a collection efficiency of 70.7%, a total yield of 4.3 x 10(11) and a contamination of 0.9 x 10(6) WBC. Presently the machine cannot collect red blood cells (RBC), along with platelets, as does the DFC Haemonetics MCS3p. With this machine in mandatory single-needle procedures lasting 80-85 min, approximately 4.5 x 10(11) platelets can be collected with a WBC contamination of 3.1 x 10(6). Along with the platelet cells (PC), with the SDP PRC protocol at the end of each pass it is possible to collect aliquots of plasma red blood cells (PRBC) (10-50 mL) with a haematocrit of 65%. Depending on the number of passes and on the donor's body weight (BW), from 1 and 2 U/PRBC containing approximately 55 g of haemoglobin (Hb) each can be obtained. This method not only costs less but also immunological and viral risks for the patients are more efficiently addressed since it is possible to reduce the number of allogeneic exposures in the same transfusion event. PRBC can also be collected with the Fresenius AS104 and the Dideco Excel apparatuses. Dideco is presently using the "Genova" separation chamber, which can collect an average of 4.8 x 10(11) platelets contaminated with 0.5 x 10(6) WBC, in approximately 55 min. The ACD-A-to-blood ratio may be as low as 1/20 since the very last belts and lines are coated with non-thrombogenic substances. Therefore, there is no alteration of the quality of the platelets as evaluated with monoclonal antibodies (MoAbs) anti-CD 62, 63, 36 and 51, no extra haemorrhagic risk for the donors and citrate reactions and microaggregate formation are totally eliminated. In 1995 there has also been some rejuvenation of the older cell separators. The CS3000+ uses the TN6/PLT 30 combination of separation collection chambers. Platelets are collected in only 30 mL of plasma, and non-plasma solutions containing acetate can be used for their resuspension. The Fresenius AS204 is ready for better-quality combined platelet collection, and RBC-PC collection with Cobe Spectra is under evaluation along with the leucocyte reduction system that apparently brings leucocyte contamination down to 0.35 x 10(5). This interest in thrombocytapheresis is also the result of the challenge that thrombopoietin phase I and II trials have promoted.
Humans↗
Mobilization/transplantation of peripheral blood progenitor cells for aggressive non-Hodgkin's lymphoma with marrow involvement.
Thirty-five aggressive non-Hodgkin's lymphomas (NHL) with marrow involvement received high-dose cyclophosphamide (7 g/m2) and G-CSF in order to collect peripheral blood progenitor cells (PBPC). Fourteen patients were in partial remission, 16 patients were in relapse ('sensitive', 12; 'resistant', 4) and five patients were refractory to conventional treatment. A good yield of PBPC was obtained in 30 patients, while a low number of CD34+ cells and of CFU-GM was seen in two cases. Two patients entered progression and one patient died. Thirty patients underwent PBPC autografting. Twenty-nine out of 35 (83%) patients entered complete remission (CR). Two patients died in CR of infection following marrow aplasia 3 and 6 months after autografting. At 3 years the probability of survival and disease-free survival (DFS) are 62 and 51%, respectively.
Adult↗
Antioxidant vitamins and immunodeficiency.
On the plasma of 20 children from 1 to 6 years old with HIV infection, the following analyses were carried out: vitamin assays (vitamin E and beta-carotene), hematochemical assays, and immunoassays. From the body of our results it emerged that in the seropositive children considered, in addition to the already well-known alterations of the hematic and immune situation, there is a state of hypovitaminosis involving the most important antioxidant vitamins.
Antioxidants↗
[Cystic nephroma].
The authors describe a rare case of cystic nephroma treated by partial surgical excision. Because there is concurrence in the association of multilocular cysts with Wilms tumors and others tumors of the kidney, it is underlined the importance of a differential diagnosis to avoid nephrectomy for treatment of this benign neoplasm.
Adult↗
Recurrence of WHV integration in the b3n locus in woodchuck hepatocellular carcinoma.
Frequent occurrence of woodchuck hepatitis virus DNA (WHV DNA) integration into or in proximity to myc oncogenes and in the win locus of cellular genome in woodchuck hepatocellular carcinomas (HCC) has been described by several authors. We report a further cellular locus as a recurrent target for WHV integration in woodchuck HCCs. A WHV DNA integration and its cellular flanking regions were cloned from a HCC developed in a chronically WHV-infected woodchuck. Sequence analysis showed integration of rearranged C, PreS1, and 5' truncated X regions of the WHV genome, located in a cellular locus previously described for WHV integration in another woodchuck HCC. The two integration sites are only about 0.5 kb apart. In addition to Alu-like repeats and a gag-like coding region, previously described, we found several features of MAR (matrix attachment region) chromosomal sequences in the normal cellular locus, leading us to predict that part of it might be a previously unrecognized MAR.
Animals↗
Sequence and phylogenetic analysis of the VP1 gene in two cell culture-adapted HAV strains from a unique pathogenic isolate.
The nucleotide sequences of the VP1 coding region of two newly characterized, cell culture-adapted hepatitis A virus (HAV) strains (RG-SB11 and RG-SB16) were analyzed and compared with homologous regions of previously characterized HAV strains of human or monkey origin, and at different levels of tissue-culture adaptation. In particular, HM175wt and its derivative strains and MBB, LCDC1, PA21, and AGM27 isolates were considered. RG-SB11 and RG-SB16 HAV strains were derived from a pathogenic isolate from an acutely infected patient, purified from stool, and subjected to different strategies of adaptation. Several nucleotide differences were observed, but high conservation was found in the predicted VP1 protein sequences, which confirms structural constraints for this region. Furthermore, comparative amino-acid sequence analysis of VP1 from all HAV isolates studied has shown, particularly for those from naturally infected monkeys, that differences are limited to the amino and carboxy-terminal part of the molecule. The results of phylogenetic analysis have confirmed the common origin of the RG-SB11 and RG-SB16 strains. The complete nucleotide sequences of the VP1 coding region of the RG-SB11/16, HM175 derivative strains and of other HAV strains has shown that branch-length evolution can give a measure of the evolution of HAV during adaptation processes.
Amino Acid Sequence↗
A PCR-based strategy for rapid mapping of hepadnavirus integrated sequences in hepatocellular carcinomas.
A methodology based on polymerase chain reaction (PCR) and restriction analysis for rapid mapping of woodchuck hepatitis virus (WHV) integrations in hepatocellular carcinoma (HCC) tissues is described. Conventional PCR with viral primer pairs is not suitable for mapping WHV-integrated regions because the presence of minimum amounts of non-integrated (PCR amplifiable) WHV genome and replicative intermediates cannot be excluded. The first relevant part of the strategy is the identification of the cellular sequences flanking the WHV integration in order to select one (or more) integration-specific primer. The cellular flanking sequence can be rapidly obtained by means of inverse-PCR amplification of the viral/cellular junction and sequencing of the product. Mapping of the integrated regions is carried out by fixed flanking primer PCR (FFP-PCR) using the cellular primer as a 'fixed' primer in PCR association with each of an available set of WHV primers. Amplification of episomal WHV sequences is thus avoided. PCR products can also undergo restriction analysis. PCR-positive viral primers and specific WHV restriction sites are assembled into a map, based on the size and restriction pattern of the PCR products. The results of WHV integration mapping in a woodchuck HCC are described.
Animals↗
Autologous stem cell transplantation as adjuvant treatment vs no further therapy for poor-risk Hodgkin's disease in first complete remission after MOPP/ABVD.
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Adolescent↗
Vibrio cholerae in the horn of Africa: epidemiology, plasmids, tetracycline resistance gene amplification, and comparison between O1 and non-O1 strains.
The prevalence of Vibrio cholerae O1 and non-O1 has been investigated in numerous Somali regions of the Horn of Africa from 1983 to 1990. From January 1983 to January 1985 and between December 1986 and December 1990, no strains of V. cholerae O1 and 226 strains (5.3%) of V. cholerae non-O1 were isolated from 4,295 diarrhea cases. During a cholera epidemic in 1985 and 1986, the overall case-fatality rate was 13% and the attack rate was 3-3.5 per 1,000 population. Matched case-control studies identified a waterborne route of transmission. A drug-susceptible Ogawa strain from Ethiopia caused the introduction of the disease into northern Somalia. There were two major resistant derivatives of the original strain, and the one resistant to ampicillin, kanamycin, streptomycin, sulfonamide, and tetracycline (TC) predominated in the spreading disease. In 1986, susceptible Ogawa strains quickly displaced this resistant strain. The two incompatibility group C plasmids responsible for the resistance patterns had complex and scattered differences in their structures. Physical analysis of the plasmid DNA region coding for TC resistance demonstrated its genetic amplification in highly resistant variants of Ogawa strains.
Adolescent↗