Study on the reinitiation of transcription in 37 RC cells after actinomycin D inhibition. Spectrum of major RNA species resynthesized after maximal suppression by the drug.

The reinitiation of the synthesis of major RNA species has been studied in 37 RC cells after maximal inhibition of RNA synthesis by actinomycin D (AMD). During the period of recovery from AMD, resynthesized RNA (rec-RNA) is initially composed of almost exclusively light (4-14S) heterogeneous RNA species. All normal species of RNA can be detected in the rec-RNA spectrum as early as 3 h after AMD removal. The synthesis of low molecular weight methylated RNA species increases slightly during the early period after AMD removal, while the increase of low molecular weight unmethylated species is more significant during the same period. Much of the radioactivity in the polyribosomal fraction is EDTA and puromycin sensitive. Since polysomal, puromycin-sensitive RNA is polyadenylated (as evidenced by the binding of poly-U filters), and is heterogenous in size, it belongs to the m-RNA class. The synthesis of m-RNA increases immediately after AMD removal, whereas the reinitiation of the r-RNA synthesis occurs after a lag period of about 2 h. The kinetics of recovery of the synthesis of major RNA species from AMD inhibition show a size dependency comparable to the size-related sensitivity to AMD inhibition in other cellular systems. This dependency is most clearly seen in HnRNA, the AMD sensitivity of which is measured by the length of the lag period between AMD removal and the appearance of HnRNA fractions in a sucrose density gradient. Low molecular weight HnRNA reappears first, whereas heavier fractions of HnRNA appear in the spectrum after a lag period, the length of which is in direct relation to the position of the HnRNA fraction in the gradient.

Alfa-interferon in the treatment of essential thrombocythemia: clinical results and evaluation of its biological effects on the hematopoietic neoplastic clone. Italian Cooperative Group on ET.

The efficacy of alfa-interferon (alfa-IFN) in essential thrombocythemia (ET) patients has been reported by several authors. The aim of this study is to assess the magnitude of the effect of alfa-IFN on the neoplastic clone. As of December 1993, 11 ET patients received alfa-IFN at a dose of 3-6 MU/s.c./day for 6 months. Ten of 11 obtained complete hematological remission (CHR) and one achieved partial hematological remission. Megakaryocyte concentration was reduced in six cases. The spleen,which was enlarged in four patients, decreased in size in two patients. Seven of eight patients who were symptomatic at diagnosis obtained resolution of symptoms. In order to obtain indications about the structural modifications induced by alfa-IFN in ET megakaryocytes (Mks), Fourier-transform infra-red microspectroscopy analysis performed on 10 single Mks of each patient, was done in seven of 11 patients; the analysis showed a reduction of A1/A2 ratios (A1 integrated area of the band at 1080 cm(-1) due to the nucleic acids absorption; A2 integrated area of the band at 1540 cm(-1) due to proteic components absorption) in five cases, and in three of these five patients A1/A2 ratios achieved normal values. After alfa-IFN treatment we did not observe any change in the methylation pattern of DNA from the granulocyte fraction. Our results confirm the efficacy of alfa-IFN in ET patients, and the decrease of A1/A2 ratios in several patients is a demonstration of the depth of the effect of alfa-IFN on the neoplastic clone. The results of clonality studies showed the persistence of clonal hematopoiesis. Whether higher alfa-IFN dose and/or more prolonged alfa-IFN therapy may allow a restoration of polyclonal hematopoiesis remains to be determined and should be explored in future clinical trials.

Bone marrow donors other than HLA genotypically identical siblings for patients with thalassemia.

This report describes the successful bone marrow transplantation of three children with thalassemia who received bone marrow, one from an HLA identical but mixed lymphocyte culture-reactive sibling, the other two from an HLA phenotypically identical parent. Evidence of engraftment was detected early (19-21 days) in all three children and only grade II acute GvHD was observed in one patient. Our report indicates that thalassemic patients can be cured by bone marrow transplantation from selected donors other than HLA genotypically identical siblings.

Allogeneic marrow transplantation for thalassemia.

Thirteen patients with homozygous beta thalassemia underwent allogeneic marrow transplantation from sibling donors, 12 of whom were heterozygous for beta thalassemia. Six patients were transplanted in an advanced stage of their disease while seven were transplanted early in their disease course. Donors and recipients were genotypically identical for the HLA-A, -B and -D loci in 11 cases and mismatched for the D locus in two. A variety of preparative regimens was utilized involving high doses of busulphan (Bu) and/or cyclophosphamide (CY) and/or total body irradiation (TBI). Failure of engraftment or autologous hematologic recovery after transient engraftment was seen after intensive preparative regimens including: CY 200 mg/kg and 800 rad of TBI; Bu 8 mg/kg and CY 200 mg/kg; and Bu 8 mg/kg, CY 200 mg/kg, and 300 rad of TBI. A regimen of Bu 16 mg/kg, CY 200 mg/kg, and 400 rad of TBI resulted in deaths from transplant-related causes in the three patients treated with this regimen. Seven of the 13 patients are surviving 363-665 days after transplant. Five of the seven failed to achieve engraftment or had autologous reconstitution after transient engraftment. Five of the six deaths were transplant related, and one patient died of cardiac failure one year after an unsuccessful transplant attempt. Two patients are surviving with engraftment and without thalassemia major 363 and 491 days after transplantation. Both of these patients were transplanted early in their disease course.

DNA methylases separated through the HeLa cell cycle methodology show allosteric properties.

Two DNA methylases (DNAmets) can be separated through the cell cycle. The first appears as a minor peak in G1, the second as a major peak in S. Both enzymes protect from HpaII a plasmid (H31), constructed with the pBR322 vector (4.3 kbp) and the inverted A gamma fragment of the human globin gene (3.5 kbp), inserted at its HindIII site (the vector carries several HpaII sites, the insert only one HpaII site). DNAmets G1 and S show distinct Km values and different kinetics vs the ionic strength of the medium, while their Michaelis-Menten and Lineweaver-Burk plots are sigmoidal and hyperbolical curves, respectively. This is the first suggestion about the allosteric nature of the eukaryotic DNAmet system.

Organisation of reciprocal connections between trigeminal and vestibular nuclei in the rat.

In order to study the connection patterns between the sensory trigeminal and the vestibular nuclei (VN), injections of anterogradely and/or retrogradely transported neuronal tracers were made in the rat. Trigeminal injections resulted in anterogradely labelled fibres, with an ipsilateral preponderance, within the VN: in the ventrolateral part of the inferior nucleus (IVN), in the lateral part of the medial nucleus (MVN), in the lateral nucleus (LVN) with a higher density in its ventral half, and in the superior nucleus (SVN), more in the periphery than in the central part. Moderate trigeminal projections were observed in the small vestibular groups f, x and y/l and in the nucleus prepositus hypoglossi. Additional retrogradely labelled neurones were seen in the IVN, MVN, and LVN, in the same regions as those receiving trigeminal afferents. Morphological analysis of vestibular neurones demonstrated that vestibulo-trigeminal neurones are relatively small and belong to a different population than those receiving projections from the trigeminal nuclei. The trigeminovestibular and vestibulo-trigeminal relationships were confirmed by tracer injections in the VN. The results show that, in the VN, there is sensory information from facial receptors in addition to those reported from the neck and body. These facial afferents complement those from the neck and lower spinal levels in supplying important somatosensory information from the face and eye muscles. The oculomotor connections of the respective zones of the VN receiving trigeminal afferents suggest that sensory inputs from the face, including extraocular proprioception, may, through this pathway, influence the vestibular control of eye and head movements.

Response to first-line chemotherapy and long-term survival in patients with multiple myeloma: results of the MM87 prospective randomised protocol.

In this study we evaluated whether a good response to conventional chemotherapy, i.e. a significant tumour reduction, is a prerequisite for improved survival in multiple myeloma (MM). Between January 1987 and March 1990, 341 consecutive previously untreated patients with MM received chemotherapy within the prospective, multicentre, randomised Protocol MM87. Of these, 258 patients were evaluable for both response and long-term survival and 244 (94.6%) have died. The median survival of all patients was 40 months (6-162 months). The median survival did not differ between patients who had complete response (CR) (50 months (9-162 months)), partial response (PR) (46 months (8-147 months)) or stable disease (SD) (41 months (7-135 months)). The median survival was shorter (13.6 months (6-135 months)) (P<0.0001) in patients whose disease progressed while they were receiving first induction chemotherapy. Causes of death were more frequently (P=0.04) related to MM in patients who had progressive disease (PD) than in patients who had a CR or PR or SD. The main clinical and laboratory characteristics were similar in the four groups. These data indicate that patients who maintain SD during first-line chemotherapy have a prognosis similar to that of patients who attain a response. Only patients whose disease progresses have a distinctly worse outcome.

Genotypic analysis of engraftment in thalassemia following bone marrow transplantation using synthetic oligonucleotides.

DNA hybridization with synthetic oligonucleotide probes was used to assess engraftment in 19 thalassemic patients who received bone marrow grafts from their respective healthy HLA-identical siblings. Three oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed so as to produce simple RFLP (restriction fragment length polymorphism) patterns. Each probe hybridizes to one or two bands in HinfI-digested genomic DNA. The combined use of these three probes allowed a discrimination between all the HLA-identical siblings tested. Both donor-specific and recipient-specific DNA fragments existed in 18 out of the 19 sibling pairs studied. One pair possessed only a donor-specific fragment. DNA analysis at an early stage after the graft detected donor-specific fragments in 15 out of 19 patients, recipient-specific fragments in three patients and a mix of recipient and donor fragments in one patient. At a later stage this patient possessed donor-specific fragments only. Follow-up DNA analysis confirmed these findings. Thus 16 patients continued to display donor-specific fragments over 60 days post-transplant. These DNA data showed strong correlation with the clinical status of the patients as well as with other markers of engraftment including cytogenetics and hemoglobin synthesis. The patients who showed donor-specific fragments over 60 days have been free of thalassemic symptoms for over 300 days. Moreover, in 11 cases it was possible to predict the fate of the graft within 15 days after transplantation. In conclusion, the use of the three synthetic oligonucleotide probes provides a powerful tool in documenting engraftment in bone marrow transplantation.

Increased serum levels of circulating intercellular adhesion molecule 1 predict the risk of graft rejection after bone marrow transplantation for thalassemia.

Beta thalassemia is a hereditary anemia curable by bone marrow transplantation (BMT). Class 3 patients have a much worse outcome with a high incidence of rejection after BMT. Adhesion molecules, including the intercellular adhesion molecule 1 are thought to play an essential role in the rejection process. To investigate whether increased levels of soluble intercellular adhesion molecule 1 (sICAM-1) may be predictive of graft rejection, the pretransplant serum concentration of sICAM-1 of 27 beta thalassemic patients who rejected the graft was compared to that of 68 beta thalassemic patients who achieved a sustained engraftment. Fifty serum samples, obtained from marrow donors, matched for age and sex, served as controls. Beta thalassemic patients had significantly higher sICAM-1 concentrations as compared to controls (P = 0.0001). Significantly increased levels of sICAM-1 were found in the patients who subsequently rejected the graft (mean (95% CI) = 490 ng/ml (440; 540)) as compared to those with sustained engraftment (400 ng/ml (384; 415)), (P = 0.004). The mean level of sICAM-1 in patients with early rejection was significantly higher than that in patients with late rejection (P = 0.04). This may indicate a transfusion-mediated role of sICAM-1: some beta thalassemic patients with high sICAM-1 levels, induced by the transfusion support, may remain immunologically active, despite the conditioning regimen, therefore such patients are likely to have an early graft rejection. Our findings indicate that sICAM-1 could be a useful indicator of immune activation in polytransfused patients with beta thalassemia who have a high risk of rejection. Determination of sICAM-1 has potential clinical implications in predicting which patients may reject after BMT.

Serum amyloid A protein concentration in bone marrow transplantation for beta thalassaemia.

AIMS: To investigate whether serum amyloid A protein (SAA) and C-reactive protein (CRP) concentrations could be used in the management of beta thalassaemic patients undergoing bone marrow transplantation (BMT). METHODS: Serum SAA and CRP concentrations were determined in paired samples from 66 patients with beta thalassaemia before and after BMT. Serum SAA concentrations were determined by an enzyme linked immunoassay (EIA); serum CRP concentrations were determined by a nephelometric assay. RESULTS: Serum SAA concentrations before transplantation were significantly higher in the group that subsequently rejected the transplant than the group without complications. SAA concentrations increased after BMT in acute graft versus host disease (GvHD) and rejection. No significant increase in SAA or CRP was found in chronic GvHD. Increases in serum in SAA and CRP concentrations were not related to concomitant infection episodes. CONCLUSIONS: The different acute phase response in acute GvHD and rejection compared with chronic GvHD suggests that different immunopathogenic mechanisms are responsible.

Increased serum concentrations of tumour necrosis factor in beta thalassaemia: effect of bone marrow transplantation.

AIMS: Serum concentrations of tumour necrosis factor-alpha (TNF) were determined in beta thalassemic patients before and after bone marrow transplantation (BMT) to evaluate whether changes in TNF concentrations after BMT were related to immune mediated complications. METHODS: Serum TNF concentrations were determined by enzyme linked immunoassay (EIA) in paired samples from 71 patients with beta thalassemia before and after BMT. Serial samples from 13 patients were also studied for up to six months after BMT. Forty one normal healthy children matched for sex and age were studied as controls. RESULTS: beta thalassemic patients had high serum TNF concentrations before transplantation compared with controls. These were not related to sex, age, duration of disease, number of blood transfusions, transferrin concentrations or splenectomy. DQw1 positive patients showed significantly lower TNF concentrations than non-DQw1 cases. Patients with severe liver fibrosis had significantly higher TNF concentrations. No correlation was found between TNF values and BMT outcome before transplantation but TNF alpha values fell significantly after BMT. The decrease persisted only in patients with successful engraftment. In serial samples studied for up to six months after BMT, TNF values decreased but in four out of five patients with graft rejection and in all five with acute graft versus host disease (GVHD) sharp increases occurred at the time of clinical symptoms. No correlation was found between the degree of GVHD and serum TNF-alpha concentrations nor between TNF-alpha concentrations after BMT and the presence of bacterial, viral, and fungal infections. CONCLUSIONS: About 50% of beta thalassemic patients have increased serum TNF, and the changes after BMT are related to the occurrence of immune mediate complications. The persistence of low TNF concentrations after successful engraftment may be due to the preparative regimen and the lack of adverse immune reactions.

PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.

In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.

Fastigiovestibular projections in the rat: retrograde tracing coupled with gammaamino-butyric acid and glutamate immunohistochemistry.

In this study, a double labeling technique using retrograde tracing with protein-gold complex (gold-HRP) in conjunction with a gammaamino-butyric acid (GABA) and glutamate immunohistochemical procedure was performed to identify GABA (GABA-IR) and glutamate (Glu-IR) immunoreactive neurons in the cerebellar fastigial nucleus (FN) that projects to the vestibular nuclei (VN). The results show that FN neurons projecting to the VN consist of both GABA-IR and Glu-IR neurons with a predominance of glutamatergic ones. Because GABAergic neurons in the cerebellar nuclei project to the inferior olive (IO), double retrograde labeling experiments were performed with injections of gold-HRP in the IO and of biotilynated dextran amine in the VN. This showed that the GABA-IR fastigiovestibular neurons project by axon collaterals to both the VN and the IO.

Definitive evidence for the actual contribution of yeast in the transformation of neutral precursors of grape aromas.

Experiments were designed to demonstrate the actual contribution of yeast in the formation of the primary aroma during the vinification of neutral grapes. Ruché was chosen as the model wine to study because of its unique fragrance. A yeast strain specific for Ruché was selected using a new and rapid isolation method for red wines. The results of this study can be summarized as follows: Skins from nonaromatic white or red grapes apparently contain most of the primary aroma compounds that are revealed in the must only after contact with yeast cells under defined conditions. Similar results were obtained with the pulp and seeds fractions; however, the olfactory notes, although well characterized, differed from those obtained with skins alone. Clarification, filtration, and centrifugation of the pulp and seed fractions or sonification of the skins produce different and well-characterized olfaction notes during the contact with yeast. The primary aroma of nonaromatic white and red grapes contained in the skins can be revealed within 24-48 h of yeast contact in a synthetic nutrient medium (SNM). The primary aroma precursors extracted from the skins with methanol, water-saturated butanol, or aqueous buffer at pH 3.2, concentrated and eluted from a C18 resin column, can be transformed to the free form wine aroma markers within 6 h of contact with yeast cells in SNM. By contrast, prolonged maceration of the skins in aqueous alcoholic buffer at pH 3.2 or 1.1, at 50 or 70 degrees C did not release primary odors typical of wine. The individual primary aroma compounds, identified by GC-MS analysis in Ruché wine samples or in Ruché skin-yeast-SNM samples, could not explain the complexity of the typical Ruché wine odor. Only odors common to many wine varieties were identified by GC-olfactometry analysis.

Effectiveness of fludarabine in advanced B-cell chronic lymphocytic leukemia.

AIMS: The study was carried out to investigate the efficacy and toxicity of fludarabine phosphate in the treatment of B-cell chronic lymphocytic leukemia (B-CLL) in previously treated patients. METHODS: Sixteen patients, 11 males and 5 females, 9 in stage B and 7 in stage C, according to the Binet Staging System, were treated with a maximum of 6 cycles of fludarabine (25 mg/m2) for 5 days, every 4 weeks. All patients had been pretreated, 10 were refractory to standard regimens, 5 were in relapse, and 1 patient was in partial remission. RESULTS: Thirteen patients were judged suitable for evaluation. Overall 9 patients were responsive to treatment; 4 complete and 5 partial responses were observed. Of the 4 patients in complete remission, 3 were alive at 6, 10 and 13 months, respectively, from the beginning of treatment. One patient died after 11 months for acute graft-versus-host disease after allogenic bone marrow transplantation by an HLA sibling donor. Two of the 5 patients in partial remission were alive at 7 and 17 months, respectively, and the other 3 died (2 of disease reexpansion after 14 and 16 months and 1 of septic shock following pneumonia). Four patients were not responsive to treatment: 1 died from disease progression after 8 months from the beginning of therapy, 1 from cardiac failure after 9 months, 1 from septic shock following meningitis, and 1 was alive after 7 months of follow-up. Treatment was well tolerated, with nausea and vomiting in only one patient. We observed two episodes of pneumonitis, without any evidence of the responsible agent, a tumor lysis syndrome with acute renal failure, a recurrence of autoimmune thrombocytopenia, and a Coombs-positive hemolytic anemia. CONCLUSIONS: Fludarabine phosphate is effective in the treatment of patients with advanced B-CLL, even in those refractory to multiple chemotherapy regimens.