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Patients with hematological malignancies who relapse after autologous stem cell transplantation (auto-SCT) generally have poor prognosis. Salvage treatment is often associated with severe toxicities. The aim of our study was to evaluate retrospectively the toxicity and outcome of rescue therapy in patients with acute leukemias, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD) and multiple myeloma (MM) relapsing after auto-SCT. Fifty-four of the 62 patients who relapsed received some form of salvage chemotherapy. Six (10%) patients were treated by second stem cell transplantation, which was allogeneic in 5 cases. Toxicity of the salvage therapy was significant. As a result of adverse effects, salvage therapy had to be discontinued or reduced in 14 patients (26%). The outcome of salvage was evaluated after 90 days. Of the treated patients, 14 (26%) entered into complete remission with another 5 (9%) reaching partial response. The disease was stabilized in 5 patients (9%) but 30 (56%) patients were in progression or dead. Overall survival of the patients was poor with the median survival of 8.7 months after relapse and the leading cause of death being progressive disease. In conclusion, the development of new, more efficient regimens is critical if disease-free survival is to be increased in patients who relapse after auto SCT.

The large number of Alu repeats in the human genome provides abundant opportunities for unequal homologous recombination events that are responsible of several human diseases. We here describe a novel large FVIII gene deletion from a severe hemophilia A patient in which Alu-repetitive elements are directly involved in the origin of the mutation. Using a long-fragment PCR method, a approximately 23 kb deletion was delimited between introns 24 and 25. The resulting FVIII gene had a hybrid 2317-bp intron and lacked exon 25. Absence of exon 25 was confirmed at the RNA level. Multiple sequence alignment of this hybrid intron and normal introns 24 and 25 provided evidence of an homologous recombination event between two Alu repeats and the exact breakpoints were delimited to a 16 bp region. To our knowledge, this is the first report of hemophilia caused by unequal homologous Alu/Alu recombination. This mechanism, commonly related to genetic human disorders, may be involved in a significant number of hemophilia cases considering that FVIII is coded by an Alu-rich gene.

Background and Objectives: FK506 (tacrolimus) is a potent immunosuppressive agent that inhibits interleukin-2 (IL-2) and interferon-g production by CD4+ cells. The effect of this agent on dendritic cells (DCs), the highly professional antigen-presenting cells for T-cells, has not been completely defined. We investigated the effect of FK506 on DC differentiation from monocytes, and on the shift from immature to mature immunophenotypes., Design and Methods: DCs were generated in vitro from monocytes of healthy donors. Cells were exposed to lipopolysaccharide (LPS) and two doses of FK506, with variations in time of exposure and sequence of FK506 and LPS addition. Immunophenotype analysis in immature and mature DCs under FK506 treatment was performed by flow cytometry at the end of cell culture. The Student's t-test was used for statistical analyses., Results: FK506 did not affect dendritic cell generation or viability. There were no changes in cell surface markers with addition of FK506 at physiologic concentrations (10 ng/mL). We found a decrease in CD1a median fluorescence intensity (MFI) and an increase in percentage of CD86-positive cells with lengthy exposure (6 days) to FK506 at 5000 ng/mL. In the sequential study, 5000 ng/mL FK506 before LPS addition resulted in a significant decrease in CD1a MFI and in the percentage of cells co-expressing CD83 and CD86., Interpretation and Conclusions: Our results indicate that lengthy exposure to 5000 ng/mL FK506 modified the expression of some DC-cell surface markers, maintaining DCs in a low maturity stage.

Objective: To study the esophageal motor disorders in patients with Barrett's esophagus after surgical treatment., Design: From January 1993 to September 1998 a prospective study with 25 patients referred to our service for surgical treatment of Barrett's esophagus was conducted. Barium transit, endoscopy, 24-hour monitoring of intraluminal pH and stationary esophageal manometry were carried out in all patients pre- and postoperatively. The results were compared before and after surgery. A p < 0.05 was considered statistically significant., Patients: 18 male (72%) and 7 women (28%). Mean age was 54.20 +/- 13.29 years (range: 25-71 years). The most frequent clinical manifestation was heartburn (92%). A laparotomy procedure was performed in 68% (n = 17) and laparoscopy in 32% (n = 8) of patients. A 360 degrees fundoplication was always performed., Results: 96% of patients presented a defective lower esophageal sphincter. The statistical study demonstrated significant differences after surgery for all pH-metric parameters and lower esophageal sphincter (p < 0.01), except for relaxation (p = 0.465). In the esophageal body, the statistical study only demonstrated significant differences for mean pressure of the peristaltic waves in segment I (p = 0.038) and mean rate of non-transmitted waves in esophageal segment IV-V (p = 0.031)., Conclusions: Antireflux surgery in Barrett's esophagus contributes to the control of gastroesophageal reflux improving esophageal clearing and with significant differences for the mean rate of non-transmitted waves in the distal esophagus.

Background: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein has been evaluated. The microcapsules are implanted intraperitoneally. In order to prevent cell immune rejection, cells are enclosed in non-antigenic biocompatible alginate microcapsules prior to their implantation into mice. It has been shown that encapsulated myoblasts can deliver therapeutic levels of Factor IX (FIX) in mice. The delivery of human Factor VIII (hFVIII) in mice using microcapsules was evaluated in this study., Methods: Mouse C2C12 myoblasts and canine MDCK epithelial kidney cells were transduced with MFG-FVIII (B-domain deleted) vector. Selected recombinant clones were enclosed in alginate microcapsules. Encapsulated recombinant clones were subsequently implanted intraperitoneally into C57BL/6 and immunodeficient SCID mice., Results: Plasma of mice receiving C2C12 and encapsulated MDCK cells had transient therapeutic levels of FVIII in immunocompetent C57BL/6 mice (up to 20% and 7% of physiological levels, respectively). In addition, FVIII delivery in SCID mice was also transient, suggesting that a non-immune mechanism must have contributed to the decline of hFVIII in plasma. Quantitative RT-PCR analysis confirmed directly that the decline of hFVIII is due to a reduction in steady-state hFVIII mRNA, consistent with transcriptional repression. Furthermore, encapsulated cells retrieved from implanted mice were viable, but secreted FVIII ex vivo at three-fold lower levels than the pre-implantation levels. In addition, antibodies to hFVIII were detected in immunocompetent C57BL/6 mice., Conclusions: Implantable microcapsules can deliver therapeutic levels of FVIII in mice, suggesting the potential of this gene therapy approach for haemophilia A. The findings suggest vector down-regulation in vivo., (Copyright 2002 John Wiley & Sons, Ltd.)

Barrett's esophagus is today, one of the digestive pathologies that raises more interest in all the meetings and congresses of the specialty, in spite of have been described 50 years ago. The definition has changed; the rising incidence of adenocarcinoma has been recognized; a most effective therapy to control gastroesophageal reflux has been developed (proton pump inhibitor v/s laparoscopic fundoplication); appropriate surveillance intervals of patients with dysplasia have been protocolized; new treatment strategies are being investigating. Although, numerous controversies still persist. The exact and accurate knowledge of physiopathology constitutes the base of treatment and prevention for gastroesophageal reflux disease and their complications.

Disparity for the minor histocompatibility antigen HA-1 between patient and donor has been associated with an increased risk of acute graft-versus-host disease (GvHD) after allogeneic human leucocyte antigen (HLA)-identical sibling donor stem cell transplantation (SCT). However, no data concerning the impact of such disparity on chronic GvHD, relapse or overall survival are available. A retrospective multicentre study was performed on 215 HLA-A2-positive patients who received an HLA-identical sibling SCT, in order to determine the differences in acute and chronic GvHD incidence on the basis of the presence or absence of the HA-1 antigen mismatch. Disease-free survival and overall survival were also analysed. We detected 34 patient-donor pairs mismatched for HA-1 antigen (15.8%). Grades II-IV acute GvHD occurred in 51.6% of the HA-1-mismatched pairs compared with 37.1% of the non-mismatched. The multivariate logistic regression model showed statistical significance (P: 0.035, OR: 2.96, 95% CI: 1.07-8.14). No differences were observed between the two groups for grades III-IV acute GvHD, chronic GvHD, disease-free survival or overall survival. These results confirmed the association between HA-1 mismatch and risk of mild acute GvHD, but HA-1 mismatch was not associated with an increased incidence of chronic GvHD and did not affect relapse or overall survival.

We here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.

The present study was undertaken to assess the feasibility, toxicity and antileukemic activity of sequential chemotherapy including mitoxantrone, etoposide, carboplatin and intermediate-dose cytarabine in adult patients with refractory and relapsed acute myelogenous (AML) or lymphoid (ALL) leukemia. Fifty-one patients with poor-risk AML and ALL received 64 courses of MECA therapy. The overall response in the entire group was 51% (43% complete remission). The stage of the disease (relapsed or primarily refractory) and the age of the patients did not strongly affect the response rate. MECA therapy was more effective in ALL than in AML, and in those patients who presented at salvage treatment with a bone marrow infiltration lower than 25% blasts. Hematological and extra-hematological toxicities were tolerable and there were 6 deaths related to the treatment (11%). The incidence of documented infectious episodes was 71%. MECA therapy is a safe treatment and has a high antileukemic activity in relapsed and primarily refractory AML or ALL.

We have developed a simple, sensitive and cost-effective direct DNA sequencing procedure for the molecular diagnosis of haemophilia B. All factor IX gene essential regions were amplified under identical thermocycling parameters allowing mutation identification in less than 15 h from blood sample collection. Identical results in terms of accuracy and speed were obtained when using a single hair as the source of DNA. Using this approach, we have found mutations in 10 out of 10 haemophilia B patients, two of which are novel (P287T and S123C). Very suitable for individual studies, this procedure can be easily scaled up for higher throughput.

Background and Objectives: Chimerism studies after allogeneic transplantation are usually performed using cytogenetic analysis, PCR-VNTR or PCR-STR. Here, we report an alternative method for following the chimerism status after an HLA-mismatched stem cell transplantation (SCT), detecting the presence of non-shared HLA alleles by reference-strand mediated conformation analysis (RSCA)., Design and Methods: We tested this new approach on allogeneic related haploidentical SCT, unrelated cord blood transplantation, and HLA-mismatched unrelated donor SCT. The quantification of the chimerism was performed by laser detection of fluorescent-labeled primers on an automated DNA sequencer., Results: In all cases this technique was able to detect mixed chimeras. The technique detected above 5% of residual cells when the analysis was based on HLA-class I and above 3% for HLA-class II. This sensitivity is similar to that of the PCR-VNTR analysis., Interpretation and Conclusions: This method avoids the need to search for an informative locus (which is essential for PCR-VNTR or -STR). Moreover, we did not find the phenomenon of preferential amplification that is observed with most VNTR, thus avoiding the need for construction of standard curves to quantify mixed chimeras. We conclude that the detection of the non-shared HLA alleles by RSCA is a useful approach for chimerism follow-up after HLA-mismatched SCT.

Cytotoxic T lymphocytes (CTLs) reactive against the disparity between HLA-B*4402 and HLA-B*4403 have been reported after unrelated donor bone marrow transplantation. These CTLs have been associated with acute graft-versus-host disease and graft rejection. This study describes the HLA-B44-subtyping in the Catalan population using reference-strand mediated conformation analysis. It has been performed on 297 unrelated HLA-B44+ cord blood units from the Barcelona Cord Blood Bank (Barcelona, Spain). We have found a predominance of HLA-B*4403 (66.04%) over HLA-B*4402 (33.02%), whereas the predominant HLA-B44 allele in Northern Europe and the United States is HLA-B*4402. This inverted proportion between HLA-B44 subtypes in Mediterranean populations compared with other Caucasian populations suggests that HLA-B44 subtyping should be performed when an HLA-B44+ unrelated donor marrow is identified.

Disparities in minor histocompatibility antigen (mHAg) HA-1 are involved in the development of acute graft-versus-host disease (GvHD) in adult recipient after HLA-identical sibling donor hematopoietic stem cell transplantation. The mHAg HA-1 is an HLA-A*0201-restricted nonapeptide, which derives from the cleavage of a protein encoded at chromosome 19. The sequence analysis of HA-1 cDNA identified two alleles, termed HA-1H and HA-1R, which differ in only two nucleotides at 3' end of exon A, at positions 500 and 504. DNA-based methods for HA-1 typing were developed in 1998, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). Here, we report the usefulness of reference strand mediated conformation analysis (RSCA), which was developed for mutation detection and typing of polymorphic loci, to discriminate between the two HA-1 alleles. We performed genomic typing of HA-1 locus in 203 HLA-A*0201-positive samples using RSCA and we confirmed these results by PCR-SSP. The results demonstrate the high reproducibility of this method and their strong correlation with the results obtained by PCR-SSP (99%). Only two samples showed disparity between the RSCA typing and the PCR-SSP. Direct sequencing of these samples confirmed that the correct allele assignment was that obtained by the RSCA typing. Furthermore, HA-1- RSCA-based typing provides additional information about the intronic structure of both alleles. With this approach, we describe the almost constant presence (99.2%) of a 5-bp deletion at intronic position 214-218 associated to the HA-1H allele, previously unidentified. We conclude that HA-1 genomic typing by RSCA is easy to perform and that could be used as a routine typing method.

Background/aims: Intraductal papillary-mucinous tumor of the pancreas is currently considered to be a tumor which is an entity of its own, different from classic pancreatic ductal carcinoma. It is basically characterized by slow growth and low malignancy potential, as well as by the production of mucin. The aim of this study is to contribute to world literature some clarification of its natural history, clinical presentation, the most useful diagnostic tests, methods of detection of stromal invasion and handling of treatment., Methodology: Of 297 pancreatectomies undertaken at the "12 de Octubre" hospital between May 1985 and January 1998, only 1 case of Intraductal papillary-mucinous tumor was found. We have revised 127 cases published in 10 series over the last 10 years. We also contribute a review of our own case., Results: These tumors, which are very infrequent, produce non-specific symptoms, with long latency periods from the first symptom up to stromal invasion. Endoscopic retrograde cholangiopancreatography showed alterations in 100% of cases where this was undertaken. Tumor-related mortality was zero amongst patients with non-invasive tumor who underwent surgery. None of the cases published presented upper gastrointestinal hemorrhage. This indicated the correct surgery and led us to our diagnosis., Conclusions: We confirm the low frequency and difficulty of diagnosis, the sensitivity of endoscopic retrograde cholangiopancreatography, the difficulty of early detection of stromal invasion, and the high survival rate in cases where resection is done before this occurs. Early diagnosis and treatment is therefore of utmost importance.

A pilot, open, comparative study was performed on patients with locally advanced cervical cancer to investigate the efficacy and safety of amifostine. Twenty patients with a histologic diagnosis of squamous cervical cancer were treated with radiotherapy and randomized in two groups. Group A received cisplatin at 20 mg/m2 for five days in two cycles during intracavitary radiotherapy and 100 mg/m2 x 2 cycles during external radiotherapy, and amifostine 825 mg/m2 15 min before the cisplatin infusion. Patients in group B received cisplatin in the same doses without amifostine. All patients had complete responses during a median follow-up of 20 months. Grade three neutropenia was present in two patients in group A and in four of the control group, P = 0.31; grade 2 neurologic toxicity was seen in four patients in group B and in one of the patients in group A, P = 0.15. One patient needed temporary interruption of amifostine due to hypotension. Eight of 10 patients in group A developed hypocalcemia during the treatment with amifostine. Our findings indicate that amifostine was well tolerated. In this series a mild neurologic and hematologic protection was found in patients that received amifostine, although this was not statistically significant. No differences in disease-free survival response and overall survival was seen between the two groups.

The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven beta3-galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressing breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at G1, with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressing cells. Because it is well known that failure of adequate p21WAF1 induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WAF1 transcript and protein are not sustained in HER-2-overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAF1 transcript and protein at 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAF1 transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAF1 in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. (ABSTRACT TRUNCATED)

Background and Objective: The relationship between cytokine concentrations and transplant-related complications has been studied in bone marrow transplant patients. The changes in TNF-alpha, IL-1 and IL-6 concentrations after transplantation are well documented in the literature but this is not the case for IL-8. The purpose of the present study was to investigate prospectively the plasma concentration of these cytokines and their relationship to transplant-related complications., Design and Methods: Pro-inflammatory cytokine (TNF-alpha, IL-1, IL-6 and IL-8) levels in plasma were determined in a group of 53 patients undergoing hematopoietic progenitor transplantation. Plasma samples were collected weekly from day -7 to day +35 and stored at -70 degrees C until assayed by ELISA. The major transplant-related toxicities registered were: veno-occlusive disease (VOD), acute graft-versus-host disease (GVHD), infectious episodes, renal failure and mucositis., Results: In spite of the great variability of plasma cytokine profiles between the different patients, we came to various conclusions. Patients' TNF-alpha and IL-1 concentrations correlated well over time. IL-6 and IL-8 profiles were similar and correlated well with febrile episodes. In some cases, an increase in IL-6 preceded hematologic recovery. In our study, increased levels of TNF-alpha, IL-6 and especially IL-8 correlated with hepatic or renal dysfunction as evaluated by increased bilirubin and creatinine in plasma, while pulmonary complications correlated only with increased IL-6 levels. Allogeneic transplant patients had a tendency to have higher TNF-alpha concentrations than autologous transplant patients, probably because an allogeneic transplant is associated with more transplant-related toxicity. Basal disease usually had no effect on cytokine profiles., Interpretation and Conclusions: IL-6 and IL-8 were the only cytokines studied whose increase correlated with febrile episodes. High IL-8 values may be a useful predictor of renal dysfunction and pulmonary disease and seems to trigger off high IL-6 levels. Plasma TNF-alpha and IL-1 concentrations during the posttransplant period have not been shown to be predictive of the development of transplant-related complications, and none of the profiles was recognized to be specific for a particular complication in this study.

Variations, such as nucleotide substitutions, deletions and insertions, within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is important for determining disease and gene associations. Techniques which target specific mutations such as restriction enzyme polymorphism and oligonucleotide probe or PCR primer reactivity are useful for the detection of specific mutations, but these techniques are not generally effective for the identification of new mutations. Approaches for measuring changes in DNA conformation have been developed, based on the principle that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electrophoresis (PAGE). Here we describe a conformation-based mutation detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci. We demonstrate the application of DSCA to detect genetic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the application of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.

Based on previous experiences in animals and humans, low doses of CD8+ lymphocytes infused together with the marrow graft seem to enhance engraftment after allogeneic T cell-depleted marrow transplantation. From April 1994 to February 1997, 12 patients with chronic myelogenous leukemia in first chronic phase receiving a bone marrow transplant (BMT) from an HLA-identical sibling were included in a pilot study of T cell subset depletion. Total depletion of CD4+ cells of the marrow graft and partial depletion of CD8+ cells was performed by immunomagnetic separation. In order to improve the engraftment rate, we infused a low fixed number of CD8+ lymphocytes (0.25 x 10(6)/kg). All the patients were at high risk of developing acute graft-versus-host disease (GVHD), with a recipient age of >30 years, and/or donor sensitized by previous pregnancies or transfusions. All of them received cyclosporin A and methotrexate post-BMT. No graft failure was observed. The grade III-IV GVHD rate was 16.6%, and the actuarial survival at 3 years is 81.8%. Immunological recovery showed persistent CD8+ HLA-DR+ lymphocytosis 8 months after transplant. Relapses were not observed. This experience shows the importance of CD8+ cells to ensure correct engraftment, decreasing the GVHD rate.

Bouveret's syndrome is a rare entity consisting in a duodenal obstruction due to the passage of gallstones from the gallbladder to the duodenum through a cholecystoduodenal fistula. Approximately 225 cases are reported in the literature. It is most common in old women with a previous history of biliary tract disease. The clinical picture is nonspecific and pre-operative diagnosis is not easy. Oral endoscopy is the main diagnostic procedure and sometimes, a therapeutic option, too. Surgery is the elective treatment specially when endoscopy is unsuccessful. We report a new case of this syndrome successfully treated by surgery, and an extensive review of the literature concerning this issue, focusing mainly on the clinical findings, diagnosis, therapeutic procedures and results. We conclude that Bouveret's syndrome is rare but more frequent in older females with previous biliary disease, better diagnosed by pyloric obstruction syndrome, plain abdominal x-ray, ultrasonography, contrast gastric study and/or gastroscopy (confirming and best procedure). When conservative endoscopic procedure fails, surgical treatment must be carried out, thus obtaining good results.

Neurological complications are not usually considered among the most important complications that may appear after allogeneic bone marrow transplantation (BMT). We have analyzed the occurrence of neurological manifestation in 27 recipients of allogeneic BMT. Ten patients (37%) developed neurological symptoms, and 14 episodes were registered. The most frequent manifestations were due to the use of cyclosporin A or prednisone for prophylaxis or treatment of graft-versus-host disease (GVHD). Cerebrovascular events (infarction or hemorrhage) and CNS infections were the most severe complications: they represented 26% of cause of death in our series. In conclusion, neurological complications are frequent in these patients, and represent an important cause of morbidity and mortality.

The p53 tumor suppressor gene product is known to be active in mediating radiation-induced G1-S cell cycle arrest and apoptosis in a number of normal cell lines. These functions are compromised by inactivation of p53, which promotes tumor progression. Because the p53 gene appears to play an important role in the cellular response to radiation, wild-type p53 gene replacement might be expected to increase the sensitivity of malignant cells with mutant p53 to the cytotoxic effects of ionizing radiation. This study demonstrates that adenovirus (AdV)-mediated transfer and expression of the wild-type p53 in malignant cells lacking the p53 gene results in an increase in cellular radiosensitivity in vitro and tumor radioresponsiveness in vivo. Cultures of the p53 double deletion mutant ovarian cell line SK-OV-3 were infected with nonreplicative adenoviral vectors containing either the wild-type p53 gene (AdVp53) or the luciferase gene (AdVluc). Cultures infected with AdVp53 efficiently expressed wild-type p53 protein and were more sensitive to radiation than uninfected cultures or cultures infected with AdVluc. The ability of AdVp53 to radiosensitize tumors in vivo was tested using SK-OV-3 tumors growing in the flanks of severe combined immune-deficient mice. Intratumoral injection with AdVp53, but not AdVluc, led to enhanced radioresponsiveness and 45% long-term tumor control. These studies demonstrate the ability of AdVp53 to effectively transfer and express p53 protein in established tumors with a resultant increase in radiation responsiveness.

We report a young woman with acute promyelocytic leukemia who showed primary resistance to chemotherapy and who responded to ATRA treatment. During the neutropenic period she developed Curvularia sp infection and was finally successfully consolidated with autologous bone marrow transplantation.

Molecular genetic analysis has been performed on a patient with Bernard-Soulier syndrome (BSS). The patient had characteristically giant platelets and was deficient in the glycoprotein (GP) Ib/IX/V complex, the von Willebrand factor (vWf) receptor on platelets. Previous studies with monoclonal antibodies directed against GP Ib alpha (CD 42b) and GP IX (CD 42a) demonstrated the absence of GP Ib alpha and presence of small amounts of GP IX on the surface of the patient's platelets. In this study the presence of GP V (CD 42d) is also demonstrated. This indicates a defect in the alpha-subunit of glycoprotein Ib. Therefore polymerase chain reaction (PCR)-amplification of the genomic DNA coding for GP Ib alpha was performed. Nucleotide sequence analysis of the entire coding region of GP Ib alpha revealed a homozygous single base pair mutation T-->A, leading to a single amino acid substitution cysteine-->serine at position 209 of the mature protein. We took advantage of the Mse I target site in the mutant allele, created by the T-->A mutation, to analyse all available family members. PCR-ASRA (allele-specific restriction enzyme analysis) using the restriction enzyme Mse I, revealed the heterozygosity of the mother and the two children of the patient, whereas homozygosity of the patient for the Cys209Ser mutation was confirmed. The sister of the patient was not found to be a carrier of the mutant allele. The mutation identified in the family studied, responsible for the deficiency of the GP Ib/IX/V complex, suggests that the cysteine at amino acid position 209 may be involved in disulphide bonding.

GP Ia/IIa (also called VLA-2 or alpha 2 beta 1) is the primary receptor for collagen on platelets. The human platelet alloantigens HPA-5a(Brb) and HPA-5b(Bra) have been found to reside on the platelet GP Ia/IIa complex. In order to establish the molecular basis of the HPA-5 system, platelet RNA was isolated form HPA-5 (a+, b-) and HPA-5(a-, b+) individuals. After reverse transcription, cDNA coding for glycoprotein Ia (GP Ia) was amplified by the polymerase chain reaction (PCR). Nucleotide sequence analysis of the PCR products revealed an A-->G polymorphism at base pair 1648 of the coding region of the mature protein, resulting in a substitution of lysine (AAG) in HPA-5b(Bra) by glutamic acid (GAG) in HPA-5a(Brb) at amino acid 505. Subsequent PCR-ASRA (allele-specific restriction enzyme analysis) with Mnl I using cDNA derived from three HPA-5 (a+, b-), one HPA-5 (a+, b+) individuals demonstrated that HPA-5a and -5b alleles are distinguishable by DNA typing. In addition to the A-->G substitution at base pair 1648, three silent mutations were identified, G-->C (195 bp), C-->T (837 bp), G-->A (1041 bp).

Interleukin 4 (IL-4) suppresses the interleukin 2 (IL-2) induced lymphokine-activated killer (LAK) cell development from human peripheral blood mononuclear cells (PBMC). Suppression is observed at high (1,000 U ml-1) as well as low (10 U ml-1) concentrations of IL-2. IL-4 needs to be present at the beginning of the IL-2 culture to exert the suppressive effect. IL-4 also inhibits the development of CD25 (Tac) antigen on the PBMC cultured in IL-2. Interleukin 1 (IL-1) can reverse the suppressive effect of IL-4 on LAK induction when added at the early phase of the IL-2 culture. IL-1 enhances IL-2 induced LAK development, which may partially explain the reversion of IL-4 inhibition by IL-1. IL-1 also reverses the inhibitory effect of IL-4 on the development of CD25 antigen expression, although IL-1 alone does not enhance the induction of CD25 expression in PBMC cultured by IL-2. Furthermore, IL-4 suppresses IL-2 induced IL-1 production in PBMC. Thus, suppression of CD25 may be a pathway for the suppression of LAK induction. The expression of CD56 is not directly associated with the expression of LAK activity. IL-4, IL-1 or combination of the two cytokines has no effect on IL-2 induced expression of CD56. These results indicate that IL-4 has an antagonistic effect and IL-1 has a synergistic effect on IL-2-induced LAK development.

The characterization of specific DNA sequences by means of hybridization techniques is of great relevance in the development of molecular biology. Because of its simplicity, the method described by Southern in 1975 (1) is widely used for such purposes. This method, also known as the Southern blot, is based on the hybridization of specific probes to DNA fragments immobilized onto a membrane filter after their separation by agarose gel electrophoresis. The identification of specific DNA fragments is possible since their relative positions are maintained during the transfer from the gel to the filter.

We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even though their light chains were also underestimated, because goat monospecific antibodies were more efficient at recognizing their heavy chains. The molecular basis for the observed difference in antigenicity is not yet known. These findings have important implications for the analysis of the binding of IgG Hu-MAbs. A direct binding assay with the label directly conjugated to the Hu-MAb should be used in preference to an indirect assay with a labeled detecting antibody to maximize the sensitivity of the assay. The altered antigenicity of IgG Hu-MAbs may also imply decreased immunogenicity when they are given in vivo as carriers for radionuclides or cytotoxic antitumor materials.

A rapid and efficient 2-step procedure is described for covalently attaching proteins to cell surfaces by using a heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethylcyclohexane)-1-carboxylate (SMCC). In the first step, protein is derivatized for about 30 min with a 1:1 (mole:mole) stoichiometric ratio of SMCC which creates 4-(N-maleimidomethylcyclohexane)-1-amidyl-protein (MCA-protein) covalent linkages with the primary amino groups of proteins. In the second step, cell-MCA-protein conjugates are rapidly prepared (in less than 30 min) by reacting MCA-protein with 'reduced' cells which have been pre-incubated (for about 1 h) with dithiothreitol (DTT). Stable protein-cell conjugates result from the covalent thioether linkages formed between the maleimido groups of the derivatized protein and the cell surface thiol groups created by DTT reduction. Protein-cell conjugates have been made in this way with several different proteins using several different types of cells. Such conjugates are characterized in this study by complement plus antibody-mediated cytotoxicity (CAMC) and by antibody-dependent cellular cytotoxicity (ADCC) with human effector lymphocytes. Because these protein-cell conjugates are shown to remain viable and to retain significant levels of coupled protein for at least 24 h in culture, they are potentially useful for probing various membrane-related phenomena such as the recognition of cell surface antigens by immune effector cells.