Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes.

Relapse of chronic myeloid leukemia (CML) in chronic phase after allogeneic stem cell transplantation (SCT) can be successfully treated by donor lymphocyte infusion (DLI). However, relapse of accelerated phase CML, blast crisis, or acute leukemia after allogeneic SCT are resistant to DLI in the majority of cases. In vitro-selected and expanded leukemia-reactive T-cell lines may be more effective in inducing an antileukemic response in vivo. To treat a patient with accelerated phase CML after allogeneic SCT, leukemia-reactive cytotoxic T-lymphocyte (CTL) lines were generated from her HLA-identical donor. Using a modification of a limiting dilution assay, T cells were isolated from the donor, selected based on their ability to inhibit the in vitro growth of CML progenitor cells, and subsequently expanded in vitro to generate CTL lines. Three CTL lines were generated that lysed the leukemic cells from the patient and inhibited the growth of leukemic progenitor cells. The CTL did not react with lymphocytes from donor or recipient and did not affect donor hematopoietic progenitor cells. The 3 leukemia-reactive CTL lines were infused at 5-week intervals at a cumulative dose of 3.2 x 10(9) CTL. Shortly after the third infusion, complete eradication of the leukemic cells was observed, as shown by cytogenetic analysis, fluorescence in situ hybridization, molecular analysis of BCR/ABL-mRNA, and chimerism studies. These results show that in vitro cultured leukemia-reactive CTL lines selected on their ability to inhibit the proliferation of leukemic progenitor cells in vitro can be successfully applied to treat accelerated phase CML after allogeneic SCT.

Sublocalization of the breakpoints of a t(5;16) in myelodysplasia.

As a first step in characterizing a t(5;16)(q31;p11.2) in a patient with the diagnosis refractory anemia with ring sideroblasts, a cell fusion was carried out between bone marrow cells from the patient and the Chinese hamster cell line A3. Using PCR and FISH analysis on hybrid lines containing the human derivative 16 chromosome, the breakpoints could be mapped between the markers TCF-7 and IL-9 on chromosome 5 and OL-7 and s30A4 on chromosome 16, both regions spanning approximately 1 Mb. Since the breakpoint on 5q has occurred in a region that is frequently deleted in myeloid malignancies, the gene disrupted by this translocation could also be implicated in this aberration.

Visceral neuropathy in slow transit constipation: an immunohistochemical investigation with monoclonal antibodies against neurofilament.

PURPOSE: The aim of this study was to investigate neuropathologic changes in the colonic wall of patients with slow transit constipation using monoclonal antibodies raised against neurofilament. METHODS: In a prospective study, 227 patients with severe, long-standing constipation and intractable defection disorders were analyzed according to a standard protocol. Slow transit constipation was diagnosed in 65 patients (29 percent). Forty-three patients (7 men and 36 women; mean age, 46 years; range, 16-76 years) underwent a partial (n = 20) or subtotal (n = 23) colectomy. In 39 patients (5 with megacolon and 34 with normal-sized colon) the cause of their constipation remained unexplained (idiopathic slow transit constipation). All resected colon specimens were investigated with the monoclonal antineurofilament antibody NF2F11 and compared with those of 20 control patients. RESULTS: In all controls the myenteric plexus revealed a moderate and diffuse axonal staining. In 29 of 39 patients with "idiopathic" slow transit constipation, the apparently normal axon bundles in the myenteric plexus stained markedly less than normal or failed to stain at all with the monoclonal antibody. In 17 patients this reduced or absent neurofilament expression was found along the entire length of the colon, whereas in 12 patients only a portion of the colon was affected. CONCLUSION: These findings indicate that a visceral neuropathy seems to be present in the majority of patients with severe, so-called idiopathic slow transit constipation.

Multiple minor histocompatibility antigen disparities between a recipient and four HLA-identical potential sibling donors for bone marrow transplantation.

A patient with acute leukemia and her family including four HLA-identical siblings were analyzed to select a donor who was not only HLA- but also minor histocompatibility (mH) antigen compatible for allogeneic bone marrow transplantation (BMT). The HLA-A2 restricted mH antigen-specific HA-1, -2, -4, and -5 cytotoxic T-lymphocyte (CTL) clones were used to type the family members for expression of these mH antigens. The patient and one HLA-identical sibling were compatible for these mH antigens. This sibling was selected as the bone marrow donor. The patient engrafted promptly but developed acute and chronic graft-versus-host disease. To study the presence of other mH antigen disparities between recipient and donor, host-versus-graft CTL lines and clones were generated by stimulation of recipient peripheral blood lymphocytes (PBLs) with donor bone marrow cells, and graft-versus-host CTL lines were generated after BMT by stimulation of PBLs of donor origin with recipient bone marrow cells. These CTL lines were cytotoxic to cells from the bone marrow donor and from the recipient, respectively, and to cells from several other family members. T-cell lines, generated from the patient after BMT by stimulation of recipient-derived PBLs with donor bone marrow cells, exhibited no specific cytotoxicity to donor or recipient cells. Chimerism studies after BMT revealed that the PBLs and T-cell lines generated after BMT were of donor origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Order of human hematopoietic growth factor and receptor genes on the long arm of chromosome 5, as determined by fluorescence in situ hybridization.

A large number of human hematopoietic growth factor and growth factor receptor genes are localized at the long arm of chromosome 5. In this study we have determined the order of the human interleukin-3 (IL3), IL4, IL5, IL9, granulocyte macrophage-colony stimulating factor (GMCSF), and the MCSF receptor (MCSFR) genes by fluorescence in situ hybridization. Genomic lambda-clones were isolated using polymerase chain reaction (PCR)-generated probes and labeled with biotin and/or digoxigenin. These clones were first individually mapped: IL3, IL4, IL5, IL9, and GMCSF to 5q31 and MCSFR to 5q33. For ordering purposes multiple probe combinations were hybridized to metaphase chromosomes and interphase nuclei. The interphase hybridizations were evaluated by image analysis, which also allowed the measurement of the physical distance between the hybridization spots. These mapping results suggest the gene order 5cen-IL3/GMCSF-IL5-IL4-IL9-MCSFR+ ++-qter. The known genomic distance between the IL4 and IL5 genes allowed the estimation of the physical distances between the 5q31-specific genes, demonstrating that they are all within about 1.5 Mb of DNA.

The human interleukin-6 receptor alpha chain gene is localized on chromosome 1 band q21.

The human interleukin-6 receptor alpha chain (IL6R alpha) gene was regionally mapped to chromosome 1 band q21 by fluorescence in situ hybridization. As hybridization probes, partially overlapping lambda clones encompassing 28 kb of the genomic region of the gene were used. These clones were isolated using a polymerase chain reaction (PCR)-generated fragment of the 3' non-coding region of the gene. This localization confirms and extends the provisional assignment of the IL6R alpha gene to chromosome 1, for which a panel of somatic cell hybrids was used.

Factors influencing release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes.

Mononuclear phagocytes play an important role in the regulation of hematopoiesis, not only by producing regulatory monokines such as prostaglandins, tumor necrosis factor and interleukin-1 (IL-1), but also by the production of colony-stimulating activity (CSA). Previously, we have demonstrated that granulocyte-macrophage CSA (GM-CSA) production by mononuclear phagocytes can be induced by IL-1. In the present study, the influence of culture conditions on the production of GM-CSA was studied. It was found that both human sera and fetal bovine sera contain constituents - at present undefined - that induce GM-CSA production. These factors are distinct from IL-1 and lipopolysaccharide. In selected experiments, no GM-CSA-inducing effect of serum was found, suggesting that the effect may be donor-related. GM-CSA release in the presence of serum could be reduced by 40% after incubation of mononuclear phagocytes at low cell concentrations in methylcellulose, indicating that intimate cell-cell contact is an additional factor that enhances GM-CSA release.

Efficacy of antibody NF2F11 staining in the investigation of severe long-standing constipation. A preliminary report.

A retrospective study of 7 adult patients (6 with severe, long-standing constipation and 1 with chronic idiopathic intestinal pseudoobstruction) was carried out to test the diagnostic potential of the antineurofilament monoclonal antibody NF2F11. In 4 of the cases of constipation and in the 1 case of pseudoobstruction, paraffin sections of resected colon revealed an anomaly in that the axon bundles of both plexus systems remained unstained. This picture differed from that found in the control population. The results are discussed in relation to previous studies of congenital neurogenic abnormalities of the digestive tract in children.

The influence of the stage of differentiation of the gut on the migration of neural cells: an experimental study of Hirschsprung's disease.

Based on experimental studies in mutant mouse strains, an imbalance between the rate of migration of neural crest cells and the rate of differentiation of the mesenchyme of the distal gut has been proposed as an etiological factor in Hirschsprung's disease. We studied the influence of the stage of differentiation of embryonal chick gut on the migration of neural crest cells in an in vivo culture system: the chorioallantoic membrane. Neural crest cells in cultured gut were demonstrated with antibodies directed against the HNK-1 epitope. Enteric neurons were demonstrated with neurofilament immunoreactivity. By culturing isolated gut segments of E4 embryos, we obtained aneuronal (neurofilament-negative) embryonal chick gut up to 25 days of development. In cocultures of aneuronal gut and the neural anlage (neural tube and neural crest) neural crest cell colonization was observed, even in advanced stages of differentiation. The significance of the results is discussed in terms of the etiology of Hirschsprung's disease.

A comparative investigation of the bowel wall in gastroschisis and omphalocele: relation to postoperative complications.

In gastroschisis the protruding bowel is generally covered by a fibrous coating as a result of prolonged exposure to amniotic fluid but no such coating is found on the exposed bowel loops in a ruptured omphalocele. In patients with gastroschisis, the postoperative course is often complicated by periods of hypoperistalsis. Based on studies of animal models of gastroschisis, various authors have suggested that the postoperative complications might be due to structural defects of the bowel wall. In our own experimental investigation of gastroschisis using chick embryos, we found no evidence of a structural defect of the bowel wall. With a view to clinical substantiation of our experimental findings, we conducted a comparative investigation of patients material, involving 10 cases of gastroschisis, 5 cases of ruptured omphalocele, and 9 cases of intact omphalocele. The investigation revealed an entirely normal structure of the bowel wall in all 10 cases of gastroschisis. On the serosa we noted an inflammation infiltrate rich in collagen fibers, frequently containing squamous cells (vernix) and lanugo. In the cases of ruptured omphalocele, the bowel wall specimens revealed a subacute inflammatory reaction. No major abnormalities were found in the bowel wall of patients with an intact omphalocele. Normal development of the autonomic nervous system was found in all patient material. Severe ischemic changes of the bowel wall were found in 4 of the gastroschisis cases and these were the 4 patients who suffered from postoperative hypoperistalsis. Thus the complications occurring during the postoperative phase of gastroschisis are most probably due to ischemic bowel damage.

Monoclonal antibodies for diagnosis and research in enteric nervous system pathology. A review.

Monoclonal antibodies (MAb) directed against neuron-specific epitopes are valuable tools in the diagnosis of congenital and acquired enteric nervous system anomalies. MAb raised against cytoskeleton proteins (neurofilaments) revealed a characteristic staining pattern in patients with various motility disorders of the gut. Application of MAb in the study of the development of the enteric nervous system in the chicken embryo provided new insights into the fate of migrating neural crest cells. The relationship between mesenchymal target cells in the gut and proliferating neural crest cells was studied by means of MAb raised against cell surface markers (HNK-1) in combination with characterization of the microenvironment using monoclonal antibodies raised against cell adhesion molecules (N-CAM).

Interleukin-1 (22-K factor) induces release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes.

An electrophoretically pure preparation of natural human interleukin-1 (IL-1) was shown to stimulate in vitro colony formation in human bone marrow cultures. Day 4 myeloid cluster-forming cells (CFC), as well as early (day 7) and late (day 10) granulocyte-macrophage colony-forming units (CFU-GM) were stimulated in a dose-dependent fashion. At optimal concentrations of IL-1, the number of day 4 CFC reached 72%, the number of day 7 CFU-GM reached 32%, and the number of day 10 CFU-GM reached 80% of the respective numbers of colonies obtained by addition of crude leukocyte-conditioned medium (LCM). The IL-1-induced stimulatory effect on CFU-GM growth could be completely neutralized by a rabbit anti-IL-1 antiserum. Colony growth was abrogated by depleting the marrow cell suspensions of phagocytic cells prior to IL-1 addition. Conversely, the effect could be reintroduced by addition of marrow-derived adherent cells to bone marrow cell suspensions that had been depleted of both phagocytic and E rosetting T cells. Furthermore, media conditioned by bone marrow-derived adherent cells or by peripheral blood mononuclear phagocytes in the presence but not in the absence of IL-1, stimulated in vitro colony growth of phagocyte-depleted bone marrow cell suspensions. These results indicate that IL-1 induces release of granulocyte-macrophage colony-stimulating activity (GM-CSA) from human mononuclear phagocytes.

Developmental aspects of gastroschisis.

The development of gastroschisis was studied experimentally as well as clinically, particularly concerning the characteristic fibrous coating of the protruding bowel loops, associated intestinal atresia, and postoperative hypoperistalsis without intestinal obstruction. Experimental investigation was carried out in the chicken embryo. The clinical study was a joint one, involving a total of 50 patients with gastroschisis seen at the Sophia Children's Hospital, Rotterdam, and the Royal Hospital for Sick Children, Glasgow, between 1972 and 1985. Some patients were followed antenatally. The results, correlating with previous experiments, were compared with data from the literature. The fibrous coating of the protruding bowel loops appeared to be a late occurrence and directly related to changes of the amniotic fluid secondary to the onset of renal function. Associated intestinal atresia and postoperative hypoperistalsis in the absence of an obstruction both appeared to be due to another late gestational event, consisting of ischemic changes of the bowel wall secondary to the compression of bowel loops and mesentery in the small abdominal wall defect.

Disturbed defecation after colectomy for aganglionosis investigated with monoclonal antineurofilament antibody.

Experience with 108 neonates treated for aganglionosis at the Sophia Children's Hospital, Rotterdam, between 1975 and 1983 has been reviewed. Twenty two of them suffered from disturbed defecation postoperatively, involving 16 cases of classical Hirschsprung's, 4 long-segment aganglionosis, 1 case of aganglionosis up to the cecum, and 1 total aganglionosis. The monoclonal antineurofilament antibody NF2F11 was used to investigate ganglionated as well as aganglionic bowel specimens of these patients as well as 17 patients from the remainder without postoperative constipation serving as controls. The original diagnosis was confirmed in all cases. Proximal ganglionated bowel of all 17 controls appeared normal, while the antibody revealed normal bowel in only 4 out of the 22 patients with postoperative constipation. In 18 cases this new staining technique revealed the picture of pseudo-obstruction. Early recognition of pseudo-obstruction in proximal ganglionated bowel would provide early warning of postoperative complications.

Pathologic explanation for postoperative obstipation in Hirschsprung's disease revealed with monoclonal antibody staining.

Until now, no pathologic explanation could be found for the postoperative obstipation occurring in some patients with intestinal aganglionosis. Twenty-two of 108 infants treated for intestinal aganglionosis suffered from postoperative obstipation. Resected material from these 22 patients and from 17 control subjects was investigated with monoclonal anti-neurofilament antibody staining. An abnormal staining pattern was revealed in 18 of the constipated patients. Consequently, this new immunohistochemical staining technic has revealed a hitherto unsuspected cause for postoperative obstipation in aganglionosis. The monoclonal antibody may provide early warning of such postoperative constipation.

The natural history of gastroschisis during fetal life: development of the fibrous coating on the bowel loops.

Patients with gastroschisis have a paraumbilical defect of the anterior abdominal wall through which bowel loops protrude. These bowel loops are edematous and covered by a fibrous coating. The moment of occurrence of gastroschisis as well as the development of the fibrous coating are unknown. This prompted an investigation of 26 human embryos and fetuses with gastroschisis at various developmental stages (crown-rump length 25-240 mm) and two stillborn fetuses (gestational age 30 weeks) with gastroschisis, as well as resected material from ten newborns (gestational age 33-40 weeks) operated for gastroschisis. Progressive changes of the serosa were only noted after the 30th week of gestation and consisted of an amniotic fluid peritonitis and progressive fibrosis. The changes are in agreement with experimental data and correlate with changes in the composition of amniotic fluid. The findings prove that gastroschisis is a very early occurrence, while the fibrous coating is a late development.