Adherent cells in cultures of blast progenitors in acute myeloblastic leukemia.

Blast cells from patients with acute myelogenous leukemia (AML) grow exponentially in suspension culture and form colonies in cultures "stiffened" with methylcellulose; under both culture conditions, cells are generated which have the ability to adhere to plastic or glass. These adherent cells lack the capacity to form colonies, to proliferate in liquid culture or to support growth. Adherent cells are generated in parallel with changes in the frequency of clonogenic cells and express surface markers of AML blasts but with a higher frequency of an antigen recognized by the monoclonal MO1, a late stage marker associated with the macrophage differentiation lineage. While the appearance of adherent cells provides further evidence that blast cells follow differentiation programs in culture, the generation of adherent cells does not indicate that normal differentiation is occurring; rather the data is consistent with the view that components of normal monocytic differentiation programs are assembled abnormally in the programs of some blast progenitors.

Use of written cases to study factors associated with regional variations in referral rates.

The major purpose was to explore the use of written case scenarios to investigate factors associated with regional variations in referral for consultation. Data were collected in Nova Scotia, where extensive computer-based records of utilization of health services are kept, and the nine health care regions are in sufficient proximity to make visits and interviews feasible. Interviews with 9 x 25 = 225 randomly-selected family physicians, using scenarios analogous to ICD-9 codes, permitted testing of hypotheses about the effects of selected variables on referral rates for hypothetical cases. The family physicians' self-reported referral behavior for the written case scenarios was compared, region by region, with the actual referral of analogous cases by physicians in the same region, as determined from Nova Scotia Health Services and Insurance Commission records. The results indicated that written scenarios provide a useful tool for studies of such variables. Generally, the family physicians responded appropriately to the information that described the hypothetical patients ("case cues"). However, comparisons of hypothetical and actual referral rates indicated that appropriate information about the environment within which referral decisions take place ("environmental cues") may be needed to explain actual regional variations in referral rates.

A regional autologous bone marrow transplant network: transfers to designated centers on the day after transplant.

Autologous bone marrow transplantation (ABMT) is becoming increasingly prevalent for treatment of advanced malignant disease. In order to increase the availability and utility of this therapy, we assessed the feasibility of transferring patients to their regional referral centers on the day after marrow infusion (day 1), for management post-transplant. This prospective study compares the outcome of 77 patients either transferred the day after marrow transplant for subsequent management at one of six selected Canadian regional centers closest to their domicile, or treated entirely at The Toronto Hospital, according to a common protocol. Study end-points included frequency of complications during transfer, transplant-related morbidity and mortality and hematopoietic recovery. Assessment of eligibility for transplant, bone marrow harvesting, autograft cryopreservation, administration of intensive therapy and marrow infusion were conducted in all cases at The Toronto Hospital. Thirty patients received marrow transplants and were transferred on day 1. There were no complications during transfer. Compared with 47 consecutive patients treated entirely at The Toronto Hospital, there were no differences in treatment-related morbidity or mortality, use of intravenous antifungal therapy or total days of hospitalization. We conclude that day 1 transfer of patients after ABMT to designated centers is feasible and safe. The operation of a regional ABMT network appears to benefit patients, relatives, referring physicians, the transplant center and may also improve health care delivery.

Outcome of Patients with Relapsed or Refractory Non-Hodgkin's Lymphoma Referred for Autologous Bone Marrow Transplantation.

Fifty-one patients with relapsed or refractory intermediate- or high-grade non-Hodgkin's lymphoma were referred for autologous bone marrow transplantation (ABMT). The primary criterion for eligibility was sensitivity to conventional-dose salvage chemotherapy. Of 47 patients who received salvage chemotherapy, 30 demonstrated chemotherapy-sensitive disease. Six eligible patients did not undergo ABMT for various reasons. A total of 24 patients underwent ABMT, with etoposide, melphalan ± total body irradiation as the intensive therapy regimen. There was one early treatment-related death and three non-responders. Of the remaining patients, 9 relapsed, while 11 remain in continuous complete remission (CR) at a median follow-up of 21 months after transplant (range 5-37 months). Two patients with chemosensitive disease and bone marrow involvement underwent allogeneic BMT with marrow from HLA-identical siblings. Both are in continuous CR at 6 and 12 months follow-up. Of the 25 patients who did not undergo ABMT, all have died (median survival 5 months). The results indicate that approximately one-half of relapsed or refractory aggressive histology lymphoma patients referred for ABMT eventually undergo transplantation, if chemotherapy-sensitive relapse is the major criterion for eligibility. Approximately 25% of the referred patients may become long-term disease-free survivors with this approach. Reports of marrow transplant series should include all patients referred for ABMT as the denominator for calculating disease-free survival in order to reduce the bias of patient selection.

Detection of a R173W mutation in the porphobilinogen deaminase gene in the Nova Scotian "foreign Protestant" population with acute intermittent porphyria: a founder effect.

OBJECTIVES: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG-->TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. DESIGN AND METHODS: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger's dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an Ncil (also Mspl) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. RESULTS: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the "Foreign Protestants," who were recruited to Nova Scotia in the 1750s. CONCLUSION: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

Differential kinetics of drug resistance in human leukaemic cells measured by SCGE/CLSM.

BACKGROUND: New analogues of DNA directed chemotherapy moieties are available for comparative efficacy testing in human neoplastic disease. In addition to MTT testing direct assessment of DNA excision repair activity after direct exposure of marrow cells may provide information on relative DNA effects in vitro. AIMS: To assess the ability of SCGE/high resolution CLSM to detect differences in drug resistance between human neoplastic cell lines in the DNA excision repair response to chemotherapy. METHODS: Eight human leukaemia samples (4 childhood, 4 adult) were exposed to 1 hour of single concentrations of daunorubicin, DaunoXome (courtesy NeXstar Pharmaceuticals Inc, USA), cyclophosphamide and 4-hydroperoxycyclophosphamide (4-HC, courtesy Dr. M. Colvin, Duke University, USA), followed by SCGE/high resolution CLSM with quantitation of total excised DNA. Differences between cases/drug moieties/exposures were analysed. RESULTS: Although generally equal effect dose levels for DaunoXome were lower than for standard daunorubicin, patients/individual neoplastic cells differed considerably in optimal dose levels. Conventional cyclophosphamide in comparison to 4-HC showed inconsistent results indicating considerable differences in the level of drug resistance to the conventional product. CONCLUSIONS: Direct testing for drug resistance patterns in DNA directed drug moieties by SCGE/CLSM reveals individual variability of human malignant cell lines warranting comparison with results of MTT testing and in-vivo patient response.

Demonstration of differences in drug resistance by direct testing of DNA excision repair activity following standard and liposomal daunorubicin exposure in normal paediatric marrow using high resolution CLSM.

BACKGROUND: High resolution Confocal Laser Scanning Microscopy (CLSM) may be applied to testing of drug resistance in vitro in clinical setting. Rapid analysis of DNA damage by precise quantitation of excised DNA in bone marrow samples exposed to potential treatment moieties directly after isolation but the relative sensitivity of the integrated method is as yet untested. AIMS: To test the clinical applicability of SCGE/high resolution CLSM for differences in drug resistance in marrow cells. METHODS: Cells from normal bone marrow samples were exposed for identical periods and at 4 concentrations to either 1 hour of standard Daunorubicin (.5, 1, 1.5, 2 micrograms/ml) or 8 hours DaunoXome (courtesy of NeXstar Inc, USA) (.05, .1, .15, .2 microgram/ml). After 2 and 6 hours recovery, cells were harvested for SCGE, randomization, analysis of tail length, total excised DNA and fragment size distribution using high resolution CLSM. RESULTS: Tail length and fragment size distribution was not, but total excised DNA was significantly increased after 0.1 microgram/ml Liposomal Daunorubicin (DaunoXome) compared to 1.0 microgram/ml Daunorubicin. CONCLUSION: SCGE/high resolution CLSM effectively demonstrated differences in Daunorubicin resistance of human marrow cells to alternative formulations. The method has potential for use in clinical testing of neoplastic cell drug resistance.

Thalassemia in Canadians.

Thirteen Canadians with a mild hypochromic anemia were found to have beta thalassemia trait. The families of these individuals had resided in Canada for two to five generations and, where known, had not emigrated from areas with a high incidence for the thalassemia gene. A Negro family with abnormal erythrocyte morphology was suspected to be carrying the thalassemia gene although the hemoglobin A(2) concentration was normal and abnormal minor components were not detected. Thalassemia trait has been detected in practically every ethnic group, and its sporadic occurrence among Canadians without Mediterranean ancestry can be expected.

Observations on a medical teacher training program.

An increasing interest in educational science as applied to medical education is apparent in the past decade. Recently, a six-week teacher training program was held at the Center for the Study of Medical Education at the University of Illinois. Fifteen medical faculty members, including five Canadians, participated. During this period there was an opportunity to engage in discussions and independent study of several aspects of medical education, using the personnel from the Center's four major divisions as resource people. Training in educational science is important for all teachers, and a centre to provide this type of instruction should be available in Canada.

The Canadian general internist: education and future role.

Available manpower data indicate that for the forseeable future there will be a continuing requirement in Canada for specialists in general internal medicine. While these specialists will be located predominantly in community hospitals, they will also be needed in university medical centres. The major roles of the general internist will be (a) to provide consultative service to primary care physicians and to other specialists, (b) to provide continuing care to patients with complex serious illness and (c) to participate in intensive care, particularly in community hospitals. Therefore training programs in this specialty must provide adequate experience in consultative medicine in both university and community hospitals, an opportunity to follow up patients with chronic serious illness over long periods, and experience in a variety of intensive care settings including surgical intensive care units. In some university departments the organization and supervision of training programs in this discipline have been carried out by a division of internal medicine that has equal status with other specialty divisions within the department. This seems to have been a salutory development.

Exemplary family physicians and consultants: empirical definition of contemporary medical practice.

To identify the characteristics of exemplary family physicians and consultants, we interviewed 25 family physicians and 25 consultants (5 each in the specialties of internal medicine, obstetrics and gynecology, pediatrics, psychiatry and surgery) selected by their peers as being exemplary in their own practice setting. The results indicated that the participants had well-formulated concepts of exemplary practitioners, defining five main categories of performance: clinical competence, relationship with patients, availability, family physician-consultant relationship and a fifth category that included organizational ability and personality attributes. The family physicians and the consultants placed different values on these categories and indicated that these values might change under different clinical circumstances. Their concepts appear to be compatible with, but not restricted to, a model of contemporary medical practice based on an ethic specific to medicine.

The diagnosis of anemia and its cause.

Screening patients for anemia and determining its cause is challenging and rewarding. Ordinarily, the cause can be readily diagnosed and treatment is available. A clinical approach to the diagnosis of anemia is presented. After clinically determining whether the anemia is due to decreased production of red blood cells, increased destruction, or loss of blood, initial laboratory investigations are specifically chosen to confirm the clinical impression or to answer basic clinical questions. The clinical features and initial laboratory findings should direct subsequent diagnostic and/or treatment approaches.

A cell suspension agar diffusion test using Neutral Red release to assess the relative irritancy potential of cosmetic ingredients and formulations.

Synopsis An established cytotoxicity test for plastic materials in medical devices has been adapted and used to assess the relative potential irritancy of cosmetic ingredients and formulations during product development. Serum-free medium containing a novel protein supplement supported growth in suspension of LS mouse fibroblast cells. Release of the vital dye Neutral Red from pre-loaded cells suspended in agar was the endpoint. Test substances and reference standards were applied to a central well cut into the agar, a sensitive method which allowed accurate dose application and yielded consistent results. Relative irritancy potential was measured quantitatively by comparing the diameters of the clear zones of damaged cells which surround the central well. The test has been used with raw materials such as surfactants, preservatives and herbal extracts, as well as finished products ranging from shampoos and conditioners to creams, lotions and coloured cosmetics. The method is practical, versatile, reproducible and economic to use. Résumé Un test de cytotoxicité utilisé pour la détection de toxines dans les plastiques des appareils médicaux a été adapté pour prévenir le potentiel d'irritation relative. Le point mesuré est la libération de la teinture vitale rouge neutre des cellules chargées au préalable, résultant de l'incubation avec des substances tests. La mesure des diamétres de la zone claire de dilutions sequentielles de substances tests permet de calculer l'irritation potentielle d'un produit testé par rapport à un standard. Parmi les modifications, on a pu constater à l'aide d'une ligne de cellules en suspension dérivées de fibroblastes de souris L-929, la pousse en milieu sans sérum pour éviter les produits d'abattoir. A la place du sérum de veau, on a ajouté au milieu une nouvelle protéine innovatrice supportant la pousse normale en suspension. Durant la procédure de test les cellules se trouvaient en suspension dans une solution agar et les substances tests ont été appliquées sur une cellule centrale bien délimitéc sur la gélose plutôt qu'à l'aide d'un disque de papier filtre. L'application par une cellule centrale était plus sensible qu'avec un disque, permettant l'application d'une dose précise et fournissant des résultats plus fiables. Le test modifié a été utilisé sur une période de 4 ans pour des matiéres premières telles que tensioactifs, conservateurs et extraits de plantes, ainsi que des formules de shampooings, savons, lotions hydratantes et cosmétiques colorés. Ces produits ont étéévalués sur des panels de volontaires, et les produits testés sont commercialisés depuis 2 ans au moins. Le test est un moyen pratique, versatile, reproductible et économique de calculer le potentiel d'irritation relative d'une gamme de produits et ingrédients cosmktiques.

Increased T-cell reactivity to leukemic B cells in chronic lymphocytic leukemia with change of stable disease to its progressive form.

Some patients with chronic lymphocytic leukemia (CLL) have a relatively stable clinical course without treatment, but many of these eventually develop progressive disease. We have followed 68 patients over 5 1/2 years by employing conventional surface markers and lymphocyte reactivity, including that of separate enriched T cells to their own leukemic B cells, to phytohemagglutinin (PHA), and to normal allogeneic B cells; we have observed an unusual response in each of the seven patients who developed progressive disease over this period of time. During the stable phase of the disease the T cells from 27 leukemic subjects did not respond to their own leukemic B cells in culture. In the seven patients who developed progressive disease, a significant reactivity of their enriched T cells to their own leukemic B cells occurred. There was no consistent change in T-cell reactivity to PHA or to allogeneic B cells, suggesting a change in the leukemic B-cell populations that was not detected morphologically. The change in reactivity of T cells to leukemic B cells occurred prior to evidence of clinical or laboratory deterioration in one of the seven cases; there was increasing lymphocytosis and lymphadenopathy in six and two instances, respectively; and anemia and thrombocytopenia in the three and one cases, respectively.

Regional variation in nonmedical factors affecting family physicians' decisions about referral for consultation.

OBJECTIVE: To determine whether there is regional variation in environmental (non-medical) factors affecting referral decisions of family physicians (FPs). DESIGN: Cross-sectional interview survey. SETTING: Nova Scotia. PARTICIPANTS: A random sample of 125 FPs grouped into 1 of 5 functionally defined geographic regions of Nova Scotia (25 in each group). Groupings were based on access to general hospital beds through active staff hospital appointments or to specialist consultants in the community, or both. Participants were personally interviewed on site. No physician refused an interview. In 9 cases the physician indicated that he or she did not fit the profile of the assigned group; the physician was excluded from the study and the next doctor on the list was substituted. OUTCOME MEASURES: The questionnaire was designed to test several hypotheses about factors known to potentially influence decisions about referral. Geographic differences in factors affecting referral and in decisions about 5 hypothetical cases were assessed with the use of significance tests for proportions that were sensitive to specific orders across groups. RESULTS: Three factors affecting referral showed unequivocal variation across the 5 groups. Access to hospital facilities and remoteness from specialist care, leading to local styles of practice or treatment policies, and the FP's relationship with specialist consultants appeared to be important nonmedical factors affecting referral decisions. For similar case scenarios the physicians living in rural areas would refer only half as often overall as those living in urban areas with tertiary care hospitals; for some cases, such as a severe asthma attack, the difference was more than 7-fold. CONCLUSIONS: Significant differences in nonmedical factors affecting referral, and in referral decisions about hypothetical cases, were found between the groups of FPs. Differences in access to resources, creating local styles of practice, appeared to explain most of the variation. The results may account for previously observed differences in actual rates of referral for these particular groups.

Immunoregulation of B lymphocyte colony formation by T cell subsets in patients with chronic lymphocytic leukemia.

Normal B lymphocytes are activated, proliferate, and then differentiate into plasma cells and secrete immunoglobulin (Ig). We have reported that chronic lymphocytic leukemia (CLL) T4 cells help and CLL T8 cells lack suppressor effects on Ig synthesis by normal B cells (Blood 62:767, 1983). We have now explored the earlier phase, proliferation, using B cell colony formation; in semisolid media. B lymphocyte colonies from normal individuals and from patients with CLL were grown in 0.3% agarose overlayed with T cells or T cell subsets and the B cell mitogen staphylococcal protein A. Enriched T cells, OKT4 or OKT8, were obtained either by sheep erythrocyte rosettes or depletion of OKT8 or OKT4 cells by monoclonal antibody or complement, respectively. Twenty thousand B cells from normal subjects yielded 65 +/- 9, 64 +/- 7, and 19 +/- 6 colonies with autologous unfractionated T-, OKT4-, or OKT8-positive cells, respectively. This compared to 29 +/- 11, 81 +/- 11, and 15 +/- 4 colonies from patients with CLL with added autologous unfractionated T-, OKT4-, or OKT8-positive cells. To determine whether the fewer number of colonies in both normal subjects and patients with CLL with OKT8-positive cells was due to suppression or lack of help, the number of OKT4-positive cells was held constant, and OKT8-positive cells were added in increasing numbers. No suppression of colony formation could be demonstrated. Furthermore, the addition of increasing numbers of concanavalin A (Con A)-activated OKT8-positive cells did not suppress colony formation. These results suggest that the CLL T cell subsets behave in a functionally similar manner to normal T cell subsets, namely, (1) that normal and CLL B cell colony growth is helped by OKT4 cells; and (2) in contrast to immunoglobulin secretion by B cells, neither normal nor CLL OKT8 cells, unstimulated or activated by Con A, suppress B cell colony growth.

Separation of T lymphocytes from normal individuals and patients with B lymphocyte chronic lymphocytic leukaemia.

Previous studies have shown that lymphocytes from patients with chronic lymphocytic leukaemia have a diminished response to mitogens which stimulate T cells. Chronic lymphocytic leukaemia is most often a disease of accumulating B cells so that T lymphocytes are diluted by large numbers of leukaemic cells. Direct comparison with the responses of normal lymphocytes to mitogenic stimulation is therefore suspect. To circumvent this difficulty, a method of isolating T cells from normal individuals and patients with chronic lymphocytic leukaemia was developed. Lymphocytes containing an average of 16.1 per cent B cells from normal individuals were applied to IgG-anti-IgG-coated Degalan bead columns and held at 4 degrees for 2 hours. The eluted cells contained less than 2 per cent B cells. When chronic lymphocytic leukaemic lymphocytes, containing an average of 68.6 per cent B cells, were applied to IgG-anti-IgG columns, the eluted cells contained 36.4 per cent B cells. To improve the purification of T lymphocytes, columns of uncoated Degalan beads were used to remove non-specifically adherent cells. Eluted lymphocytes were then applied to IgG-anti-IgG columns. This resulted in the recovery of purified populations of T cells with less than 2 per cent contamination with B cells. Patients with chronic lymphocytic leukaemia were found to have lymphocytes with either a normal density or a low density of surface immunoglobulins. B cells were successfully removed from lymphocyte suspensions in all cases of chronic lymphocytic leukaemia with a normal density of lymphocyte surface immunoglobulins. In the three cases of chronic lymphocytic leukaemia with low density surface immunoglobulins, separation by this method was unsuccessful. However, an enriched T-cell population was obtained when leukaemic lymphocytes which had lost all detectable surface immunoglobulins were passed through a column coated with heat-aggregated IgG.

Sclerotic lesions of bone in myeloma.

Osteolytic defects and osteoporosis are common in myeloma, while sclerotic lesions of bone are rare. Eighteen patients with increased bone density have been described in the literature and five patients are presented in this report. Diffuse increase in skeletal density, similar to that seen in the myelofibrosis-myelosclerosis syndrome, occurred in two patients, and progressive multiple focal areas of sclerosis with splenomegaly in a third. Two patients had solitary areas of sclerosis. Although there was increased cortical and trabecular bone, osteoblastic activity was normal on histological sections. Whether the sclerosis was due to new bone formation or interference with bone resorptive processes could not be determined. Patients with polycythemia, myelofibrosis and myelosclerosis have been found to have, or later develop, myeloma. This has led to the suggestion that myeloma be included among the myeloproliferative disorders. At present the evidence for this interrelationship is the frequency of the association of these diseases.