Molecular genetic characterization of a locus that contains duplicate Adh genes in Drosophila mojavensis and related species.

Drosophila mojavensis and other species of the mulleri subgroup contain a duplicate gene encoding the enzyme alcohol dehydrogenase (ADH). Studies on the genetic relationship of the two genes using electrophoretic variants show them to be closely linked. We have cloned a 13.5-kb fragment of D. mojavensis DNA into the lambda vector, Charon 30. This fragment contains both Adh genes separated by approximately 2 kb of DNA. The clone hybridized to a single position on chromosome 3 in D. mojavensis following in situ hybridization. It is likely that the genes are tandemly arranged in the genome. One of the two genes shows a complexity in its structure that suggests the close linkage of a pseudogene or part of a gene. The structure of the Adh locus in five species of the mulleri subgroup have been compared by constructing restriction maps of genomic DNA. Two of these species D. arizonensis and D. mojavensis express Adh-1 in the ovaries; the others do not. In comparing these species it is evident that there has been one or two insertions into the region between the Adh genes. It is possible that one of these structural changes is related to the change in Adh tissue-specific expression that has occurred during the evolution of these species.

Structure and evolution of the Adh genes of Drosophila mojavensis.

The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.

Mature polymorphonuclear leukocytes express high-affinity receptors for IgG (Fc gamma RI) after stimulation with granulocyte colony-stimulating factor (G-CSF).

The high-affinity receptor for the constant region of immunoglobulin G IgG (Fc gamma RI; CD64) is virtually undetectable on mature polymorphonuclear neutrophils (PMNs) in healthy individuals but is expressed on PMNs in patients with certain infections and in patients treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). The induction of Fc gamma RI by rhG-CSF has previously been reported to result from effects on immature granulocyte progenitors. To evaluate the G-CSF effect on mature PMNs, we studied the correlation between G-CSF plasma concentration and expression of Fc gamma RI on PMNs in vivo as well as the effect of G-CSF on Fc gamma RI expression on mature PMNs in vitro. Fc gamma RI expression on PMNs correlated (R = 0.79; p < .001) with plasma concentrations of endogenous or recombinant G-CSF in healthy volunteers and in patients undergoing high-dose chemotherapy and autologous bone marrow transplantation. PMNs exhibited a unimodal distribution for elevated Fc gamma RI expression, suggesting that G-CSF induced increased expression of Fc gamma RI on mature as well as on immature PMNs. In vitro, incubation of mature PMNs with G-CSF induced mRNA for Fc gamma RI. Significant Fc gamma RI surface expression was induced in a time- and dose-dependent manner. Thus, G-CSF can act on mature PMNs to increase Fc gamma RI expression and may be useful for stimulating antibody mediated immune functions of PMNs in vivo.

The effect of recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor on Fc gamma receptor expression and antibody-dependent cellular cytotoxicity of hematopoietic progenitor cells during in vitro myeloid maturation.

Enriched progenitor cell fractions from human bone marrow were induced to undergo myeloid maturation in culture using recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony stimulating factor (rhGM-CSF). A negative selection method using the murine monoclonal antibodies (MABs) PM81 (anti-CD15), AML-2-23 (anti-CD14), PC251 (anti-CD33), OKT11 (anti-CD2), and SCCL-1 (anti-CD71) and immunomagnetic beads coated with sheep anti-mouse IgG (Dynal A.S., Oslo, Norway) was used to remove the more mature cellular components of mononuclear cells from normal donor bone marrow samples. The resulting fraction of cells contained 35 to 40% CD34-positive cells, and less than 1% of cells expressed the receptors for the constant portion of immunoglobulin G Fc gamma RI or Fc gamma RII. A small population (3-5%) expressed Fc gamma RIII on day 0, and these cells were found by two-color flow cytometry to be primarily natural killer (NK) cells. The level of Fc gamma R expression was determined every 2 to 3 days on aliquots of the differentiating cells. Thirteen percent of the cultured bone marrow cells expressed Fc gamma RII after 48 hours in liquid culture with rhIL-3 and rhGM-CSF. The percent of cells expressing Fc gamma RII increased to a peak of 78% of the gated population on day 10. The mean fluorescence intensity (MFI) remained low for the first 8 to 10 days of culture, but at that time the MFI more than doubled. Fc gamma RI and Fc gamma RIII expression remained low throughout the culture period. The ability of the differentiating cells to perform antibody-dependent cellular cytotoxicity (ADCC) was determined at a single-cell level in a modified plaque assay using monolayers of ox erythrocyte (oxE) target cells. The purified progenitor cells, when placed in oxE monolayers sensitized with polyclonal rabbit anti-oxE antibody (AB), showed no plaque formation over control oxE layers. No increase in ability to generate cytolytic plaques in antibody-sensitized oxE layers was seen compared with unsensitized oxE layers until after 10 days of incubation in liquid culture. At that time, the percent of cells forming plaques in the AB-sensitized oxE layers was 34.4 +/- 10.7% (average +/- standard error of the mean [SEM]; n = 4) compared with 10.0 +/- 0.7% on the control oxE layers. The peak plaque formation appeared to coincide with the increase in MFI of a large population of the cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Autologous bone marrow transplantation for acute myeloid leukemia following in vitro treatment with neuraminidase and monoclonal antibodies.

Although monoclonal antibodies (MoAbs) to CD15, especially PM-81, react with leukemic blasts from the majority of patients with acute myeloid leukemia (AML), a small subset of patients have cells that are CD15 negative or dim. We determined previously that neuraminidase will increase the reactivity of PM-81 with AML blasts, as well as blasts from many patients with acute lymphoblastic leukemia (ALL). In this report, we describe the laboratory results and clinical course of the first patient with AML whose harvested bone marrow was treated with neuraminidase prior to MoAbs and complement treatment. Neuraminidase increased the percentage of the patient's leukemia cells that reacted with PM-81 from 18% to 90% and more than doubled the percentage of AML blasts that were lysed by PM-81 and complement. The patient suffered no acute toxicity, engrafted rapidly, and was transfusion independent by day 21 post-ABMT. This report demonstrates the probable safety and efficacy of pretreatment of bone marrow with neuraminidase, and increases the number of patients with AML or ALL who may benefit from ABMT using marrow purging with MoAb to CD15.

Anti-body-dependent cellular cytotoxicity (ADCC) function of peripheral blood polymorphonuclear neutrophils (PMN) after autologous bone marrow transplantation (ABMT).

Rapid recovery of the number and function of polymorphonuclear neutrophils (PMN) is critical to recovery from bone marrow transplantation. Although it is relatively easy to measure PMN number recovery, the evaluation of the functional recovery of these cells has not been adequately examined. The ability of peripheral blood PMNs to perform antibody-dependent cellular cytotoxicity (ADCC) was assessed in 25 patients undergoing autologous bone marrow transplantation (ABMT). PMNs were evaluated at a single cell level for ADCC function as measured by their ability to form plaques in antibody-sensitized ox erythrocyte (oxE) monolayers. The PMNs demonstrated low or absent ADCC function in the first week after completion of high-dose chemotherapy, regardless of primary diagnoses or myeloablative regimens. Although recovery to a neutrophil count of 500/microliters was prolonged in patients with AML (mean 40.2 days; range 25-67 days), functional activity of PMNs appeared much earlier (mean 19.6 +/- 6.1 days; range 2-65 days) in this group of patients compared to the group of patients with other diagnoses in which recovery to a neutrophil count of 500/microliters and the recovery of functional activity of PMNs occurred at roughly the same time. This single cell assay provided a useful method for determining ADCC functional ability of recovering PMNs post-BMT since few cells were required for each assay. This approach may also be useful in determining optimal timing of immune therapies post-ABMT, relying on myeloid cells as effector cells.

Isodicentric chromosome 18q as sole abnormality in a histologically normal bone marrow from a patient with diffuse large-cell non-Hodgkin's lymphoma.

Cytogenetic analysis was performed on the histologically and immunophenotypically normal bone marrow (BM) of a 33-year-old woman with non-Hodgkin's lymphoma (NHL) before BM harvest. Unstimulated 24- and 48-hour cultures produced only normal metaphases. A pokeweed mitogen (PWM)-stimulated 48-hour culture, however, showed a clonal isodicentric chromosome 18q as the sole abnormality, suggesting a role for this approach in detection of submicroscopic BM involvement by B-cell NHL.

Role of ipsilateral forebrain in lateral hypothalamic stimulation reward in rats.

The forebrain was ablated unilaterally to a level dorsal to the thalamus and anterior commissure. Ipsilateral lateral hypothalamic electrodes were then implanted and the animal was tested for self-stimulation behavior. Tests included an initial test for behavioral reactivity to changes in reward level and then two estimates of the quantitative relationships between stimulation parameters: the number-current and charge-duration relationships. Comparison between these findings and those known for intact rats suggest that the substrate for unilateral hypothalamic stimulation reward is not impaired by removal of the ipsilateral tissue.

Quality of life of long-term adult survivors of autologous bone marrow transplantation.

PURPOSE/OBJECTIVES: To extend the knowledge of quality of life (QOL) of survivors of allogeneic bone marrow transplantation (BMT) to include survivors of autologous BMT. To determine the appropriateness of using a reliable, valid, allogeneic BMT QOL instrument for survivors of autologous BMT. DESIGN: Cross-sectional, descriptive, replication survey. SETTING: An autologous BMT program in a National Cancer Institute-designated comprehensive cancer center. SAMPLE: All survivors of autologous BMT (N = 37) at the center were recruited; 29 survivors participated (85% response rate). METHODS: Three mailed questionnaires: the City of Hope quantitative QOL-BMT instrument and qualitative questionnaire and an investigator-developed demographic questionnaire. MAIN RESEARCH VARIABLES: QOL, autologous BMT treatment, disease type, age, gender, employment, and date of transplant. FINDINGS: Global QOL was high (mean = 8.17 on a 1-10 scale). Most respondents experienced few long-term physical disruptions and had only mild psychological distress. Fatigue, sexuality concerns, and family distress created by the illness were the most negatively rated items. Content, face, and construct validity of the QOL-BMT instrument in the autologous BMT population were acceptable. Overall internal consistency reliability of the tool, as measured by Cronbach's alpha, was 0.82. Themes of uncertainty, concern about relapse, and pain were reported in the qualitative data but not revealed by responses to the QOL-BMT instrument. CONCLUSIONS: The majority of survivors of autologous BMT reported few physiologic disruptions and above-average QOL. The City of Hope QOL-BMT instrument had acceptable reliability and validity when adapted for survivors of autologous BMT. Addition of items related to uncertainty, pain, and concern about relapse could further strengthen its validity. IMPLICATIONS FOR NURSING PRACTICE: Most survivors of autologous BMT can expect above-average QOL. Incorporating results of QOL evaluation in the informed consent process may help BMT candidates in making the decision to undergo the procedure by providing a more complete picture of life after BMT. However, since a minority of patients will experience continued disruptions in any one of the QOL domains, healthcare providers need to conduct comprehensive follow-up evaluations to determine which patients may need referral to the specialized services of a survivor clinic.

Autologous bone marrow transplantation in the treatment of acute myeloid leukemia: the Dartmouth experience and a review of literature.

Autologous bone marrow transplantation is increasingly being investigated as a treatment for patients with acute myelogenous leukemia. Review of the literature demonstrates that much of the data are incomplete. Most reports contain small numbers of patients, making analysis of any particular regimen difficult to assess. The morbidity and mortality of the procedure appear to be substantially less than that seen in the allogeneic setting. The major complications relate to problems with engraftment. Recovery of platelet production to normal levels is frequently cited as delayed, and in some patients, does not occur. This phenomenon may be heightened by marrow manipulation during purging or posttransplant drug therapy. It is not known if this is a problem related to stem cells or related to the changes in the hematopoietic microenvironment. The results of autologous bone marrow transplantation for patients with acute myeloid leukemia suggest that, as with standard chemotherapy, there is little survival benefit when patients are in relapse at the time of transplantation. There are few long-term survivors, and relapse within 5 months is the rule. It should be noted that the vast majority of the studies reported here have used marrow that has not been treated in an attempt to remove occult leukemia cells. The use of purged bone marrow has not yet been adequately studied. In patients in second or subsequent remission, ABMT appears to offer a chance for long-term survival not seen with present second-line standard chemotherapy regimens and should be considered a viable option for patients under the age of 55. The results to date do not define whether marrow purging is beneficial, and most studies being carried out at the present time are not evaluating this question. The majority of studies are examining different methods of purging. The result of our study in patients in second and third remission using in vitro purging of bone marrow with monoclonal antibodies PM-81 and AML2-23 are encouraging, as are the studies of purging with 4-HC. The Cancer and Leukemia Group B has just begun a study for patients with AML in second remission using the protocol we piloted at Dartmouth. We are also evaluating the feasibility of using this therapy in patients at the time of first relapse, as studies in the allogeneic setting have suggested the results are similar to those achieved in second remission (60).(ABSTRACT TRUNCATED AT 400 WORDS)

Approach and withdrawal analysis of the effects of hypothalamic stimulation and lesions in rats.

In a paradigm in which rats would both initiate and terminate hypothalamic stimulation, "purely" rewarding lateral hypothalamic and "purely" aversive medial hypothalamic electrodes were identified. Subjects were then given a series of tests designed to assess the effects of brain stimulation on approach and withdrawal behaviors. Lateral hypothalamic stimulation facilitated approach behavior and suppressed withdrawal behaviors, whereas medial hypothalamic stimulation produced largely the opposite effects. No serious motor deficits due to stimulation were detected with either type of electrode. In a second experiment, the approach-withdrawal effects of bilateral lateral hypothalamic lesions were tested and found to suppress approach behaviors and facilitate withdrawal behaviors. Other neurological examinations revealed motor deficits, but these deficits do not account for the specific pattern of results observed on the approach-withdrawal tests. These approach-withdrawal phenomena are interpreted in terms of altering a natural balance between approach and withdrawl behavior facilitating systems in the lateral and medial hypothalamus, respectively.

Spontaneous factor VIII inhibitor occurring in association with chronic graft-versus-host disease.

The development of spontaneously acquired Factor VIII inhibitors is rare and may lead to serious hemorrhagic sequelae. We report here the case of a patient who acquired a Factor VIII inhibitor two years after an allogeneic bone marrow transplant for CML. This occurred in association with a flare of chronic graft versus host disease (GVDH). He responded to corticosteroid therapy. A review of autoimmune phenomena associated with chronic GVDH and the treatment of Factor VIII inhibitors is discussed.

The first decade of the American Board of Internal Medicine certification in critical care medicine: an overview of examinees and certificate holders from 1987 through 1996.

OVERVIEW: This study reviews the first decade of critical care medicine (CCM) certification by the American Board of Internal Medicine (1987-1996). Included are the characteristics of examinee and certificate-holder groups; examination performances from different underlying disciplines of internal medicine, with or without formal CCM training; and the influence of background and a training program as correlates of examination performance. DATA SOURCES: The CCM certification examination has been offered biennially since November 1987. Performance data on the American Board of Internal Medicine examinations in internal medicine and its subspecialties and added qualifications were available for candidates taking the CCM examinations. For examinees with formal CCM training, residency program director ratings, and information regarding the program characteristics of size and percentage of United States and Canadian medical graduates were also available. STUDY SELECTION: All examinees who ever attempted certification were included in this study. The study cohort for each of the five examination administrations consists of all first-time takers. CONCLUSIONS: Cohort sizes have decreased since formal training became an admission requirement in 1993. Percentages of International Medical Graduates and women attempting and achieving certification have increased steadily. Examination performance was positively associated with formal training, internal medicine examination performance, recent medical training, and pulmonary disease certification. For those with formal training, performance was also positively associated with training program director ratings of overall clinical competence and completion of a training program with a higher proportion of United States and Canadian medical graduates.

Crystallocryoglobulinemia resulting from human monoclonal antibodies to albumin.

Two patients had a previously unrecognized form of crystallocryoglobulinemia. Their clinical presentations were similar, consisting of necrotizing vasculitis and purpura involving the legs. Analysis of each cryoglobulin complex showed that two components, albumin and a monoclonal IgG-lambda, were present, and both components were needed in a fixed ratio for precipitation. In addition, cryoprecipitation occurred in serum, but not plasma, due to citrate inhibition of complex formation. Our findings suggest that the monoclonal IgGs have the properties of antibodies directed specifically against a calcium-dependent antigenic site on human albumin, and that the resultant IgG-lambda-albumin immune complexes crystallized in the cold.

Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid cell-specific monoclonal antibodies.

Second or third chemotherapy-induced remissions in acute myelogenous leukemia (AML) are limited by early relapse of the leukemia. We developed monoclonal antibodies (MoAbs) that are cytotoxic to myeloid leukemia cells to treat bone marrow from these patients ex vivo for autologous transplantation. In this pilot study, bone marrow was harvested from ten patients with AML in remission, treated with one or two complement-fixing MoAbs, PM-81 and AML-2-23, which react with myeloid differentiation antigens, incubated with rabbit complement, and cryopreserved. These MoAbs were chosen because they have broad reactivity with AML cells but not with pluripotent progenitor cells. At the time of transplant, 6 patients were in second complete remission, 1 each was in third complete or partial remission, and 2 were in early first relapse. The patients were treated with cyclophosphamide (60 mg/kg a day for 2 days) and total body irradiation (200 cGy twice a day for 3 days) and given infusions of MoAb-treated bone marrow. Full bone marrow reconstitution was observed in eight patients; two patients did not recover platelets. Seven of the ten patients are surviving and disease-free at 21.0, 15.0, 13.0, 10.0, 6.0, 3.0, and 2.0 months posttransplant. Treating bone marrow with MoAbs to myeloid differentiation antigens does not interfere with pluripotential stem cell engraftment. Longer follow-up and a controlled study are necessary to prove that the apparent efficacy of this therapeutic approach in some patients is attributable to MoAb-mediated killing of leukemia cells.

Pregnancy and hormonal influences on malignant melanoma.

Halo cutaneous melanoma developed in a 30-year-old woman. Following wide excision of the melanoma, she remained clinically free of tumor for 5 years. In a subsequent pregnancy, she developed metastases to the liver which became evident in the immediate postpartum period. Long-term survival associated with cutaneous hypopigmentation has been reported and occurred in our patient. The interaction between hormonal and immunologic factors and melanoma is explored.

PIP: This article presents the case of a 30-year-old woman who developed halo cutaneous melanoma. She had been taking oral contraceptives (Norinyl) for about 5 years before diagnosis. Following wide excision of the melanoma, the patient remained clinically free of tumor for 5 years. However, in a subsequent pregnancy, she developed metastases to the liver that became evident in the immediate postpartum period. Long-term survival associated with cutaneous hypopigmentation has been reported and occurred in this patient. There is considerable debate as to whether oral contraceptives or pregnancy can influence the occurrence and course of melanoma. Also unclear is whether oral contraceptive use or a subsequent pregnancy in women with a history of melanoma will accelerate the growth of latent metastases, stimulate a benign pigmented lesion to become malignant, or cause a previously removed melanoma to recur and metastasize. Given the lack of uncertainty in this area, it is recommended that women with a history of melanoma use a nonhormonal method of contraception. Frequent follow up and thorough physical examinations during pregnancy are essential, and any suspicious skin lesions should be biopsied early. To better answer the questions raised by cases such as this, establishment of an organized mechanism for the registry of patients with melanoma who subsequently become pregnant is suggested. A cooperative prospective melanoma study could accumulate the necessary data on tumor site and thickness, staging, parity, and the use of hormonal contraception.

Mitoxantrone in relapsed or refractory acute nonlymphocytic leukemia.

Seventeen patients with relapsed or refractory acute nonlymphocytic leukemia were treated with 14 mg/m2 of mitoxantrone given in a 30-minute infusion daily for three days. If the day fourteen bone marrow showed residual leukemia, a second course was given at the same dose for two days. Eight patients (47%) entered complete remission. Three patients (17%) had a partial response, four (24%) did not respond, and two (12%) died with hypoplastic marrows during treatment. Seven of the 12 relapsed patients entered a complete remission, as did one of the five refractory patients. Toxicity was acceptable; prolonged myelosuppression, moderate hepatic toxicity, and stomatitis were the only problems. Several dose schedules of mitoxantrone have been studied by other investigators with varying results. The three-day schedule in the present study is similar to the schedule used for common induction regimens employing anthracycline drugs. On the basis of its activity and acceptable toxicity in relapsed and refractory ANLL patients, we feel that this schedule could be safely combined with other agents in future studies.

Improved outcome for high-risk acute myeloid leukemia patients using autologous bone marrow transplantation and monoclonal antibody-purged bone marrow.

We have conducted a 9-year multicenter trial of autologous bone marrow transplantation (ABMT) for acute myeloid leukemia (AML). Remission BM was purged in vitro using monoclonal antibodies (MoAbs; PM-81, AML-2-23) and complement targeting myeloid differentiation antigens (CD15, CD14). In 1988, the preparative regimen changed from 60 mg/kg/d cyclophosphamide x 2 and fractionated total body irradiation (TBI) total dose, 1,200 cGy (Cy/fTBI), to 4 mg/kg/d busulfan x 4 and 60 mg/kg/d Cy x 2 (Bu/Cy2). Recent analysis (October 1, 1993) shows that the Bu/Cy2 regimen along with the same MoAb purging method yields an improved outcome. Seven first complete-remission (CR) (CR1), 45 second- or third-CR (CR2/3), and 11 first-relapse (R1) patients were treated with chemotherapy and TBI or chemotherapy alone followed by ABMT with MoAb-purged BM. Median age at ABMT for those patients in CR 2/3 and R1 patients was 36 years. Twenty-nine CR 2/3 and R1 patients were conditioned with Cy/fTBI, and 27 CR2/3 and R1 patients were conditioned with Bu/CY. Using the Kaplan-Meier method, the CY/fTBI, CR2/3, and R1 patients have a 3-year disease-free survival (DFS) of 21%. On the other hand, the Bu/Cy2, CR2/3, and R1 patients have a 3-year DFS of 48%. Nineteen CR2/3 and R1 patients relapsed post-ABMT. On analysis by conditioning regimen, those treated with Cy/fTBI have a 3-year relapse rate (RR) of 58%, whereas the patients conditioned with Bu/Cy2 have a 39% 3-year RR. Long-term DFS can be achieved in about 50% of patients with advanced remissions and relapsed AML using Bu/Cy2 with MoAb-purged BM.

Spurious E rosette formation in B cell chronic lymphocytic leukemia due to monoclonal anti-sheep RBC antibody.

The apparent simultaneous presence of surface markers characteristic of both B and T cells is a phenomenon being described with increasing frequency in patients with chronic lymphocytic leukemia (CLL). We describe a patient with CLL whose B lymphocytes possessed surface immunoglobulin reactive with neuraminidase-treated sheep erythrocytes (SRBCs) and produced E rosette formation. Cytofluorography using monoclonal antibodies demonstrated the B cell nature of these cells and the absence of the SRBC receptor. Further documentation that the binding of SRBCs was mediated through immunologic reaction included E rosette formation inhibition by monospecific antisera and hemagglutination of SRBCs by a paraprotein isolated from the patient's serum. Fusion of the CLL cells with a human hypoxanthine-aminopterin-thymidine-sensitive plasma cell line resulted in the production of human hybridomas that secreted the SRBC-reactive IgM antibody. An analysis of clinical histories of CLL patients whose cells exhibited this phenomenon from both immunologic and clinical perspectives is presented.