Acute stress-induced facilitation of the hypothalamic-pituitary-adrenal axis: evidence for the roles of stressor duration and serotonin.

Stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis stimulates the release of both facilitatory and inhibitory components. We proposed that the transient removal of the inhibitory component, corticosterone, during a stressor would leave the HPA axis in a state of hyper-responsiveness (facilitated state). Consistent with this expectation, we have previously observed that aminoglutethimide (AG)-induced removal of corticosterone during an immobilization stressor resulted in the hypersecretion of both ACTH and corticosterone to a subsequent stressor. In the present study we determined the effect of stressor duration on the magnitude of facilitation. AG plus a 10-min immobilization (IMM(10)) stress on day 1 resulted in facilitation of the HPA axis. This was reflected in higher ACTH and corticosterone responses to an injection stress on day 2 as compared to appropriate control rats. AG plus a 60-min immobilization (IMM(60)) stress on day 1 resulted in significantly greater facilitation as compared to the AG+IMM(10) pretreatment. It is apparent that facilitation of the HPA axis is dependent on the duration of stress. Stress can alter plasma corticosterone-binding globulin levels and AG administration can cause accumulation of the corticosterone biosynthetic precursor, adrenal cholesterol. In order to rule out these peripheral reasons for the hypersecretion of ACTH and corticosterone in our paradigm, we measured the plasma free fraction of corticosterone and adrenal mitochondrial cholesterol levels on day 2 after different pretreatments on day 1. AG+IMM(60) pretreatment caused a significant increase in the plasma free fraction of corticosterone. Hypersecretion of ACTH and corticosterone in this group, despite an enhanced feedback signal, suggests central loci for the origin of facilitation. Also, AG treatment on day 1 did not result in accumulation of free or esterified adrenal cholesterol levels on day 2, and therefore cannot account for the hypersecretion of corticosterone. In our final study we attempted to determine if serotonin released during the first stressor is partially responsible for stress-induced facilitation of the HPA axis. We administered 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a 5HT(1A) agonist, either alone or in conjunction with stress and examined the effects of these pretreatments on the magnitude of facilitation. Interestingly, DPAT administered in lieu of stress produced facilitation similar in magnitude to that produced by IMM(10). DPAT administered in conjunction with IMM(10) augmented stress-induced facilitation. Our results suggest that stress-induced facilitation of the HPA axis is associated with the release of serotonin during stress.

Acute-stress-induced facilitation of the hypothalamic-pituitary-adrenal axis.

It has been hypothesized that the hypothalamic-pituitary-adrenal (HPA) axis responds to a stressor by secreting facilitatory and inhibitory factors. During a stressor, the relative magnitude of secretion of these factors determines the responsiveness of the HPA axis to a subsequent stressor. Previous studies have suggested that corticosterone (B) secreted during the first stressor is an inhibitory factor. We hypothesized that the transient removal of the inhibitory factor, B, during the first stressor would result in the secretion of only facilitatory factors. This would cause the HPA axis to exist in a state of hyperresponsiveness, and to hypersecrete corticotropin (ACTH) and B in response to a second stressor. Therefore, our primary objective was to demonstrate stress-induced facilitation of the HPA axis response to a subsequent stressor. Male Sprague-Dawley rats were subjected to a 1-hour physical immobilization stressor (IMM) or administered a single dose of ACTH on day 1. B response during these treatments was markedly but transiently attenuated with an 100 mg/kg i.p. dose of aminoglutethimide (AG). Twenty-four hours later, rats were subjected to an intraperitoneal saline injection stressor. B and ACTH levels were measured 15 min after the injection stressor. Rats treated with AG plus IMM on day 1 hypersecreted B and ACTH after the injection stressor on day 2. These results suggest that immobilization stress induces facilitation of both pituitary and adrenal responses. Exogenous administration of ACTH- to AG-pretreated rats on day 1, in lieu of immobilization stress, did not affect the responsiveness of the HPA axis on day 2. This suggests that ACTH secreted during the first stressor does not play an important role in acute-stress-induced facilitation.

Therapy for childhood acute lymphoblastic leukemia.

Recent advances in therapy for childhood acute lymphoblastic leukemia have come primarily from the intensive laboratory study of patient's lymphoblasts. DNA-ploidy determination, the analysis of specific chromosomal translocations, and in vitro chemosensitivity studies now facilitate the stratification of risk groups and the prediction of treatment outcome. More is known about the heterogeneity of molecular defects involved in leukemogenesis, and this information is being exploited to devise sensitive tests for minimal residual disease. Conventional chemotherapy of childhood acute lymphoblastic leukemia is associated with adverse neuropsychological sequelae and second malignancies when intensive epipodophyllotoxin therapy is used. Treatment of relapsed acute lymphoblastic leukemia remains problematic. The development of alternative donor marrow sources will expand the role of bone marrow transplantation, which has provided better outcomes for a limited number of patients. We are still waiting to spawn novel therapeutic agents that are more effective and less toxic than present chemotherapy.

Etoposide, cyclophosphamide, cisplatin, and doxorubicin as neoadjuvant chemotherapy for osteosarcoma.

The authors evaluated the combination of etoposide/cyclophosphamide (VP/CY) as initial, presurgical therapy for patients with osteosarcoma and found an 88% response rate for the primary tumor and any metastases. After definitive, limb-salvage surgery and adjuvant chemotherapy with etoposide, cyclophosphamide, cisplatin, and doxorubicin, patients without metastases at diagnosis whose cases were followed for a median of 2 years from diagnosis achieved a relapse-free survival (RFS) probability of 78% +/- 9%. This result is equivalent to the best adjuvant chemotherapy results reported to date. Patients without metastases at diagnosis had significantly better RFS probability (78% +/- 9%) than those with metastases at diagnosis (0%). Transient, severe myelosuppression has been the only major toxicity of the VP/CY courses. No irreversible organ damage or toxic deaths have been seen in patients enrolled in this study. The authors conclude that the combination of VP/CY is effective treatment for osteosarcoma, and when combined with cisplatin/doxorubicin (CIS/DOX), is as effective as any previously reported chemotherapy for osteosarcoma.

Mesenchymal specificity of three new monoclonal antibodies generated to the osteosarcoma cell line 4444T.

We have generated three murine monoclonal antibodies to the new human osteosarcoma cell line 4444T. Analysis of their binding patterns to tumor cell lines, normal human tissues, and surgical tumor specimens indicates that the antibodies recognize a subset of human sarcomas and stromal tissues but fail to react with carcinomas or normal human epithelial tissue. These mesenchyme-specific monoclonal antibodies bind to antigens in the extracellular matrix. One antibody is specific in its binding to the muscularis arteriosus. We expect these antibodies to aid in the identification of sarcomas and to extend our knowledge of the components of the extracellular matrix and its interaction with tumors.

Alternative method of gestational age assessment by the measurement of human erythrocyte differentiation antigen expression.

Standard estimates of gestational age are dependent on such subjective data as maternal recollection of last menstrual period, ultrasound examination of the fetus, and the postnatal physical and neurological examination (Ballard score). We hypothesized that a quantitative, objective laboratory test using flow cytometric analysis of erythroid differentiation antigens could be useful to predict gestational age. For this study erythrocyte samples were obtained within 24 hours of birth from 25 infants (gestational ages 20 to 41 weeks) who met the criteria that traditional estimates of gestational age, such as the Ballard score, fetal ultrasound, and maternal estimate of last menstrual period all agreed within 1 week for the assessed infant's gestational age. Study measurements included reticulocyte count and determination of the percentage of erythrocytes that expressed the 5F1 and 20.3 erythropoietic differentiation antigens. Linear regression analysis indicated that the best correlations with gestational age were reticulocyte count (R2 = .354) and the reciprocal of the percentage of erythrocytes expressing the 5F1 antigen (R2 = .470). When both variables were incorporated into a linear regression model, the predictability of gestational age achieved an R2 = .608. Through this study we have established the feasibility and methodology of using fetal and newborn erythrocytes to provide an objective assessment of gestational age by flow cytometric analysis of erythroid differentiation antigen expression. This methodology will allow for an independent assessment of gestational age when fetal blood sampling is performed for other prenatal diagnostic studies. Further investigation is needed to identify other erythroid differentiation markers that would improve the accuracy of our model to predict gestational age.

A pilot study of monoclonal antibody targeted radiotherapy in the treatment of central nervous system leukaemia in children.

A pilot study was performed to investigate the toxicity, pharmacokinetics and therapeutic effect of intrathecally administered radiolabelled monoclonal antibody (MAb) in patients with meningeal acute lymphoblastic leukaemia (ALL). Six children aged 3-16, in second or subsequent central nervous system (CNS) relapse of ALL, received between 629 and 1480 MBq of 131Iodine conjugated to either MAb HD37 (CD19, n = 2) or WCMH15.14 (CD10, n = 4). Conjugate was administered as a single injection either via an Ommaya reservoir (n = 4) or by lumbar puncture (n = 2). Acute toxicity was manifest by headache (n = 4), nausea and vomiting (n = 4) and pyrexia (n = 2). All acute symptoms resolved within 72 h. Transient myelosuppression occurred in three patients. Pharmacokinetic studies included investigation of whole body, blood and CSF clearance of isotope. 131I was seen to clear from the CSF by biexponential kinetics. Five patients responded to therapy. In four, the CSF became clear of blast cells at both 2 and 4 weeks following antibody injection, but evidence of relapse was seen at 6 weeks. The fifth patient, with blast cells present on a cytospin preparation, responded to therapy over an 8-week period but relapsed at 12 weeks. This study demonstrates the potential of targeted radiotherapy in CNS ALL, but further studies are necessary to increase the length of remission.

Intravenous ribavirin therapy for adenovirus cystitis after allogeneic bone marrow transplantation.

Acute hemorrhagic cystitis due to adenovirus infections may be more severe or protracted in immunocompromised patients. This patient with chronic graft-versus-host disease following allogeneic marrow transplantation for acute myelogenous leukemia developed painful hematuria due to adenovirus infection that failed to respond to diuresis and narcotic analgesics. Intravenous ribavirin, administered for a total of 9 days, produced rapid resolution of symptoms while urine cultures became negative for adenovirus. No adverse drug reactions were observed. We conclude that controlled clinical trials of intravenous ribavirin therapy for serious adenovirus infections following bone marrow transplantation are warranted.

Graft-versus-host disease.

Graft-versus-host disease is an uncommon but serious illness, caused by foreign, immunocompetent T lymphocytes attacking an immunoincompetent host. It is encountered following allogeneic bone marrow transplantation and in congenital cellular immunodeficiency disorders. Diagnostic features, clinical grading system, therapy and prevention are reviewed.

Characterization of a new T-lineage glycoprotein expressed in mature T-cell leukemias and lymphomas.

We identified a new human, T-lineage restricted glycoprotein of molecular weight 120 Kd that is expressed primarily in mature T-cell malignancies. The antigen, named TCA-1 (T-cell cytoplasmic antigen), is an intracellular glycoprotein found mainly in the Golgi stacks, although a few cell lines also display surface membrane TCA-1. Many but not all T-cell neoplasms express this antigen. The antigen is absent from neoplastic and normal human tissue outside the T-lymphocyte lineage. TCA-1 was identified by murine monoclonal antibodies produced after immunization of mice with T-cell chronic lymphocytic leukemia cells. The glycoprotein is a monomer containing approximately 4% N-linked carbohydrate with terminal D-galactose residues. Partial amino acid sequence analysis of TCA-1 shows homology with an immunoglobulin heavy chain region, which suggests that TCA-1 may belong to the immunoglobulin supergene family of receptor and adhesion molecules.

Response of osteogenic sarcoma to the combination of etoposide and cyclophosphamide as neoadjuvant chemotherapy.

A Phase II study of the combination of etoposide (VP-16) and cyclophosphamide (CPM) was conducted in an attempt to identify active and potentially less toxic agents for treating patients with osteogenic sarcoma (OS). VP-16 was given as a 72-hour infusion for a total dose of 600 mg/m2. CPM was given as six pulses of 300 mg/m2 every 12 hours for a total dose of 1800 mg/m2. Seventeen newly diagnosed patients, including five (29%) with metastatic disease, were evaluated before and after two courses of VP-16 and CPM for clinical, radiologic, and biochemical (serum alkaline phosphatase [SAP]) responses of the primary tumor and metastases. Fifteen (88%) patients achieved complete or partial clinical responses. Fourteen (82%) patients achieved radiologic responses. Thirteen (87%) of 15 patients with higher than normal SAP levels for their age showed partial or complete responses. Three (60%) of the five patients with metastatic disease achieved complete or partial responses. The only major toxicity was myelosuppression, which led to 21 (62%) brief admissions after 34 courses of chemotherapy for intravenous antibiotic therapy for fever and neutropenia, without associated mortality. It was concluded that the combination of VP-16 and CPM is effective chemotherapy for both primary and metastatic OS. Although myelosuppression is inevitable, it is rapidly reversible in the drug dosages used. Further studies are needed to evaluate the effect of these drugs in combination with established agents in improving the disease-free survival of patients with OS.

Relative efficiency of leukemic cell depletion using anti-murine-IgG1(Fc) or anti-murine-IgG coated immunomagnetic microbeads.

Microbeads for immunomagnetic bone marrow purging, to which sheep anti-mouse-IgG1(Fc) antibodies have been linked, and beads linked with antibodies against whole murine immunoglobulin were compared. Competitive binding studies, in which Fc fragments and Fab fragments were titrated onto the microbeads, followed by incubation with 125I-labeled whole mouse Ig, revealed that the beads linked with anti-mouse-IgG1(Fc) specifically bound the Fc region of the murine immunoglobulin molecules. The total amount of antibody of either IgG1 or IgG2 isotype that would adhere to microbeads linked with either type of antibody, as revealed by secondary binding with 125I rabbit antimouse Ig, was identical, suggesting that similar numbers of antibody binding sites were available. In cell depletion studies, it was found that if IgG1 isotype monoclonal antibodies were employed as binding intermediaries between the target cells and the microbeads, the efficiency of target cell depletion was superior with the anti-mouse-IgG1(Fc)-coated beads, even if the amount of MoAb coating the target cells was suboptimal. However, if the intermediary antibodies were of the IgG2 isotype, the efficiency of target cell depletion with these beads was inferior. These findings indicate that the efficiency of immunomagnetic bone marrow purging is dependent upon matching of the targeting MoAb and the secondary antibodies that link to the surface to the microbeads.

Establishment of donor hematopoiesis after hydroxyurea-induced aplasia following allograft failure in a patient with monosomy 7 variant of childhood chronic myelogenous leukemia.

Monosomy 7 variant childhood chronic myelogenous leukemia (CML) is a rare, fatal leukemia that usually terminates in blast crisis. We report successful marrow transplantation in a patient with this disease using his one HLA locus mismatched mother. Initially following transplant the patient exhibited mixed hematopoietic chimerism, cytogenetic relapse, and clinical relapse of leukemia. However, following recovery from a period of hydroxyurea-induced aplasia, marrow studies showed elimination of the mixed chimerism, absence of the 45,XY,-7 leukemic clone and full engraftment with donor marrow (46,XX). The ability of hydroxyurea to eliminate mixed chimerism in favor of donor hematopoiesis and to eliminate the persistent leukemic clone in this patient with CML suggests treatment approaches worthy of future investigation.

Murine monoclonal anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays and immunohistologic staining.

To improve the sensitivity of conventional immunoassay methods using avidin-biotin-enzyme complex reagents, we have developed several murine monoclonal antibodies to the egg white avidin glycoprotein. These anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays by selectively enlarging the avidin-biotin-enzyme complex through bridging avidin to a second layer of avidin-biotin-enzyme complex, thereby increasing the signal from substrate conversion. Six hybridomas producing murine monoclonal antibodies which bind avidin-biotin-enzyme complexes were isolated and cloned. Two of these anti-avidin antibodies, WC19.10 and WC19.7, have been shown to produce a four-fold enhancement of signal obtained in avidin-biotin-enzyme ELISAs and qualitative enhancement of immunohistologic staining procedures.

Metastatic retinoblastoma successfully treated with immunomagnetic purged autologous bone marrow transplantation.

There are no documented cases of long-term, disease-free survival in retinoblastoma (RB) metastatic to the bone marrow. The following study details successful outcome in a 3-year-old child with extensive marrow replacement 7 months postenucleation in an otherwise untreated group V RB. Therapy consisted of combinations of vincristine, doxorubicin, and cyclophosphamide for 3 months. After demonstrating cross-reactivity by a panel of six monoclonal neuroblastoma (NB) antibodies with the patient's RB cells, her marrow was purged by using microsphere-linked monoclonal antibodies, and then reinfused as rescue therapy after ablative doses of etoposide and cyclophosphamide. The authors conclude that short-term induction therapy followed by marrow ablative combination chemotherapy and immunomagnetically purged autologous marrow rescue can (1) effect successful outcome in widely metastatic RB, and (2) eliminate the risk of therapy-induced second malignancies.

Clonal trisomy 8 is associated with myeloid phenotype rather than the neoplastic transformation in acute leukemia.

Our studies of an acute leukemia with a clonal t(1;11) marker have demonstrated a conversion from pre-B cell to myelomonocytic phenotype associated with the acquisition of trisomy 8. This finding suggests that trisomy 8, a frequent clonal abnormality in acute myeloid leukemias, may be associated with the myeloid phenotype rather than the neoplastic transformation. A permanent myelomonocytic cell line, designated UF-SK1, has been established from leukemic cells with a 47,XX,+8,t(1;11) (p31;q25) karyotype and shown to have myelomonocytic characteristics, including phorbol ester-induced differentiation to macrophages. This new cell line will be a valuable tool in the study of leukemogenesis and lineage commitment.