Analysis of the dynamic mutation in the SCA7 gene shows marked parental effects on CAG repeat transmission

The gene for spinocerebellar ataxia 7 (SCA7) includes a transcribed, translated CAG tract that is expanded in SCA7 patients. We have determined expansions in 73 individuals from 17 SCA7 kindreds and compared them with repeat lengths of 180 unaffected individuals. Subjects with abnormal expansions comprise 59 clinically affected individuals and 14 at-risk currently unaffected individuals predicted to carry the mutation by haplotype analysis. For expanded alleles, CAG repeat length correlates with disease progression and severity and correlates inversely with age of onset. Increased repeat lengths are seen in generational transmission of the disease allele, consistent with the pattern of clinical anticipation seen in these kindreds. Repeat lengths in expanded alleles show somatic mosaicism in leukocyte DNA, suggesting that these alleles are unstable within individuals as well as between generations. Although dynamic repeat expansions from paternal transmissions are greater than those from maternal transmissions, maternal transmission of disease is more common, suggesting germline or embryonic effects of the repeat expansion.      

Human neutrophil dysfunction with giant granules and defective activation of the respiratory burst

We describe a patient whose peripheral blood neutrophils and bone marrow precursors (beyond promyelocytes) contained multiple large azurophilic granules. There were also giant granules in eosinophils, basophils, melanocytes, renal tubules, thyroid, and neurones, but not lymphocytes or monocytes. His clinical course included recurrent (ultimately fatal) infections and severe neurologic impairment. Immunofluorescent staining with fluoroscein- and rhodamine-conjugated antisera to primary and secondary granule markers showed virtually all of the granulocyte granules and rare monocyte granules to be fusion products containing both markers. Electron microscopy showed the granules to be large peroxidase-containing lysosomes. Only rare normal primary and secondary granules were present. Superoxide generation in response to opsonized zymosan was 7.3 nmole/min/10(6) cells (control 8.9); but in response to phorbol myristate acetate, only 2.2 (control 9.4). Nitroblue tetrazolium slides showed 3+ dye reduction in response to opsonized zymosan by 90% of granulocytes (control 91%) and to phorbol myristate acetate by 22% (control 99%), with 71% producing only a minimal 1+ response. Cellular contents of myeloperoxidase and beta- glucuronidase were elevated, but the percent release during exocytic degranulation was equivalent to control. Ingestion of complement- opsonized Staphylococcus aureus and zymosan was also normal. Killing of Staphylococcus aureus was 60% at 90-min incubation (control 92%). Granulocyte cyclic adenosine monophosphate (AMP) content was 4 pmole/10(7) cells (control 3.1). In order to determine whether these characteristics derived from the cells' genetic program or their environment, the patient's bone marrow was grown in long-term culture. Granulocytes produced in vitro demonstrated the same morphology, same defect in activation of nitroblue tetrazolium reduction, and same normal cyclic AMP level as those harvested from peripheral blood. These studies describe a new disorder of granulocytes; the structural similarity to, but biochemical differences from, Chediak-Higashi disease indicate the probable heterogeneity of mechanisms for the same morphological abnormality.     

Dependency on intercellular adhesion molecule recognition and local interleukin-2 provision in generation of an in vivo CD8+ T-cell immune response to murine myeloid leukemia

The immune response to a murine myeloid leukemia (cell line C1498) was studied in vitro and in vivo. Natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (CTL) were shown to lyse C1498 in vitro through the binding of leukocyte function antigen-1 (LFA-1) on effectors and intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on C1498 target cells. However, the ability of nonimmunized mice to resist an in vivo challenge of a low dose (10(4)) of C1498 was NK-cell, but not T-cell dependent. The failure of T cells to participate in the immune surveillance of a low leukemia burden appeared, in part, because of a lack of expansion of leukemia reactive CTL precursors (CTLp). Leukemia reactive CTLp frequency estimations in naive and leukemia bearing mice were not significantly different (range, 1:20,600 to 1:74,000) in contrast to immunized mice (range, 1:1,400 to 1:4,400). Leukemia reactive CTLp could be expanded to a level that could apparently mediate in vivo immune surveillance of 10(5) leukemia cells by injection of irradiated leukemia cells intraperitoneally (IP) or subcutaneously (SC), but not intravenously (IV). However, IV injection of 10(5) live leukemia cells engineered to secrete interleukin-2 (IL-2) resulted in systemic immunity mediated primarily by CD8+ T cells. We conclude that NK cells can mediate immune surveillance of a low leukemia burden. CD8+ CTL-mediated immune surveillance can eliminate a higher leukemia burden than NK cells, but requires T-cell help, which can be delivered by local IL-2. Both NK and CTL-mediated immune surveillance of C1498 murine myeloid leukemia is dependent on recognition through the LFA-1:ICAM adhesion pathway.     

Inflammatory and Metabolic Dysregulation and the 2-Year Course of Depressive Disorders in Antidepressant Users

Scarce evidence suggests that inflammatory and metabolic dysregulation predicts poor response to antidepressants, which could result in worse depression outcome. This study prospectively examined whether inflammatory and metabolic dysregulation predicted the 2-year course of depressive disorders among antidepressant users. Data were from the Netherlands Study of Depression and Anxiety, including 315 persons (18–65 years) with a current depressive disorder (major depressive disorder, dysthymia) at baseline according to the DSM-IV criteria and using antidepressants. Inflammatory (C-reactive protein, interleukin-6 (IL-6), tumor-necrosis factor-α) and metabolic (waist circumference, triglycerides, high-density lipoprotein (HDL) cholesterol, blood pressure, fasting glucose) factors were measured at baseline. Primary outcome for course of depression was indicated by whether or not a DSM-IV depressive disorder diagnosis was still/again present at 2-year follow-up, indicating chronicity of depression. Elevated IL-6, low HDL cholesterol, hypertriglyceridemia, and hyperglycemia were associated with chronicity of depression in antidepressant users. Persons showing ⩾4 inflammatory or metabolic dysregulations had a 1.90 increased odds of depression chronicity (95% CI=1.12–3.23). Among persons who recently (ie, at most 3 months) started antidepressant medication (N=103), having ⩾4 dysregulations was associated with a 6.85 increased odds of depression chronicity (95% CI=1.95–24.06). In conclusion, inflammatory and metabolic dysregulations were found to predict a more chronic course of depressive disorders among patients using antidepressants. This could suggest that inflammatory and metabolic dysregulation worsens depression course owing to reduced antidepressant treatment response and that alternative intervention treatments may be needed for depressed persons with inflammatory and metabolic dysregulation.     

Adherent cell inhibition of human leukemic in vitro agar clonal growth by short-term cell-to-cell contact

The effects of peripheral blood adherent cells from normal donors on human myeloid leukemic cluster growth in agar were studied. A prior co- incubation of nonadherent leukemic cells with adherent cell monolayers from 9 out of 10 donors in liquid cultures over a 4-hr period was sufficient to reduce subsequent leukemic growth in semisolid agar cultures. Inhibition was seen with adherent to leukemic cell ratios of as low as 0.5:1. Conversely, identical numbers of adherent cells in agar cultures but separated from the leukemic cells enhanced growth more than the cultures containing human placental conditioned media alone. Because leukemic cell exposure to adherent cells was brief, a cytotoxic mechanism appeared likely; however, this could not be detected by 51Cr release. Human peripheral blood adherent cells not activated by any in vitro mechanism suppress clonal growth of human myeloid leukemic cells by a mechanism requiring cell to cell contact. Examination of the inhibition of clonal growth appears to be more sensitive than 51Cr release as an indicator of adherent cell effects on myeloid leukemia.     

Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.     

Interleukin-8-stimulated polyphosphoinositide hydrolysis in human peripheral blood lymphocytes

We have attempted to provide evidence for the production of inositol phosphate (IP) metabolites as an indication of specific receptor- mediated signal transduction in human peripheral blood lymphocytes (PBL) in response to interleukin-8 (IL-8). IP metabolites were measured, after loading of PBL with [3H]-D-myo-inositol, by anion exchange high-performance liquid chromatography (HPLC) and liquid scintillation counting of collected fractions. In addition, inositol- 1,4,5-trisphosphate (IP3), in extracts from unlabeled cells, was measured using a specific radioligand binding assay. Compared with phytohemagglutinin (PHA), which stimulated an increase in IP metabolites and, specifically, IP3 by greater than threefold, human recombinant (hr) IL-8 (1 nmol/L) also stimulated an increase in IP metabolites, as measured by HPLC, and a greater than threefold increase in IP3. The increase in IP3 was observed as early as 15 seconds after stimulation with hrIL-8, reaching maximal levels by 30 seconds. To further assess the signal transduction mechanism involved, the protein tyrosine kinase inhibitor genistein was added to the cells 10 minutes before stimulation with hrIL-8. After preincubation of PBL with this inhibitor, the generation of IP3 in response to PHA (5 micrograms/mL) and hrIL-8 (1 nmol/L) was inhibited by 42% and 51% of control values, respectively. In contrast to the production of IP metabolites, there were only small increases in intracellular calcium in response to hrIL- 8 when compared with PHA.     

Premature chromosome condensation studies in human leukemia: 5. Prediction of early relapse

Previous reports have suggested that the technique of premature chromosome condensation (PCC) is useful for predicting relapse in patients with acute leukemia. However, these studies involved patients had been in complete remission (CR) for various periods of time and had heterogeneous expectations for relapse. The purpose of this study was to further determine the value of PCC in predicting relapse by examining the PCC characteristics of bone marrow specimens from patients with acute leukemia on a common therapeutic regimen after similar periods in CR. The remission durations after the PCC determinations were compared between patients with high or low proliferative potential indices (PPI, or the fraction of G1 cells in late G1 phase). Of 60 patients studied between two and eight weeks after achieving CR, 14 of the 16 patients exhibiting high PPI values (greater than or equal to 35) have relapsed. The mean time from PCC measurement to relapse was 23 weeks. In contrast, only 19 of the 44 patients exhibiting low PPI values have relapsed, with an estimated mean time to relapse of 68+ weeks. Likewise, of 38 patients studied between nine and 15 weeks of CR, nine of the ten patients exhibiting high PPI values have relapsed (mean time to relapse, 23 weeks), while only 16 of 28 patients with low PPI values have relapsed (estimated mean time to relapse, 54+ weeks). The predictive value of the PCC technique was found to be independent of other prognostic factors for the duration of CR, and it identified those patients within the poor prognostic category with a high likelihood of imminent relapse. While similar trends were observed at later time intervals in CR, the differences in relapse rate between patients with high or low PPI values is not significant. These results confirm the usefulness of the PCC technique in predicting relapse in acute leukemia and could aid in the identification of patients who might benefit by an alteration of therapeutic strategy.     

Identification of risk groups for development of central nervous system leukemia in adults with acute lymphocytic leukemia

The risk of development of CNS leukemia was investigated in 153 adults with acute lymphocytic leukemia (ALL) who received systemic combination chemotherapy without CNS prophylaxis. Overall, 31 patients (20%) developed CNS leukemia after a median of 6 months of therapy; the estimated 1-year incidence of CNS leukemia was 21% (SE, 3.9%). Characteristics significantly associated with CNS involvement included the presence of elevated hemoglobin creatinine, alkaline phosphatase, fibrinogen, and lactic dehydrogenase levels; B-cell leukemia; and high leukemic cell proliferative activity. Multivariate analysis identified lactic dehydrogenase levels of greater than or equal to 600 U/L and greater than or equal to 14% of cells in the S + G2M compartment to have independent additive poor prognostic significance. Patients were categorized into different risk groups for CNS leukemia with 1-year incidences ranging from 4% to 55%. While related to a high occurrence of CNS leukemia at diagnosis (33%) and subsequently (100%), the low incidence of B-cell disease excluded it from the multivariate analysis. The use of systemic chemotherapy containing multiple agents with good CNS penetration and in high doses (VAD regimen) in 90 patients was associated with a trend for lower CNS leukemia at 1 year (15% v 31%), especially in the low-risk category. We propose to develop future therapies for adults with ALL that include risk-oriented CNS prophylactic approaches.     

Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti- CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.