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Spectrin, a heterodimer of alpha and beta subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid beta-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2, chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the alpha/beta subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid beta-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.

Angiostatin, a product of the proteolytic cleavage of plasminogen, possesses potent antitumor and antiangiogenic properties in vivo. Studies with cultured endothelial cells suggest that under certain conditions, angiostatin inhibits the migration and proliferation of these cells or, alternatively, increases their rate of apoptosis. In general, the effects of angiostatin have been considerably less potent in vitro than in vivo. One potential explanation for this disparity is that the in vivo target of angiostatin is not the mature endothelial cell. Recently, evidence has accumulated to show that circulating endothelial progenitor cells (EPCs) contribute to neovascularization. In this study, we have isolated EPCs from human subjects and demonstrated that, in contrast to that of mature endothelial cells, the growth of EPCs is exquisitely sensitive to angiostatin. These results suggest that angiostatin and related compounds may exert their biological effects by inhibiting the contribution of EPCs to angiogenesis and not by altering the growth of mature endothelial cells.

X-linked severe combined immunodeficiency (XSCID) is a life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in the IL2RG gene, which encodes the common cytokine receptor gamma chain, gamma(c). Previously, we generated gamma(c)-deficient mice as a murine model of XSCID. We have now used lethally irradiated gamma(c)-deficient mice to evaluate a gene therapeutic approach for treatment of this disease. Transfer of the human gamma(c) gene to repopulating hematopoietic stem cells using an ecotropic retrovirus resulted in an increase in T cells, B cells, natural killer (NK) cells, and intestinal intraepithelial lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and of serum Ig levels. In addition, the restored cells could proliferate in response to interleukin-2 (IL-2). Thus, our results provide added support that gene therapy is a feasible therapeutic strategy for XSCID. Moreover, because we used a vector directing expression of human gamma(c) to correct a defect in gamma(c)-deficient mice, these data also indicate that human gamma(c) can cooperate with the distinctive cytokine receptor chains such as IL-2Rbeta and IL-7Ralpha to mediate responses to murine cytokines in vivo.

The hemoglobin-deficit mouse mutant (hbd) is characterized by a severe microcytic anemia that is inherited in an autosomal-recessive manner. Previous results from our laboratory indicated that normal mice develop anemia if they are transplanted with bone marrow from mutant animals. Furthermore, we demonstrated a delay in erythroid reconstitution from hbd marrow compared to normal marrow. Although these experiments show a defect that is intrinsic to hbd marrow, it is unclear if the hbd phenotype is solely the result of a bone marrow-derived defect. To exclude an environmental influence on hbd anemia, we attempted to cure the defect by transplanting normal marrow into the hbd mouse. We observed that the transplanted animals converted to a normal phenotype. These results indicated that the defect is bone marrow derived. In contrast to the microcytosis mutant whose defective gene is ubiquitously expressed, our data suggest that the defective gene product is specific to hematopoietic cells.

The mouse mutant hemoglobin deficit (gene symbol hbd) is characterized by a severe microcytic anemia that is inherited in an autosomal-recessive manner. To assess the mutation's effect on hematopoiesis, unfractionated bone marrow (BM) from either a mutant C57BL6/J-hbd/hbd, Gpi1b/Gpi1b (phenotype symbol HBD), or normal C57BL6/J-+hbd/+hbd, Gpi1b/Gpi1b mouse was injected intravenously into irradiated congenic C57BL6/J-+hbd/+hbd, Gpi1a/Gpi1a, lgh(a)/lgh(a), Thy1a/Thy1a mice. The congenic recipients of mutant or normal marrow obtained complete red blood cell (RBC) and leukocyte reconstitution, with the exception of one recipient of HBD marrow. After 24 weeks posttransplantation, the normal recipients of HBD marrow obtained a microcytic anemia similar to the donor. These results suggest that the HBD phenotype is caused by a BM defect. We observed that the erythroid lineage derived from donor HBD marrow repopulated more slowly than the normal marrow at 4 weeks post-transplantation. To determine if this difference was a result of an erythropoietic defect, competitive repopulation was performed using either mutant or normal marrow competed against normal congenic marrow. For the erythroid lineage, no significant contribution from HBD marrow was observed. To assess if the RBC block was based on a deficiency of myeloid progenitors, both in vitro and in vivo assays were performed: absolute numbers of bone progenitors were increased, suggesting that the defect results in a late block to erythroid differentiation.

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroid beta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains and we suggest the position of several functional domains including f-actin, synapsin I, ankyrin and spectrin self association sites. Analysis of deduced amino acid sequences indicated striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging.

Chronic lymphocytic leukemia is recognized as having a variable prognosis, but its staging has depended exclusively on anatomical sites of involvement and the presence or absence of anemia and thrombocytopenia. The recent availability of techniques permitting cytogenetic analysis of malignant B lymphocytes led us to examine the karyotypic abnormalities in chronic lymphocytic leukemia and to correlate them with clinical stage, progression of disease, and survival. Of 53 patients with metaphases adequate for study who were followed for a minimum of one year, 21 (40 per cent) had abnormal karyotypes, of which trisomy 12 was the most frequent (25 per cent). Abnormal karyotypes were found to be significant correlates of advanced clinical stage (P less than 0.005) and of shortened survival (P less than 0.05). We conclude that cytogenetic analysis provides useful clinical and prognostic information in patients with chronic lymphocytic leukemia.

In the present study, there was a complete lack of autologous MLR between responding T cells or T subsets and unirradiated or irradiated leukemic B cells or monocytes in all 20 patients with CLL, regardless of disease status, stage, phenotype, or karyotype of the disease. The stimulating capacity of unirradiated CLL B cells and CLL monocytes or irradiated CLL B cells was significantly depressed as compared to that of respective normal B cells and monocytes in allogeneic MLR. The responding capacity of CLL T cells was also variably lower than that of normal T cells against unirradiated or irradiated normal allogeneic B cells and monocytes. The depressed allogeneic MLR between CLL B cells or CLL monocytes and normal T cells described in the present study could be explained on the basis of a defect in the stimulating antigens of leukemic B cells or monocytes. The decreased allogeneic MLR of CLL T cells might simply be explained by a defect in the responsiveness of T lymphocytes from patients with CLL. However, these speculations do not adequately explain the complete lack of autologous MLR in these patients. When irradiated CLL B cells or irradiated CLL T cells were cocultured with normal T cells and irradiated normal B cells, it was found that there was no suppressor cell activity of CLL B cells or CLL T cells on normal autologous MLR. Our data suggest that the absence or dysfunction of autoreactive T cells within the Tnon-gamma subset account for the lack of autologous MLR in patients with CLL. The possible significance of the autologous MLR, its relationship to in vivo immunoregulatory mechanisms, and the possible role of breakdown of autoimmunoregulation in the oncogenic process of certain lymphoproliferative and autoimmune diseases in man are discussed.

The chromosomes of unstimulated and stimulated blood lymphocytes from 5 cases with B-cell prolymphocytic leukemia (PLL) were examined following the use of polyclonal B-cell activators (PBA). Banding techniques revealed a common and specific chromosome abnormality to be present in each of the cases, which was due to a translocation between chromosomes 6 and 12 (t(6;12)(q15;p13]. The fact that this specific chromosome change has not been reported in other lymphoproliferative disorders may indicate that PLL is a distinct clinical entity and different from other lymphoproliferative disorders, whether it occurs de novo or complicates chronic lymphocytic leukemia (CLL).

The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.

Bone marrow biopsies were prospectively performed on 75 patients with chronic lymphocytic leukemia (CLL). There was a highly significant relationship (p less than 0.002) between clinical stages and bone marrow infiltration patterns. Ten (50%) of 20 patients with diffuse patterns died; the estimated median survival time for these patients was 87 months. In contrast, only six (11%) of 55 patients with nondiffuse patterns died; the estimated median survival time for these patients could not be computed. When both clinical stage and infiltration pattern were evaluated for survival, a highly significant association between clinical stage and survival time was still observed (p less than 0.003) whereas bone marrow infiltration pattern was no longer significant. A significant association was also observed between bone marrow infiltration patterns and absolute lymphocyte counts (p less than 0.0005), Fc-receptor-positive cells (p less than 0.002), 3H-thymidine uptake of leukocytes (p less than 0.01), serum alkaline phosphatase levels (p less than 0.05), monoclonal urinary-free light chain status (p less than 0.05), and cytogenetics of leukemic cells (p less than 0.05). These observations lead to the conclusion that in an overall prognostic evaluation of patients with CLL, although bone marrow histopathology may have no additional value over a well-established clinical staging system, as a whole, it may be of clinically predictive value in disease progression of patients with stage I and II.

From 1951 through 1978, we have seen 20 cases of stage O chronic lymphocytic leukemia (CLL) without disease progression for 6.5–24 years. The cohort included 7 males and 13 females, aged 48–77 years at the time of diagnosis. None presented with anemia, thrombocytopenia, or neutropenia nor developed cytopenias during follow-up. Mean total lymphocyte count in these patients was 20,100/microL, with ranges from 10,000 to 43,700 at the time of diagnosis, and was 20,600, with ranges from 1,000 to 47,200, at last follow-up. Of 12 patients studied, 8 and 4 were phenotyped as heavy chain mu delta- and mu-type, respectively, with 7 kappa- and 4 gamma-type (no light chain was detectable in one patient). Of 13 patients studied, one had a slightly elevated IgG level and two had slightly depressed serum IgA and IgM levels. All patients had positive delayed hypersensitivity responses to at least one of five skin test antigens. Each of seven patients studied for an in vitro leukocyte thymidine uptake had a low level of [3H]thymidine incorporation. Nine of 12 patients studied had elevated total T cells, and the remaining 3 had normal T cell counts. In vitro unseparated lymphocyte response to phytohemagglutinin showed normal kinetics of DNA synthesis, with a peak response on day 3 or 4 of culture in 4 and slightly or moderately depressed and/or delayed kinetics in 8 patients studied. Cytogenetic analyses by Q- or G-banding techniques of polyclonal B cell mitogen-stimulated lymphocytes in all six patients studied showed normal karyotypes. These data are consistent with a previously undescribed syndrome involving a monoclonal B cell lymphocytosis, a prolonged asymptomatic or benign clinical course, and essentially normal humoral and cellular immunity and normal karyotype. Our observations indicate that these 20 patients with stage O CLL have a benign clinical course and that they may also be designated as benign monoclonal B cell lymphocytosis ( BMBL ), a benign variant of CLL.

Cytogenetic analyses by G-banding and/or Q-banding techniques of polyclonal B cell mitogen-stimulated peripheral blood lymphocytes in 77 patients with chronic lymphocytic leukemia were carried out in the present study. Adequate metaphases were obtained in 65 patients (84%). Of 29 patients with abnormal karyotypes, ten (34%) had trisomy 12 as the sole abnormality, eight (28%) had trisomy 12 in combination with other karyotypic changes, and the remaining 11 had various karyotypic changes other than trisomy 12. There was a significant relationship between the abnormal karyotype and disease status, clinical stage, lymphocyte count, bone marrow infiltration pattern, monoclonal IgM gammopathy, and urinary monoclonal-free light chain status. Six of seven patients (87%) with trisomy 12 only had stage 0-11 disease, whereas all eight patients with trisomy 12 with other changes had stage III or IV disease (P less than .02). However, of nine patients with other karyotypic changes without trisomy 12, five had stage 0-II and four had stage III or IV disease. These observations suggest that trisomy 12 may be the primary or the earliest karyotypic change in a majority of aneuploid patients with chronic lymphocytic leukemia, and that other karyotypic changes in addition to trisomy 12 may develop as a result of clonal evolution, dedifferentiation, or therapy. Of nine patients in whom autopsy studies were carried out, four were found to have diffuse histiocytic lymphoma or Richter's syndrome (three with trisomy 12 in combination with other chromosome changes and one with normal karyotype). Our findings clearly demonstrate that cytogenetic study may be of value in the clinical and prognostic evaluation of patients with chronic lymphocytic leukemia.

From 1951 through 1978, we have seen 20 cases of stage O chronic lymphocytic leukemia (CLL) without disease progression for 6.5-24 years. The cohort included 7 males and 13 females, aged 48-77 years at the time of diagnosis. None presented with anemia, thrombocytopenia, or neutropenia nor developed cytopenias during follow-up. Mean total lymphocyte count in these patients was 20,100/microL, with ranges from 10,000 to 43,700 at the time of diagnosis, and was 20,600, with ranges from 1,000 to 47,200, at last follow-up. Of 12 patients studied, 8 and 4 were phenotyped as heavy chain mu delta- and mu-type, respectively, with 7 kappa- and 4 gamma-type (no light chain was detectable in one patient). Of 13 patients studied, one had a slightly elevated IgG level and two had slightly depressed serum IgA and IgM levels. All patients had positive delayed hypersensitivity responses to at least one of five skin test antigens. Each of seven patients studied for an in vitro leukocyte thymidine uptake had a low level of [3H]thymidine incorporation. Nine of 12 patients studied had elevated total T cells, and the remaining 3 had normal T cell counts. In vitro unseparated lymphocyte response to phytohemagglutinin showed normal kinetics of DNA synthesis, with a peak response on day 3 or 4 of culture in 4 and slightly or moderately depressed and/or delayed kinetics in 8 patients studied. Cytogenetic analyses by Q- or G-banding techniques of polyclonal B cell mitogen-stimulated lymphocytes in all six patients studied showed normal karyotypes. These data are consistent with a previously undescribed syndrome involving a monoclonal B cell lymphocytosis, a prolonged asymptomatic or benign clinical course, and essentially normal humoral and cellular immunity and normal karyotype. Our observations indicate that these 20 patients with stage O CLL have a benign clinical course and that they may also be designated as benign monoclonal B cell lymphocytosis ( BMBL ), a benign variant of CLL.

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The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.

In the present study, there was a complete lack of autologous MLR between responding T cells or T subsets and unirradiated or irradiated leukemic B cells or monocytes in all 20 patients with CLL, regardless of disease status, stage, phenotype, or karyotype of the disease. The stimulating capacity of unirradiated CLL B cells and CLL monocytes or irradiated CLL B cells was significantly depressed as compared to that of respective normal B cells and monocytes in allogeneic MLR. The responding capacity of CLL T cells was also variably lower than that of normal T cells against unirradiated or irradiated normal allogeneic B cells and monocytes. The depressed allogeneic MLR between CLL B cells or CLL monocytes and normal T cells described in the present study could be explained on the basis of a defect in the stimulating antigens of leukemic B cells or monocytes. The decreased allogeneic MLR of CLL T cells might simply be explained by a defect in the responsiveness of T lymphocytes from patients with CLL. However, these speculations do not adequately explain the complete lack of autologous MLR in these patients. When irradiated CLL B cells or irradiated CLL T cells were cocultured with normal T cells and irradiated normal B cells, it was found that there was no suppressor cell activity of CLL B cells or CLL T cells on normal autologous MLR. Our data suggest that the absence or dysfunction of autoreactive T cells within the Tnon-gamma subset account for the lack of autologous MLR in patients with CLL. The possible significance of the autologous MLR, its relationship to in vivo immunoregulatory mechanisms, and the possible role of breakdown of autoimmunoregulation in the oncogenic process of certain lymphoproliferative and autoimmune diseases in man are discussed.

It has been shown that the nitrosoureas are substrates for hepatic microsomal enzymes in vitro and that phenobarbital (PB) administered in multiple doses prior to nitrosourea administration significantly reduces the activity of the nitrosoureas in murine brain tumor models. In the present study, the effect of PB on 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was assessed by determining the CCNU dose which would result in the long-term survival of 50% of the treated mice, and the CCNU dose which would result in the toxic death of 50% of the treated mice, with or without PB pretreatment in C56BL/6J mice. The therapeutic index, the CCNU dose which would result in the long-term survival of 50% of the treated mice, per the CCNU dose which would result in the toxic death of 50% of the treated mice, without PB pretreatment was 2.1; the therapeutic index of CCNU after PB pretreatment was 1.7. There is no significant difference between the therapeutic indices. Thus, the reduction in the tumoricidal activity of CCNU after PB pretreatment was restored by increasing the dose of CCNU without a significant change in its lethal toxicity.l

The chromosomes of unstimulated and stimulated blood lymphocytes from 5 cases with B-cell prolymphocytic leukemia (PLL) were examined following the use of polyclonal B-cell activators (PBA). Banding techniques revealed a common and specific chromosome abnormality to be present in each of the cases, which was due to a translocation between chromosomes 6 and 12 (t(6;12)(q15;p13]. The fact that this specific chromosome change has not been reported in other lymphoproliferative disorders may indicate that PLL is a distinct clinical entity and different from other lymphoproliferative disorders, whether it occurs de novo or complicates chronic lymphocytic leukemia (CLL).