Lactoferrin gene promoter: structural integrity and nonexpression in HL60 cells.

Lactoferrin is a member of the transferrin family of iron-binding proteins. It is found in several glandular epithelial tissues and human neutrophils, where it is localized to secondary granules. To examine the mechanisms controlling lactoferrin gene expression in neutrophils and defects in its expression in acute leukemia, we have cloned a lactoferrin cDNA from a chronic myelogenous leukemia library, and used it to obtain genomic clones representing the chromosomal lactoferrin gene. Using polymerase chain reaction, primer extension, and S1 analysis, we have identified the 5' end of the lactoferrin mRNA. We have defined a putative promoter region for the gene, and characterized its first two exons. In addition, we have examined the structure of these regions in DNA from HL60 cells. HL60 is a leukemic cell line that undergoes phenotypic neutrophil maturation on exposure to dimethyl sulfoxide (DMSO). However, the cells cannot be induced to express any secondary granule protein genes. We have shown that the 5' end of the lactoferrin gene, including the putative promoter region, is entirely normal in HL60. By Northern analysis, nuclear run-on studies, and primer extension assays we have shown that the gene is not transcribed in DMSO-induced HL60 cells. This supports the hypothesis that the defect in HL60 is an abnormality in the production or activity of a transacting regulator of lactoferrin gene expression.     

Plasminogen activator inhibitor (PAI-1) antigen levels in primary TTP and secondary TTP post-bone marrow transplantation

Our objectives were to measure and compare plasminogen activator inhibitor levels (PAI-1) in primary adult thrombotic thrombocytopenic purpura (TTP) and in secondary TTP associated with bone marrow transplantation (BMT)-TTP. PAI-1 antigen levels were measured by an enzyme linked immunosorbent assay on platelet poor plasma samples obtained from patients at the time of diagnosis of the TTP disorder and from a group of normal volunteers. The samples were frozen at –70°C. Patients with TTP secondary to bone marrow transplantation had their grade determined by percentage fragmented cells and lactate dehydrogenase levels. The primary TTP samples were contributed by investigators in the multi-institutional North American TTP Group, and the bone marrow transplant samples were obtained from an adult bone marrow transplant program. Nineteen patients with adult TTP, and 47 patients with bone marrow transplant-TTP were evaluated. Of the latter, 14 had Grade 2, 13 had Grade 3, and 20 had Grade 4 BMT-TTP. PAI-1 levels were elevated compared to control volunteers in both primary adult TTP and BMT-TTP, P < 0.001. Levels did not differ from normal in Grade 2 BMT-TTP (median = 16 ng/ml; quartiles = 9–20). PAI-1 levels were similar in primary TTP (median = 32 ng/ml; quartiles = 25–51) and Grade 3 BMT-TTP (median = 35 ng/ml; quartiles = 19–48 ng/ml), P = 0.7. However, PAI-1 levels were significantly higher in Grade 4 BMT-TTP (median = 83 ng/ml; quartiles = 60–143) than Grade 3 BMT-TTP, and primary TTP, P < 0.001. PAI-1 levels are high in primary TTP and secondary bone marrow transplant-TTP (Grades 3–4). In contrast, normal levels are seen in Grade 2 BMT-TTP, which is a self-limited disorder. Therefore, high PAI-1 levels may contribute to hypofibrinolysis in the pathogenesis of primary TTP and of moderate to severe TTP (Grades 3–4) following bone marrow transplantation. Am. J. Hematol. 59:9–14, 1998. © 1998 Wiley-Liss, Inc.     

Hypotetraploidy in a patient with small cell carcinoma

While numerical and structural chromosomal abnormalities characterize many hematopoietic and nonhematopoietic malignancies, the occurrence of polyploidy is by and large rare. We report here an interesting patient with small cell carcinoma (SCC) and hypotetraploidy initially referred to us because of a question of acute nonlymphocytic leukemia, M3 subtype, with a question of a 15;17 translocation characteristic of acute promyelocytic leukemia. However, the patient did not have a 15;17 translocation and the final hematopathologic analysis of the bone marrow aspirates and immunohistochemistry studies subsequently revealed the patient to have SCC. Small cell carcinoma is a highly malignant and a very aggressive neoplasm. A review of the literature, using Medline, Cancerlit, and the Science Citation Index, revealed that in most, if not all, reports, the presence of polyploidy is noted as a rare entity. In leukemia, reports of polyploidy point to a distinct category of patients with a poor risk for which more intensive treatment is needed. Limited information is currently available to assess the risk of polyploidy in small cell carcinoma. Our case is important not only because of the relative rarity of polyploidy, but also because insights gained from the study of this and other similar patients may help shed additional light on the mechanism of carcinogenesis, which is not fully known to date. As polyploidization is a manifestation of genetic instability and as genetic instability has been implicated in the genesis and progression of many cancers, it is perhaps not too surprising that polyploidy in our case was associated with a poor disease outcome. The patient has since expired.     

Primary Cutaneous Natural Killer Cell Blastic Lymphoma

An 81 yo male presented with several asymptomatic firm 1–5 cm red purple plaques on the trunk and lower extremities associated with a mild pancytopenia. Histological examination revealed a diffuse, monotonous dermal infiltrate of atypical medium sized cells with fine chromatin and scanty cytoplasm. Immunoperoxidase staining demonstrated positivity for CD 45, CD43, CD4, Bcl‐2, and TdT; but was negative for cytokeratin, melan‐A, CD30 and hematopoietic lineage specific markers. A subsequent bone marrow aspirate demonstrated a dense population of cells that were morphologically consistent with blasts. Immunophenotyping by flow cytometry revealed lesional cells that expressed CD56, CD4, CD7, CD5, HLA‐DR and TdT. However, lineage specific markers for B‐cells (CD19, CD20, cCD79a, and CD10), T‐cells (sCD3 and cCD3), and myeloid cells (CD13, CD33, CD117, cCD13, CD14, CD41, CD61, myeloperoxidase, and alpha napthyl butyrate esterase) were not expressed. Molecular studies by PCR exhibited no evidence of T‐cell receptor or heavy chain gene rearrangements. Collectively, these findings are consistent with a primary cutaneous blastic natural killer cell lymphoma. Blastic natural killer cell lymphomas are characterized by a high incidence of cutaneous involvement and an aggressive clinical course. Our patient responded dramatically to one cycle of CHOP chemotherapy with resolution of his cutaneous tumors.     

Management of persistent B19 parvovirus infection in AIDS

Summary An HIV⁺ 26-year-old white man with a CD4 count of 006 × 10⁹/1 was found to have red blood cell aplasia secondary to B19 parvovirus infection. Regular infusions of intravenous immunoglobulin (IVIG) were begun and resulted in marked reticulocytosis and correction of anaemia. The patient has been followed for over 4 years and has become anaemic and reticulocytopenic whenever IVIG was interrupted. Serial dot blot analysis of the patient's sera for B19 parvovirus DNA showed absence of DNA immediately following IVIG treatments but reappearance within 3–6 weeks. Regular IVIG was effective in controlling but not eradicating B19 parvovirus infection in this HIV⁺ patient.     

Cryoprecipitate poor plasma does not improve early response in primary adult thrombotic thrombocytopenic purpura (TTP)

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease that is treated with plasma exchange and typically with replacement with fresh frozen plasma (FFP). This approach results in an approximate 50% response rate following 1 week of therapy and 80% survival. Cryoprecipitate poor plasma (CPP) is plasma from which the cryoprecipitate fraction is removed. CPP has been reported to be successful as salvage therapy in refractory TTP and has been suggested to be superior to FFP in retrospective studies. The present report compares initial therapy of TTP with exchange using replacement with either FFP or CPP in a multi-institutional prospective randomized study performed by the North American TTP Group (NATG Group) from 1993 to 1995. Initial therapy also included corticosteroids. Antiplatelet drugs or vinca alkaloids were not employed. A severity score index, response score, and individual clinical parameters (platelet count, LDH x upper limit of normal, hemoglobin level, and creatinine) were compared at their nadir or peak values, baseline, and days +6 and +13 of therapy. Thirteen patients were randomized to FFP exchange and 14 to CPP exchange. Results were equivalent for all parameters. Survival was equal with three deaths in each group. These data indicate that the efficacy of FFP and CPP are the same in the initial treatment of TTP in adults.     

Plasma von Willebrand factor antigen (vWF:AG) and thrombomodulin (TM) levels in adult thrombotic thrombocytopenic purpura/hemolytic uremic syndromes (TTP/HUS) and bone marrow transplant-associated thrombotic microangiopathy (BMT-TM)

Endothelial damage is thought to be a contributing factor in the pathogenesis of Thrombotic Thrombocytopenic Purpura/Hemolytic Uremic Syndromes (TTP/HUS). The present studies measured two markers of endothelial cell stimulation and/or activation [von Willebrand Factor (vWF:Ag) and thrombomodulin (TM)] in patients with TTP/HUS disorders and compared them to controls. The patient groups consisted of adults with TTP/HUS, with (n = 13) and without (n = 14) peak Cr levels >2.0 mg/dl. Additionally, 52 patients with Bone Marrow Transplant-associated Thrombotic Microangiopathy (BMT-TM) following allogeneic BMT were evaluated. Both vWF:Ag and TM were elevated in all patient groups compared to controls. TTP/HUS patients with peak Cr >2.0 mg/dl had higher TM levels (P <0.001) than did those with peak Cr levels below 2 mg/dl. However, thrombomodulin/creatinine (TM/Cr) ratios did not differ in these two groups nor did they differ from controls. BMT-TM pts had higher vWF:Ag levels and higher TM/Cr ratios than controls and TTP/HUS, P < 0.001. The median TM/Cr ratio in BMT-TM was 91 (range = 34–229) compared to 38 (range = 29–50) in controls, P < 0.001 and 38 (range = 6 to 156) in TTP/HUS, P < 0.001. Additionally both TM (P < 0.001) and TM/Cr (P < 0.02) were higher in patients with Grades 3 and 4 BMT-TM compared to those with Grade 2 BMT-TM. These results suggest that endothelial cell activation occurs in TTP/HUS and BMT-TM. Since TM/Cr ratios were higher in BMT-TM compared to TTP/HUS, these findings suggest that the mechanism of elevated TM in BMT-TM cannot be explained solely by altered renal excretion. Taken together, these findings strongly indicate a role of endothelial cell damage in BMT-TM. © 1996 Wiley-Liss, Inc.