Successful percutaneous coronary intervention in a patient with combined deficiency of FV and FVIII due to novel compound heterozygous mutations in LMAN1.

Percutaneous coronary intervention (PCI) in patients with congenital coagulation factor deficiencies presents a unique challenge. They are not only at increased risk of perioperative bleeding but can also suffer thrombosis of the stent as preventive anticoagulation and antiplatelet therapy is difficult. Several cases of successful PCI have been described in patients with haemophilia A and B, but there are no reports in patients with combined coagulation factor deficiencies. We used PCI to treat the coronary artery disease in a patient with the combined deficiency of factor V and factor VIII (F5F8D) and analysed the molecular basis of the disorder for this patient. A 68-year-old patient was admitted for urgent PCI with bare metal stent placement after the diagnosis of the F5F8D. Peripheral blood DNA was extracted for the sequence analysis of LMAN1 and MCFD2 genes. Mutations in LMAN1 was confirmed by molecular cloning of the PCR product and resequencing of the resulting clones. The patient underwent successful PCI with good long-term outcome. Our patient tolerated anticoagulation therapy well, with unfractionated heparin, and double antiplatelet therapy while he was initially supported with fresh frozen plasma and recombinant FVIII. Molecular analysis revealed that the patient carries unusual compound heterozygous frameshift mutations on the same microsatellite repeat region in exon 8 of LMAN1, one of which is a novel mutation (c.912delA). Our results suggest that patients with F5F8D can safely undergo PCI for coronary artery disease, with the treatment individualized to the specific patient.

Antilymphoma activity of human gamma delta T-cells in mice with severe combined immune deficiency.

Human Burkitt lymphoma (Daudi) cells grow as disseminated tumors in mice with severe combined immune deficiency (SCID) after either i.v. or i.p. injection. These cells are lysed in vitro by human V gamma 9/V delta 2 T-cells that recognize the groEL homologue on the Daudi cell surface. We report that both Daudi cell-stimulated peripheral blood mononuclear cells (Daudi-PBMC) containing 41-95% of V gamma 9/V delta 2 T-cells and V gamma 9/V delta 2 T-cell clones prolong the survival of SCID mice given inoculations of a lethal dose of Daudi cells. Groups of 6-8-week-old SCID mice were given inoculations i.v. or i.p. of 10(5) Daudi cells followed (through different injection sites) by: (a) 10(7) Daudi-PBMC; or (b) 10(7) unstimulated PBMC; or (c) 0.9% saline solution. All animals in groups (b) and (c) died of disseminated lymphoma, and their survival was significantly shorter than that of mice in group (a) (P < 0.001 for both i.v. and i.p. routes). Significant antitumor effects were also detected when Daudi-PBMC were injected 4 days before or 4 days after Daudi cells (P < 0.05). In vivo depletion of murine natural killer cells by anti-asialo GM-1 rabbit antiserum did not affect survival, suggesting that these cells did not contribute to lymphoma killing. Daudi-PBMC did not exert in vivo antitumor activity against the control Raji lymphoma. Mice receiving i.p. injections of Daudi cells followed by cytotoxic V gamma 9/V delta 2 T-cell clones also survived significantly longer (P < 0.05 for 3 different clones) than animals given Daudi cells alone or Daudi cells followed by noncytotoxic gamma delta T-cell clones. Our results indicate that this model system can be used for studies of human antilymphoma T-cell responses in vivo.

Mechanism of suppression of normal hemopoietic activity by lymphokine-activated killer cells and their products.

Interleukin 2 (IL-2)-activated lymphocytes (lymphokine-activated killer [LAK] cells) have been shown to inhibit the formation of autologous human granulocyte-macrophage hemopoietic progenitors (granulocyte-macrophage colony-forming units, CFU-GM) in vitro. Effects of LAK cells on these progenitors may include a number of different mechanisms. LAK cells are potent cytotoxic lymphocytes capable of lysing certain normal autologous cells. They also produce cytokines known to inhibit hemopoiesis (interferon gamma [IFN-gamma] and tumor necrosis factor alpha [TNF-alpha]) or enhance it (granulocyte-macrophage colony-stimulating factor, GM-CSF). In our current study we analyzed the mechanism of suppression of autologous CFU-GM by LAK cells. Our results suggest that LAK cells are not directly cytotoxic to normal CFU-GM. We show that it is possible to abolish the hemopoiesis-inhibiting activity of LAK cells without abrogating their cytotoxicity against tumor cell lines using inhibitors of DNA synthesis, namely hydroxyurea or irradiation.

Prospects for interleukin-2 therapy in hematologic malignant neoplasms.

Interleukin-2 (IL-2) is a regulator of diverse functions of the immune system that can induce regressions in some experimental and human tumors. These early findings suggest a potential role for IL-2 in the treatment of certain malignant neoplasms including lymphomas and leukemias. Advanced, rapidly growing tumors are generally not amenable to immunotherapy. Therefore, it is more likely that protocols with IL-2 will be used to prolong remission and prevent relapse in leukemia patients with minimal tumor load. Several approaches are currently being tested in animal experiments and clinical trials. Activation of tumor-reactive lymphocytes (specific or nonspecific) by IL-2 in vivo may eradicate residual leukemia in patients with occult disease. In vitro-propagated autologous or allogeneic leukemia-reactive T cells may be infused with IL-2 to facilitate the tumor destruction. The IL-2 enhances monoclonal antibody-dependent effector systems, such as antibody-dependent cell-mediated cytotoxicity in vivo. Monoclonal antibodies recognizing epitopes on leukemia/lymphoma cells could therefore be used with IL-2 to target nonspecific effectors to destroy tumor cells. Other cytokines appear to potentiate various antitumor activities of IL-2, including cytotoxicity of antigen-specific T lymphocytes or lymphokine-activated killer cells in vitro, and these combined effects may be exploited in clinical trials in which more than one cytokine is used simultaneously or in sequence. Finally, a stepwise completion of clinical protocols testing this immunologic approach in combination with other treatment modalities is necessary.(ABSTRACT TRUNCATED AT 250 WORDS)

Concurrent Pneumocystis carinii and cytomegalovirus pneumonia after autologous peripheral blood stem cell transplantation.

A 46-year-old woman developed concurrent CMV and Pneumocystis carinii pneumonia (PCP) 140 days after autologous peripheral blood stem cell transplantation (APBSCT) for AML. She was seropositive for CMV before undergoing APBSCT and had required prednisone for immune thrombocytopenia and allergic dermatitis for 9 weeks prior to the onset of pneumonia. She had also been receiving PCP prophylaxis with pentamidine aerosol every month for 3 months before developing symptoms. The pneumonia was complicated by severe hypoxia, requiring ventilator support and pneumothorax requiring chest tube thoracostomy. She recovered following treatment with trimethoprim-sulfamethoxazole (TMP-SMX), prednisone, gancyclovir and intravenous immunoglobulin. Although the overall incidence of severe CMV disease is low after APBSCT, preventive measures such as surveillance culture and secondary prophylaxis with gancyclovir may be warranted in patients whose cellular immune response is further compromised by corticosteroid use or other factors.

In vivo effects of multiple cycles of recombinant interleukin-2 (IL2) on peripheral granulocyte-macrophage hematopoietic progenitors circulating in the blood of cancer patients.

The numbers of peripheral blood (PB) granulocyte-macrophage colony forming units (CFU-GM) were evaluated in five patients treated with multiple weekly cycles of recombinant interleukin-2 (IL2). A 4.5-12 fold increase in the number of CFU-GM was evident within 8 days after the beginning of the treatment. The maximal increase in the absolute numbers of CFU-GM/ml blood caused by the IL2 treatment, ranged from 14 to 57 times the baseline values and was reached after two or three cycles of IL2. IL2-activated PBMC, added in vitro to the PBMC of a normal donor did not modify the number of CFU-GM present in the donor PBMC. CFU-GM were also recovered from frozen samples of in vivo IL2-activated PBMC.

Human T cells in hu-PBL-SCID mice proliferate in response to Daudi lymphoma and confer anti-tumour immunity.

In vitro culture of human peripheral blood lymphocytes (PBL) with Daudi (Burkitt lymphoma) cells results in selective proliferation of V gamma 9/V delta 2 T cells with high cytotoxicity against Daudi cells. After adoptive transfer into severe combined immunodeficient (SCID) mice, these cells exert specific anti-tumour activity against Daudi lymphoma. To test whether cytotoxic V gamma 9/V delta 2 T cells are induced in SCID mice, human PBL injected intraperitoneally were stimulated with irradiated Daudi cells (PBL/Daudi-SCID). After 7-14 days, PBL/Daudi-SCID had a significantly higher percentage of human gamma delta T cells in their peritoneal cavity, lymph nodes and blood than controls (PBL-SCID). DNA content analysis of T cell subsets from PBL/Daudi-SCID showed a significantly higher percentage of cells in S + G2 + M phases of the cell cycle in the TCR-gamma delta-1+ than in CD3+ cell population. Human cells recovered from PBL/Daudi-SCID showed specific cytotoxicity against Daudi cells. PBL/Daudi-SCID inoculated with a lethal dose of Daudi lymphoma survived significantly longer than controls. This protection was specific for Daudi cells and was not mediated by murine natural killer (NK) cells. Thus human peripheral blood T cells grafted in SCID mice proliferate in response to antigen and confer specific immunity.

Direct effect of interleukin 2 on chronic lymphocytic leukaemia B cell functions and morphology.

The functional and morphological changes induced by recombinant interleukin 2 (IL-2) were studied in purified B cells from patients with untreated B chronic lymphocytic leukaemia (B-CLL). In eight of nine patients, purified B-CLL cells increased their DNA synthesis in response to IL-2 without preactivation in vitro. This response, studied in detail in three patients, was dose dependent and reached a maximum on day 5 or 6. IL-2 induced or increased IgM secretion in cultures from five of the nine patients studied. Two of this responsive group were particularly interesting as IL-2 not only stimulated IgM secretion but also induced the secretion of IgG. Immunoglobulin production was invariably monoclonal. B CLL cells incubated with IL-2 showed distinct morphological changes including an increase in the size of cytoplasm and enlargement of nuclei together with the appearance of nucleoli. These changes were present in all IL-2 treated cultures but were more pronounced in those containing immunoglobulin secreting cells. None of the IL-2 induced changes appeared to correlate with the clinical stage of the disease or the level of Tac antigen expression on the freshly isolated CLL B cells.

Impaired mitogen responses of the non-leukaemic B cells from patients with chronic lymphocytic leukaemia.

We investigated the immunological mechanism of the low level of circulating immunoglobulin and depressed primary and secondary responses of patients with chronic lymphocytic leukaemia (B-CLL) using purified non-leukaemic B cells in vitro. To assess the function of the non-leukaemic B cells we separated them from the much larger leukaemic population, which expresses the pan-T cell marker CD5, by immunoabsorption using anti-CD5 antibodies and Dynabeads. Immunoglobulin production was measured after the cells had been cultured with the B cell mitogens, pokeweed mitogen (PWM) and Staphylococcus aureus Cowan strain 1 (SAC). Autologous T cells that were found to function normally in our systems were added to cultures containing PWM. Non-leukaemic B cells from 15 B-CLL patients produced 539 ng/ml, immunoglobulin (mean value) with SAC and 162 ng/ml with PWM compared with 14,182 and 5513 ng/ml, respectively, from B cells from normal, age-matched control patients. Most of the immunoglobulin produced in the non-leukaemic B cell cultures carried the light chain associated with the leukaemic clone. We conclude that even at early stages in the disease (12 patients were Rai stage 0 patients) when the total serum immunoglobulin levels are still near normal, the B cells respond poorly to B cell mitogens.

BCR-ABL and v-abl oncogenes induce distinct patterns of thymic lymphoma involving different lymphocyte subsets.

The human BCR-ABL oncogenes encoded by the Philadelphia chromosome (Ph) affect the pathogenesis of diverse types of leukemia and yet are rarely associated with T-lymphoid leukemia. To determine whether BCR-ABL kinases are inefficient in transforming T lymphocytes, BCR-ABL-expressing retroviruses were injected intrathymically into mice. Thymomas that expressed BCR-ABL kinase developed after a relatively long latent period. In most thymomas, deletion of 3' proviral sequences resulted in loss of tk-neo and occasionally caused expression of kinase-active carboxy-terminally truncated BCR-ABL oncoprotein. In contrast, deletion of 3' proviral sequences was not observed in thymomas induced with Abelson murine leukemia virus (A-MuLV). BCR-ABL viruses induced distinct patterns of disease and involved different thymocyte subsets than A-MuLV and Moloney murine leukemia virus (Mo-MuLV). While Mo-MuLV only induced Thy-1+ thymomas, v-abl- and BCR-ABL-induced thymomas often contained mixed populations of B220+ and Thy-1+ lymphocytes in the same tumor. In most v-abl and BCR-ABL tumors, Thy-1+ lymphoid cells expressed CD8 and a continuum of CD4 ranging from negative to positive. Conversely, Mo-MuLV thymomas contained distinct populations of CD4+ cells that were either CD8+ or CD8-. A-MuLV-transformed T-lymphoid cells did not express the CD3/T-cell receptor complex, while BCR-ABL tumors were CD3+. Thus, BCR-ABL viruses preferentially induce somewhat more differentiated T lymphocytes than are transformed by A-MuLV. Furthermore, rare B220+ lymphocytes may represent preferred v-abl and BCR-ABL transformation targets in the thymus.

Transient pancytopenia associated with parvovirus infection in paroxysmal nocturnal haemoglobinuria.

A 25 year old woman with a 15-year history of paroxysmal nocturnal haemoglobinuria developed transient pancytopenia following infection with human parvovirus B19. This is the first report of transient pancytopenia in a patient with an acquired haemolytic anaemia due to parvovirus. The possible mechanism of pancytopenia in such a case is discussed.

Increased gamma-delta T-lymphocyte response to Mycobacterium bovis BCG in major histocompatibility complex class I-deficient mice.

Mice with a homologous deletion of the beta 2-microglobulin gene (beta 2m-) are deficient in class I major histocompatibility complex molecules (MHC) and consequently are deficient in CD8+ T cells. These beta 2m- mutant mice control the intraperitoneal growth of an avirulent vaccine strain of mycobacteria, Mycobacterium bovis BCG, after intraperitoneal infection similarly to normal mice. We show that beta 2m- mice have an increased gamma-delta (gamma delta) T-cell response after infection with live avirulent mycobacteria. beta 2m- mice have an earlier and more sustained rise in the proportion of intraperitoneal gamma delta T cells, averaging 17% of T cells, compared with 6% in normal mice, at 28 days after infection. Compared with the population in normal mice, gamma delta T cells in the spleens of beta 2m- mice averaged a higher proportion of the total T-cell population of the spleen on days 5, 8, and 14 after intraperitoneal infection. These data document the kinetics of gamma delta T cells reactive to mycobacterial antigens in vivo without class I MHC restriction and support a role for class I MHC and CD8+ T cells in the in vivo regulation of gamma delta T cells.

In vitro preactivated human T cells engraft in SCID mice and migrate to murine lymphoid tissues.

Mice with severe combined immunodeficiency (SCID) accept grafts of human T and B lymphocytes derived from resting peripheral blood mononuclear cells (PBMC). We wished to determine whether activated human T cells engraft and migrate into lymphoid tissues in SCID mice. PBMC (50 x 10(6)) activated in vitro in a 4-day mixed lymphocyte culture (MLC) were injected into the peritoneum of 12 SCID mice. In 11 of 12 animals killed at 3 or 4 weeks after injection, human cells were detected in cells pooled from lymphoid organs by flow cytometry and by immunohistochemical staining of frozen tissue sections. The percentage of CD45+ cells in the 11 mice ranged from 2% to 45% and the absolute numbers of CD45+ cells recovered from lymphoid organs ranged from 4 x 10(6) to 90 x 10(6). Up to 93% of the human cells expressed the CD3 antigen together with either CD4 or CD8. Human T cells were localized in periarteriolar areas in murine spleens, whereas in the lymph nodes and gut mucosa, the T cells did not show the pattern for T-dependent areas found in human lymphoid tissue. Numerous human plasma cells were detected in the spleen and gut mucosal crypts of engrafted SCID mice. Human IgG was detected in the serum of all 11 engrafted SCID mice. The functional activity of human T cells recovered from murine splenic tissue was very low 3-4 weeks after engraftment.

Lymphoma in a patient with rheumatoid arthritis receiving methotrexate treatment: successful treatment with rituximab.

A 55 year old man with chronic lymphocytic leukaemia (CLL) and rheumatoid arthritis (RA), treated for four years with methotrexate (MTX), who developed a B cell non-Hodgkin's lymphoma (B-NHL), is described. The tumour was localised to the shoulder and axillary lymph nodes, and positive for Epstein-Barr viral antigens. After failure of radiation and chemotherapy, a complete remission was achieved with a combination of antibody treatment (rituximab) and EPOCH. The development of a second malignancy in a patient with RA receiving MTX has not been described before. The summation of T cell deficiencies induced by MTX, CLL, and RA may all have contributed to the development of the B-NHL.

In vivo binding and antitumor activity of Ch14.18.

The melanoma reactive chimeric 14.18 (ch14.18) antibody can mediate enhanced in vitro lysis of human M-21 melanoma cells. This study analyzes the antitumor effects and the in vivo binding of ch14.18 antibody with M-21 melanoma cells in severe combined immunodeficiency (SCID) mice. Outgrowth of tumors was prevented in 6/6 animals by the simultaneous subcutaneous injection of peripheral blood mononuclear cells (PBMC) [3 x 10(6) cells (2 animals); 10 x 10(6) cells (2 animals); and 30 x 10(6) cells (2 animals)], with 0.5 mg ch14.18, 1,500 U interleukin 2 (IL-2), and 10(6) M-21 cells. In contrast, 7 of 7 control mice that received M-21 cells alone, 7 of 7 mice that received M-21 cells and ch14.18, and 5 of 6 mice that received M-21 cells plus PBMC plus IL-2, grew subcutaneous tumors. The in vivo localization of ch14.18 was then evaluated in an intraperitoneal (i.p.) tumor model, where 0.3 cm melanoma nodules develop within 3 weeks after the i.p. administration of M-21 cells. Flow cytometric and immunohistochemical analysis revealed the GD2 antigen present throughout the tumor nodule. Intraperitoneal administration of 0.01, 0.1, or 1.0 mg of ch14.18 to SCID mice previously engrafted i.p. with M-21 cells resulted in detectable ch14.18 binding to tumor cells in vivo within 10 hours of antibody administration. Ch14.18 penetration was limited to approximately 20 cell layers, demonstrating that ch14.18 has limited access to some cells in large tumor nodules. This study demonstrates that the addition of ch14.18 to IL-2 and human effector cells can result in significant antitumor activity by preventing the establishment of tumor nodules. These results suggest that clinical testing of IL-2 plus ch14.18 might be most effective if used in the setting of microscopic residual disease. Therapies that enhance ch14.18 penetration into tumor nodules should be evaluated with ch14.18 for patients with advanced melanoma.

Lymphocyte sensitization detected by the macrophage electrophoretic mobility assay in patients with renal cell carcinoma: Theophylline increases the sensitivity of the assay.

An antigen-induced release of a macrophage slowing factor (MSF) by peripheral blood lymphocytes was used to evaluate lymphocyte sensitization to various antigens in 30 patients with renal cell carcinoma (RCC) and in 14 normal individuals. Twenty-three of 30 (77%) patients with RCC, but no healthy controls were found to be sensitized to a soluble antigen prepared from an allogeneic kidney tumor by 3 M potassium chloride extraction. Peripheral blood lymphocytes from some patients with RCC displayed sensitization to protein isolated from fetal kidney (6 of 24; 25%), control "normal" kidney (6 of 30; 20%) and urinary bladder carcinoma (3 of 21; 14%) tissues. It has been suggested that cyclic adenosine 3' ,5'-monophosphate (cyclic AMP) could play a role in the mechanism of the MSF action (24). In agreement with this idea, the presence of 10-4 M theophylline enhanced the macrophage electrophoretic mobility (MEM) reduction caused by MSF. Furthermore, the DNA synthesis in lymphocytes (monitored by measuring the uptake of tritiated thymidine) on contact with phytohemagglutinin (PHA) was depressed in 9 of 21 (43%) patients with RCC as compared with healthy controls.

Generation of lymphokine-activated killer cells does not require DNA synthesis.

We studied the role of DNA synthesis in the induction of lymphokine-activated killer (LAK) cells by recombinant interleukin-2 (IL-2) and the dependence of this phenomenon on DNA synthesis. Doses of gamma-irradiation (1000-5000 rads) that profoundly reduced DNA synthesis in human peripheral blood mononuclear leucocytes (PBL) also effectively suppressed the development of cytotoxic activity in the absence of IL-2. However, the same doses of irradiation affected the induction of LAK activity by IL-2 to a much lesser extent. Blocking the formation of deoxyribonucleotides by hydroxyurea, which resulted in a complete inhibition of DNA synthesis in PBL or purified T lymphocytes, had virtually no effect on the generation of LAK cells. These results indicate that the expression of LAK activity is not dependent on DNA synthesis.