Long-term establishment of a human plasmacyte cell line derived from a patient with IgD multiple myeloma. I. Requirement of a plasmacyte-stimulating factor for the proliferation of myeloma cells in tissue culture.

Cell line LA-49, derived from pleural fluid cells of a patient with IgD multiple myeloma, was established in culture and maintained for more than 1 yr. The D-myeloma protein produced in culture was similar to the serum D-myeloma protein in electrophoretic mobility and in delta- and lambda-chain antigens. The plasma cell tumor culture, LA-49, differed from numerous immunoglobulin-producing B-lymphoblastoid cell lines established in this laboratory in: (a) Morphology (revealing various stages of maturation); (b) type of immunoglobulin produced (IgD vs. IgM, IgG, and/or, rarely, IgA); (c) growth characteristics (requirement of plasmacyte-stimulating factor); and (d) chromosomal features (polyploid vs. pseudodiploid). A growth factor was needed for cell division and maintenance of culture viability. This factor was supplied readily by irradiated feeder layers of normal human fibroblasts or conditional media from fibroblast cultures. Preliminary characterization of this factor revealed it to be a protein with a mol wt of approximately 150,000 daltons.

2 types of lambda polypeptide chains in human immunoglobulins.

Two antigenic subtypes of human lambda polypeptide chains were distinguished by rabbit antiserum produced to a lambda Bence Jones protein. Lambda Bence Jones proteins and G myeloma proteins with lambda light chains were identified as being in one or the other subtype. The Oz (+) lambda chain subtype is present in light chains from pooled normal human immunoglobulin G and in whole normal immunoglobulin G molecules.

Limited periods of gene expression in immunoglobulin-synthesizing cells.

In synchronized human lymphoid cell lines, production of immunoglobulins G and M is greatest during the late Gl and S phases of the cell cycle. Little immunoglobulin appears immediately before, during, and immediately after mitosis. The results indicate that transcription of immunoglobulin genes takes place during a limited part of the mitotic cycle.

Immunoglobulin synthesis in vitro by established human cell lines.

Several lines of human lymphoid cells derived from malignant lym phoma produce immunoglobulins in vitro. Immunoglobulins G(I(g)G, (gamma)G) are synthesized in two cell lines and immuno-lobulin M (I(g)M, (gamma)M) in one.

Molecular heterogeneity of human lymphoid (HL-A) alloantigens.

Soluble preparations of HL-A alloantigens were separated by gel filtration into components having either the "LA" series or the "4" series of alloantigenic determinants. This separation may indicate that several different structural cistrons within the HL-A (locus) control the expression of these two series of determinants. The alternative possibility, in which one structural cistron with multiple mutational sites controls the synthesis of a single molecule cannot be excluded.

Continuing medical education on antiretroviral therapy in HIV/AIDS in India: needs assessment and impact on clinicians and allied health personnel.

BACKGROUND: Clinicians and associated health professionals charged with prescribing antiretroviral therapy (ART) deal with continuously evolving new drugs and combinations. To meet the needs of clinicians in India for ongoing education in this field, continuing medical education (CME) programmes on ART for HIV/AIDS were developed, conducted, evaluated and revised. Over a 2-year period, 2005-2007, 3 CME programmes for ART were conducted for physicians and a fourth (predominantly) for paediatricians. METHODS: Both 1- and 2-day CME programmes on various aspects of ART were held on weekends for professionals treating patients with AIDS in Delhi and adjacent states. Topics included characteristics of ART drugs, their dosages, monitoring and toxicity management, adherence, complications of therapy, dealing with treatment failure and HIV co-infections. These topics were addressed in lectures and group discussions and via case presentations. Programmes were evaluated by anonymous response to questionnaires, by a 1-year follow up of participants and by informal discussions with participants and faculty. Detailed analyses and a recommended format for these programmes are presented. RESULTS: The CMEs were attended primarily by clinicians (physicians and paediatricians). Nurses, laboratory scientists, and others involved in the treatment of AIDS also attended the programmes. An interactive workshop format was evolved with substantial time devoted to discussions and case analyses. One-day programmes such as the one included here can be comprehensive and effective. The educational needs of healthcare professionals who provide care and support to patients receiving ART were similar to those of the prescribing doctors. Because of new drugs being made available and with continued clinical experience, updated programme content was required each year. Participants preferred case-based interactive discussions rather than didactic lectures. Participants suggested that there should be more time for discussion after each talk. CONCLUSION: Annual CME programmes focused on ART are required to meet the professional needs of clinicians in India for providing quality care management to patients with AIDS.

Biosynthetic and structural studies of a heavy chain disease protein.

A new heavy chain disease protein ((gamma)HCD-JM) has been characterized by antigenic and structural criteria. The protein belongs to the IgG3-subclass and is closely related to Fc-fragment of G3-immunoglobulins. The predominant N-terminal amino acid of this protein is glutamic acid in the uncyclized form, and that of another (gamma)HCD is glycine. Studies of the N-terminal peptides indicate that the N-terminal portion of the (gamma)3-heavy polypeptide chain is absent from the (gamma)HCD-JM. These findings rule out a process of normal heavy chain initiation and a large deletion of the Fd region as being responsible for these two heavy chain disease proteins. The (gamma)HCD-JM is a secretory product of cells from bone marrow as shown by studies of in vitro incorporation of amino acids-(14)C. Bone marrow and lymph node have a population of lymphoplasmacytic cells which by immunofluorescence contain (gamma)-heavy chain antigens in the absence of light chain antigens.

Acute effects of orally administered levamisole on random monocyte motility and chemotaxis in man.

Cellular immune functions were studied in patients with early bladder cancer 2 hours after ingestion of either levamisole or a placebo. Random monocyte motility was significantly increased (P less than 0.025) in 13 of 17 patients receiving levamisole. Monocyte chemotaxis was significantly increased (P less than 0.025) in 16 of the 17 patients. Random monocyte motility and monocyte chemotaxis did not change in either 8 patients on the placebo or in 15 normal controls. Monocytes from normal donors showed increased random motility and chemotaxis after incubation with levamisole in vitro. These results indicated that increases in peripheral blood monocyte motility followed oral administration of levamisole. Kinetic studies indicated that these effects were rapid in onset and short lived.

Secondary in vitro lymphocyte-proliferative responses to syngeneic plasma cell tumors.

Two characteristics of immune responses to weakly immunogenic plasma cell tumors were demonstrated in this study. (a) Elevated lymphoproliferative responses following in vivo inoculation of sublethal doses of plasma cell tumors were detected by a mixed lymphocyte-tumor interaction (MLTI) assay. Specificity of elevated MLTI responses correlated with resistance to in vivo tumor challenge. These responses were dependent upon the presence of thymus-derived cells. (b) Spleen and lymph node cells showed markedly different patterns of MLTI activity following plasma cell tumor immunization. Spleen cell responses were depressed for the first 30 days postimmunization, whereas lymph node cells showed augmented MLTI reactivity immediately after immunization. These observations indicated that lymphocyte populations from different lymphoid organs may show widely dissimilar responses in vitro at the same point in the in vivo development of tumor resistance.

Immunological responsiveness in patients with bladder cancer.

Delayed cutaneous hypersensitivity tests, especially skin tests with dinitrochlorobenzene, are impaired increasingly as the amount of tumor increases. Recall antigens are less sensitive indicators of disease. Therapy, especially radiotherapy, also depresses cell-mediated immunity. Removal of tumor, however, allows these tests to return to normal. Dinitrochlorobenzene skin testing can contribute significantly to prognostic evaluation. An important facet of the tumor-host relationship is measured, and this reflects factors that are independent of tumor staging. Combination of tumor staging and dinitrochlorobenzene-delayed hypersensitivity testing can provide a strong indication of the clinical course, especially for the year following initial treatment of invasive or metastatic transitional cell carcinoma of the bladder.

Antigens shared by leukemic blast cell and lymphoblastoid cell lines detected by lymphocyte-dependent antibody.

Lymphocyte-dependent antibodies (LDA's) directed against antigenic determinants present on lymphoblastoid cell lines as well as human leukemia blast cells were demonstrated in heterologous antisera obtained by immunizing rabbits with a membrane fraction from RPMI-4265 (a lymphoblastoid cell line derived from a patient with chronic myelogenous leukemia). LDA was present at high titers against B-lymphoblastoid, myelomonocytic, and stem cell lines. The T-lymphoblastoid cell line MOLT-4, however, did not react. LDA was demonstrated against acute myelogenous as well as lymphoblastic leukemia cells. The reactivity was not directed against phytohemagglutinin-induced blastoid antigens, fetal antigens, or fetal calf serum. Absorptions with lymphoblastoid cell lines removed all LDA reactivity. Similar results were obtained by absorbing the rabbit antiserum with acute lymphoblastic and/or acute myelogeneous leukemia cells. These findings indicate the presence of cross-reactive antigens between lymphoblastoid cell lines and leukemia cells. Furthermore, cross-reactivity between acute lymphoblastic and acute myelogenous leukemia cells was demonstrated.

Critical evaluation of lymphocyte functions in urological cancer patients.

One hundred three patients with varying stages of urological cancer (bladder, prostate, kidney) were investigated with regard to the following lymphocyte functions. T-cells were assessed numerically (E rosettes), their blastogenic response to phytohemagglutinin (pHA) was determined, and their cytotoxic potential against heterologous target cells in short-term presence of PHA (e.i., PHA-dependent cellular cytotoxocity) was evaluated. Similarly, B cells were numerically assessed (EA rosettes), and their function was evaluated by antibody-dependent cellular cytotoxicity against antibody-coated deterologous target cells. The data on cancer patients, divided on the basis of extent of disease and prior radiation therapy, were compared to those of normal young and age-matched controls. Our investigations emphasize the importance of the following factors: (a) comparison of data with age-matched controls, since several lymphocyte functions appear to change with age; (b) use of multiple controls to compensate for the inherent variability found in certain tests; (c) minimized contamination by nonlymphoid cells in the purified cell preparation; and (d) the influence of certain treatment regimens (radiation, chemotherapy, etc.) on the results. Radiotherapy significantly depressed T-cell number with a depression of PHA blastogenic responses as well as PHA-dependent cellular cytotoxicity. When all of these conditions were taken into account, the urological cancer patients as a group were found to have a lower proportional value of E rosettes (T-cells) and a reduced PHA blastogenic responsiveness. Certain cancer patients displayed an elevated PHA-dependent cellular cytotoxicity as compared to age-matched controls, which may indicate the presence of activated cells in the presence of tumors. With this identification of a group of cancer patients with markedly depressed E rosette values and PHA responsiveness, it will now be possible to follow them clinically in comparison with a group of cancer patients with normal T-cell functions.

Depression of the generation of cell-mediated cytotoxicity by macrophage-like suppressor cells in bladder carcinoma patients.

Depressed T-lymphocyte function as assessed by delayed-type hypersensitivity reactions and in vitro proliferative response to mitogens is a characteristic finding in many types of solid tumors, including bladder carcinoma. Peripheral blood leukocytes from 16 patients with transitional cell carcinoma of the bladder were compared with age-matched, control subjects. Both the unfractionated leukocytes containing 10 to 30% monocytes and the lymphocyte-enriched preparations, obtained by monocyte depletion with iron filing ingestion, were analyzed. Mixed leukocyte culture-induced cytotoxicity was depressed in the patient group; the amount of depression was directly correlated to the extent of the disease. In patients who underwent surgical removal of tumor, the mixed leukocyte culture-induced cytotoxicity appeared normal. This mixed leukocyte culture-generated cytotoxic response was a more sensitive indicator of tumor effect than was the induced proliferative response. Removal of phagocytic or adherent monocytes from the responding cell population caused a significant increase in the generated cytotoxicity, especially in those patients with invasive disease. These suppressive effects could be partially reconstituted by quantitative addition of the separated monocytes back to the responding lymphocyte culture. The depressed lymphocyte-mediated cytotoxicity present in these bladder cancer patients was due, in a major part, to a circulating macrophage-like cell with active suppressor function.

Serum-free medium for the in vitro growth of normal and malignant urinary bladder epithelial cells.

A serum-free medium, DH-S1, is described which is valuable for the establishment of primary cultures of normal and malignant transitional epithelium (transitional cell carcinoma of the bladder). Growth of epithelial cells in DH-S1 is facilitated but that of fibroblasts is suppressed. Another established human transitional cell carcinoma cell line, 647V, has grown continually in DH-S1 for over 36 passages. Morphological and antigenic studies comparing 647V cells growing in serum-containing and serum-free medium reveal differences which are pronounced at low cell density but which almost disappear at higher densities. A new human transitional cell carcinoma cell line, LA-B1, is described whose initial growth was supported by this serum-free medium.

Isolation and partial characterization of plasma membranes bearing human fetal-associated antigens.

A rabbit antiserum to first-trimester human fetal tissue had greater reactivity in complement fixation and saturation binding assays with fetal tissues than with both a pool of normal adult lung, liver, and kidney and pools of the individual organs. This anti-fetal membrane reactivity was only partially inhibited by carcinoembryonic antigen. The serum still reacted strongly with human fetal and tumor cells after rendering it specific for plasma membrane components by adsorption to and elution from intact human fetal tissue culture cells. This plasma membrane-specific serum was then used to monitor the purification of the fetal membrane-associated antigens. The fetal antigens copurified with the putative plasma membrane enzymatic markers 5'-nucleotidase and Mg2+-adenosinetriphosphatase through differential and density gradient centrifugation. Insulin-binding activity only partially copurified with the antigenic activity. Little antigenic activity was found in nuclear and mitochondrial fractions. The isolation protocol gives fetal plasma membrane-associated antigens in approximately 50% yield with moderate purification. The sera and isolation procedures described should have general utility for the detection of human oncofetal antigens.

Leukemia derived growth factors produced by human malignant T-lymphoid cell lines.

An autocrine (noninterleukin 2) growth factor, which we term leukemia derived growth factor (LDGF), has previously been found in the culture supernatant of the human malignant T-lymphoid cell line MOLT-4f. We now show that two other human malignant T-lymphoid cell lines, CCRF-CEM and CCRF-HSB-2 also produce such a factor. All three factors, i.e., the LDGF from MOLT-4f, CCRF-CEM, and CCRF-HSB-2 are similar to each other both in biological activity and in physicochemical characteristics. In addition to their autocrine activity, these LDGFs stimulate the growth of other malignant T-lymphoid cell lines, but they do not stimulate B-lymphoblastoid or myeloid cell lines. The results therefore suggest that these LDGFs are T-cell specific.

Prediction of systemic fungal infection in allogeneic marrow recipients: impact of amphotericin prophylaxis in high-risk patients.

PURPOSE: To identify risk factors that might predict for systemic fungal infections in marrow transplant recipients within the first 100 days and to assess the efficacy of low-dose amphotericin B used as prophylaxis for candidemia and infection with invasive Aspergillus species in patients at risk. PATIENTS AND METHODS: A retrospective analysis of transplant outcomes for 331 allogeneic marrow recipients transplanted between 1983 and 1989 was performed to identify patients who might be at increased risk of fungal infection. Factors analyzed included disease, remission status, transplant regimen, graft-versus-host disease (GVHD) prophylaxis, duration of neutropenia, and development of GVHD. A trial of low-dose amphotericin (5 to 10 mg/d) begun on day +1 and continuing for 2 to 3 months posttransplant was begun in 1987 to evaluate its utility in reducing systemic mycoses. RESULTS: There were 18 episodes of candidemia and 18 systemic mycoses documented by blood or tissue culture or by biopsy. The initiation of high-dose (0.5 to 1 mg/kg/d) corticosteroids early as a component of GVHD prophylaxis in 1986 was identified as the most important risk factor for fungal infections, with a sixfold increase in infections as compared with the previous GVHD regimen (P < .0001); this was despite a significant decrease in the incidence of grade II to IV GVHD (7% v 43%; P = .0001). Low-dose amphotericin B initiated before the start of high-dose corticosteroid GVHD prophylaxis reduced the incidence of fungal infections from 30% to 9% (P = .01) without renal toxicity. Cyclosporine levels were lower in the patients who received amphotericin, leading to an increase in the rate of GVHD to 19% (P = .02). Controlling for GVHD prophylaxis, prolonged neutropenia (P = .00), and grade II to IV GVHD (P = .01) were also identified as risk factors for fungal infection. CONCLUSION: Amphotericin B can be used in low doses as prophylaxis for fungal infections early in the posttransplant course. However, cyclosporine doses need to be monitored to maintain target levels.

Allogeneic bone marrow transplantation as therapy for primary induction failure for patients with acute leukemia.

The survival of patients with acute leukemia who do not achieve a remission with primary therapy is very poor. High-dose chemoradiotherapy followed by allogeneic bone marrow transplantation (BMT) has been shown to be effective therapy for patients with acute and chronic leukemia. Therefore, we determined the long-term disease-free survival of patients who did not achieve a remission and were then treated with high-dose therapy and bone marrow allografting from matched sibling donors. Twenty-one patients (median age, 28 years) who did not achieve a remission with induction chemotherapy were subsequently treated with allogeneic BMT. After BMT, 90% achieved a complete remission. Six died of complications of the therapy, and six patients relapsed between 27 and 448 days after BMT. Nine patients (43%; median age, 25 years) are alive between 556 and 4,174 days after BMT. The cumulative probability of disease-free survival at 10 years is 43%. This study suggests that allogeneic BMT can be an effective therapy to achieve long-term control of acute leukemia, even in those patients who do not achieve a remission with primary therapy.

Bone marrow transplantation for hematologic malignancies in patients aged 30 years or older.

During the past 10 years, 86 patients 30 to 54 years of age with hematologic malignancies were prepared with high-dose radiochemotherapy and received histocompatible bone marrow grafts. Thirty-four of these patients are surviving for 4 months to 9 years (median, 26 months) following marrow transplantation and 32 of them are in continuing complete remission (CR). Disease-free survival is 44% for 37 patients who were in first remission of acute leukemia or in the chronic phase of chronic granulocytic leukemia (CGL), 23% for 39 patients whose leukemia had relapsed at least once before transplantation or who had advanced stages of CGL, and 60% for ten patients who had hematologic malignancies other than leukemia. The median age of the surviving 34 patients is 36 years (range, 30 to 43 years). The incidence of moderate to severe acute graft-v-host disease (GVHD) was 48% and of chronic GVHD, 26%. The major causes of failure were interstitial pneumonia in 31 patients (24 of whom had antecedent acute GVHD) and recurrent leukemia in 12 patients (11 of whom had either never entered a CR or had relapsed at least once with acute leukemia or had progressive CGL before transplantation). Our data warrant further prospective studies in patients with hematologic malignancies who are older than 30 years.

Bone marrow transplantation for myelodysplastic and myeloproliferative syndromes.

Twenty patients (age range, 4 to 48 years; median age, 36 years) with de novo or drug-induced myelodysplastic syndromes or myeloproliferative disorders were treated with myeloablative immunosuppressive therapy followed by bone marrow transplantation (BMT). Four preparative regimens were used; three regimens consisted of combined total body irradiation (TBI) and chemotherapy and one of combination chemotherapy only. One patient received marrow from his identical twin brother, whereas the other 19 patients were grafted with marrow from histocompatible siblings. In 19 patients the abnormal clone was at least temporarily ablated, while in one patient the congenital myelodysplasia persisted. Eight patients are alive and well for +108 to +3,359 days post-transplantation. Nine patients died of transplant-related complications (six of interstitial pneumonia, two of gastrointestinal bleeding, and one of fungal sepsis) and three patients died with persisting or recurring disease. One patient with a late recurrence has undergone a second successful bone marrow transplant procedure. Outcome of BMT was not related to French-American-British (FAB) type, marrow fibrosis, cytogenetic abnormalities, or preparation regimen. Marrow transplantation as a means of providing long-term disease-free survival and possible cure should be considered in patients if a suitable donor is available.