Louis A. Fernandez

Immunologic changes after blood transfusion in patients undergoing vascular surgery

Immunologic changes after blood transfusions cannot be studied ethically in normal individuals. We therefore studied two comparable groups of patients with atherosclerotic cardiovascular disease who received similar drug treatment and experienced a similar degree of surgical trauma, except that one group received an average of 2.5 units of packed red cells at one time period during surgery. We conducted immunologic tests preoperatively and 5, 10, 45 to 60, 90, 180, and 360 days postoperatively. There was no significant difference in all indices tested preoperatively between the two groups. Five and 10 days postoperatively, the absolute numbers of CD3, CD4, CD8, and B cells decreased in both groups; however, the decrease was significantly greater in the transfused group than in the nontransfused group 5 days postoperatively. There was no significant difference in these parameters between the two groups at other time periods tested. At 5 and 10 days postoperatively, the lymphocyte responses to phytohemagglutinin, concanavalin A, and allogeneic lymphocytes in autologous serum were decreased in both groups. However, at 60 days postoperatively, the responses of the nontransfused group became significantly increased, whereas those of the transfused group remained relatively unchanged. By days 90, 180, and 360, the lymphocyte responses of the nontransfused group had dropped to levels seen at earlier time intervals and were comparable to responses in the transfused group. There were no significant differences between the groups in the number of T-cell colonies formed, the number of immunoglobulin-producing cells obtained, and the lymphokine responses (migration inhibitory factor/migration stimulation factor) at all times tested. The major immunologic perturbations attributed to blood transfusions were an exaggerated decrease in the numbers of circulating lymphocytes, particularly those with markers associated with T-helper cells, and failure to demonstrate a rebound increase in the proliferative response 45 to 90 days later.

Decreased autologous mixed lymphocyte reaction with aging

The responses of T cells to autologous non-T mononuclear cells is called the autologous mixed lymphocyte reaction (AMLR). It seems to be an immunological response, as there is evidence of both immunologic specificity and memory. The AMLR is absent in systemic lupus erythematosis and chronic lymphocytic leukemia, and our data in normal human show that AMLR decreases with aging. Reactive T cells in the AMLR are subsequently cytotoxic to autologous B cells. We propose that the AMLR may be a protective phenomenon particularly against aberrant or neoplastic clones of B cells; and its decrease in the elderly may play an important permissive role for the development of either increased autoantibodies and/or B cell neoplasias.

Impaired T-cell responses in chronic lymphocytic leukemia: Lack of suppressor cell effect

Abstract T-Lymphocyte responses in B-cell chronic lymphocytic leukemia (CLL) are abnormal by several criteria. We investigated the possibility that impaired T-cell responses might be due to increased suppressor cell activity by: (a) measuring the responses to phytohemagglutinin (PHA) stimulation of normal mononuclear cells cocultured with CLL lymphocytes compared to normal cells alone, (b) preincubation of lymphocytes in tissue culture medium prior to the addition of PHA, (c) measuring the responses of lymphocytes to PHA in the presence of indomethacin. Cells from 8 patients with CLL cocultured with normal cells did not suppress responses to PHA. Both preincubation in tissue culture medium and addition of indomethacin to the cell cultures have been shown to block different suppressor cell activities. As expected, both of these procedures significantly increased the PHA responses of lymphocytes from 7 normal individuals in contrast to essentially no change in the responses of lymphocytes from 9 and 11 patients with CLL. Since T cells are diluted by the predominant B lymphocytes in CLL, the mononuclear cells were fractionated with enrichment of T lymphocytes to 75–85%. No increases in PHA responses of the enriched T cells were observed after preincubation in two cases or with the addition of indomethacin in four cases. We were therefore unable to demonstrate global suppressor cell activity in CLL affecting PHA responses, and there was evidence of less than normal activity in two specific suppressor cell systems. We suggest that decreased T-cell responses to PHA in CLL represent an innate cellular abnormality.

Multiplex Ligation-Dependent Probe Amplification Versus Multiprobe Fluorescence in Situ Hybridization To Detect Genomic Aberrations in Chronic Lymphocytic Leukemia: A Tertiary Center Experience

Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH, genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%), trisomy 12 (7%), ATM deletion (6%), 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes, the most common being 19p13 (LDLR and CDKN2D). Moreover, the cost for MLPA analysis, including technical time and reagents, is 86% less than FISH. In conclusion, cytogenetic abnormalities are a common finding in CLL patients, and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis.

Adhesion and spreading behaviour of human peripheral blood mononuclear cells (PBMC) in vitro.

Abstract While the in vitro adhesion and spreading behaviour of fibroblastic cells have been extensively documented, similar studies on the cells of immunological importance are still lacking. We report here the in vitro adhesion and spreading behaviour of normal human peripheral blood mononuclear cells (PBMC) isolated by the Ficoll-Hypaque technique. Since the presence of serum retarded cell adhesion by 25%, serum-free RPMI-1640 medium was used in these studies. Based on the light and scanning electron microscope studies, glass-immobilized PBMC were classified on the basis of their spreading behaviour as follows: (A) Cells with non-deformable nucleus: I, Cytoplasm non-spread; II, Cytoplasm partially spread; III, Cytoplasm well spread. (B) Cells with deformable nucleus: IV, Nucleus partially spread; cytoplasm well spread; V, Nucleus and cytoplasm well spread. These different cell types occur in the normal human peripheral blood in the relative frequency of 24 ± 3: 27 ± 4: 13 ± 2: 20 ± 5: 16 ± 2 percent. Cell types I–III show the characteristics of lymphocytes and the cells of type V were identified as monocytes, while the nature of cell type IV is not known at present. The immobilized PBMC contained T and B cells as confirmed by sheep erythrocyte (SE) and mouse erythrocyte (ME) rosette formation and surface immunoglobulin. The spreading behaviour of these cells appears to be limited by factors such as available surface membrane area, number and density of adhesion sites, other membrane receptors and the deformability of cell and nuclear membranes. Further characterization of these cell types would aid as a behavioural marker for the subpopulations of lymphocytes and monocytes in health and disease.

Production of Interferon by Peripheral Blood Mononuclear Cells from Normal Individuals and Patients with Chronic Lymphocytic Leukemia

Peripheral blood mononuclear cells (PBMC) from normal individuals were studied to identify which cells produce alpha-interferon (IFN-alpha) in response to a virus stimulus. It was found that cells both adherent and nonadherent to plastic formed IFN-alpha after induction by any one of several viruses studied. When nonadherent cells were separated on discontinuous Percoll gradients, only the cells in the less dense Percoll fractions produced IFN, whatever the virus used. By indirect immunofluorescence with monoclonal antibodies to HLA-DR and to Leu 11b, the distribution of the HLA-DR+ cells was shown to resemble most closely that of the IFN-producing population. Elimination of these cells (by complement-mediated lysis with the same antibodies) abrogated the IFN response, but NK cells remained and thus do not produce IFN-alpha. In confirmation, elimination of the Leu 11b+ cells had no effect on the amount of IFN produced. PBMC preparations from patients with chronic lymphocytic leukemia (CLL) appeared incapable of producing IFN-alpha but were shown to contain identifiable IFN-producing cells. The low or absent IFN levels in CLL are probably due to the relative scarcity of IFN-producing cells in their PBMC.

Tumor localization and therapeutic potential of an antitumor-anti-CD3 heteroconjugate antibody in human renal cell carcinoma xenograft models

A heteroconjugate (HC) antibody, constructed with the monoclonal antibody (MoAB) Dal K29 to human renal cell carcinoma (RCC) and an anti-CD3 MoAb, could induce a very high level of lysis of human RCC cells when incubated with human peripheral blood lymphocytes (PBL) in vitro (Kerr et al., 1990, J. Immunol., 144, 4060-4067). We now report that this HC antibody selectively localizes in RCC xenografts in nude mice and could inhibit RCC in an ascites tumor xenograft model when administered intraperitoneally together with PBL.

Migration Inhibition Produced by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride

Guinea pig peritoneal exudate cells were harvested 3 to 4 days after the intra‐peritoneal injection of Marcol oil. The washed cells were exposed to various concentrations of sodium periodate in phosphate‐buffered saline (PBS) at pH 7.4 for 10 min at +4°C. The cells were then used in the in vitro migration assay, and migration was consistently inhibited at concentrations from 10−3 to 10−5M. The viability of the macrophages was not affected by this treatment. Sodium borohydride (10−3 to 10−5M) in PBS for 10 min at pH 7.4 reversed the periodate effect. Experiments with purified macrophages showed that sodium periodate has a direct effect on macrophage function rather than an indirect effect via the potentiation of migration inhibition factor. In support of this, the in vitro spreading of macrophages on glass substrate foe 1 h has been shown to be inhibited. This spreading inhibition can also be reversed by treatment with sodium borohydride. These results provide a new approach to understanding the biological significance and role of macrophage migration inhibition.

Prolonged complete remission of myelodysplastic syndrome treated with danazol, retinoic acid and low-dose prednisone

Myelodysplastic syndrome (MDS) is a diverse group of clonal hematologic neoplasms. Different medications have been tried in MDS; however, no effective treatment has been yet established. We report a patient with MDS who achieved a complete remission in response to combination therapy of danazol, retinoic acid, and prednisone. A 53‐year‐old female presented with pancytopenia, macrocytosis, and hypercellular bone marrow with erythroid hyperplasia and dysplasia and 10% ringed sideroblasts. Cytogenetic studies revealed the presence of two abnormal clones. She was diagnosed as having MDS‐refractory anemia and was given blood transfusions to maintain blood cell counts at acceptable levels. At the same time, she was started on a combination of danazol (600 mg/day), retinoic acid (100 mg/day), and prednisone (10 mg every other day). Fourteen months later, the patient was in complete hematologic remission; she had normal peripheral blood count, and the blood smear showed normal morphology. Bone marrow studies revealed normal trilineage hematopoiesis. She was continued on the same combination treatment for 86 months, and she remained in complete clinical remission. Eighty‐eight months from diagnosis, she relapsed with acute myeloid leukemia. This is the first reported case of MDS‐RA that sustained a complete hematologic remission for a prolonged period in response to this combination treatment. This report indicates that restoration of normal hematopoiesis, prolongation of disease‐free survival, and delay in the transformation to acute leukemia may be achieved by this combination of treatment in a subset of patients with MDS, especially refractory anemia with severe thrombocytopenia. Am. J. Hematol. 64:306–310, 2000.

Normal T cell colony numbers in untreated patients with chronic lymphocytic leukaemia (CLL)

Summary. T cell colony formation in normal individuals and untreated patients with chronic lymphocytic leukaemia (CLL) was studied to determine if there is a deficiency in the number of T cells capable of forming colonies in this disease. In normal individuals, assays using enriched T cells did not reveal the total number of potential colony forming cells, preventing accurate assessment in patients with B cell CLL where enriched T cells are mandatory. Conditions were therefore optimized to obtain the maximal number of colonies, so that accurate comparisons could be made between the potential of normal individuals and patients with CLL. Addition of normal autologous non‐T cells to enriched T cells enhanced colony numbers in normal individuals to those seen in the whole mononuclear population. However, addition of normal non‐T cells to CLL T cells would have caused a mixed lymphocyte reaction (MLR), which influences T cell colony numbers. Therefore the MLR was bypassed by adding to the enriched T cells, supernatants having characteristics of Interleukin‐1 or commercially obtained Interleukin‐2. The supernatants with Interleukin‐1 activity enhanced T cell colony numbers comparably in both normal individuals and patients with CLL. Interleukin‐2 did not increase T cell colony numbers in normal individuals and the increase seen in patients with CLL were not significant. Thus the effect of the lymphokines on T cell colony numbers was comparable in both normal individuals and untreated patients with CLL. We therefore concluded that there are normal numbers of cells which have the potential to form T cell colonies in untreated patients with CLL.