A comparison of prestorage WBC-reduced whole-blood-derived platelets and bedside-filtered whole-blood-derived platelets in autologous progenitor cell transplant.

BACKGROUND: Prestorage WBC-reduced platelet concentrates (PCs) can be manufactured from platelet-rich plasma (PRP) by in-line filtration of PRP. There are few published data on the clinical use of these products, as compared to bedside-filtered pools of standard PCs (S-PCs) manufactured from PRP. STUDY DESIGN AND METHODS: A prospective, randomized trial was conducted in autologous progenitor cell transplant patients requiring platelet transfusions with each patient as his or her own control who was given a pool of 5 units of WBC-reduced PCs and a pool of 6 units of S-PCs within a 3-hour period. The pools were characterized before transfusion for platelet and WBC content, P-selectin expression, and IL-8. The patients were monitored with platelet counts and vital signs and observed for reactions. Data were analyzed using Mann-Whitney U tests. RESULTS: Thirty-three transfusions were administered to 13 patients. Median platelet content in the WBC-reduced PC pools was lower than that in the S-PC pools (3.3 vs. 4.0 x 10(11), p<0.01). Median WBC content was 4 to 5 log less in the WBC-reduced PC pools (2.5 x 10(4) vs. 4.6 x 10(8), p<0.01). Median IL-8 levels (pg/mL) were lower in the WBC-reduced PC pools (2 vs. 36, p<0.01). No differences were observed in CCI, but the median absolute increase after transfusion of the S-PC pools was higher (25 vs. 19 x 10(9)/L, p<0.01), which reflected the larger size of the S-PC pools. No overall differences in vital signs were recorded. Two reactions were observed, both in temporal association with the transfusion of pools of S-PCs. CONCLUSIONS: A pool consisting of 5 units of WBC-reduced PCs gave a median platelet increment of 19 x 10(9) per L in these thrombocytopenic patients and has a median WBC content 1 to 2 log below the accepted threshold for primary alloimmunization or CMV transmission.

Prediction of well-conserved HIV-1 ligands using a matrix-based algorithm, EpiMatrix.

This preliminary study was undertaken to identify new human leucocyte antigens (HLA) ligands from human immunodeficiency virus type 1 (HIV-1) which are highly conserved across HIV-1 clades and which may serve to induce cross-reactive cytotoxic T lymphocytes (CTLs). EpiMatrix was used to predict putative ligands from HIV-1 for HLA-A2 and HLA-B27. Twenty-six peptides that were both likely to bind and also highly conserved across HIV-1 strains in the Los Alamos HIV sequence database were selected for binding assays using the T2 stabilization assay. Two peptides that were also highly likely to bind (for A2 and B27, as determined by EpiMatrix) and well conserved across HIV-1 strains, and had previously been described to bind in the published literature, were also selected to serve as positive controls for the assays. Ten new major histocompatibility complex (MHC) ligands were identified among the 26 study peptides. The control peptides bound, as expected. These data confirm that EpiMatrix can be used to screen HIV-1 protein sequences for highly conserved regions that are likely to bind to MHC and may prove to be highly conserved HIV-1 CTL epitopes.

Modulation of cytokine production by carnitine.

The ability of carnitine congeners to modulate cytokine production by human peripheral blood mononuclear cells (PBMC) was investigated. Modulation of cytokine production by PBMC of young (30 years of age or younger) and old (70 years of age or older) normal donors was first compared. The PBMC were collected over Ficoll-Hypaque and incubated in the presence of various concentrations of acetyl L-carnitine for 24 h. Subsequently the supernatants were collected and examined for cytokine production. The presence of cytokines in tissue culture supernatants was examined by ELISA. The cytokines measured included IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, TNFalpha, GM-CSF, and IFNgamma. The results showed that at 50 mug/ml of acetyl L-carnitine the most significant response was obtained for TNFalpha. In this regard four of five young donors responded, but only one of five old donors responded. More recently these studies were expanded to examine the ability of L-carnitine to modulate cytokine production at higher doses, 200 and 400 mug/ml, in young donors. The results of these studies showed that in addition to TNFalpha, significant production of IL-1beta and IL-6 was observed. These preliminary studies provide evidence that carnitine may modulate immune functions through the production of selected cytokines.

Plasma adenosine deaminase2: a marker for human immunodeficiency virus infection.

Plasma concentrations of the two isoenzymes of adenosine deaminase (ADA, E.C. 3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), were measured in a cohort of ambulatory patients infected with the human immunodeficiency virus (HIV) and controls. A sensitive isoenzyme-specific radioisotopic assay system was developed for these studies. Among 22 HIV-infected patients, plasma ADA2 was significantly elevated as compared with 16 control subjects (p less than 0.01) and 6 uninfected subjects having a risk factor for HIV infection (p less than 0.01). Plasma ADA2 was not associated with the stage of disease as defined by clinical status (p greater than 0.05) or helper (CD4) lymphocyte count (p greater than 0.05). Available evidence suggests that elevated plasma ADA2 could be a useful surrogate marker for HIV infection that occurs early in the disease process.

Thymic hormone modulation of age-induced changes in the induction of graft-versus-host disease by DBA/2J lymphocytes.

Aging induces a number of changes in the immune system, including the involution of the thymus which results in the loss of thymic hormone production and alteration in T cell function. One age-dependent change in immune response is the increasing risk of developing acute or chronic form of graft-versus-host disease (GVHD) following bone marrow transplantation as the age of the recipient increases. A murine model of GVHD that has been extensively studied is one in which injection of C57BL/6 spleen cells into unirradiated B6D2F1 mice results in an acute form of GVHD characterized by cytolytic T lymphocytes (CTL), suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells results in a chronic form of GVHD characterized by a lack of CTL and hyperproduction of immunoglobulin and autoantibodies. This study shows that the GVHD response of DBA/2J spleen cells is dependent on the age of the donor DBA/2J mice. If spleen cells from DBA/2J mice older than 3 months are injected into B6D2F1 recipients, CTL and lack of immunoglobulin production indicative of acute GVHD result. Administration of thymosin fraction 5, a collection of thymic hormones, to DBA/2J mice older than 3 months caused spleen cells from these treated mice to give a GVHD response characteristic of the chronic form of GVHD in B6D2F1 recipients. Thus, thymic hormones were able to modulate the changes in GVHD responses of DBA/2 lymphocytes that occur as the mice age. Preliminary fractionation of TF5 has indicated that there are at least two active thymic peptides present in TF5.

Thymosin-like peptides as potential immunostimulants. Synthesis via the polymeric-reagent method.

This paper reports our attempt at designing new immunostimulating peptides which are chemically related to the bioactive peptides thymosin alpha 1 and thymopentin. Three peptides were synthesized, Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr (3), Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (2), and Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (1), each of which contains the thymopentin sequence and portions of the bioactive sequence of thymosin alpha 1. Peptides 1-3 were assembled from selected blocked fragments that were synthesized by the polymeric-reagent method, using PHBT (polystyrene-bound 1-hydroxybenzotriazole) as the activating polymer. The ability of peptides 1-3 to enhance the activation (RNA synthesis) and proliferation (DNA synthesis) of human T lymphocytes was determined. In comparison to thymosin alpha 1, thymosin alpha 1 (15-28), and thymopentin, peptides 1-3 did not show significant enhancement of these processes.

Thymomodulin: biological properties and clinical applications.

Thymomodulin (Ellem Industria Farmaceutica s.p.a., Milan, Italy) is a calf thymus acid lysate derivative, composed of several peptides with a molecular weight range of 1-10 kD. Thymomodulin did not exhibit any mutagenic effect. Furthermore, thymomodulin used in animal studies showed no toxicity even when used at high concentrations. Of major significance are the observations in murine and human systems that thymomodulin remains active when administered orally. In vitro and in vivo administered thymomodulin was able to induce the maturation of T-lymphocytes. Additionally, studies in vitro showed that this thymic derivative can enhance the functions of mature T-lymphocytes with cascading effects on B-cell and macrophage functions. Extensive human clinical trials with thymomodulin showed that this agent can improve the clinical symptoms observed with various disease processes, including infections, allergies and malignancies, and can improve immunological functions during ageing.

Lymphokine activity in malignant effusions after intracavitary viral oncolysate.

Six patient with malignant effusions (five with ascites and one with malignant pleural effusion) due to refractory ovarian carcinomatosis, received intracavitary injections of ovarian viral oncolysate (VO). Measurements were made of the intracavitary activities of T cell growth factor (TCGF-IL-2) and B cell growth factor 12-kD (BCGF) and these were correlated with the clinical activity and cytologic changes in the malignant effusions. Both BCGF and IL-2 activities were demonstrated in malignant effusions prior to therapy although these were relatively higher for BCGF. Increases in the activities of both lymphokines were observed in the post VO treatment samples ranging from 4.1% to 45.0% for BCGF and 4.8% - 33.9% for IL-2. One patient who exhibited marked clinical improvement accompanied by mononuclear cell infiltrates and diminution of ascitic fluid malignant cells, also had elevation of lymphokine activities after repeated VO injections. These data provide further support for the role of VO as inducers of regional immunity.

Production of human B and T cell growth factors is enhanced by thymic hormones.

The thymic preparations thymosin fraction 5 (TF5) and synthetic thymosin alpha 1 (T alpha 1) were examined for their ability to enhance growth factor production by human peripheral blood mononuclear cells (PBMC). The results showed that both TF5 and T alpha 1 were capable of enhancing the production of a B cell growth factor (BCGF-12kD) and T cell growth factor (TCGF; IL-2). Enhancement by T alpha 1 could be obtained at 100-200-fold lower concentrations than that seen with TF5. In contrast, no enhancement of growth factor production was obtained with control preparations of non-thymic tissue extracts at any concentrations used. It was observed that stimulation of BCGF-12kD and IL-2 was most significantly obtained when the PBMC were activated with lectin. Furthermore, no direct effect of thymic hormones on test B and T cells was observed. These observations provide the first direct evidence that production of B cell growth factors can be enhanced by thymic hormones. In addition, these studies suggest that thymic hormones may regulate B cell responses by acting on mature activated T lymphocytes.

Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients.

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.

Update and future perspectives of a thymic biological response modifier (Thymomodulin).

Thymomodulin (Ellem Industria Farmaceutica spa, Milan, Italy) is a calf thymus acid lysate with immunomodulating activities. It is composed of several peptides with a molecular weight range of 1-10kD. Extensive studies in animal systems showed that Thymomodulin exhibited no, or very little toxicity even when used at high doses. Studies done in vitro and in vivo demonstrated that Thymomodulin is a biologically active compound which regulates the maturation of human and murine pre T lymphocytes, as well as modulate the functions of apparently mature human and animal B and T lymphocytes. It was observed that Thymomodulin can promote myelopoiesis as demonstrated by an increase of granulocyte-macrophage colonies in agar. Although additional studies to examine its target cell lineage are required, it appears that Thymomodulin exhibits specificity toward T cells. Therefore, enhancement of other cell lineage functions by Thymomodulin may be indirect, and mainly due to its effect on T cells. Of major importance is to note that Thymomodulin is prepared in a manner which allows it to maintain its biological activity when administered orally.

Rearrangement in the breakpoint cluster region and the clinical course in Philadelphia-negative chronic myelogenous leukemia.

We have followed one patient with Philadelphia (Ph)-negative chronic myelogenous leukemia and identified an additional four patients from the literature who showed the rearrangement in the breakpoint cluster region (bcr) on chromosome 22 characteristic of Ph-positive chronic myelogenous leukemia. The clinical course of these five patients was similar to that of Ph-positive patients, with easily controlled leukocyte counts, a prolonged benign phase, and prolonged survival. Furthermore, we have shown, for the first time, that bcr rearrangement in Ph-negative chronic myelogenous leukemia can result in expression of the aberrant 210-kilodalton bcr-abl fusion protein, which has been strongly implicated in Ph-positive leukemogenesis. Research data pertaining to possible cytogenetic mechanisms leading to production of p210bcr-abl in the absence of the Ph chromosome are reviewed. Molecular analysis provides an important tool for classifying and predicting prognosis of some patients with Ph-negative chronic myelogenous leukemia.

Growth factor-mediated proliferation in B cell non-Hodgkin's lymphomas.

The non-Hodgkin's lymphomas (NHLs) are a heterogeneous group of human lymphoid tumors, primarily of B cell lineage, which appear to represent arrested stages in B lymphocyte differentiation. Control of cell proliferation is a fundamentally important but poorly understood area of study in these tumors. We have studied a representative group of B cell NHLs to assess their potential for growth factor-mediated proliferation in vitro. Our results show that purified monoclonal NHL B cells of the small cell (well-differentiated lymphocytic lymphoma, nodular poorly differentiated lymphocytic lymphoma, etc) type, that were positive for the human malignancy-associated nucleolar antigen could be stimulated by human B cell growth factor (BCGF) to proliferate in vitro. Other B cell activators such as insoluble anti-Ig and the mitogen protein A also could stimulate thymidine incorporation in the lymphoma cell populations. In vitro lymphoma cell growth could be maintained in the presence of the growth factor for up to five weeks. The large B cell type NHL, however, appeared to be refractory to in vitro stimulation by BCGF as well as other stimulators of normal B cells. These studies suggest that human B cell lymphoid tumors are not only phenotypically similar to their normal B lymphocyte counterparts, but are also sensitive in some cases, to the same types of immunoregulatory molecules that control normal lymphoid cell growth.

Lymphokines and monokines as regulators of human lymphoproliferation.

The development of a competent immunoregulatory response in the face of an antigenic challenge is modulated by soluble proteins of relatively low molecular mass. Lymphokines and monokines, secreted by cells of T lineage and cells of the monocyte/microphage series, respectively, function in a bimodal amplification network that results in the proliferation and differentiation of the immunoregulatory cells. Interleukin 1 is typically assayed by its effect on thymocytes or by its ability to promote the T cell-dependent release of interleukin 2. Interleukin 2 is routinely measured by its ability to support the long-term growth of cultured T cells, whereas B cell growth factor is measured by its ability to support the long-term growth of cultured B lymphocytes. The availability of homogeneous purified factors and the subsequent availability of monoclonal antibodies against these reagents should allow for the development of rapid quantitative assays for these analytes in diverse biological fluids. In addition, large quantities of purified reagents will promote studies to determine therapeutic efficacy in several immunodeficiency syndromes.

Association of an interleukin abnormality with the T cell defect in Hodgkin's disease.

The cellular immune defect in untreated Hodgkin's disease (HD) has long been recognized. This defect appears to be responsible for at least some of the morbidity and ultimately the mortality associated with the disease. In recent years, many studies have shown that the T cell component of the immune response is the apparent site where the defect in HD exists and where the immunoregulatory abnormalities that may account for the deficit are observed. The discovery of the lymphokines and monokines, comprising the human interleukin system, has elucidated some aspects of the regulatory control of the functional pathways involved in T lymphocyte activation and proliferation. The interleukin system can therefore provide the framework to dissect immunodeficiency states, such as that seen in HD. The present study indicates that HD patients' interleukin 1 (IL1) response appears to be normal, as is their T cell proliferative response to exogenous IL2. Interleukin 2 production by HD patients' peripheral blood mononuclear cells, however, is decreased when compared with age/sex-matched controls. The inability to generate IL2 after appropriate stimulation may reflect either a primary cellular defect or a regulatory defect, such as excessive immunosuppression, giving rise to the characteristic T cell hyporesponsiveness seen in HD.

Changes in the compositions of the null cell compartment with murine autoimmunity.

The subpopulations that comprise the null cell compartment were examined sequentially in various strains of autoimmune-prone mice. Different patterns emerged that were consistent within strains but differed from strain to strain. Abnormalities appear earlier in life in short-lived mice, such as male BXSB and MRL/l mice, than in relatively long-lived strains, such as female BXSB and NZB mice. The accumulation of T cells in MRL/l mice was accompanied by null cell changes that contrasted with those that developed in AKR/J mice after their spleens were infiltrated with leukemic T cells. It would seem that lymphocyte perturbations with murine autoimmunity also involve their precursor cells and that these precursor cell changes vary in different strains, perhaps in relation to different genetic factors.

Long-term growth of human B cells and their use in a microassay for B-cell growth factor.

Normal human B lymphocytes, prepared from peripheral venous blood, have been stimulated with intact anti-IgM (mu chain specific) bound to an insoluble matrix. The activation event, in a subfraction of human B cells, was associated with subsequent receptivity to the mitogenic effects of exogenously added B-cell growth factor. The ability of the cell population to specifically absorb the B-cell growth factor was dependent upon the time of stimulation with the anti-IgM. Continuous replenishment of the growth factor resulted in the ability to maintain long-term growth-factor-dependent human B-cell populations. These cultured B lymphocytes were shown to specifically absorb the B-cell growth factor, suggesting the presence of membrane receptors for it. The cultured B lymphocytes were routinely maintained in logarithmic-phase growth, in the presence of growth factor, with a population doubling time of 36 hr. These cultured B cells have been utilized in a microassay for the assessment of B-cell growth factor activity that is accurate, sensitive, and precise.

Null cell senescence and its potential significance to the immunobiology of aging.

The null cell compartments of human bone marrow and mouse spleen were arbitrarily divided into three subpopulations based upon the ability of cells to acquire T or B cell membrane markers when incubated with poly A:U or ubiquitin. There was an accumulation of T cell precursors with congenital absence of the thymus. In contrast, T cell precursors were reduced and there was an accumulation of uninduced null cells with old age. These observations suggest that there is an intrinsic defect of null cell differentiation with a drift towards more differentiated precursors in T cell differentiation with aging. This could result in a diminution in the range of responses by their progeny, mature T lymphocytes.