Pedro Gascon

Immunologic Abnormalities in Patients Receiving Multiple Blood Transfusions

Concern for the potential risk of the acquired immunodeficiency syndrome among blood cell recipients led us to measure immunologic functions in patients who had received multiple transfusions. Abnormalities of two immunologic tests, natural killer cell function and helper/suppressor (T4/T8) lymphocyte subpopulation ratio, have characterized patients with the acquired immunodeficiency syndrome and are prevalent in populations at risk for this syndrome. Natural killer cell function was severely depressed in multiply transfused patients. However, T4/T8 ratios were normal in this population. The role of chronic antigenic stimulation was studied by measurement of HLA-DR expression on T cells. The expression of HLA-DR antigen is markedly elevated in multiply transfused patients. These results show that chronic exposure to foreign antigens may be associated with abnormalities of immunologic function, but that chronically transfused patients do not have the same immunologic profile as reported in some homosexuals and hemophiliacs.

A Prospective Clinical and Immunologic Analysis of Patients with Serum Sickness

We prospectively evaluated the clinical and immunologic features of serum sickness in 12 patients with bone-marrow failure treated for 10 days with intravenous infusions of horse antithymocyte globulin. Eleven of the 12 patients had signs and symptoms of serum sickness 8 to 13 days after beginning therapy with antithymocyte globulin. Eleven patients (including 10 of 11 with signs and symptoms of serum sickness) acquired circulating immune complexes, with peak levels occurring at 10 to 12 days. Serum C4 and C3 levels fell precipitously, with nadirs on the 10th day. Plasma levels of C3a anaphylatoxin were elevated in the four patients in whom it was measured. Eight of the 11 patients with signs and symptoms of serum sickness had a characteristic serpiginous erythematous and purpuric eruption on the hands and feet at the junction of palmar and plantar skin. Direct immunofluorescence of skin lesions revealed immune deposits (IgM, IgE, IgA, or C3) in the blood vessels of three of five patients. This study documents the immunopathology of serum sickness in human beings and describes a cutaneous marker for the disease.

Hematopoietic Regulation Mediated by Interactions Among the Neurokinins and Cytokines

This review summarizes the current data regarding the mechanisms by which two mammalian neurokinins (tachykinins), substance P (SP) and neurokinin-A (NK-A) are involved in hematopoiesis. SP and NK-A are derived from the preprotachykinin-I (PPT-I) gene which can be induced by cytokines and neurotrophic factors. In the bone marrow (BM), nerve fibers and stroma are potential sources for the PPT-I gene products. SP and NK-A interact with either of three cloned receptors, neurokinin-1 (NK-1), NK-2 or NK-3, although SP and NK-A exhibit binding preferences for NK-1 and NK-2 respectively. Through specific receptors, SP and NK-A exert dichotomous hematopoietic effects mediated mostly by the BM stroma. SP enhances the proliferation of primitive BM stem cells and progenitors and these effects correlate with the induction of stimulatory hematopoietic growth factors. NK-A appears to be protective to stem cells through the induction of TGF-beta. Proliferation of myeloid progenitors is inhibited by NK-A, effects which correlate with the induction of two suppressive factors, TGF-beta and MIP-1alpha. Stimulation of NK-2 leads to partial blunting of the enhanced stimulatory effects mediated by NK-1. Furthermore, stimulatory hematopoietic cytokines upregulate NK-1 expression and downregulate the constitutively expressed NK-2 in BM stroma. Together, the experimental evidence suggests that NK-A-NK-2 interactions could be a feedback to hematopoietic stimulation. Expression of NK-1 and NK-2 in CD34+ cell lines and also, the presence of SP binding sites on primary CD34+ cells suggest that the neurokinins could be interacting directly with BM progenitors and stem cells. In BM stroma, cytokines and neurokinins regulate the expression of each other and also, their respective receptors. In summary, the current literature pertaining to hematopoietic regulation indicates the involvement of a complex network that includes, but not exclusive of the cytokines and neurokinins. The current models that pertain to stem cell proliferation and differentiation should therefore add neuropeptides to the list of hematopoietic modulators.

Human Serum Sickness: A Prospective Analysis of 35 Patients Treated with Equine Anti-Thymocyte Globulin for Bone Marrow Failure

We have prospectively evaluated the clinical and immunological features of serum sickness in 35 patients treated for bone marrow failure with anti-thymocyte globulin (ATG 15 mg/kg/day) and methylprednisolone (1 to 1.5 mg/kg/day). Twenty-one patients were treated for 10 days and 14 were treated for 28 days. Clinical evidence of serum sickness developed in 30 patients (86%) and included fever and malaise (100%), cutaneous eruptions (93%), arthralgias (67%), gastrointestinal complaints (67%), cephalgia (57%), blurring of vision (37%), arthritis, (30%) and lymphadenopathy (13%). Clinical serum sickness began on day 7 +/- 1 (X +/- S.E.M.) and lasted for 10 +/- 2 days in the 18 affected patients receiving the shorter course of ATG. In the 12 affected patients receiving the longer course of ATG, serum sickness began on day 9 +/- 1. The earliest manifestations of serum sickness were fever, malaise, and cutaneous eruptions. Cutaneous findings consisted of morbilliform eruptions (n = 19) and urticaria (n = 1) or a combination (n = 8) that lasted 10 to 14 days. Twenty-one patients (75%) developed a highly characteristic serpiginous band of erythema and purpura along the sides of the fingers, toes, palms and soles 12 to 48 hours before other symptoms of serum sickness. Biopsies of lesional skin during the course of serum sickness revealed immune deposits (IgM, IgE, IgA and C3) in dermal vasculature in 7 of 9 patients. Immunological changes that occurred during the course of serum sickness included increased serum levels of IgG, IgM, IgA, and IgE. Circulating immune complexes, as measured by the C1q-binding assay, increased from a mean value of 12% to 45% on day 13 +/- 1. Complement levels (C3, C4, and CH50) decreased 50 to 80% from their baseline levels on day 10 +/- 2. Acute phase reactants increased: erythrocyte sedimentation rate, C-reactive protein and beta-2 microglobulin. Abnormal urinalysis developed in 17 patients (57%) over the course of serum sickness and included proteinuria, hematuria and hemoglobinuria on day 10 +/- 3. Hematopoietic response occurred in 43%. All 5 patients who did not develop serum sickness recovered from bone marrow failure. Our data document the clinical and immunopathological findings in human serum sickness and suggest that the principles of antigen-antibody interaction, complement activation, and resultant inflammatory response as seen in the previous animal studies are directly applicable to studies of patients with serum sickness.

Cloning of Human Preprotachykinin-I Promoter and the Role of Cyclic Adenosine 5′-Monophosphate Response Elements in Its Expression by IL-1 and Stem Cell Factor

Preprotachykinin-I gene (PPT-I) encodes several peptides with organ-specific functions that link the neuroendocrine-immune-hemopoietic axis. We cloned upstream of the initiation site of human PPT-I promoter and identified consensus sequences for two cAMP response elements (CRE). PPT-I is induced by cytokines including those that signal through the cAMP pathway. Therefore, we studied the role of the two CRE in IL-1α and stem cell factor (SCF) stimulation of bone marrow stroma because both cytokines induce endogenous PPT-I in these cells and activate the cAMP pathway. Furthermore, bone marrow stroma expresses the transcription factors regulated by the cAMP pathways such as the repressor (ICERIIγ) and activator (CREMτ). Mutagenesis of the two CRE and/or cotransfection with vectors that express ICERIIγ or CREMτ indicated that the two CRE have major roles in PPT-I expression. The two CRE are also required for optimal promoter activity by SCF and IL-1α. A particular cytokine could concomitantly induce PPT-I and the high affinity G protein-coupled receptor for PPT-I peptides, NK-1R. We showed that SCF, a representative cytokine, induced PPT-I and NK-1R leading to autocrine and/or paracrine cell activation. Because NK-1R activates cAMP through the G protein, the results suggest that the presence of CRE sequences within PPT-I promoter could be important in the regulation of PPT-I expression by cytokines, irrespective of their ability to signal through cAMP. As PPT-I is implicated in hemopoietic regulation, immune responses, breast cancer, and other neural functions, these studies add to the basic biology of these processes and could provide targets for drug development.

Hematopoietic growth factor inducible neurokinin-1 type: a transmembrane protein that is similar to neurokinin 1 interacts with substance P.

Neurokinin 1 (NK-1) is a member of seven transmembrane G protein-coupled receptors. NK-1 interacts with peptides belonging to the tachykinin family and showed preference for substance P (SP). NK-1 is induced in bone marrow (BM) stroma. NK-1-SP interactions could lead to changes in the functions of lymphohematopoietic stem cell (LHSC). This report describes the cloning and characterization of a cDNA clone isolated after screening of three cDNA libraries with an NK-1-specific probe. Based on its expression, the cDNA clone was designated hematopoietic growth factor inducible neurokinin-1 type (HGFIN). Computational analyses predicted that HGFIN is transmembrane with the carboxyl terminal extracellular. Proteomic studies with purified HGFIN and SP showed noncovalent interactions. HGFIN-SP interactions were supported by transient expression of HGFIN in CHO cells. Transient expression of HGFIN in unstimulated BM fibroblasts led to the induction of endogenous NK-1. Since NK-1 expression in BM fibroblasts requires cell stimulation, these studies suggest that there might be intracellular crosstalk between NK-1 and HGFIN. Northern analyses with total RNA from different BM cell subsets showed that HGFIN was preferentially expressed in differentiated cells. This suggests that HGFIN might be involved in the maturation of LHSC. HGFIN was detected in several other tissues, but not in brain where NK-1 is constitutively expressed.

Lymphocyte phenotype and lymphokines following anti-thymocyte globulin therapy in patients with aplastic anaemia

Twenty‐two patients with adult onset aplastic anaemia were analysed before and after therapy with antithymocyte globulin (ATG). Lymphocyte phenotype, lymphokine levels or production, and haematopoietic progenitor cell number were measured 3 months after therapy: clinical response was determined 1 year post‐therapy. By flow cytometry there was a significant reduction in both the proportion and absolute number of peripheral blood lymphocytes expressing activation antigen Tac (IL‐2 receptor) and in the proportion of HLA‐DR+ lymphocytes. For T cells bearing HLA‐DR, there were proportional decreases in both activated helper and suppressor cells. There was no statistically significant difference pre‐ATG to post‐ATG in the absolute numbers of total, helper and suppressor lymphocytes. In all 10 haematologic responders the number of Tac bearing lymphocytes after ATG therapy was in the normal range, but half of 12 non‐responding patients continued to have abnormally elevated numbers of Tac+ T cells. The proportion of Tac+ cells were not related to transfusion history. γ‐interferon levels in serum by radioimmunoassay were elevated in almost half the aplastic patients; post‐ATG. γ‐interferon was detectable in only three patients. Haematologic response to ATG therapy was associated with increased numbers of haematopoietic progenitors post‐treatment, but pre‐treatment values were not predictive of a response. These results are consistent with a pathogenic role for activated T‐cells and their lymphokine products and suggest that the target of ATG therapy may be a Tac+ lymphocyte.

Serum sickness and haematopoietic recovery with antithymocyte globulin in bone marrow failure patients

We have evaluated 3 3 patients with various bone marrow dyscrasias treated with horse antithymocyte globulin (ATG) (Upjohn) to determine the relationship of haematopoietic response to the occurrence of serum sickness. Patients received ATG intravenously over 10 or 28 d at a dose of 15 mg/kg/d. Total or partial haematological responses were noted in 12 of 33 patients. Twenty‐eight patients developed clinical signs of serum sickness 6–14 d after the first infusion of ATG, while five patients did not. Twenty‐five of these patients were evaluated by immunochemical assays for circulating immune complexes (Clq‐binding assay) and 21 patients for serum complement (C3, C4 and CH50 assay) levels. There was a direct correlation between increases in immune complex levels, decreases in serum complement levels, and the development of the clinical signs and symptoms and serum sickness. Twenty‐one of 28 patients who developed serum sickness failed to show haematological improvement. However, haematopoietic recovery occurred in all five patients who did not manifest serum sickness (P <0.05) and in four patients who failed to develop immune complexes. These findings indicate that the development of serum sickness is not required for a haematopoietic response with ATG and may indeed impair recovery.

Neural Regulation of Hematopoiesis by the Tachykinins

The neuromodulators/neurotransmitters, substance P (SP) and neurokinin-A (NK-A) belong to the tachykinin family. Both peptides are widely distributed in the central and peripheral nervous systems. Although lymphoid organs such as the bone marrow (BM) are innervated by peptidergic fibers, non-neural sources for these peptides are possible. Specific cells (endothelial, macrophages and eosinophils) produce SP. In addition, we have found that cytokines induce SP-immunoreactivity (SP-IR) in BM stroma. Although both peptides interact with each of the three cloned NK- receptors (NK-1R, NK-2R, NK-3R), SP and NK-A exhibit preferences for NK-1R and NK-2R respectively. SP and NK-A interact with the G-protein coupled NK-like receptors on hematopoietic cells (CD34+, stroma) to regulate hematopoiesis. Our data indicate the presence of NK-1 and NK-2 like receptors, as well as subtypes of these receptors in BM cells. We have reported that SP stimulates in vitro hematopoiesis (erythroid and myeloid). Compared to SP, NK-A is less stimulatory to erythroid and inhibits myeloid colonies. The regulatory effects by the tachykinins are partly determined by the particular type of NK-Rs being stimulated. NK-1R appears to mediate a stimulatory response whereas, NK-2R inhibits hematopoiesis. Most of the effects by the tachykinins are indirect through the induction of growth factors. We have shown that SP induces IL-1, IL-3, IL-6, GM-CSF and c-kit ligand in BM cells and NK-A induces MlP-1α and TGF-s. Our data indicate that the stroma is involved in most of the tachykinin-mediated effects through the induction of cytokines which in turn regulate the induction, expression and binding affinities of NK-R on BM cells. These studies suggest that the tachykinins can mediate a network of regulatory interactions among BM cells and, that this can lead to “fine-tuned” hematopoiesis. The results also provide evidence that in addition to being neuro-transmitters/-modulators, the tackykinins may be a link of a putative neurohematopoietic axis.

Differential expression of neurogenes among breast cancer subtypes identifies high risk patients

The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p < 0.0001) and distant metastasis-free survival (p < 0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p < 0.0001) and metastasis-free survival (p < 0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes.