Requirement of MIP-1 alpha for an inflammatory response to viral infection.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a chemokine that has pro-inflammatory and stem cell inhibitory activities in vitro. Its biologic role in vivo was examined in mice in which the gene encoding MIP-1 alpha had been disrupted. Homozygous MIP-1 alpha mutant (-/-) mice were resistant to Coxsackievirus-induced myocarditis seen in infected wild-type (+/+) mice. Influenza virus-infected -/- mice had reduced pneumonitis and delayed clearance of the virus compared with infected +/+ mice. The -/- mice had no overt hematopoietic abnormalities. These results demonstrate that MIP-1 alpha is an important mediator of virus-induced inflammation in vivo.

Biosynthesis of proteochondroitin sulfate by HL-60 human promyelocytic cells.

Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/10(5) viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCl gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCl gradient (rho greater than 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/10(5) cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.

Impact of marketing work-place diversity on employee job involvement and organizational commitment.

Much of the debate about managing work-force diversity concerns the rationale for the procedures used and the outcomes produced by programs perceived to be unfair. The authors explored the role of organizational justice in diversity initiatives; they examined which of 6 diversity arguments (T. H. Cox & S. Blake, 1991) were most often used by U.S. firms and accepted by employees. They also surveyed U.S. workers about diversity issues at work; the problem-solving diversity argument was related to higher employee job involvement and organizational commitment, even though the respondents ranked the resource-acquisition argument as the most acceptable. The authors also found that many organizational-justice issues still need to be resolved, even in those organizations with diversity-management programs.

The therapeutic potential of erythropoietin receptor transgenes.

Allogeneic bone marrow transplantation is an effective curative therapy for both malignant and heritable diseases. The use of genetically altered autologous hematopoietic stem cells (HSC) is being increasingly investigated as a treatment for a variety of non-malignant but significantly morbid diseases, including hemoglobinopathies, immunodeficiencies and autoimmune diseases. Other hematopoietic cells capable of proliferation, such as antigen-specific T cells and dendritic cells, have also been used for adoptive immunotherapy. Genetic procedures to modify these various therapeutic cells so that they can be selectively amplified either in vitro or in vivo could enhance their efficacy. For example, HSC that contain a gene that confers a survival, selection or growth advantage may enhance their engraftment. Such enhancement could be expected to reduce graft failures and the intensity of the required conditioning regimen, thereby decreasing the toxicities of transplantation. In this review, the functions of cytokine receptor transgenes coding for erythropoietin receptors (EpoR) are analyzed. The characteristics of these transgenic cells and animals are discussed with regard to the possible therapeutic use of EpoR transgenes in the transplantation of hematopoietic cells.

Bone marrow: target for gene transfer.

Marrow cells are currently the most widely utilized target in human trials of gene therapies, and hematopoietic stem cells may be the best target of all. Able both to self-replicate and to engender blood cells in all lineages, they could be a lifelong source of therapeutic genes for disorders in any lineage. Hematopoietic stem cells may also portend the therapeutic use of even more versatile embryonic stem cells.

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies.

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.

Proteoglycan synthesis in two murine bone marrow stromal cell lines.

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3-2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3-2A does not. Cells were labeled with 35S-sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3-2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3-2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

A prospective crossover trial of direct current electrotherapy in symptomatic hemorrhoidal disease.

Many new modalities have been developed to treat symptomatic hemorrhoidal disease. In order to determine whether direct current electrotherapy therapy is effective, a prospective, crossover study was done. Sixteen patients referred for treatment of symptomatic hemorrhoids underwent a 16-week treatment protocol. Patients were randomized to receive either medical therapy with a sham therapy, or medical therapy with active direct current electrotherapy at 4-week intervals. All patients were instructed in medical therapy including sitz baths, hydrocortisone suppositories, topical preparations, and a high fiber diet with an educational booklet. Patients who did not respond to initial therapy were crossed over at 8 weeks, receiving alternate therapy twice. Improvement in symptoms, or lack thereof, was not statistically different between the two treatment groups. Comparisons of the internal sphincter pressure, external sphincter pressure, maximum squeeze pressure, and rectal sensitivity to balloon insufflation (volume) were not statistically different before, during, or after any treatment modality (p = 0.46, 0.60, 0.33). The size and grade of hemorrhoids on entry into the study had no bearing on the outcome. In a prospective crossover trial, no difference could be found between standard medical therapy and direct current electrotherapy in the treatment of symptomatic-hemorrhoidal disease.

Bone marrow stromal proteoglycan heterogeneity: phenotypic variability between cell lines and the effects of glucocorticoid.

Hematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component. Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation. Proteoglycan synthesis in the hematopoietic-supportive D2XRII, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S-sulfate precursor labelling and ion-exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC. The effects of glucocorticoid were also investigated. A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation. All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio-Sil TSK-400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media. The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers. CS/DS species were the major proteoglycans in all cultures. Hydrocortisone-free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell-associated form (HS1), partially excluded from the TSK-400 column, and a secretory form (HS2), eluting at Kd = 0.15. D2XRII cells also secreted an apparently-unique, high-density proteoglycan, Kd = 0.65, into the culture medium. Hydrocortisone at 10(-6) M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells.

Biochemical and functional characterization of proteoglycans produced by Sl/Sld murine bone marrow stromal cell lines.

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.

Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma.

In multiple myeloma, karyotopic 14q32 translocations have been identified at a variable frequency (10-60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q+), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus (i) are frequent (karyotypic 14q32 translocations and/or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); (ii) occur mainly in switch regions; and (iii) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.

Murine T lymphocytes incapable of producing macrophage inhibitory protein-1 are impaired in causing graft-versus-host disease across a class I but not class II major histocompatibility complex barrier.

The routine use of bone marrow transplantation is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). Current approaches to decreasing the occurrence of GVHD after allogeneic transplantation use T-cell depletion, use immunosuppressive agents, or block costimulatory molecule function. The role of proteins in the recruitment of alloreactive lymphocytes has not been well characterized. Chemokines are a large family of proteins that mediate recruitment of mononuclear cells in vitro and in vivo. To investigate the role of T-cell production of the chemokine macrophage inhibitory protein-1 (MIP-1) in the occurrence of GVHD, splenocytes either from wild-type or from MIP-1-/- mice were administered to class I (B6.C-H2(bm1)) and class II disparate mice (B6-C-H2(bm12)). The incidence and severity of GVHD was markedly reduced in bm1 mice receiving splenocytes from MIP-1-/- mice as compared with mice receiving wild-type splenocytes. Bm1 mice receiving MIP-1-/- splenocytes had significantly less weight loss and markedly reduced inflammatory responses in the lung and liver than mice receiving C57BL/6 splenocytes. Bm1 mice receiving MIP-1-/- splenocytes had a markedly decreased production of antichromatin autoantibodies and impaired generation of bm1-specific T lymphocytes versus wild-type mice. However, MIP-1-/- splenocytes easily induced GVHD when administered to bm12 mice. This data show that blockade of chemokine production or function may provide a new approach to the prevention or treatment of GVHD but that chemokines that recruit both CD4(+) and CD8(+) lymphocytes may need to be targeted.

Transdermal nicotine patches do not cause clinically significant gastroesophageal reflux or esophageal motor disorders.

Transdermal nicotine delivery systems are widely used in smoking cessation. The purpose of this study was to determine whether common symptoms of pyrosis and dyspepsia associated with these patches are related to gastroesophageal reflux or esophageal dysmotility. Twenty-seven paid volunteer cigarette smokers (> 15 cigarettes/day) without symptomatic gastroesophageal reflux disease participated in this single-blinded, placebo-controlled study. Twenty subjects completed the study. Subjects underwent three sequential 24-h intraesophageal pH/motor studies (Synectics model T32342084, Shore View, MN). The pH/motility probe was positioned 5 cm above the manometrically determined LES. A placebo patch was applied for the first 24-h study and a 15-mg nicotine patch (Nicotrol) was applied for the initial 16 h (removed for remaining 8 h) of the second 24-h period. A 21-mg nicotine patch (Nicoderm) was applied for another 24-h study period. All subjects consumed an identical, defined diet documented by meal receipts, and refrained from smoking and tobacco use throughout the study periods (CO breath test confirmation). The Wilcoxon, paired t-test, exact McNemar statistical methods were used. The results showed that there were no significant differences in reflux symptoms (pyrosis, chest pain, nausea, dysphagia), supine gastroesophageal reflux (number of episodes, duration, or cumulative acid exposure), or the total number of reflux episodes between placebo and nicotine patch treatment periods. The number of post-prandial upright acid reflux episodes (p = 004) and number of upright acid reflux episodes lasting more than 5 min (p = 0.007) were statistically higher with the placebo patch compared to the active nicotine patches. No differences in intraesophageal pH or motility indices were noted between the two transdermal nicotine patches (Nicotrol, Nicoderm). It was concluded that dyspeptic symptoms in subjects utilizing transdermal nicotine patches are not related to gastroesophageal reflux or to esophageal motor abnormalities.

Comparison of granulocyte colony-stimulating factor (G-CSF)--mobilized peripheral blood progenitor cells and G-CSF--stimulated bone marrow as a source of stem cells in HLA-matched sibling transplantation.

HLA-identical bone marrow or stem cell transplantation from a sibling is the preferred treatment for patients with chronic myelogenous leukemia, bone marrow failure syndromes, relapsed acute leukemia, and specific inborn errors of metabolism. Several groups have shown that granulocyte colony-stimulating factor (G-CSF)--mobilized peripheral blood progenitor cells (PBPCs) obtained from HLA-matched siblings are effective in reconstitution of marrow function after marrow ablative conditioning therapy. To evaluate whether G-CSF treatment before bone marrow harvest leads to enhanced recovery of PBPC counts and recovery from limited graft-versus-host disease (GVHD), we assessed the outcome of a sequential cohort of patients treated identically and then given either G-CSF--mobilized PBPCs or G-CSF--stimulated bone marrow from HLA-identical siblings. We show that the time to neutrophil engraftment is identical in the 2 cohorts, whereas platelet engraftment is earlier with the use of PBPCs. The incidence of acute GVHD was decreased, and that of chronic GVHD significantly decreased, in the group receiving bone marrow. Overall survival was not different between the 2 groups. Thus, G-CSF--stimulated bone marrow offers a source of stem cells that allows for early neutrophil engraftment with a decreased risk of GVHD.