The immunoregulatory and fibrotic roles of activin A in allergic asthma.

Activin A, a member of the TGF-ß superfamily of cytokines, was originally identified as an inducer of follicle stimulating hormone release, but has since been ascribed roles in normal physiological processes, as an immunoregulatory cytokine and as a driver of fibrosis. In the last 10-15 years, it has also become abundantly clear that activin A plays an important role in the regulation of asthmatic inflammation and airway remodelling. This review provides a brief introduction to the activin A/TGF-ß superfamily, focussing on the regulation of receptors and signalling pathways. We examine the contradictory evidence for generalized pro- vs. anti-inflammatory effects of activin A in inflammation, before appraising its role in asthmatic inflammation and airway remodelling specifically by evaluating data from both murine models and clinical studies. We identify key issues to be addressed, paving the way for safe exploitation of modulation of activin A function for treatment of allergic asthma and other inflammatory lung diseases.

Early hematopoietic events during tumor growth in mice.

Lewis lung carcinoma (LLC) of C57BL/6 mice, a transplantable tumor widely metastatic by 6-7 days post implant (PI), caused hematopoietic alterations such as progressive anemia (hemoglobin: day 1 PI, 11.0 g/dl; day 19 PI, 7.8 g/dl), neutrophilia (neutrophils: day 1 PI, 2 X 10(3)/microliter; day 19 PI, 22 X 10(3)/microliter), and marrow and splenic myeloid hyperplasia (marrow myeloid-to-erythroid ratio: day 1 PI, 1:1; day 7 PI, 3:1). Accompanying these changes were an increased concentration of marrow granulocyte-macrophage colony-forming units (culture) (GM-CFUC) (day 3 PI, LLC 185 +/- 27% of control; day 19 PI, LLC 265 +/- 10% of control) and accelerated cycling of these myeloid progenitors [day 3 PI, LLC 45.3 +/- 6.5% GM-CFUC in cycle vs. sham (media) injected 17.5 +/- 10.5%; day 7 PI, LLC 52.2 +/- 2.5% vs. sham (media) injected 29.8 +/- 9.8%; day 11 PI, LLC 56.2 +/- 4.4% vs. sham (media) injected 22.2 +/- 14%]. This study questioned whether enhanced hematopoiesis was a result of progressive tumor growth or whether the injection of tumor cells could evoke the response. By use of groups of C57BL/6 mice given an injection of live LLC cells, x-irradiated killed LLC cells, or media, the hematopoietic response to live LLC cells versus dead LLC cells could be dissected. A biphasic colony-stimulating activity (CSA) response in the sera of tumor bearers was found to account for the myelopoietic changes. The first wave of CSA from days 1 to 3 PI stimulated 168 +/- 3.7% more GM-CFUC than control sera and was likely released by dead cells of the tumor inoculum; the second wave from day 7 onward stimulated 220 +/- 7.6% more colonies and was a result of the enlarging tumor mass. Tumor growth was necessary for GM-CFUC proliferation, and the declining growth fraction at day 19 in LLC-bearing mice suggested that hematopoietic exhaustion was a consequence of tumor growth.

Adhesive interaction of hemopoietic progenitor cell membrane with the RGD domain of fibronectin.

The binding of two cloned hemopoietic progenitor cell lines, B6Sut (multipotential) and FDCP-1 (bipotential) to dishes coated with fibronectin or its chymotryptic fragments was studied by labeling the cells with 51Cr or [35S]methionine. Intact fibronectin molecule and its 120 kDa fragment, containing the Arg-Gly-Asp (RGD) sequence motif, as well as a synthetic RGD-containing peptide Peptite 2000 all bound progenitor cells. However, the 40 or 45 kDa fragments, containing the heparin-binding and CS-1 domains, failed to bind the cells in a comparable magnitude. The binding of intact fibronectin and its 120 kDa fragment was inhibited in a dose-dependent fashion with increasing concentration of RGD-containing Gly-Arg-Gly-Asp-Ser peptide, but not with Gly-Arg-Gly-Glu-Ser control peptide that does not contain the RGD sequence motif. To explore the nature of the receptor for this fragment of fibronectin, membrane proteins were labeled with 125I and subjected to affinity chromatography using a matrix to which the 120 kDa fragment of fibronectin was covalently bound. Specific competitive elution with RGD yielded two bands with molecular masses of 160 and 110 kDa, corresponding, respectively, to those of alpha 5 and beta 1 chains of integrin molecule. Western blotting of whole-cell-lysate proteins with a monospecific, polyclonal serum specific for vertebrate beta 1 integrins identified a beta 1 integrin in these cells. Thus, it appears that an interaction involving alpha 5 beta 1 integrin with 120 kDa fragment of fibronectin may be involved between hemopoietic progenitor cells and the fibronectin component of extracellular matrix.

Restorative effect of IL-3 on adherence of cloned hemopoietic progenitor cell to stromal cell.

Mammalian hemopoiesis results from a complex interaction between hemopoietic progenitor cells, stromal cells and extracellular matrix components, orchestrated by specific glycoprotein growth factors. Recently, these growth factors have been shown to possess an important function, apart from stimulation of proliferation, and that is suppression of an active cellular process of programmed cell death, or apoptosis. Highly specific biochemical and morphologic changes have been shown to occur during apoptosis, but their reflections on cellular functions are poorly understood. Interleukin-3 (IL-3)-dependent FDCP-1 (factor-dependent cell lines cloned in Paterson Laboratories) cells were studied for their ability to adhere to hemopoietic stroma in a temporal fashion under conditions of apoptosis and following rescue from apoptosis with growth factor. It was found that cloned FDCP-1 cells always maintained, in the presence of a source of IL-3 (either WEHI conditioned medium or rm-IL-3), bound cloned hemopoietic stromal cell GB1/6 in a constant fashion for 20 hours, while cells starved of IL-3 experienced a 50% time-dependent decrement in binding. If IL-3 were added back to FDCP-1 that had been starved of growth factor for 8 hours, but not 12 hours, adherence to stroma was restored to that of control cells always in the presence of IL-3. Granulocyte/macrophage colony-stimulating factor (GM-CSF), Interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) did not restore cytoadherence. By transmission electron microscopy, nucleus and cytoplasm of IL-3-replenished cells resembled that of control cells. These data indicate that at least some events related to apoptosis were reversible for a period up to 8 hours, but not 12 hours, in cells that had been rescued by readdition of IL-3. These findings offer important insight into a way in which bone marrow progenitor cells may be maintained in a condition that optimizes their ability to engraft stroma during transplantation.

A cytoadhesion assay for the binding of cloned hemopoietic progenitor cells to stroma.

Cell adhesion molecules responsible for the specific recognition and adhesion that necessarily occur between hemopoietic progenitor cells (HPC) and stromal cells within the bone marrow are likely of multiple nature in the cell membrane. Systems less complex than intact bone marrow, in which the interactions between adhesion molecules and their ligands may be studied, is greatly needed. Using 4 cloned murine IL-3-dependent HPC lines, B6Sut, FDCP-1, FDCP-2 and FDCP-Mix, a system of co-culture has been established and standardized with 2 murine stromal cell lines, GB1/6 and 3T3. HPC were radiolabeled with 51Cr, and an optimal set of conditions was established for the adherence of HPC to stromal cells. It was found that a source of IL-3, whether supplied as recombinant murine IL-3 or WEHI-conditioned medium, was a necessary component of the labeling and assay medium to achieve maximal adherence to stroma. Likewise, the presence of serum also resulted in overall better cytoadhesion than did serum-free conditions. All 4 cell lines bound GB1/6 in a reproducible manner from approximately 63% for FDCP-1 to 20% for FDCP-Mix; binding to 3T3 was higher than to GB1/6 for all HPC. Approximately 25 to 30% of the cell populations were not able to adhere to stroma, indicating a fairly constant degree of heterogeneity with respect to expression of adhesion molecules. Cytoadhesion was found to have at least one component that was temperature-sensitive, as adhesion of FDCP-1 to GB1/6 was 41.5 +/- 1.3% at 4 degrees C compared with 63.2 +/- 1.1% at 37 degrees C. The adhesion reaction itself occurred independently of metabolic energy production and relied on the presence of receptor-ligand molecules. This very standard and reproducible system should allow closer examination of the individual cytoadhesive events that occur between HPC and marrow stromal cells using cloned cell lines.

Distribution of homing protein on hemopoietic stromal and progenitor cells.

Homing receptor is a membrane lectin of 110 kd molecular weight that recognizes galactosyl and mannosyl residues of an as yet unknown glycoconjugate. It is responsible for recognition and selective homing of hemopoietic progenitor cells after these cells are transplanted intravenously. Consequently, it is present on the surface of hemopoietic progenitor cells. To determine the distribution of this receptor on other cell types we performed standard binding assays in many cell types using galactosyl and mannosyl residues covalently bound to bovine serum albumin (G-BSA and M-BSA) as an index of homing receptor. BSA moiety was then labeled with 125I. The three cloned hemopoietic cell lines B6Sut, FDCP-1, and FDCP-mix all showed combined binding of G-BSA and M-BSA, whereas the lymphoid cell line L1210 showed only M-BSA, not G-BSA binding and, therefore, was considered to lack homing receptors. Similarly, stromal cell lines D2X and GB1/6 as well as primary marrow stroma (progenitor cell-depleted) did not show homing receptors as evidenced by combined binding of G-BSA and M-BSA. Nor did the nonhemopoietic stromal cell line Swiss 3T3 show the presence of homing receptors by these criteria. We conclude that homing receptors are distributed narrowly and are present on hemopoietic progenitor cells, but absent on hemopoietic stroma.

Characterization of FDCP-2, a cloned hemopoietic progenitor cell deficient in homing protein.

The progenitor cell clone, FDCP-2, was found to lack the expression of membrane homing lectin that recognizes galactosyl and mannosyl residues of glycoconjugate on the surface of hemopoietic stroma. Adherence of these cells to hemopoietic stroma is significantly less than that of either normal clones B6SUt or FDCP-1, although their adherence to nonhemopoietic stroma 3T3 is preserved. As determined by electron microscopy, the cells lack microvilli, which in their normal counterparts serve to mediate the contact and adherence to hemopoietic stroma. This cell line can be useful as a negative control in elucidating the molecular basis of the homing phenomenon and its function in the regulation of hemopoiesis.

Homing of a cloned multipotential stem cell line in spleen and intraperitoneal membrane.

B6SUT is a murine cell line cloned from nonadherent cells of viral-infected long-term marrow cultures. In soft agar it can form colonies of three different hemopoietic lineages and is considered to be a hemopoietic stem cell line. We studied its ability to "home" in the spleen of lethally irradiated mice. These cells formed large surface colonies on days 8 and 12, the colonies on day 12 being greater than 3 mm in diameter. Microscopically, these colonies consisted of undifferentiated cells, suggesting that B6SUT cells are capable of homing and proliferation in hemopoietic tissues, but not differentiation. To confirm this, cells were labeled with 51Cr and infused into the peritoneum of mice bearing cellulose ester membranes (CEM). We noted significant uptake of radioactivity that could be inhibited in the presence of excess unlabeled B6SUT cells. Electron-microscopic studies showed binding and migration of these cells into the CEM coat. The membrane of B6SUT cells was mapped for lectin and sugar-recognizing receptors and found to possess receptors for PHA, Con A, WGA, and RCA, but not UEA, fucosyl, galactosyl, or mannosyl residues. We conclude that cell line B6SUT is capable of homing into hemopoietic tissues, and that surface glycoproteins may be responsible for this phenomenon. This cell line permits the study of the "homing" phenomenon apart from proliferation and differentiation.

Reduction in immunosuppression as initial therapy for posttransplant lymphoproliferative disorder: analysis of prognostic variables and long-term follow-up of 42 adult patients.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is an Epstein-Barr virus-associated malignancy that occurs in the setting of pharmacologic immunosuppression after organ transplantation. With the increased use of organ transplantation and intensive immunosuppression, this disease is becoming more common. We explore reduction in immunosuppression as an initial therapy for PTLD. METHODS: We analyzed our organ transplant patient database to identify patients with biopsy-proven PTLD who were initially treated with reduction of their immunosuppressive medications with or without surgical resection of all known disease. RESULTS: Forty-two adult patients were included in this study. Thirty patients were treated with reduction in immunosuppression alone. Twelve patients were treated with both reduction in immunosuppression and surgical resection of all known disease. Thirty-one of 42 patients (73.8%) achieved a complete remission. Of those patients who were treated with reduction in immunosuppression alone, 19 of 30 (63%) responded with a median time to documentation of response of 3.6 weeks. Multivariable analysis showed that elevated lactate dehydrogenase (LDH) ratio, organ dysfunction, and multi-organ involvement by PTLD were independent prognostic factors for lack of response to reduction in immunosuppression. In patients with none of these poor prognostic factors, 16 of 18 (89%) responded to reduction in immunosuppression in contrast to three of five (60%) with one risk factor and zero of seven (0%) with two to three factors present. The analysis also showed that increased age, elevated LDH ratio, severe organ dysfunction, presence of B symptoms (fever, night sweats, and weight loss), and multi-organ involvement by PTLD at the time of diagnosis are independent prognostic indicators for poor survival. With median follow-up of 147 weeks, 55% of patients are alive with 50% in complete remission. CONCLUSIONS: Reduction in immunosuppression is an effective initial therapy for PTLD. Clinical prognostic factors may allow clinicians to identify which patients are likely to respond to reduction in immunosuppression.

Factors controlling levels of CD8+ T-cell lymphocytosis associated with murine gamma-herpesvirus infection.

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8+ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4+ CD8+ T cells. The Vbeta4+ CD8+ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4+ CD8+ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8+ T cell activation and Vbeta4+ CD8+ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4+ CD8+ T-cell response correlates with the degree of CD8+ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4+ CD8+ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.

Autogenous vaccine treatment of juvenile laryngeal papillomatosis.

Seventeen cases of juvenile laryngeal papillomatosis have been seen and treated with microlaryngoscopy, removal of papillomas, and administering of autogenous vaccine. Holinger's original findings could be confirmed. The operation frequency of 9 patients was significantly reduced, 5 were improved, and 3 unchanged. In no case was an undesirable reaction to the vaccine observed. Electron microscopy showed no virus-like particles in the papilloma but a section of a vulvar wart did show the virus.

The homing of hematopoietic stem cells to the bone marrow.

In this article, the author discusses some of the most notable aspects of the work of Mehdi Tavassoli and others on the homing of intravenously transplanted hematopoietic stem cells to the marrow. It is well-recognized that homing of stem cells is a highly selective process, perhaps similar to the homing of lymphocytes to lymphoid tissues. The nature of the selectivity of stem cell homing is unclear, however, and may be mediated through a specific homing receptor or through a method of selective capture, retainment, or survival advantage afforded by the marrow. In this article, the focus is on current research in the identification of a specific homing receptor, the potential regulation of such a receptor by cytokines, the homing phenomenon as a multi-step process, and secondary adhesive interactions mediated by known adhesive molecules. These interactions may serve to strengthen the initial recognition and engraftment of stem cells within the hematopoietic compartment of the marrow.

Endothelial mediation is necessary for subsequent hepatocyte uptake of transferrin.

The authors previously have reported on the presence of transferrin (TF) receptors on liver endothelial cells and have shown that hepatic uptake of transferrin-iron (TF-Fe) complexes in the liver is mediated by the endothelium. We now provide evidence that this endothelial cell mediation may be necessary for hepatocyte uptake of TF-Fe complexes. Transport of TF-Fe from endothelial cell to hepatocyte was studied in mixed cell suspensions in which radiolabeled TF-Fe complexes were incubated at 37 degrees C with the two cell populations purified and then mixed in equal ratios. The mixtures were then refractionated at various times after incubation and cell-associated radioactivities measured. Radiolabeled TF was rapidly taken up by the endothelial cell fraction, but radioactivity began to decline in this fraction as it increased in the hepatocyte fraction. In double labeling experiments with 125I-TF-59Fe, both radiolabels moved across the endothelium in parallel fashion, indicating that Fe remains associated with TF during transcytosis. However, in hepatocytes the two radiolabels became dissociated, with Fe remaining cell-associated and TF being recycled. Hepatocyte uptake of processed TF was partially inhibitable by galactan and asialofetuin, indicating that hepatocyte uptake may occur via asialoglycoprotein receptors of hepatocyte.

Homing of hemopoietic stem cells to hemopoietic stroma.

The existing knowledge of the molecular mechanism that underlies the successful engraftment of hemopoietic progenitor cells in their specific stromal microenvironment has been discussed. It appears that membrane lectins on the surface of the stem cell with specificity for galactose with and/or mannose-bearing glycoconjugates on the surface of the stromal cell are involved. This recognition and binding of hemopoietic stem cells which is called "homing" initiates the processes of differentiation, proliferation, and maturation of hemopoietic cells.

Systemic treatment of cancer in the elderly.

The goal of this review is to provide a readable and exhaustive reference in three major areas of geriatric oncology: complications of chemotherapy and radiotherapy, responsiveness of cancer to systemic treatment, social issues in the care of elderly patients with terminal illnesses. The conclusions of this study are: 1. Progressive deterioration of renal function is the most consistent change of aging. Adjustment of doses of renally excreted drugs to individual creatinine clearance may prevent life-threatening myelotoxicity in the elderly. 2. Intensive chemotherapy regimens (acute leukemia, non Hodgkin's lymphoma) cause more serious and prolonged myelotoxicity in the elderly. Elderly are more susceptible than younger patients to cardiotoxicity and central and peripheral neurotoxicity. Age is a poor predictor of complications in other organs or systems. 3. The prognosis of patients with Hodgkin's disease worsens with aging, possibly due to increased prevalence of mixed cellularity histology. It is controversial whether the prognosis of other neoplasias is poorer. Prognosis is not age-related in multiple myeloma. In general, elderly in good performance status may benefit from systemic cancer treatment to the same extent as younger patients, except for Hodgkin's disease. 4. The Informal Support Network, epitomized by the family, appears the most suitable environment to care for the elderly with cancer.

Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma.

BACKGROUND: Activin A is a member of the transforming growth factor-beta superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin. OBJECTIVE: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2-driven mucosal inflammation in a murine model of allergic asthma. METHODS: Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin-sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes. RESULTS: Follistatin was released concurrently with activin A suggesting it acts as an endogenous regulator: peak BAL concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in mediastinal lymph nodes but induction lagged behind the onset of inflammation. Follistatin and activin A immunoreactivity were lost in airway epithelial cells in parallel with goblet cell metaplasia. Exogenous follistatin inhibited the allergen-specific Th2 immune response in mediastinal lymph nodes and mucus production in the lung. CONCLUSION: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Disturbance of the fine balance between activin A and its endogenous inhibitor follistatin may be a determinant of the severity of allergic inflammation or tissue phenotypic shift in asthma.

Specificity of Hematopoietic Stem and Progenitor Cell Homing to Bone Marrow: A Perspective.

Little is known about the hematopoietic stem and progenitor cell membrane recognition and adhesion molecules which mediate their specific patterns of movement into and out of the marrow compartment during steady state hematopoiesis and during pathological conditions. Implicit in the cellular targeting of these cells to marrow stroma, or "homing", is a high degree of molecular specificity. Identification of homing determinants and knowledge of their function in conferring specificity to these events may provide new insight into the localization of hematopoietic stem cells within the bone marrow, directly impacting clinical stem cell transplantation. In addition, a homing protein gene/promoter complex, or a stromal counter-receptor gene, may provide a valuable target for driving expression of gene constructs in early hematopoietic cells.

Advanced Lewis lung carcinoma cured by tiazofurin as a system to study delayed hemopoietic effects of cancer.

Lewis lung carcinoma (LLC) induces a range of hemopoietic alterations in its murine host including progressive anemia, thrombocytopenia, splenomegaly, neutrophilia, and marrow and splenic myeloid hyperplasia. Concentrations of both pluripotent and committed marrow hemopoietic progenitors is increased and the cycling fraction of granulocyte-macrophage progenitors is accelerated. We have developed a way to study whether these hemopoietic effects become long-term consequences of cancer, using LLC-bearing mice with advanced tumor treated with the antineoplastic agent tiazofurin, 2-beta-D-ribofuranosyl-thiazole-4-carboxyamide, NSC 286193 (TZ). LLC mice were treated with a single dose of TZ either 150, 300, or 600 mg/kg, intraperitoneally on day 6 posttumor implant when lung metastases are present and all hemopoietic effects of the tumor are recognizable. Even a single dose of 150 mg/kg of TZ produced a significant survival advantage, and 600 mg/kg resulted in 30% of the animals remaining disease free during a 5-month follow-up. A 6-week treatment schedule was devised, administering TZ intraperitoneally, 600 mg/kg, weekly beginning on day 6. In this group, median survival was not reached after 9 months of follow-up. The only evidence of myelotoxicity produced by intermittent administration of TZ was a mild anemia which was fully reversible 2 weeks after discontinuance of the drug. No difference in white blood cell count, differential count, or platelet count was detected in tumor bearers and controls treated with TZ. Both pluripotent and committed marrow hemopoietic precursors remained unchanged in TZ-LLC, TZ-controls and untreated controls throughout treatment and 2 weeks thereafter. This study demonstrates that TZ-cured LLC mice are suitable to explore late hemopoietic effects of cancer.

High proliferation of early hemopoietic progenitors in tumor-bearing mice.

During the progression of Lewis lung carcinoma (LLC) in C57Bl/6 mice, a neutrophilia with granulocytosis develops. This heightened myeloid response is further reflected in a progressively increasing concentration of precursors CFU-GM (colony-forming unit, granulocyte-macrophage) in the marrow. However, during the final days of tumor growth (day 19) the cycling fraction of CFU-GM declines in LLC mice, although their concentration remains high. It was of interest to explore in the present study whether this decline in proliferation of committed myeloid progenitors was due to exhaustion of the pool of early and late pluripotential stem cells CFU-S (colony-forming unit, spleen). The results indicate that early stem cells experience an initial proliferative burst (day 14) during tumor growth, but fail to replenish their own pool by day 19, suggesting that their self-replicative ability may become exhausted during growth of this cancer. The implications of these findings to conditions of chronic hemopoietic stress are discussed.